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Biotechnology Advances 47 (2021) 107694
Available online 1 January 2021
0734-9750/© 2021 Elsevier Inc. All rights reserved.
Research review paper
Bio-synthesis of food additives and colorants-a growing trend in future food
Lichao Sun
a
, Fengjiao Xin
a
,
*
, Hal S. Alper
b
,
c
,
*
a
Institute of Food Science and Technology, Chinese Academy of Agricultural Sciences, Beijing 100193, People’s Republic of China
b
Institute for Cellular and Molecular Biology, The University of Texas at Austin, 2500 Speedway Avenue, Austin, TX 78712, United States
c
McKetta Department of Chemical Engineering, The University of Texas at Austin, 200 E Dean Keeton St. Stop C0400, Austin, TX 78712, United States
ARTICLE INFO
Keywords:
Food additives
Food colorants
Synthetic biology
Metabolic engineering
Bioproduction
Future food
ABSTRACT
Food additives and colorants are extensively used in the food industry to improve food quality and safety during
processing, storage and packing. Sourcing of these molecules is predominately through three means: extraction
from natural sources, chemical synthesis, and bio-production, with the rst two being the most utilized. How-
ever, growing demands for sustainability, safety and “natural” products have renewed interest in using bio-based
production methods. Likewise, the move to more cultured foods and meat alternatives requires the production of
new additives and colorants. The production of bio-based food additives and colorants is an interdisciplinary
research endeavor and represents a growing trend in future food. To highlight the potential of microbial hosts for
food additive and colorant production, we focus on current advances for example molecules based on their
utilization stage and bio-production yield as follows: (I) approved and industrially produced with high titers; (II)
approved and produced with decent titers (in the g/L range), but requiring further engineering to reduce pro-
duction costs; (III) approved and produced with very early stage titers (in the mg/L range); and (IV) new/po-
tential candidates that have not been approved but can be sourced through microbes. Promising approaches, as
well as current challenges and future directions will also be thoroughly discussed for the bioproduction of these
food additives and colorants.
1. Introduction
Food additives comprise a wide range of functional categories such
as antioxidants, thickeners, stabilizers and emulsiers, avorings,
sweeteners, nutritional additives and colorants and are most broadly
dened as substances added to food or food ingredients to preserve
avor, enhance taste, and/or alter appearance (Martins et al., 2019). A
further class of additives, colorants or color additives, are specically
dened by U.S. Food and Drug Administration (FDA) as “any dye,
pigment or substance which when added or applied to food, drug or
cosmetic, or to the human body, is capable (alone or through reactions
with other substances) of imparting color” (FDA, 2020). Together, these
additives and colorants are critical components that enhance food
safety, quality, and appearance across the entire life-cycle of processing
to consumption. While these molecules are all collectively evaluated by
the joint FAO/WHO Expert Committee on Food Additives (JECFA),
specic rules and regulations vary country. As examples, more than
3,000 food additives and colorants can be found in the EAFUS list
(Everything Added to Food in the United States) of FDA, over 2,000 food
additives and colorants are designated under Chinese Number System
(CNS) and National Food Safety Standard for Uses of Food Additives,
and only 402 substances are assigned to the catalog of European Union
inventory number (“E”-number) as approved by the European Food
Safety Authority (EFSA).
Despite differences in adoption across countries, there is a renewed
interest in developing safe, natural, and sustainable food additives.
Traditionally, these molecules are mainly produced through chemical
synthesis or extraction from natural sources. In contrast, microbial
production of food additives can provide advantages over chemical
synthesis and natural extraction including low-cost starting materials,
controllable cultivation processes and product specicity, and higher
production yields and robustness. Indeed, some commonly used food
additives and colorants have been derived from microbial fermentation
such as Arpink red
TM
from Penicillium oxalicum, riboavin from Ashbya
gossypii, and microalgal astaxanthin from Haematococcus pluvialis.
Recently, an expanded list of about forty European Union-approved food
additives and colorants was reported as compatible with microbe-based
production (Kallscheuer, 2018).
* Corresponding authors.
E-mail addresses: sun2004go@163.com (L. Sun), 2002hongzhi30@163.com (F. Xin), halper@che.utexas.edu (H.S. Alper).
Contents lists available at ScienceDirect
Biotechnology Advances
journal homepage: www.elsevier.com/locate/biotechadv
https://doi.org/10.1016/j.biotechadv.2020.107694
Received 7 September 2020; Received in revised form 24 December 2020; Accepted 27 December 2020
Biotechnology Advances 47 (2021) 107694
2
Over the past few decades, advances in synthetic biology and
biotechnology in general have led to the emergence of many companies
that produce bio-based food additives and ingredients. In fact, among
the top ten funded synthetic biology startups in the rst half of 2020,
four of these companies focus on food biotechnology including Impos-
sible Foods, Apeel Sciences, Memphis Meats, and Nature’s Fynd (Cum-
bers, 2020). These companies all focus on biotechnological additives /
alternatives for food production and enhancement. One of the quickest
rising companies, Impossible Foods, makes plant-based meat alterna-
tives with similar taste, texture and color of animal meat using bio-
engineered heme and soy protein. Additional companies are entering
this space as well. For example, Endless West is a beverage startup
producing products such as articial whiskey directly by mixing edible
alcohol with avor and aroma molecules sourced from nature such as
yeasts. Other start-up companies such as Perfect Day and Clara Foods
have developed technologies to create dairy proteins including casein,
whey, and egg albumen via fermentation for the application as food
ingredients. Notably, the global market of synthetic biology in food area
is projected to reach USD 2,575.2 million by 2024 from 213.1 million in
2019 with a high CAGR (64.6%). To help meet this demand, the engi-
neering and industrialization of microbial cell factories to sustainably
produce food additives and colorants should be strengthened and
accelerated.
In this review, we highlight the current state and potential of mi-
crobial hosts for food additive and colorant molecule production. To do
so, we focus here on current advances for approved food additives and
colorants as well as highlight newer/potential candidate molecules and
conclude with future directions. To organize the broad scope of mole-
cules covered here (Fig. 1), we have coarsely divided these molecules
based on their utilization stage and bio-production yield as follows: (I)
approved and industrially produced with high titers; (II) approved and
produced with decent titers (in the g/L range), but requiring further
engineering to reduce production costs; (III) approved and produced
with very early stage titers (in the mg/L range); and (IV) newer/po-
tential candidates that have not been approved but can be sourced
through microbes. A list of key compounds and their microbial pro-
duction level is provided in Table 1. The molecular structures and food
application area of these microbially produced food additives and col-
orants are displayed in Fig. 2.
2. Recent advance in approved food additives and colorants
2.1. Approved food additives and colorants that have been successfully
industrially sourced using microbes
Microbial production of food additives and colorants has received
attention as a scalable and economically viable manner of production. In
this regard, the industry can leverage many well-established processes
with high titers including molecules such as xanthan, erythritol, 2’-
fucosyllactose, L-glutamate,
α
-galactosidase and riboavin. We high-
light recent advances in overproduction of these molecules here as they
showcase how synthetic biology, metabolic engineering, and other
biotechnological approaches can improve overall production titers and
enabled the large-scale production.
Xanthan is an extracellular polysaccharide (EPS) hetero-
polysaccharide composed of β-1,4-linked glucose units attached by a
branched side chain of glucuronic and mannose units (Jindal and Singh
Khattar, 2018). Owing to its pseudoplastic rheological behavior and
high stability under different pH and temperature, xanthan has broad
use in food as a stabilizer, thickener/viscosier, gelling/lm-making
agent and emulsier. The market size of xanthan gum exceeded 960
million USD in 2019 and was estimated to achieve more than 1.4 billion
by 2026 (Ahuja and Rawat, 2020a). Xanthan gum was rst commer-
cialized in 1970s by CP Kelco Company, United States, and has achieved
a titer of 15-65 g/L and a productivity of 0.12-0.72 g/L/h using aerobic
natural producer Xanthomonas campesteris with an immobilized
Fig. 1. Schematic pyramid of bio-based food additives and food colorants. A simplied schematic of bio-based food additives and food colorants is provided. The
compounds are broadly categorized into four different groups based on the utilization stage and bio-production yield. Representative examples of each group are
displayed in each of the four different layers of the pyramid.
L. Sun et al.
Biotechnology Advances 47 (2021) 107694
3
Table 1
Production of bio-based food additives and colorants using microbes.
Compound Market size (USD) Carbon source or
precursor
Microbial hosts Scale Titer Yield Productivity References
Group I: High g/L bio-production
Xanthan 960 million in 2019 Glucose Xanthomonas campesteris 20-L
bioreactor
62 g/L 0.82 g/g glucose 0.72 g/L/h Amanullah et al., 1998
Erythritol 195 million in 2019 Glycerol Yarrowia lipolytica 5-L bioreactor 224 g/L 0.77 g/g glycerol 0.54 g/L/h Miro´
nczuk et al., 2015
2’-fucosyllactose 76 million in 2027 (estimated HMO
market)
Fucose and lactose Escherichia coli 2.5-L
bioreactor
47 g/L 0.52 mol/mol fucose 0.60 g/L/h Jung et al., 2019
Glucose and lactose Saccharomyces cerevisiae 2-L bioreactor 15 g/L NR 0.22 g/L/h Hollands et al., 2019
Glucose and lactose Yarrowia lipolytica 2-L bioreactor 24 g/L NR 0.44 g/L/h Hollands et al., 2019
Sucrose Escherichia coli 3-L bioreactor 64 g/L NR 0.76 g/L/h Parschat et al., 2020
L-glutamate 15.5 billion in 2023 (estimated) Glucose Corynebacterium
glutamicum
5-L bioreactor 120 g/L NR NR Zhang et al., 2014
α
-galactosidase NR Wheat bran and soybean
meal
Aspergillus foetidus Flask 2572.5 U/g NR NR Liu et al., 2007
Riboavin 7790 million in 2019 Glucose Ashbya gossypii Bioreactor >20 g/L NR NR Sahm et al., 2013
Glucose Bacillus subtilis 5-L bioreactor 26.8 g/L NR NR Lee et al., 2007
Group II: g/L bio-production
Vanillin 493 million in 2024 (estimated) Isoeugenol Bacillus fusiformis Flask 32.5 g/L NR NR Zhao et al., 2005
Ferulic acid Amycolatopsis sp. ATCC
39116
2-L bioreactor 22.3 g/L NR NR Fleige et al., 2016
Ferulic acid Escherichia coli 1-L bioreactor 13.3 g/L NR 2.1 g/L/h Ni et al., 2018a
Nisin 443 million in 2020 Glucose Lactococcus lactis 10-L
bioreactor
15367 IU/
ml
59 mg/g DCW NR Jiang et al., 2015
D-tagatose 1.55 billion in 2020 D-galactose Lactobacillus plantarum NR 255 mM 85% of D-galactose NR Bober and Nair, 2019
Lactose Saccharomyces cerevisiae 2-L bioreactor 37.69 g/L 0.33 g/g lactose 0.126 g/L/h Liu et al., 2019
Limonene 314.1 million in 2020 Glycerol Escherichia coli 3.1-L
bioreactor
3.6 g/L NR 0.15 g/L/h Rolf et al., 2020
Lycopene 126 million in 2020 Glycerol Escherichia coli Flask 925 mg/L 67 mg/g DCW NR Xu et al., 2018b
Glucose Saccharomyces cerevisiae 7-L bioreactor 3.28 g/L NR NR Shi et al., 2019
Glucose Yarrowia lipolytica 5-L bioreactor 374.4 mg/L 60 mg/g DCW NR Zhang et al., 2019
Group III: mg/L bio-production
Rebaudioside M 2 billion in 2019 (stevia sweeteners
market)
Glucose Saccharomyces cerevisiae 2-L bioreactor 2673 mg/L NR NR Mikkelsen et al., 2014
Glucose Yarrowia lipolytica Bioreactor 740 mg/L NR 6.12 mg/L/h Hoeven et al., 2015
Thaumatin 170 million in 2020 Glycerol Pichia pastoris Bioreactor 100 mg/L NR NR Masuda et al., 2010
Sucrose Aspergillus awamori 5-L bioreactor 150 mg/L NR NR Moralejo et al., 2001
Leghemoglobin NR Glycerol Pichia pastoris 2-L bioreactor NR 6%-9% of total protein content NR Shankar and Hoyt,
2019b
Curcumin 58.4 million in 2019 Ferulic acid Escherichia coli Flask 563.4 mg/L ~100% NR Rodrigues et al., 2020
Tyrosine Escherichia coli Flask 15.9 mg/L NR NR Rodrigues et al., 2020
Group IV: emerging bio-production
EPA 4.07 billion in 2019 (omega-3
market)
Glucose Yarrowia lipolytica Flask NR 56.6% of total fatty acids, 15% of
the DCW
NR Xue et al., 2013
Glucose Schizochytrium sp. S31 3-L bioreactor 1.65 g/L 1.41% of total lipids NR Li et al., 2018a
DHA 4.07 billion in 2019 (omega-3
market)
Glucose Schizochytrium sp. S31 3-L bioreactor 47.39 g/L 42.89% of total lipids NR Li et al., 2018b
Naringenin NR Glucose Yarrowia lipolytica 3-L bioreactor 898 mg/L NR NR Palmer et al., 2020
Coumaric acid Saccharomyces cerevisiae 5-L bioreactor 648.63
mg/L
15.6% of coumaric acid NR Gao et al., 2020
Raspberry
ketone
443 million in 2019 Coumaric acid Corynebacterium
glutamicum
Flask 99.8 mg/L NR NR Milke et al., 2020
γ-PGA 840.5 million in 2026 (estimated) Glucose and
L-glutamate
Bacillus subtilis 10-L
bioreactor
101.1 g/L 0.57 g/g total substrates 2.19 g/L/h Huang et al., 2011
(continued on next page)
L. Sun et al.
Biotechnology Advances 47 (2021) 107694
4
Table 1 (continued )
Compound Market size (USD) Carbon source or
precursor
Microbial hosts Scale Titer Yield Productivity References
Glucose Bacillus licheniformis 5-L bioreactor 39.9 g/L NR 0.926 g/L/h Kongklom et al., 2017
Safranal 3.2 billion in 2019 NR NR NR NR NR NR NR
Isobutyl acetate 9.5 billion in 2019 Glucose Escherichia coli 1.3-L
bioreactor
36 g/L 0.18 g/g glucose 0.5 g/L/h Tai et al., 2015
Isoamyl acetate 5344.6 million in 2019 Glucose Escherichia coli Flask 780 mg/L 0.039 g/g glucose 16.25 mg/L/h Tai et al., 2015
Hydroxytyrosol 2.5 billion in 2030 (estimated) Tyrosine Escherichia coli Flask 1243 mg/L NR NR Li et al., 2018a
Glucose Escherichia coli Flask 647 mg/L NR NR Li et al., 2018b
Tyrosine Escherichia coli 5-L bioreactor 4690 mg/L 95% NR Yao et al., 2020b
Betalains NR L-DOPA Escherichia coli 2-L bioreactor 150 mg/L NR NR Guerrero-Rubio et al.,
2019
Glucose Saccharomyce cerevisiae Flask 17 mg/L NR 0.35 mg/L/h Grewal et al., 2018
Indigoidine NR Glucose Escherichia coli Flask 7.08 g/L NR NR Xu et al., 2015
Glucose and
L-glutamine
Escherichia coli Flask 8.81 g/L NR NR Xu et al., 2015
Glucose Saccharomyces cerevisiae 2-L bioreactor 980 mg/L NR NR Wehrs et al., 2018
Glucose Rhodosporidium toruloides 2-L bioreactor 86.3 g/L 0.91 g/g glucose 0.73 g/L/h Wehrs et al., 2019
Lignocellulosic
hydrolysate
Rhodosporidium toruloides 2-L bioreactor 2.9 g/L 0.045 g/g sugar NR Wehrs et al., 2019
Violacein NR Glycerol and
L-tryptophan
Citrobacter freundii 5-L bioreactor 4.13 g/L NR 82.6 mg/L/h Yang et al., 2011
Glucose Corynebacterium
glutamicum
3-L bioreactor 5.436 g/L 0.054 g/g glucose 47 mg/L/h Sun et al., 2016
Glucose Escherichia coli 5-L bioreactor 4.45 g/L NR 98.7 mg/L/h Zhou et al., 2018
Melanin NR Glucose Escherichia coli 1-L bioreactor 3.22 g/L 0.093 g/g glucose 0.004 g/g DCW/
h
Ch´
avez-B´
ejar et al.,
2013
L-tyrosine Armillaria cepistipes Empa
655
Flask 27.98 g/L NR NR Ribera et al., 2019
L-tyrosine Yarrowia lipolytica Flask 0.5 g/L NR NR Ben Tahar et al., 2020
NR, not reported or not found; DCW, dry cell weight.
L. Sun et al.
Biotechnology Advances 47 (2021) 107694
5
fermentation process (Ju and Zhao, 1993; Amanullah et al., 1998;
Kuppuswami, 2014). In Xanthomonas, the gum operon responsible for
xanthan biosynthesis including which encode a series of glycosyl-
transferases (GTs) and this pathway has been modied to improve
production. For example, overexpression of gumB and gumC on a
multicopy plasmid increased the viscosity of xanthan polymer by at least
20% in shake ask (Patel et al., 2008). Outside of modulating pathways,
the inoculum quality, culture medium, and growth condition such as
oxygen, pH, and temperature are also critical factors that inuence the
structures, properties and the yields of xanthan. A stochastic algorithm
method based on differential evolution was applied to optimize the
initial inoculation volume, substrate concentrations and oxygen levels
for its industrial fermentation, increasing the xanthan production by
148.7% in a 2-L fed-batch bioreactor compared to that of conventional
batch cultivation (Chaitali et al., 2008). To reduce downstream puri-
cation costs (especially for the cost of alcohol precipitation), a Vitre-
oscilla globin gene was overexpressed in a xanthomonadin-decient
strain X. campesteris CGMCC15155 to obtain a xanthan gum with lower
pigment but similar yield, molecular weight and rheological properties
(Dai et al., 2019). Likewise, a cell-wall decient mutant of X. campesteris
was obtained by screening the hyper-producer strain during continuous
subculture, reaching 32 g/L of white xanthan gum with 3 times higher
conversion rate (Liu et al., 2018b). A sustainable solution for current
industrialized production of xanthan is the utilization of low-cost
glucose alternative substrates including glycerol, whey, winery waste-
water and lignocellulosic agro-industrial wastes, which has been
explored at scales up to 7-L bioreactor (Wang et al., 2017a; Ronˇ
cevi´
c
et al., 2019; Li et al., 2016; Mohsin et al., 2018; Soleymanpour et al.,
2018; da Silva et al., 2018). For example, 33.9 g/L of xanthan was
produced from glycerol by a glycerol-tolerance strain of X. campesteris in
a 7-L fermenter (Wang et al., 2017b). Synthesis of tailor-made EPSs is an
emerging area for xanthan production. For example, Wu et al. success-
fully designed eight customized xanthan variants with dened second-
ary structures and rheological properties by controlling the molecule
side chains through marker-less gene knockout and overexpression of
specic genes (Wu et al., 2019b). To enable optimized EPSs production,
further engineering of pathways through structural and biochemical
strategies, protein engineering of critical machinery using domain
swapping or targeted shufing techniques, metabolic engineering of
tailor-made EPSs pathways through synthetic biology and innovative
genetic-editing technology are necessary.
Erythritol is a four-carbon sugar alcohol and a popular food
sweetener (Mart˘
au et al., 2020). The global market size of erythritol
exceeded 195 million USD in 2019 and was estimated to reach 310
million by 2026 (Ahuja and Rawat, 2020b). Biochemically, erythritol is
the dephosphorylated and reduced version of erythrose-4-phosphate
(E4P) derived from pentoses phosphate pathway (PPP). Industrial pro-
duction of erythritol started in the early 1990s in Japan and has gained
tremendous attention with improved productivity and yield including
titers of 100-250 g/L using yeasts such as Moniliella Pollinis, Yarrowia
lipolytica, Candida magnoliae, Pseudozyma tsukubaensis, Trichosporon sp.,
Torula sp. or lamentous fungi Ustilago and Aureobasidium sp. (Carly and
Fickers, 2018; Sun and Alper, 2020). To achieve this, metabolic engi-
neering efforts have focused on elevating the carbon ux of PPP through
pathway overexpression, enhancing the uptake of substrate glycerol via
overexpression of glycerol kinase and glycerol-3-phosphate
Fig. 2. Food application areas for bio-derived food additives and food colorants. Using the classication scheme developed here, we display the basic molecules, their
application and molecular structure. Where applicable, the structures of food colorants are depicted using the same color as the colorant.
L. Sun et al.
Biotechnology Advances 47 (2021) 107694
6
dehydrogenase, and inhibiting erythritol consumption by gene deletion
of erythrulose kinase or erythritol dehydrogenase. Likewise, bioprocess
optimization has included lower pH, controlled osmotic pressure, oxy-
gen availability, suitable nitrogen sources and added vitamins and/or
metal ions, as well as fed-batch fermentation and two-stage fermenta-
tion operation. Beyond natural producers such as P. tsukubaensis,
Y. lipolytica is perhaps the most widely explored cell factory for heter-
ologous erythritol production, owing to its GRAS status, synthetic
biology toolset and unique tolerances (Bilal et al., 2020). To accomplish
this, mutagenesis strategies such as ultraviolet irradiation exposure have
been widely employed to obtain erythritol overproducers (Miro´
nczuk
et al., 2015). Qiu et al. utilized a rapid screening method for erythritol
overproducers using a biosensor-regulator in Y. lipolytica based on an
erythritol-responsive transcription repressor EryD to screen a muta-
genesis library and obtain 1.4-fold enhancement of production to yield
148 g/L in a 3-L fermenter after 95 h of fermentation (Qiu et al., 2020).
Additionally, attempts to use cheaper carbon sources such as molasses
and waste cooking oil have been explored to further reduce production
costs (Rakicka et al., 2016; Liu et al., 2017). Other representative sugar
alcohols such as xylitol, occupy 12% of the total polyol market, are also
of high interest and have been extensively studied in natural host such as
Candida tropicalis and engineered microbes Escherichia coli and Saccha-
romyces cerevisiae, reaching high titers near 200 g/L at near theoretical
yield (Park et al., 2016).
2’-fucosyllactose (2’-FL) is an
α
-1,2 linked trisaccharide consisting
of L-fucose and D-lactose (
α
-L-Fuc-(1→2)-β-D-Gal-(1→4)-D-Glc) and has
been widely used as a sugar additive in infant formula as it is the most
abundant and simplest oligosaccharide among more than 200 human
milk oligosaccharides (HMOs) in breast milk (Hegar et al., 2019). Given
that microbial-produced 2’-FL is considered as a safe food additive and
has been approved in 2016, numerous studies have been conducted to
improve 2’-FL production in the past decade (van Berlo et al., 2018). In
microbial hosts, 2’-FL is synthesized by 1,2-fucosyltransferase (fucT2),
by transferring a fucosyl residue from guanosine 5’-diphosphate-L-
fucose (GDP-L-fucose) to lactose, either from de novo GDP-mannose
pathway or from the fucose salvage pathway. Within the pathway, the
presumed rate-limiting enzyme, fucT2, is a target for further improve-
ment (Seydametova et al., 2019). Nevertheless, 2’-FL has been suc-
cessfully produced in various microbes with titers ranging from 0.49 g/L
to 64 g/L in hosts such as E. coli, S. cerevisiae, and B. subtilis (Jung et al.,
2019; Yu et al., 2018; Deng et al., 2019; Parschat et al., 2020). To
accomplish this work, these studies have utilized techniques including
the ne-tuning of the metabolic ux through dynamic control, engi-
neering the redox regeneration, inactivating competitive pathway,
inhibiting the product and substrate degradation, and optimizing cul-
ture conditions. Recently, Hollands et al. identied a promising 2’-FL
transporter, CDT2 from Neurospora crassa, and demonstrated its ef-
ciency in both S. cerevisiae and Y. lipolytica and this has led to titers of 15
g/L in S. cerevisiae and 24 g/L in Y. lipolytica via 2-L fed-batch fermen-
tation, respectively (Hollands et al., 2019). Given the high cost of the
substrate fucose, de novo GDP-mannose-dependent pathway is preferred
for the production of 2’-FL. To this end, construction of a de novo GDP-
fucose pathway in S. cerevisiae through introducing GDP-mannose-4,6-
dehydratase (Gmd) and GDP-4-keto-6-deoxymannose-3,5-epimerase-4-
reductase (WcaG) enabled the conversion of native GDP-mannose to
GDP-fucose. Further engineering of lactose transporter Lac12 and fucT2
facilitated the lactose utilization and the 2’-FL production reached 0.51
g/L from lactose and glucose in shake ask cultures via a batch
fermentation (Liu et al., 2018a). Recently, a more cost-competitive
strategy using sucrose as a sole substrate was developed in E. coli
through the overexpression of de novo UDP-galactose and GDP-fucose
pathways, expression of a novel galactosyltransferase GalTpm1141
and a suitable fucT2 WbgL along with using the Y. bercovieri sugar efux
transporter TPYb. This process reached 64 g/L in a 3-L fed-batch
fermentation (Parschat et al., 2020). These advances along with ef-
cient purication methods establish biotechnologically relevant routes
for 2’-FL production in large-scale processes. Notably, the worldwide
commercialization has been achieved by several companies such as
German company Jennewein biotechnologie GmbH with titers as high
as 180 g/L (Bych et al., 2019). Further studies on developing highly
active fucT2, maintaining the metabolic balance between GDP-fucose
and cell growth and utilization of cost-effective fucose-attached sub-
strates such as natural fucogalactoxyloglucan can improve yields and
reduce costs.
L-glutamate is the major compound offering umami taste. The L-
glutamate market size was estimated to reach more than 4 million tons
and worth USD 15.5 billion by 2023 (Global Market Insights, 2020). L-
glutamate could be converted from 2-oxo-glutarate via glutamate de-
hydrogenase, and the most popular industrial production method is
fermentation using Corynebacterium glutamicum, which was rst intro-
duced in 1957 (Sano, 2009). C. glutamicum naturally secretes low
amounts of L-glutamate via MscS-like channel such as MscCG and
MscCG2 and the secretion could be greatly enhanced via limitation of
biotin or addition of penicillin and surfactant that mainly impact
intracellular metabolism and cell envelope (Hirasawa and Wachi, 2016;
Wang et al., 2018; Kawasaki and Martinac, 2020). Previously employed
metabolic engineering strategies for overproduction of L-glutamate
including pathway engineering and evolutionary approaches have been
well reviewed by Kimura and Ma et al. (Kimura, 2003; Ma et al., 2017).
L-glutamate fermentation is a high-energy consuming process due to the
high agitation rate in bioreactors. To reduce the requirement of high-
speed agitation and the formation of unwanted by-products under
insufcient oxygen condition, Zhang et al. (2014) knocked out the L-
lactate dehydrogenase gene ldhA in C. glutamicum GDK-9, yielding an
enhanced L-glutamate production with a titer of 120 g/L and lower
levels of
α
-ketoglutarate, L-lactate and L-alanine in 5-L fed-batch
fermentation under micro-aerobic condition. In addition, developing
novel efcient high-throughput screening methods with high accuracy
and sensitivity to screen L-glutamate overproducers will be a promising
way to further improve its production. L-glutamate production was
successfully achieved from low cost lignocellulose feedstock with an
industrial strain, C. glutamicum S9114, via attenuation of
α
-oxoglutarate
dehydrogenase complex (ODHC) activity and improvement of secretion
through C-terminal truncation of glutamate channel MscCG (ΔC110),
leading to high level production of L-glutamate from corn stover with a
titer of 65.2 g/L and a yield of 0.63 g/g glucose without chemical
treatments (Wen and Bao, 2019). To reduce the cost and achieve envi-
ronmental sustainability, utilization of such biotin-rich lignocellulose
hydrolysate should be explored further.
Differing from the small molecule products above,
α
-galactosidase
is a glycoside hydrolase enzyme catalyzing the hydrolysis of galactose
residues from non-reducing end of
α
-1,6-linked galactooligosaccharides
(GOS) or branched polysaccharides. This enzyme has been widely used
as approved food additive especially for the removal of non-digestible
rafnose oligosaccharides in soy and sugar beet processing, the prebi-
otic GOS production and the lactose degradation in cheese
manufacturing (Katrolia et al., 2014).
α
-galactosidase natively exists in
many organisms including microbes, plants and animals (Bhatia et al.,
2020). This product has been commercially produced via solid state
fermentation using Aspergillus and S. cerevisiae for food use (Prasad and
Roy, 2017). Fermentation conditions including nutrient sources, inoc-
ulum size, pH and temperature are key factors affecting
α
-galactosidase
production yield, which varies in different strains but could be opti-
mized via response surface methodology analysis (Gurkok et al., 2011;
´
Alvarez-Cao et al., 2019). For example, the solid substrate and essential
elements for
α
-galactosidase production was optimized to be 8.2 g wheat
bran, 1.8 g soybean meal, 0.001 g MnSO
4
•H
2
O and 0.001 g
CuSO
4
•5H
2
O in 10 g dry matter medium via a fractional factorial design
(FFD) and the central composite experimental design (CCD), reaching
2572.5 U/g dry matter after 144-h cultivation in solid-state ask
fermentation (Liu et al., 2007). To meet the demands across various food
processing processes, numerous
α
-galactosidases with distinct
L. Sun et al.
Biotechnology Advances 47 (2021) 107694
7
characteristics such as higher thermostability, better acidic property,
increased protease-resistance and improved activity have been isolated
broadly (Lee et al., 2017; Zhang et al., 2018; Zhao et al., 2018a). Protein
engineering strategies have also been applied for improving the char-
acteristics and increasing the yield (Xu et al., 2014; Xu et al., 2018b).
Engineering of
α
-galactosidases production together with other useful
enzymes such as carbohydrases and pectinases within the same Asper-
gillus strain also provided a unique economical strategy for enzymes
production in this area (Li et al., 2020a). Besides, utilization of agro-
industrial wastes such as whey, beet molasses and even kitchen wastes
is an important topic in this area to reduce the cost (´
Alvarez-Cao et al.,
2018).
Riboavin (vitamin B2) is a heterocyclic isoalloxazine derivative
and an approved yellow food colorant and dietary supplement. Large-
scale production of industrial riboavin started in 1990 by BASF com-
pany, and total worldwide market was valued at roughly 7.8 billion USD
in 2019 and was expected to reach 10.3 Billion in 2024 (Fior Markets,
2019). Riboavin is currently produced via fermentation of natural host
Ashbya gossypii from triacylglyceride or genetically engineered Bacillus
subtilis from glucose with titers near 30 g/L (Lee et al., 2007; Sahm et al.,
2013). The industrial overproducers were established mainly through
traditional mutagenesis and selection methods as well as some modern
metabolic engineering approaches. Riboavin is synthesized via seven
enzymes cascade transcribed by rib operon by building upon the pre-
cursors of ribulose-5-phosphate and guanosine triphosphate (GTP) (Liu
et al., 2020). Overexpression of the rib genes, improvement of purine
precursors availability through enhanced synthesis of glycine precursor,
overexpression of purine pathway and rewiring of metabolic ux toward
the building blocks on the targeted strain are all effective engineering
strategies to improve the yield (Revuelta et al., 2016). A comparative
analysis showing droplet uorescence activated cell sorting (FACS)
screening method outperformed single cell FACS in both extracellular
and total riboavin production in Y. lipolytica indicated the importance
of riboavin secretion and the ability to sort for this phenotype via
droplet sorting (Wagner et al., 2018). An extensive search on avin
mononucleotide (FMN) riboswitch identied some novel riboavin
transporters of bacteria and highlighted the co-occurrence of riboavin
transporters and riboavin biosynthesis (Guti´
errez-Preciado et al.,
2015). However, most identied riboavin transporters have only been
experimentally characterized as riboavin importers but not exporters,
and their expression such as RibM from Streptomyces davawensis in B.
subtilis only enhanced riboavin yield at a slight level (5%) (Hemberger
et al., 2011). Thus, homology search and experimental validation of
specic and efcient riboavin exporters, improvement of riboavin
export and hence product secretion would be more benecial for con-
struction of robust producers. Considering the unknown aspects asso-
ciated with some of these industrial producers, approaches such as the
transposon insertional mutagenesis and omics-based techniques should
be further applied to identify potential key genes to enable the maximal
production. Extensive studies have also been conducted to optimize the
parameters of pH, aeration, temperature as well as the downstream
separation process for industrial fermentation of riboavin (Schwech-
heimer et al., 2016). For example, the optimized fed-batch production
process using B. subtilis involved a lower agitation speed (600 rpm) in
cell growth stage and a higher speed (900 rpm) in the riboavin pro-
duction stage. Although high amount of riboavin product is crystal-
lized naturally after the fermentation, additional washing and
crystallization steps are still required for the purication of food-grade
riboavin. Beyond these industrial producers, engineering of other a-
vogenic yeast such as Candida have also yielded titer of 21 g/L from
glucose using a 450-L fermentation tank (Heefner et al., 1988). Non-
avogenic strains such as E. coli have been engineered and got up to
2.7 g/L of riboavin with a yield of 137.5 mg/g glucose in shake ask
batch fermentation (Lin et al., 2014). In contrast, engineering of other
hosts such as Clostridium, Lactobacillus, Lactococcus, Aspergillus, Coryne-
bacterium, Pichia and Yarrowia resulted in less than 1 g/L. In a very
recent study, a riboavin-producing Lactobacillus plantarum was
screened, mutated and applied in the soymilk processing, resulting in a
fermented functional food rich in riboavin (2.9 mg/L) (Ge et al., 2020).
Thus, riboavin-enriched functional foods can leverage engineered or-
ganisms beyond production as a bulk additive.
2.2. Approved food additives and colorants that can be produced using
microbes with decent titers
Moving from the popularly used additives in the last section, there
are many compounds for which bioproduction is in the g/L range and
further optimization will be needed to realize full industrialization. As
expected, some companies have already established pilot plants for
testing production. For example, the biotechnology rm Solvay
launched a natural vanillin named Rhovanil produced from ferulic acid
via fermentation using yeast, and the product was labeled as a “natural
avor” in US and Europe (Watson, 2017). In this section, we highlight a
few molecules that t within this category, address many of the chal-
lenges including reducing metabolic imbalance, product toxicity, and
byproduct formation, and further provide some feasible ideas to facili-
tate the industrialization process.
Vanillin (4-hydroxy-3-methoxybenzaldehyde) is a popular aromatic
avoring compound native to vanilla bean in trace amounts (Braga and
Faria, 2020), its market was forecasted to reach 493 million USD by
2024 (Mordor Intelligence LLP, 2019). Currently, more than 99% of the
vanillin in the market comes from chemical synthesis, but there is a
growing demand for more natural sources. Many bacteria such as Ba-
cillus, Pseudomonas, Amycolatopsis and Streptomyces have the ability to
produce vanillin from ferulic acid, eugenol or isoeugenol with produc-
tion titers ranging from 0.1 to 32.5 g/L (Zhao et al., 2005; Kaur and
Chakraborty, 2013). Current bottlenecks for vanillin production are the
unwanted byproducts and the products cytotoxicity. To reduce
byproducts, specic approaches including the deletion of key vanillin
degradation enzymes, de novo production of vanillin using heterologous
hosts such as E. coli and yeast, as well as adopting cascade enzymatic
methods have been made (Fleige et al., 2016; Kunjapur et al., 2014; Ni
et al., 2015; Hansen et al., 2009; Yao et al., 2020b). To reduce cyto-
toxicity, efforts have focused on evolution to improve tolerance, con-
verting vanillin to less toxic product such as vanillin glucoside, as well as
optimizing the bioprocess using sol-gel technology (Yoon et al., 2007;
Brochado et al., 2010; Luziatelli et al., 2019). Despite ample previous
efforts in the de novo biosynthesis from glucose, vanillin production
using native precursor ferulic acid is potentially more preferred in the
food industry as this pathway is much simpler and ferulic acid can be
obtained from nature with high abundance. In this regard, current ap-
proaches have enabled the highest titers of vanillin from ferulic acid
increased to 22.3 g/L via a 2-L fed-batch fermentation of Amycolatopsis
sp. ATCC 39116, reaching a decent titer required for industrial pro-
duction (Fleige et al., 2016). However, metabolic engineering efforts are
still needed to create a cost-competitive product as current price of bio-
based vanillin is several hundreds of dollars per kilogram higher than
that of chemically synthetic vanillin (USD 10 per kg). Further systematic
studies on native producers via omics-based technology and the precise
rewiring of vanillin metabolism using overexpression and CRISPR
techniques should lead to higher yields. Managing the trade-off between
cell growth and production through dynamic control is an alternative
promising strategy to solve the toxicity problem. Recently, the use of the
uric acid regulatory protein HucR created a multi-layer dynamic control
of vanillin biosynthesis and improved the production efciency (Liang
et al., 2020). Future efforts using such dynamic control can help balance
the industrial production of toxic products including vanillin. Another
alternative promising improvement strategy is the whole-cell trans-
formation using genetically engineered strains. The best characterized
pathway of vanillin biosynthesis is a ferulic acid-based coenzyme A
(CoA)-dependent non-β-oxidative cascade, which consists of feruloyl-
CoA synthetase (FCS) and enoyl-CoA hydratase (ECH). Using the
L. Sun et al.
Biotechnology Advances 47 (2021) 107694
8
E. coli whole-cell system with new thermostable FCS and ECH under a
higher temperature (50 ◦C) that the vanillin degradation enzymes are
not active, Ni et al. was able to obtain 3.55 mM of vanillin from 5 mM of
ferulic acid with a productivity of 1.1 g/L/h productivity of 1.1 g/L/h
(Ni et al., 2018a). This productivity was further improved to 2.1 g/L/h
(with titer of 13.3 g/L) in a 1-L bioreactor through a temperature/pH-
directed strategy via the use of a highly efcient and thermostable
phenolic acid decarboxylase and aromatic dioxygenase as the
coenzyme-free catalyst (Ni et al., 2018b). Future efforts in rational
design of coenzyme-independent enzymes or searching for more robust
catalysts along with immobilization strategy should be useful for
enhancing the performance of whole-cell transformation in industrial
production of vanillin.
Nisin is an antimicrobial oligopeptide and the only bacteriocin
commercially used as approved food preservative with an estimated
market value of 443 million USD in 2020 that is projected to reach 553
million by 2025 (Markets and Markets, 2020b). As the most known
lantibiotic, nisin is naturally generated by Lactococcus lactis via enzymes
encoded by nisABTCIPRK and nisFEG. Nisin variants, including nisin A,
Z, Q, U and U2, F and H, have been isolated from L. lactis and other
bacteria such as Streptococcus uberis and Streptococcus hyointestinalis
(¨
Ozel et al., 2018). Although nisin has been industrially produced via
fermentation of L. lactis in whey followed by direct recovery from
fermentation broth with foaming fractionation, the high cost resulted
from the limited production yield restricts total supply. Efforts for
improving production have been made successfully through increasing
the biomass, relieving the feedback inhibition by nisin and lactic acid,
enhancing the expression of biosynthetic genes, strengthening the acid
tolerance and optimizing the fermentation conditions (¨
Ozel et al., 2018).
Main fermentation factors inuencing nisin production in L. lactis such
as carbon and nitrogen source, pH, aeration, temperature and immobi-
lization strategies have been summarized in a recent review (Khelissa
et al., 2020). High levels of nisin with 15367 IU/ml have been achieved
in aerobic fed-batch fermentation using a variable feeding rate (Jiang
et al., 2015). As evidenced in many cases such as cell wall remodeling,
future host engineering for better cell robustness during fermentation
may be main targets to further improve the yield (Zhang et al., 2016;
Hao et al., 2017; Cao et al., 2018; Wu et al., 2020).
D-tagatose is a fructose isomer and approved low-calorie sweetener
(Ashwell et al., 2020). The market of low-calorie sweetener including D-
tagatose has been projected to reach 1.55 billion USD by 2020 (Markets
and Markets, 2016). D-tagatose can be enzymatically produced from
galactose by L-arabinose isomerase based on the “Izumoring strategy”,
reaching a high yield of 158 g/L and productivity of 7.9 g/L/h with a
lower conversion rate of 52.7% (after 24 h) (Lim et al., 2007). An un-
precedented production with a titer of 370 g/L and a conversion rate of
74% was further obtained in this study via the addition of boric acid,
which could be removed more than 99% via Amberite IRA-743 and
Dowex X50X8 resins (4:1, v/v). Bober and Nair also developed a cellular
encapsulation system using a permeablized L. plantarum with anchored
Lactobacillus sakei L-arabinose to achieve a higher conversion rate (85%
after 48 h) but a lower productivity of 5.3 g L
-1
h
-1
(after 6 h) starting
from 54 g/L (300 mM) of galactose (Bober and Nair, 2019). Although
the highest titer of enzymatic approach meets the industrial require-
ment, this traditional manufacturing process was not favored in industry
due to the high costs of hydrolyzing lactose to galactose and removing
the remaining galactose from the product mixture. Alternatives to this
enzymatic conversion is to use microbes as tagatose factories by rewir-
ing of metabolism such as with converting lactose into galactose by
overexpression of galactose kinase, followed by the intracellular con-
version of galactose into tagatose by heterologous xylose reductase (XR)
and galactitol dehydrogenase (GDH). Through ne-tuning the expres-
sion ratio of XR and GDH in yeast strain EJ2, a nal tagatose production
titer of 37.7 g/L and productivity of 0.126 g/L/h was achieved in a 2-L
bioreactor with a tagatose to galactose ratio of 9:1, which was much
higher than the enzymatic conversion rate (Liu et al., 2019). The
remaining galactose in the fermentation broth could be easily removed
by adding galactose-consuming yeast for near-complete conversion of
galactose to D-tagatose. This engineered yeast may unlock the
commercial-scale production of D-tagatose if low-cost substrates such as
whey waste could be further utilized. Certainly more robust enzyme
catalysts with high activity and stability would be benecial to achieve
complete conversion, thus to increase productivity and decrease costs.
Limonene is a cyclic monoterpene and a GRAS versatile compound
that widely used in food and drinks industry. The global market is
valued at 314.1 million USD in 2020 and is projected to reach 379.2
million by 2026 (Market Watch, 2020a). Current production either
through plant extraction or chemical synthesis is limited and does not
meet the quality requirement, leading to its relatively high price.
Limonene can be synthesized in plants via limonene synthase from
geranyl diphosphate (GPP), which is condensed by GPP synthase using
one molecule of IPP and DMAPP through methylerythritol 4-phosphate
(MEP) pathway. In E. coli, MEP pathway employs the natively existed
pyruvate or glyceraldehyde-3-phosphate (G-3-P) as precursors to pro-
duce limonene (Willrodt et al., 2014). To further improve the produc-
tion, the mevalonate (MVA) pathway forming IPP and DMAPP from
acetyl-CoA based on intermediate neryl diphosphate (NPP) was con-
structed in E. coli and a systematic optimization strategy was employed,
leading to 1.3 g/L of limonene from glucose in shake-ask fed-batch
fermentation (Wu et al., 2019a). Using a similar strategy in the presence
of a non-toxic organic phase, 3.6 g/L limonene was obtained from in
E. coli via fed-batch fermentation using a 3.1-L stirred-tank bioreactor,
the highest titer achieved so far (Rolf et al., 2020). Yeast such as
S. cerevisiae could be an ideal host for terpene production owing to the
advantages in expression of terpene synthases and abundant pools of IPP
and DMAPP. An efcient MVA pathway consisting of NPP synthase
SlNDPS1 and limonene synthase CltLS2 was engineered into S. cerevisiae
leading to 917.7 mg/L of limonene in shake-ask fed-batch fermentation
(Cheng et al., 2019). It should be noted that although microbial pro-
duction of enantio-specic limonene has been labeled as “natural” and
presents sustainability especially when using cheap feedstock as sub-
strates, the titer still needs to be increased near two orders of magnitude
to provide a stable and scalable source with the price of plant-derived
limonene (Jongedijk et al., 2016). The pathway ux, the cofactor bal-
ance, GPP synthase and limonene synthases activity as well as efcient
secretion are all potential targets to improve the production, which
partially relies on time-consuming combinatorial tuning. Recently, a
multiplexed cell-free approach was built for rapid optimization of the 9-
step biosynthetic pathway through in vitro prototyping of cofactors
supply and enzymes performance, reaching a titer of 610 mg/L (Dudley
et al., 2019, 2020). Such methods may serve as useful tools to provide
quick design-build-test data for further cellular metabolic engineering as
well as to provide for a potentially easier process with respect to puri-
cation and removal of cell toxicity. Detailed strategies and future
perspectives for microbial production of limonene can be found in an
extensive review published recently by Ren et al. (Ren et al., 2020).
Beyond limonene, other terpenoid fragrance compounds such as
valencene and nootkatone are also of high interest in aroma industry
and have been commercially produced via fermentation by Evolva and
Isobionics companies using yeast and/or Rhodobacter sphaeroides
(Schempp et al., 2018).
Lycopene, as well as other carotenoids, are well-sought out food
colorants and dietary supplements. Lycopene in particular is a popular
C
40
carotenoid with eleven conjugated and two non-conjugated double
bonds (Ranveer, 2018). Its global market was estimated to be 126
million USD in 2020 and was projected to be 161 million by 2025
(Markets and Markets, 2020a). The biggest competition to fermentation
is commercial extraction from tomatoes. As a tetraterpenoid, lycopene
can be synthesized from three molecules of isopentenyl diphosphate
(IPP) and one molecule dimethylallyl diphosphate (DMAPP) through a
sequential condensation pathway. De novo biosynthesis of lycopene has
been extensively studied in native hosts such as carotenogenic fungus
L. Sun et al.
Biotechnology Advances 47 (2021) 107694
9
Blakeslea trispora (Sevgili and Erkmen, 2019), and haloarchaea Hal-
oferax mediterranei (Zuo et al., 2018), and heterologous microbes such as
E. coli (Xu et al., 2018a), S. cerevisiae (Shi et al., 2019), and Y. lipolytica
(Zhang et al., 2019) have been well-reviewed by others (Hern´
andez-
Almanza et al., 2016). Specically, the engineering of Haloferax medi-
terranei via redirection of carbon ux to lycopene and overexpression of
heterologous enzymes along with inhibition of bacterioruberin biosyn-
thesis resulted in a superior yield of 119.25 mg/g DCW in shake ask
fermentation (Zuo et al., 2018). However, both this strain and native
producer Blakeslea trispora that can be easily applied in industrial
fermentation without any specic growth conditions, or E. coli that also
produced high level of lycopene (925 mg/L and 67 mg/g DCW in shake
ask fermentation) are not favored in food industry due to their safety
issues raised by cyclase inhibitors addition or endotoxin release. Efforts
to improve production in other hosts especially GRAS yeast S. cerevisiae
commonly revolve around increasing precursors levels, removing
competing pathways, enhancing biosynthetic genes, overexpressing
rate-limiting enzymes, cofactor supply, engineering synthetic regulatory
circuits as well as optimizing the fermentation process. These pathway
engineering strategies in combination with the host engineering of
distant genetic loci and distinct cell mating types of S. cerevisiae resulted
in a 22-fold increase in lycopene production to achieve a high yield (55.6
mg/g DCW) with titer of 1.65 g/L in a 5-L bioreactor (Chen et al., 2016).
Ma et al. found that the combination of systematic metabolic engineer-
ing with lipid engineering strategies promoted high-level production of
lycopene in a 7-L fed-batch fermentation with a titer of 2.37 g/L and a
higher yield (73.3 mg/g DCW) (Ma et al., 2019). This study demon-
strated the benecial effect of lipid metabolism in lycopene biosynthesis
and showcased the use of non-oleaginous organisms in lycopene pro-
duction. The highest titer (3.3 g/L of lycopene in S. cerevisiae) that close
to the level required for industrialization was achieved by the same
group in a 7-L fermenter using systematic metabolic engineering and
optimized two-stage fed-batch fermentation (Shi et al., 2019). Further
hyper-production of lycopene in S. cerevisiae is limited by cell capacity
and cytotoxicity, which might be addressed by enlarged cell storage
capacity to provide more space for lycopene-containing droplets and/or
increased secretion ability through transporters. As evidenced in E. coli,
expression of a ubiquitous ABC transporter MsbA (containing a substi-
tution I89T) from Salmonella enterica ser. typhymurium successfully
resulted in a 4.3-fold increase of secreted lycopene (Doshi et al., 2013),
therefore, searching for suitable exporters with broad poly-specicity
might be helpful for lycopene secretion in different microorganisms.
Another remaining challenge with this highly hydrophobic molecule is
the extraction and separation that can be increased via secretion and/or
in situ crystallization, topics that are being explored currently. Beyond
S. cerevisiae, other GRAS yeast such as Y lipolytica showing great com-
mercial interest with a high yield of lycopene (60 mg/g DCW in 5-L
bioreactor) should also be extensively studied in a similar way (Zhang
et al., 2019).
2.3. Approved food additives and colorants with very early stage titers (in
the mg/L range)
A number of approved food additivities and colorants are sourced via
low-yielding extractions from natural sources, thus opening the door for
a microbial production approach. However, microbial bioproduction is
not always easy and the titers can still be very low initially. In this re-
gard, we highlight molecules of this class in this section including the
hyper-sweet Rebaudioside M and thaumatin, soy leghemoglobin, and
polyphenol curcumin. While heterologous production has been ach-
ieved, early stage titers in the mg/L range still provide more challenges
in the eld compared to the molecules described above. In many cases,
incomplete biosynthetic pathways, the complexity and instability of
target compounds, the promiscuity and limited activity of biosynthetic
enzymes, and the product toxicity are the predominant challenges for
molecules within this category.
Rebaudioside M (Reb M) is a hexa-glucosylated 1,2-stevioside
identied from Stevia rebaudiana, and has received approval by Health
Canada as a next-generation and non-calorie sweetener (Amyris Inc.,
2020). There are more than 35 steviol glucosides (SGs) in leaves of
S. rebaudiana, including the high abundant tetra-glucosylated rebau-
dioside A, rhamnose moiety-attached tetra-glucosylated rebaudioside C,
the less abundant penta-glucosylated rebaudioside D and Reb M (Pra-
kash et al., 2014). The global sweetener market is 90 billion USD and
stevia sweeteners are around 2 billion (Watson, 2019). In contrast to
existing stevia sweeteners such as Reb A that have been used in the food
and beverage industry for many years, low abundant SGs such as Reb D
and Reb M offer superior avors with higher sweetness intensity and
much lower lingering bitterness. Focus on such pure products highlight
the importance of understanding the biochemical basis of mixtures and
extracts. The biosynthetic pathway for SGs from greanylgeranyl
diphosphate (GGPP) has been identied from S. rebaudiana and involves
cyclization by two diterpene cyclases to form (-)-kaurene which is
further oxidized and hydroxylated prior to SG production via a series of
UDP-glycosyltransferases (UGTs). Heterologous production of Reb M in
S. cerevisiae has been achieved and enhanced via increasing the copy
numbers of rate limiting enzyme steps, thus allowing for 2.7 g/L in 2-L
fed-batch fermentation (Mikkelsen et al., 2014). To reduce the pro-
miscuity of key enzymes, the structure of UGT76G1 was modeled and
subsequently mutated to increase the accumulation of Reb M by 1.3-fold
in variants T55K and K337 along with a decrease of side-products level
(Olsson et al., 2016). Reb M can also be produced in the GRAS organism
Y. lipolytica with a production of 740 mg/L or obtained via enzymatic
conversion of Reb A or Reb D with a titer more than 30
μ
M (29 mg/L)
(Hoeven et al., 2015). The downstream purication process of SGs
including Reb M from fermentation broth has been established using
adsorbent-based recovery strategy (Galaev, 2015) and thus the future
looks promising for microbial production of high purity and high yield
Reb M despite some further strain engineering required. It should also be
noted that targeted enzyme modication of Stevia leaf extract such as
non-cognate glucosylation that add extra glucose residues to natural SGs
may facilitate the production of non-natural glycosides with important
variations on the avor and the sweetness quality.
Thaumatin is a high-intensity sweet protein consisting of 207 amino
acids with 8 disulde bonds. Its global market was expected to reach 220
million USD by 2024 from 170 million in 2020 (Market Watch, 2020b).
The natural thaumatin isolated from the Katemfe fruit of tropical plant
Thaumatococcus daniellii Benth was identied as a mixture of two major
isoforms (thaumatin I and II) and three minor isoforms (thaumatin a, b
and c) (Joseph et al., 2019). Of strong interest, thaumatin elicits a
sweetness 3000-fold higher than sucrose at a concentration of 50 nM
with negligible health concerns, thus earning its approval as a low-
calorie food sweetener and avor enhancer for over 35 years (EFSA
ANS Panel, 2015). Due to the limitation of natural thaumatin, recom-
binant thaumatin has been produced in microbial hosts including E. coli,
B. subtilis, S. cerevisiae, Pichia pastoris, Streptomyces lividans and Asper-
gillus awamori (Joseph et al., 2019). Although the highest titer has been
achieved in a 5-L fermenter (near 150 mg/L and over 5 mg/mg DCW)
using a strain of containing 3.2-fold higher level of protein disulde
isomerase, the complex physiology of A. awamori hampers the potential
as an efcient factory. (Moralejo et al., 2001). In contrast P. pastoris
appeared as a more preferred host owing to its well-established platform
for protein expression, post-translational modication, efcient secre-
tion system. In this host, current titers of thaumatin I and thaumatin II
isoforms are in a range of 15-100 mg/L and 25-50 mg/L, respectively
(Masuda et al., 2010; Joseph et al., 2019). There is still great space to
move forward as P. pastoris has strong capability to produce proteins
with quantities as high as g/L. Some innovations during the traditional
downstream separation process (mainly through protein purication)
are still requires to reduce the costs, extracellular production along with
a simple ltration-based purication process is more efcient as limited
amount of native proteins were secreted from P. pastoris. In this regard,
L. Sun et al.
Biotechnology Advances 47 (2021) 107694
10
the protein stability should be extensively studied for the secreted pro-
duction. Selection of suitable signal peptides and chaperone proteins has
been demonstrated to help secretion and folding (Healey et al., 2017).
Likewise, other well-established and widely used hosts for recombinant
proteins production in the food area (such as B. subtilis) needs to be
further explored in this area.
Leghemoglobin (LegH) is a hemoprotein consisting of one poly-
peptide globin and an iron-containing heme B cofactor with a molecular
weight of 16 kDa (Fraser et al., 2018). There is growing interest in this
protein as it exhibits function identical to animal hemoglobin and
myoglobin, which have been safely consumed in the human diet. More
specically, the increasing demand for meat alternatives (both cultured
meat and plant-based meat) has sparked an interest in bulk-production
of novel heme containing proteins such as LegH from soybean (Glycine
max) to facilitate uniquely meaty color and avor (Ismail et al., 2020).
Adding to this demand, soy LegH produced from P. pastoris has been
approved as a safe color additive by the FDA for ground beef analogue
products and other meat alternatives (Fraser et al., 2018; Jin et al.,
2018). To this end, Impossible Foods Inc. synthesized soy LegH in
P. pastoris via overexpression and genomic integration of codon opti-
mized soy LegH c2 (LGB2) gene and a series of native heme B biosyn-
thetic genes encoding 5-aminolevulinate (ALA) synthase, ALA
dehydratase, porphogilinogen deaminase, uroporphyrinogen (UPG) III
synthase, UPG III decarboxylase, coproporphyrinogen (CPG) oxidase,
protoporphyrinogen (PPG) oxidase, and ferrochelatase to achieve at
least 6% of heme-loaded LegH protein with more than 65% purity after
fed-batch fermentation and ltration-based recovery (Fig. 3) (Shankar
and Hoyt, 2019a). Alternative GRAS hosts such as Bacillus and
S. cerevisiae have been used to produce various hemoglobins including
LegH (Fraser et al., 2017; Liu et al., 2014). Novel strategies involving
current microbial synthesis of hemoglobins have been extensively
summarized in a recent review (Zhao et al., 2020). Outside of the protein
itself, high-efciency heme biosynthesis is a prerequisite for the proper
LegH production, and thus metabolic engineering efforts have explored
iron uptake and heme transmembrane transport as well as rational
design of rate limiting enzymes. Specically, Zhao et al. hyperproduced
free heme in engineered E. coli using an effective C5 ALA biosynthetic
pathway (Zhao et al., 2018b). By optimizing metabolic ux as well as
deletion of genes in the heme degradation pathway (yfeX) and lactate
and acetate formation pathway (ldhA and pta) along with over-
expression of heme exporter CcmABC allowed for secreted free heme
production to reach 73.4 and 151.4 mg/L from glucose only and glucose
added with L-glutamate in fed-batch fermentations, respectively. These
production hosts will allow for further studies into the expression of
varying hemoprotein variants that would benet from a balanced globin
expression and heme biosynthesis for proper protein maturity and
folding.
Curcumin is a set of diarylheptanoid compounds (C
6
-C
7
-C
6
) and
approved yellow colorant that is mainly isolated from the rhizome of
Curcuma longa, and can also be found in other Curcuma species such as
Curcuma zedoaria, Curcuma phaeocaulis, Curcuma aromatica, Curcuma
mangga and Curcuma xanthorrhiza. The term curcumin is usually used to
describe three distinct curcuminoids that make up curcumin (~77% of
curcumin, ~18% of demethoxycurcumin and ~5% of
Fig. 3. Biosynthesis of Leghemoglobin in Pichia pastoris. The biosynthetic pathway of heme is indicated with arrows with the enzyme name displayed adjacent to the
arrows. Mature leghemoglobin (heme-loaded) is created by the folding of apo-leghemoglobin and heme B. Both apo-leghemoglobin and mature leghemoglobin are
displayed as homodimers.
L. Sun et al.
Biotechnology Advances 47 (2021) 107694
11
bisdemethoxycurcumin) (Rodrigues et al., 2015). The global curcumin
market size was valued at 58.4 million USD in 2019 and was expected to
reach 152.0 million by 2027 (Grand View Research, 2020). Curcumi-
noid molecules can be synthesized from various hydroxycinnamic acid
derivatives in plants by type III polyketide synthases (PKSs). Although
the key PKSs involved in curcuminoids biosynthetic pathway have been
identied for over ten years, heterologous production of these curcu-
minoids is still at an early stage with primary work established in E. coli
through synthetic biology and metabolic engineering strategies (Kim
et al., 2017). Recently, Rodrigues et al. developed a combinatorial
approach using an optimized articial biosynthetic pathway along with
a co-culture system to enable 41.5 mg/L of total curcuminoids and 15.9
mg/L of curcumin produced from tyrosine, the highest values to date
(Rodrigues et al., 2020). The rst trials for bisdemethoxycurcumin
biosynthesis in Y. lipolytica and P. putida were recently conducted
achieving 0.17 mg/L and 2.15 mg/L respectively (Palmer et al., 2020;
Incha et al., 2020). Likewise, curcuminoids derivatives including un-
natural curcuminoids were also achieved through exogenous precursor
feeding (Katsuyama et al., 2010). While an additive of interest, there are
major challenges associated with the limited activity of curcuminoids
synthases as well as downstream product toxicity. Future efforts are
required here to establish more robust microbial hosts along with better
enzymatic activity for the synthases to produce titers on par with the
levels of other polyketides and avonoids. High-throughput methods
have been widely used in the screening of polyketides for the
improvement of metabolic balance and for the identication of over-
producers. Although there is no direct biosensor of curcuminoids, the
biosensors of the same precursors-derived polyketides could be applied
as an indicator in the high-throughput screening. The production of
specic curcuminoids with higher purity is another important direction
for future endeavors, which might be improved through the increased
supply of specic precursors and the targeted engineering of the CoA
ligase and/or curcuminoids synthases.
3. Recent advance in newer/potential food additives and
colorants
3.1. Newer/potential food additives
Moving beyond the list of current and long-approved food additives,
there are several popular compounds that have been widely used in the
food/supplement area and received letters of no objection concerning its
GRAS status from FDA, but not typically seen as explicit food additives.
In this section, we evaluate the production of such molecules including
nutritional additives omega-3 eicosapentaenoic acid (EPA), docosa-
hexaenoic acid (DHA), and naringenin, avoring agents raspberry ke-
tone and poly-γ-glutamic acid, fragrance compounds safranal, isobutyl
acetate and isoamyl acetate, as well as antioxidant hydroxytyrosol. In
most cases, microbes are being explored in the laboratory for the pro-
duction of these molecules, however, production yield is still low and
process scale-up remains challenging.
EPA and DHA are highly valued essential omega-3 polyunsaturated
fatty acids (PUFAs) that are recently being used as nutritional additives
and enhancers (Tocher et al., 2019). The omega-3 market has been
estimated to reach 8.52 billion USD by 2025 from 4.07 billion in 2019
(Markets and Markets, 2019b). De novo production of EPA and DHA
using microalgae such as Schizochytrium, which produces high level of
PUFAs especially high amount of DHA (42.9% of the total lipids, 26.7%
of the dry cell weight) is seen as a promising way to meet the increasing
market and environmental demands while at the same time reducing
CO
2
(Li et al., 2018b; Wang et al., 2019b). To deal with the scale-up
issues of microalgae, alternative microbial platforms have been
explored such as oleaginous yeast and oilseed crops. For examples,
DuPont succeeded in producing high levels of EPA in shake ask culture
(56.6% of total lipids, 15% of the dry cell weight) in Y. lipolytica through
EPA biosynthetic pathway integration along with β-oxidation disruption
and TCA recycling (Xue et al., 2013). Genetically modied oilseeds such
as Camelina sativa provide both EPA-only and EPA-DHA oils at a level
that is equivalent to that from sh oil (Han et al., 2020). Attempts to
produce other PUFAs such as
α
-linolenic acid (ALA) have seen successes
in Y. lipolytica with contents reaching the same level as the content seen
in axseed oil (Li and Alper, 2020). These advances demonstrate the
shift in producing lipids for fuels to lipids for food additives.
Naringenin along other interesting avone molecules is a poly-
phenolic polyketide (2S)-avanone with high interest due to anti-
inammatory, antioxidant, and neuroprotective activity and thus of
interest as a nutritional food additive (Palmer and Alper, 2019). Current
price of naringenin is about USD 404 per kg. The de novo biosynthetic
pathway of naringenin consists of the phenylpropanoid pathway and the
avonoid pathway (Milke et al., 2018). The rst de novo naringenin
synthesis was achieved in E. coli and S. cerevisiae through assembly of
tyrosine ammonia lyase or/and phenylalanine ammonia lyase, 4-couma-
rate: CoA ligase, chalcone synthase and chalcone isomerase together
with optimization of metabolic ux, producing 29 mg/L naringenin in
shake ask culture and 113 mg/L (415
μ
M) in 2-L controlled aerobic
batch bioreactor, respectively (Santos et al., 2011; Koopman et al.,
2012). Although naringenin is the rst plant polyphenol produced in
microorganism, production is still limited to the mg/L scale (Sun and
Alper, 2020). The highest production titer of de novo naringenin syn-
thesis was 898 mg/L realized in Y. lipolytica using a 3-L bioreactor
through heterologous pathway overexpression along with malonyl-CoA
increase via a β-oxidation mediated strategy (Palmer et al., 2020).
Indeed, the precursors pool and the CHS activity were identied as the
major bottlenecks for naringenin bioproduction, which could be
addressed by pathway engineering and increasing/improving CHS ac-
tivity (Gao et al., 2020; Li et al., 2020b). Additionally, systematic ap-
proaches such as dynamic metabolic control have been demonstrated for
naringenin production when Dinh et al. developed autonomous quorum-
sensing (QS)-based circuits either for the dynamic control of metabolic
ux or for the control of coculture population composition (Dinh and
Prather, 2019; Dinh et al., 2020). Despite all of these advances, further
work is required to improve production levels to reach g/L levels.
Raspberry ketone [4-(4-hydroxyphenyl)-butan-2-one] is a highly
valuable food avor compound with signicant market potential in food
industries. The ketone market size including raspberry ketone was
estimated to reach 640 million USD by 2025 from 443 million in 2019
(Markets and Markets, 2019a). Conventional production of raspberry
ketone relies on extraction from the fruits of raspberry (Rubus idaeus)
with extremely low yield and price up to 20,000 USD per kg (Beekwilder
et al., 2007). Although gram-scale synthesis has been achieved recently
via chemical alkylation and decarboxylation using a solid acid catalyst,
there is considerably increasing interest and demand in the biosynthesis
of “natural” raspberry ketone using microbes recently (Naikwadi et al.,
2020). The biosynthesis of raspberry ketone in raspberry fruits starts
from the phenylpropanoid pathway to form coumaric acid from
phenylalanine or tyrosine, then the coumaric acid is converted to
coumaroyl-CoA by 4-coumarate-coenzyme A ligase (4CL), next one
molecule of 4-coumaroyl-CoA is further condensed with malonyl-CoA by
benzalacetone synthase (BAS), and synthesized hydroxybenzalacetone
is further reduced by benzalacetone reductase (BAR) in the presence of
NADPH (Borejsza-Wysocki and Hrazdina, 1994). Before the identica-
tion of these specic enzymes, microbial production of raspberry ketone
was rst obtained from coumaric acid in E. coli by heterologous
expression of a promiscuous R. idaeus chalcone synthase (CHS) along
with using an unknown endogenous enzyme with BAR activity, reaching
5 mg/L in an 800-mL fermenter (Beekwilder et al., 2007). Identication
of a BAS from Rheum palmatum and especially a raspberry ketone syn-
thase RZS1 from R. idaeus further enabled the module pathway engi-
neering in E. coli, producing raspberry ketone with a titer of 90.97 mg/L
from coumaric acid (Abe et al., 2001; Koeduka et al., 2011; Wang et al.,
2019a). De novo production of raspberry ketone was rst achieved in
S. cerevisiae via assembly of Rhodosporidium toruloids phenylalanine/
L. Sun et al.
Biotechnology Advances 47 (2021) 107694
12
tyrosine ammonia lyase, Arabidopsis thaliana cinnamate-4-hydroxylase,
Petroselinum crispum 4CL and R. palmatum BAS, and the yield was
improved to 2.81 mg/L via synthetic enzyme fusion under anaerobic
conditions (Lee et al., 2016). Recently, raspberry ketone was also pro-
duced in Corynebacterium glutamicum via engineering of P. crispum 4CL,
R. palmatum BAS and E. coli-derived NADPH-dependent reductase CurA
in combination with increasing NADPH availability, reaching 99.8 mg/L
from coumaric acid (Milke et al., 2020). Metabolic engineering attempt
for increasing precursors and cofactors as well as balancing metabolic
ux, in combination with enzyme engineering efforts for improving the
characteristics of synthases and reductases might be useful for
improving the production of raspberry ketone.
Poly-γ-glutamic acid (γ-PGA) is a food-derived and water-soluble
polymer consisting of D- and/or L-glutamic acids that is widely used
as a texture enhancer, bitterness-relieving reagent, oil-reducing reagent,
cryoprotectant, and food supplement (Luo et al., 2016). Its global mar-
ket is expected to be more than 840.5 million USD by 2026 (Acumen
Research and Consulting, 2019). γ-PGA can be naturally synthesized in
the mucilage of natto (fermented soybeans) by organisms within the
genus Bacillus. In native producers, exogenous or endogenous L-gluta-
mic acid is converted to D-glutamic acid through glutamate racemase,
then L and/or D-glutamic acid monomers are polymerized to γ-PGA by
the membrane-embedded γ-PGA synthase complex pgsBCA (Ogunleye
et al., 2015). These polymers can range from approximately 100 kDa to
more than 1000 kDa with low molecular weight (LMW)-γ-PGA
(MW<400 kDa) being preferred as probiotic protectant (257 kDa),
antioxidant, gastrointestinal cytoprotectant (about 400 kDa) and cal-
cium absorption enhancer (11 kDa) and high molecular weight (HMW)-
γ-PGA ((MW>1000 kDa) being preferred as emulsier and texture
enhancer (>1000 kDa). As such, metabolic engineering and bio-
processing efforts are required to achieve not just high titers, but the
desired molecular weight. For example, extended fermentation time can
achieve LMW-γ-PGA owing to the activity of γ-PGA hydrolases. In doing
so, 17.6 g/L of LMW-γ-PGA with an average MW of 20-30 kDa was
successfully obtained by batch fermentation from raw inulin extract in a
7.5-L fermenter via stable overexpression of γ-PGA hydrolase PgsS in
Bacillus amyloliquefaciens NB (Sha et al., 2019). Culture media can be
used to impact the molecular weight of this product as seen in Bacillus sp.
SJ-10 under batch fermentation when 8% NaCl was added to achieve
24.7 g/L γ -PGA with constant MW of about 400 kDa (Lee et al., 2018).
Beyond MW, stereochemistry and conformational state are important
and inuenced by the activity of the various enzymes. Specically,
through multiple combinations of γ-PGA synthases and glutamate
racemases, diverse tailor-made γ-PGA can be produced from the same
microbial chassis with molecular weights ranging from 40 to 8500 kDa
(Halmschlag et al., 2019). One of the major challenges remaining in
production is the L-glutamic acid that can either be endogenously pro-
duced or exogenously fed. The highest titers of these two types of pro-
duction were achieved by B. subtilis and B. licheniformis through culture
condition control in fed batch fermentation, reaching 101.1 g/L in a 10-L
fermenter and 39.9 g/L in a 5-L fermenter, respectively (Huang et al.,
2011; Kongklom et al., 2017). Given the overall cost and ease of pro-
cessing, the glutamic-acid independent process is preferred yet im-
provements in overall productivity are still required.
Safranal (4-hydroxy-2,4,4-trimethyl 1-cyclo-hexene-1-carboxalde-
hyde) is a cyclical terpenic aldehyde and a C
10
-carotenoid-derived
compound naturally existing in saffron, which has been broadly used as
amora additive to give unique saffron odor and avor to the foods
(Ahrazem et al., 2015). Its global market was 3.2 billion USD in 2019
and will expand to reach 5.3 billion by 2025 (Reports Express, 2020a).
Typical method of saffral manufacturing relies on the manual separation
of the stigmas from the ower of Crocus sativus, which is cost-expensive,
time-consuming and seasonally dependent. Although the genes for
safranal biosynthesis have not been fully characterized, it is clear that
safranal can be synthesized in C. sativus by β-glucosidase (β-GS) from
hydrolytic deglucosylation of apocarotenoid picrocrocin, which can be
formed from zeaxanthin via carotenoid cleavage dioxygenase (CCD) and
UDP glucosyltransferase (UGT) (Yue et al., 2020). Deep transcriptome
sequencing together with in vitro and in vivo assay veried a dioxygenase
CCD2 which is responsible for zeaxanthin cleavage at 7,8 and 7′,8′
double bonds in saffron stigma (Frusciante et al., 2014; Ahrazem et al.,
2016). Additionally, saffron UGT709G1 catalyzing the specialized
biosynthesis of picrocrocin was also identied through a targeted search
for differentially expressed UGTs in Crocus transcriptomes (Diretto et al.,
2019). Recently, seven transcripts encoding β-GS were screened from an
integration analysis of the transcriptome and metabolome using
different developmental stages of stigmas (Tan et al., 2019). Identi-
cation of these critical enzymes and candidates provided promising
perspectives for the industrial production of safranal and other saffron
components. Interestingly, safranal can also be generated through the
degradation of low-cost seed extracts of Ditaxis heterantha by
S. cerevisiae, which offered an alternative way for its microbial pro-
duction and needs to be further exploited (Del Toro-S´
anchez et al.,
2006). Regarding other major saffron components such as colorant
crocin that has already been produced successfully in yeast and E. coli,
there is high value in producing mixtures of saffron components
accompanied with de novo biosynthesis of safranal as they are all
downstream products of cleaved zeaxanthin (Raghavan et al., 2014;
Wang et al., 2019c). Through constructing the β-carotene biosynthetic
pathway along with employing C. sativus zeaxanthin cleavage dioxyge-
nase (ZCD) and various UGTs, it is highly expected that customizable
saffron mixtures with picrocrocin, crocin and safranal could be pro-
duced in metabolically enhanced yeast (Palms, 2017).
Isobutyl acetate and isoamyl acetate are structurally similar and
medium branched-chain esters that are naturally produced by brewing
yeast in fermentation. These compounds are volatile fragrances and
avors with fruity odor and of high interest for decades (Forney and
Song, 2017). Their global market each is expected to reach 11.9 billion
USD and 7,991.7 million USD by 2025 from 9.5 billion and 5,344.6
million in 2019, respectively (Reports Express, 2020b; Market Study
Report, 2020). Current manufacturing methods included chemical
Fischer esterication and plant extraction. Alternatively, these esters
can be synthesized either through esterication or trans-esterication by
lipase or via condensation of branched-chain alcohols with acetyl-CoA
by alcohol acyltransferase (ATF), with the latter one being more favor-
able in aqueous fermentation. To generate a renewable and inexpensive
production process, de novo synthesis of isobutyl acetate and isoamyl
acetate was achieved in E. coli via utilization of native valine and leucine
pathway along with overexpression of ATF1 from S. cerevisiae and
deletion of byproduct pathways, yielding 2.5 g/L of isobutyl acetate and
780 mg/L of isoamyl acetate in shake ask fermentations and 36 g/L
isobutyl acetate in a 1.3-L bioreactor (Tai et al., 2015). Likewise, high-
level expression and increased ux of isobutanol pathway along with
moderate-level expression of ATF1 in E. coli produced 3.0 g/L isobutyl
acetate (Rodriguez et al., 2014). Further in situ product removal via
incorporation of a hexadecane layer alleviated isobutyl acetate toxicity
and enabled high-level production of isobutyl acetate with a titer of 17.5
g/L and a yield of 0.334 g/g glucose in shake ask fermentation. Iso-
butyl acetate biosynthesis was also studied in S. cerevisiae via subcellular
metabolic engineering strategies through mitochondrion-based expres-
sion of ATF1 and increased ux of isobutanol pathway along with seg-
mentation of the esterication step into the cytosol, achieving 260.2
mg/L in tube fermentation (Yuan et al., 2016). Other hosts such as
P. Pastoris was engineered via overexpression of endogenous valine
biosynthesis pathway and heterologous expression of ATF1, yielding
isobutyl acetate with a titer of 51 mg/L in shake ask fermentation
(Siripong et al., 2018). Recently, cellulolytic Clostridium thermocellum
harboring native ATF-dependent ester biosynthesis pathway was engi-
neered to produce isobutyl acetate directly using cellulose, and reducing
the ester degradation by disruption the endogenous carbohydrate es-
terases further improved the production, reaching a 1.6-fold higher titer
(3.1 mg/L) in tube-scale fermentation (Seo et al., 2020). Future work in
L. Sun et al.
Biotechnology Advances 47 (2021) 107694
13
esters production such as increasing the isobutanol pool and balancing
the metabolic ux via dynamic control, improving ATF enzymes via
rational design could be useful for further improvement.
Hydroxytyrosol (3,4-dihydroxyphenylethanol) is a nutritional and
functional food additive and is an ortho-diphenolic compound naturally
found in olive trees (Hassen et al., 2015). It is a potential antioxidant and
antimicrobial agent that can be used in various foods such as bakery
products, beverages, yogurt, fats and oils, vegetables and fruits as well as
the juices at a recommended level of 5.0 mg/serving (Connor, 2019).
Hydroxytyrosol market was estimated to surpass 2.5 billion USD by
2030 (Fact.MR, 2020). GRAS notices for natural and the synthesized
hydroxytyrosol (both through chemical synthesis or biotechnological
means with high purity) have been led and accepted by FDA with no
objections (Keefe, 2016; Adams-S, 2018; Carlson-S, 2020). Production of
hydroxytyrosol from tyrosol can be achieved through either enzyme like
tyrosinase or through whole cell catalysts of Pseudomonas, Serratia,
engineered E. coli and Halomonas. For example, 37.3 mM (5750 mg/L) of
hyroxytyrosol was obtained with 86.9% yield in 3.6-L fermenter via
tyrosol hydroxylating Pseudomonas by controlling the biocatalyst
loading and substrate concentration (Bouallagui and Sayadi, 2018).
Moving toward a cheaper process (as tyrosol is expensive), several
articial pathways have been proposed in E. coli including by Satoh et al.
who generated 0.08 mM (12.3 mg/L) hydroxytyrosol in ask fermen-
tation using the rst articial pathway from glucose consisting of tyro-
sine hydroxylase, L-DOPA decarboxylase, tyramine oxidase and native
alcohol dehydrogenases (Satoh et al., 2012). A novel pathway was
proposed and optimized by Li et al. in which tyrosine was converted to
hydroxytyrosol through sequential catalysis of native aminotransfer-
ases, ketoacid decarboxylase, alcohol dehydrogenases and 4-hydroxy-
phenylacetic acid 3-hydroxylase (HpaBC) (Li et al., 2018a). After
optimizing metabolic ux and medium composition, the resulting strain
produced 1243 mg/L of hydroxytyrosol from tyrosine with 48% yield in
ask feeding experiments. Recently, a avin-dependent monooxygenase
HpaBC was engineered through directed divergent evolution to reach
1890 mg/L of hydroxytyrosol with 82% yield in ask fed-batch
fermentation (Chen et al., 2019). Finally, Yao et al. developed a
highly-active tyrosine hydroxylase through structure-guided directed
evolution of HpaBC and used a biosensor to obtain a better mutant that
also de-bottlenecked rate-limiting steps thus resulting in a nal pro-
duction of 4690 mg/L with 95% yield from tyrosine in 5-L scale fed-
batch fermentation, which is the highest value to date (Yao et al.,
2020a). Thus, this rapid improvement of strains demonstrates potential
for bio-based hydroxytyrosol production.
3.2. Newer/potential food colorants
Recent consumer interest is moving away from articial colorants
and toward more natural pigments, ideally that do not change the avor
prole of the food. In this regard, natural pigments that are non-toxic,
non-allergic, non-carcinogenic, and biodegradable are generally of
particular interest (Sen et al., 2019). In this regard, there are a variety of
newer bio-based pigments of interest that exhibit an array of attractive
color hues for use as food colorants. This section attempts to review
recent advance in the production of some newer/potential food color-
ants by microbial hosts. Specically, many of these compounds were
chosen as the representative examples to exemplify each color classied
by hue. It is expected that these newer molecules will be of interest as
food additives given the move away from articial colors.
Betalains are water-soluble, N-heterocyclic compounds that are
mainly classied into two pigment groups, betacyanins (red-violet) and
betaxanthins (yellow-orange) (Martins et al., 2017). Chemically, these
molecules are conjugates of betalamic acid with either cyclo-DOPA or
with amines (or amino acids). The backbone of betalains, betalamic
acid, is formed by tyrosine hydroxylation and DOPA oxidation via a
cytochrome P450 and a DOPA-4,5-extradiol-dioxygenase (DOD) fol-
lowed by spontaneous cyclization. As the rst identied betalains-
producing bacteria, Gluconacetobacter diazotrophicus was found to har-
bor the most efcient DOD (Contreras-Llano et al., 2019). More recently,
betalains have been successfully produced in engineered hosts using this
enzyme with up to 150 mg of pure products was achieved from L-DOPA
in a 2-L bioreactor of E. coli (Guerrero-Rubio et al., 2019). Many factors
such as temperature, light, oxygen and other culture conditions can
impact betalains stability, thus inuencing product quality and yield (da
Silva et al., 2019). To improve the stability of these molecules (and often
solubility), glycosylation has been explored to create more stable beta-
lains such as red-violet betanin. As an example, Grewal et al. constructed
a betanin synthesis pathway in S. cerevisiae with a cytochrome P450
mutant CYP76AD1 from Beta vulgaris, a DOD and a cyclo-DOPA gluco-
syltransferase from Mirabilis jalapa, and de novo synthesized betanin
from glucose to obtain a titer of 17 mg/L in ask fermentation (Grewal
et al., 2018). The betalain color palette can be further expanded by
feeding (or producing) structurally diverse amines to form novel betanin
derivatives. In another recent study, a new photo-stable and metal-free
blue quasibetalain was successfully synthesized from belalamic acid via
dehydrative extension of
π
-conjugation (Freitas-D¨
orr et al., 2020). As
studies on the production and diversication of betalains are still early
stage and the total yield is far below the market demand, future work
should focus on ne-tuning the metabolic ux, identifying more efcient
cytochrome P450 enzymes and cofactor partners, better DOD enzymes,
and relevant exporters to help facilitate commercial application.
Indigoidine is a natural blue pigment belonging to the class of
bacterial non-ribosomal peptide (NRP) molecules that resembles the
synthetic food dye indigo and has attracted particular attention as a
promising alternative to synthetic blue dyes (Singh, 2017). Indigoidine
can be synthesized through condensation of two L-glutamine units by
NPR synthetase (NPRS). Gene clusters for this have been characterized
from Erwinia chrysanthemi, Photorhabdus luminescens, Vogesella sp. as
well as Streptomyces and marine roseobacter Phaeobacter. Yu et al. suc-
cessfully demonstrated indigoidine production in E. coli using the NPRS
indigoidine synthetase IndC and an associated helper protein IndB from
Streptomyces coelicolor, and produced 3.9 g/L in ask fermentation (Yu
et al., 2013). A higher titer of 8.8 g/L was obtained in ask fermentation
using the same recombinant strain with the exogenous addition of 1.5 g/
L of L-glutamine (Xu et al., 2015). De novo production of indigoidine was
further achieved through overexpression of a glutamine synthetase and
optimization of nitrogen source leading to a titer of 7.1 g/L. S. cerevisiae
was the rst fungal host explored for the production of indigoidine and a
maximum titer of 980 mg/L was obtained in a 2-L bioreactor (Wehrs
et al., 2018). To date, the highest titer of indigoidine was achieved in the
oleaginous yeast Rhodosporidium toruloides by using NRPS BpsA from
Streptomyces lavendulae reaching titers of 2.9 g/L from lignocellulosic
hydrolysate and 86.3 g/L from glucose in 2-L bioreactors (Wehrs et al.,
2019). Indeed, this study demonstrates the great industrial potential of
R. toruloides for the sustainable and scalable production of indigoidine.
Specically, the catalytic mechanism of BpasA was recently illustrated,
which provided more useful information for further engineering (Pang
et al., 2020).
Beyond indigoidine, the development of other natural blue colorants
is also of high interest. Currently, the only commercial natural blue
colorant is a protein extract from blue-green Spirulina algae, and the blue
color is mainly attributed to the blue phycobiliprotein phycocyanin.
Searching for thermostable phycocyanin from thermoacidophilic
microalgae such as Cyanidioschyzon merolae was able to discover a
molecule with higher stability (Rahman et al., 2017). Blue anthocyanin
is another desirable and promising candidate but requires improvement
of the stability and extension of short half-life still need future en-
deavors. Recently, a blue acylated A5 cyanidin was successfully pro-
duced by Arabidopsis cell through metabolic engineering, which raises
prospects for new blue colorants (Appelhagen et al., 2018).
Violacein is an indolocarbazole compound exhibiting a purple
pigment, which is soluble in water and stable across a pH range of 1-11
and temperature range of 25-60 ◦C (Venil et al., 2015). Violacein is a
L. Sun et al.
Biotechnology Advances 47 (2021) 107694
14
secondary metabolite found in natural producers such as Chromobacte-
rium, Duganella, Janthinobacterium, Psychotropic, Pseudoalteromonas,
Collimonas and Microbulbifer. The biosynthetic pathway begins from a
decarboxylated condensation of two L-tryptophan units, in which one
molecule of L-tryptophan is rst converted by VioA to form indole-3-
pyruvic acid (IPA), and then condensed with another unit of L-trypto-
phan by VioB and VioE to form prodeoxyviolacein C, and nally con-
verted to violacein through the catalysis of VioC and VioD oxygenases
(S´
anchez et al., 2006). Heterologous production of violacein was rst
achieved in a genetically engineered Citrobacter freundii where 4.1 g/L
was obtained from glycerol by 5-L scale fed-batch fermentation with
exogenous addition of tryptophan (Yang et al., 2011). Higher production
(4.5 g/L) of violacein was obtained in a 5-L fermenter from glucose using
E. coli through overexpression of the rate-limiting enzyme vioE (Zhou
et al., 2018). To facilitate the application of violacein as a food additive,
a GRAS and amino acids production host C. glutamicum was employed as
a chassis for violacein production and a yield of 5.4 g/L was achieved in
a 3-L bioreactor, which is the highest production titer to date (Sun et al.,
2016). Compared to bacterial hosts, production of violacein in fungal
systems has been limited with nal titers only reaching the low mg/L
levels, thus demonstrating the promise for bacterial systems (Wong
et al., 2017).
Melanin and melanin-like pigments are a large family of complex
phenolic or indolic-based heteropolymers that are of interest as black
food colorants (Sigurdson et al., 2017). Melanin pigment has been
widely consumed in Japan as the component of cephalopod ink, in
which the insoluble sepia melanin was characterized (Mbonyiryivuze
et al., 2015). Melanin pigments are mainly classied into three groups
based on the biosynthetic pathway and the chemical monomers:
eumelanins (black-brown), pheomelanins (yellow-red) and allomelanins
(various colors) (Pavan et al., 2020). Eumelanin is the oxidative poly-
merization product of L-tyrosine by tyrosinase or laccase, and pheo-
melanin can be produced in a similar process where L-cysteine is
incorporated into the intermediate L-DOPA to form cysteinylDOPA. In
contrast, allomelanin is the most heterogenous group, which is consti-
tuted of pyomelanin and a variety of polymers derived from dihydrox-
ynaphthalene (DHN), tetrahydroxynaphthalene (THN), catechols, 4-
hydroxyphenylacetic acid, or γ-glutaminyl-4-hydroxybenzene (GDHB).
Natural host production has been explored extensively here as melanin
biosynthesis is seen in fungi such as Streptomyces and Aspergillus, bac-
teria such as Pseudomonas and Bacillus, and yeasts such as Saccharomyces
neoformans var. nigricans and Cryptococcus neoformans (Pombeiro-
Sponchiado et al., 2017). For example, Ribera et al. screened different
basidiomycetes, and the Armillaria cepistipes strain Empa 655 was
identied to be able to produce eumelanin from tyrosine in ask culture
with a titer as high as almost 28 g/L (Ribera et al., 2019). Genetically
modied hosts such as E. coli have also been explored for melanin pro-
duction with de novo production upwards of 3.2 g/L in a 1-L bioreactor
(Ch´
avez-B´
ejar et al., 2013). Due to the variations of the colors and
physiochemical properties, there are growing interest in producing
different forms of melanin. A new type of melanin was successfully
obtained from glycerol in E. coli by integrating an improved tyrosinase
Fig. 4. Perspective of bio-based food additives and colorants. The gene clusters of natural products can be characterized from natural sources and engineered into a
suitable microbe factory to then produce bio-based food additives and colorants for food application. Key factors affecting commercial adoption of food additives and
colorants (as described in this paper) are summarized in this gure. The gure was created with BioRender.com.
L. Sun et al.
Biotechnology Advances 47 (2021) 107694
15
mutant into the chromosome of a catechol producing strain, reaching
1.2 g/L catechol melanin in a 1-L bioreactor (Mejía-Caballero et al.,
2016). Recently, a tyrosinase from Bacillus megaterium was engineered
into an extremely fast-growing marine bacterium Vibrio natriegens to
produce a melanin-like pigment from tyrosine with a titer over 0.9 g/L
within 10 h, a rate that is signicantly less time than other heterologous
hosts (Wang et al., 2020). In another study, a brown pigment pyome-
lanin was successfully obtained in Y. lipolytica from glucose through the
homogentisic acid pathway with addition of tyrosine, reaching a yield of
0.5 g/L (Ben Tahar et al., 2020). Among these microbial hosts, Bacillus
and GRAS yeasts seem like the most appealing hosts for food used
melanin production.
4. Perspectives
Natural products have gained increasing attention as the sources of
food additives due to their versatile functions. To be ultimately used as a
food additive and colorant, the characteristics of natural products such
as toxicity, solubility and stability needs to be addressed as well as
evaluating the regulatory issues and consumer acceptance. Beyond these
molecular features, environmental sustainability, productivity, and
production cost are also key factors affecting commercial adoption. To
this end, bio-manufacturing using microbes provides a promising and
economical alternative for the production of food additives and color-
ants over chemical synthesis and plant extraction. As seen in the rst
section, there are several successful examples of commercial, bio-based
food additives and colorants that can be produced by microbes. To
enable the efcient and large-scale production of other bio-based food
additives and colorants (especially those that are traditionally extracted
from plants), there are several aspects that should be considered and
addressed (Fig. 4). First, omics-based techniques should be extensively
applied to identify and characterize the biosynthetic gene clusters of
natural products. Second, microbial hosts, especially the GRAS microbes
such as B. subtilis, lactobacillus, S. cerevisiae, Y. lipolytica and P. pastoris,
should be appropriately chosen and explored to match the target
molecule. Third, metabolic engineering and synthetic biology should be
combined to enable the bioproduction of food additives and colorants.
Inherent in these technologies is both rational design strategies as well
as new approaches for dynamic metabolic control and high-throughput
screening approaches to debottleneck the metabolic imbalance, over-
come the product toxicity and reduce byproduct formation. In addition,
regulatory mechanisms of key enzymes should be thoroughly studied
and illustrated to further improve the production yield. There may be
some priorities in some cases to use non-GMO approaches for the
metabolic regulation. In many cases, transporter protein identication is
also crucial for product separation. Additionally, it may be valuable to
produce specic mixtures of related metabolites to meet the multi-
functional applications in an integrated economical biomanufacturing
process, or produce non-natural compounds with better characteristics
through targeted modication of natural precursors, such as by non-
cognate glucosylation. Finally, as with any bio-based molecule, it is
important to balance strain engineering with bioprocessing approaches
to achieve a process that is efcient and cost-effective (taking into ac-
count cheaper sources utilization and downstream processing). There-
fore, bioprocess that are performed in bioreactors range from 5 L to 10 L
is a prerequisite for scale up and industrialization. With these advances,
many of the molecules described above (in addition to countless others)
will become more competitive in the future and nd their way as food
additives as well as integrated into functional foods.
Further development of robust biosynthesis platform for food addi-
tives and colorants based on advanced synthetic technologies such as
high-throughput screening and in vitro driven biosynthesis technologies,
and combined with the implementation of machine learning can be
useful areas of focus for further development. However, in many cases,
basic pathway characterization and engineering is still required. The
maturation of bio-synthesis technology of food additives and colorants
would facilitate the production of more safe, healthy and sustainable
bio-based food products and put forward the development of food bio-
manufacturing. Based on this, it is highly expected to see a rising future
of bio-based food additives and colorants and a huge market potential as
the future trend in food.
Declaration of Competing Interest
None.
Acknowledgements
This work was supported by The Welch Foundation [grant number F-
1976-20190330]; National Natural Science Foundation of China [grant
number 31700701]; Central Public-interest Scientic Institution Basal
Research Fund [grant number S2020JBKY-13]; and China Scholarship
Council (CSC).
References
Abe, I., Takahashi, Y., Morita, H., Noguchi, H., 2001. Benzalacetone synthase. A novel
polyketide synthase that plays a crucial role in the biosynthesis of phenylbutanones
in Rheum palmatum. Eur. J. Biochem. 268, 3354–3359. https://doi.org/10.1046/
j.1432-1327.2001.02255.x.
Acumen Research and Consulting, 2019, Gamma-polyglutamic acid market-global
industry analysis, market size, opportunities and forecast, 2019 – 2026, 2020,
Available online: https://www.acumenresearchandconsulting.com/gamma-polyglu
tamic-acid-market (Accessed on 23 November 2020).
Adams-S, M. A., 2018. Agency Response Letter GRAS Notice No. GRN 000726. Phenolic
preparation from olive fruit. Available online: https://www.fda.gov/media/111692
/download (Accessed on 23 November 2020).
Ahrazem, O., Rubio-Moraga, A., Nebauer, S.G., Molina, R.V., G´
omez-G´
omez, L., 2015.
Saffron: its phytochemistry, developmental processes, and biotechnological
prospects. J. Agric. Food Chem. 63, 8751–8764. https://doi.org/10.1021/acs.
jafc.5b03194.
Ahrazem, O., Rubio-Moraga, A., Berman, J., Capell, T., Christou, P., Zhu, C., G´
omez-
G´
omez, L., 2016. The carotenoid cleavage dioxygenase CCD2 catalysing the
synthesis of crocetin in spring crocuses and saffron is a plastidial enzyme. New
Phytol. 209, 650–663. https://doi.org/10.1111/nph.13609.
Ahuja, K., Rawat, A., 2020a. Xanthan gum market size by application (oil & gas, food &
beverages, pharmaceutical, cosmetics), industry analysis report, regional outlook,
application potential, price trends, competitive market share & forecast, 2015–2026.
Available online: https://www.gminsights.com/industry-analysis/xanthan-gum-mar
ket (Accessed on 23 November 2020).
Ahuja, K., Rawat, A., 2020b. Erythritol market size by form (powder, granular), by
application (beverage, bakery, confectionery & dairy products, personal care,
pharmaceutical), regional outlook, application potential, price trends, competitive
market share & forecast, 2020–2026. Available online: https://www.gminsights.
com/industry-analysis/erythritol-market (Accessed on 23 November 2020).
´
Alvarez-Cao, M.E., Rico-Díaz, A., Cerd´
an, M.E., Becerra, M., Gonz´
alez-Siso, M.I., 2018.
Valuation of agro-industrial wastes as substrates for heterologous production of
α
-galactosidase. Microb. Cell Fact. 17, 1–13. https://doi.org/10.1186/s12934-018-
0988-6.
´
Alvarez-Cao, M.E., Cerd´
an, M.E., Gonz´
alez-Siso, M.I., Becerra, M., 2019. Optimization of
Saccharomyces cerevisiae
α
-galactosidase production and application in the
degradation of rafnose family oligosaccharides. Microb. Cell Fact. 18, 1–17.
https://doi.org/10.1186/s12934-019-1222-x.
Amanullah, A., Satti, S., Nienow, A.W., 1998. Enhancing xanthan fermentations by
different modes of glucose feeding. Biotechnol. Prog. 14, 265–269. https://doi.org/
10.1021/bp9800079.
Amyris Inc., 2020, Amyris continues market adoption of its sweetener with approval by
Health Canada, Available online: https://www.prnewswire.com/news-releases/amy
ris-continues-market-adoption-of-its-sweetener-with-approval-by-health-canada-3
01130781.html (Accessed on 23 November 2020).
Appelhagen, I., Wulff-Vester, A.K., Wendell, M., Hvoslef-Eide, A.K., Russell, J., Oertel, A.,
Martens, S., Mock, H.P., Martin, C., Matros, A., 2018. Colour bio-factories: Towards
scale-up production of anthocyanins in plant cell cultures. Metab. Eng. 48, 218–232.
https://doi.org/10.1016/j.ymben.2018.06.004.
Ashwell, M., Gibson, S., Bellisle, F., Buttriss, J., Drewnowski, A., Fantino, M.,
Gallagher, A.M., De Graaf, K., Goscinny, S., Hardman, C.A., Laviada-Molina, H.,
L´
opez-García, R., Magnuson, B., Mellor, D., Rogers, P.J., Rowland, I., Russell, W.,
Sievenpiper, J.L., La Vecchia, C., 2020. Expert consensus on low-calorie sweeteners:
Facts, research gaps and suggested actions. Nutr. Res. Rev. 33, 145–154. https://doi.
org/10.1017/S0954422419000283.
Beekwilder, J., Van der Meer, I.M., Sibbbesen, O., Broekgaarden, M., Qvist, I.,
Mikkelsen, J.D., Hall, R.D., 2007. Microbial production of natural raspberry ketone.
Biotechnol. J. 2, 1270–1279. https://doi.org/10.1002/biot.200700076.
Ben Tahar, I., Kus-Li´
skiewicz, M., Lara, Y., Javaux, E., Fickers, P., 2020. Characterization
of a nontoxic pyomelanin pigment produced by the yeast Yarrowia lipolytica.
Biotechnol. Prog. 36 (2), e2912 https://doi.org/10.1002/btpr.2912.
L. Sun et al.
Biotechnology Advances 47 (2021) 107694
16
Bhatia, S., Singh, A., Batra, N., Singh, J., 2020. Microbial production and
biotechnological applications of
α
-galactosidase. Int. J. Biol. Macromol. 150,
1294–1313. https://doi.org/10.1016/j.ijbiomac.2019.10.140.
Bilal, M., Xu, S., Iqbal, H.M.N., Cheng, H., 2020. Yarrowia lipolytica as an emerging
biotechnological chassis for functional sugars biosynthesis. Crit. Rev. Food Sci. Nutr.
0, 1–18. https://doi.org/10.1080/10408398.2020.1739000.
Bober, J.R., Nair, N.U., 2019. Galactose to tagatose isomerization at moderate
temperatures with high conversion and productivity. Nat. Commun. 10, 1–10.
https://doi.org/10.1038/s41467-019-12497-8.
Borejsza-Wysocki, W., Hrazdina, G., 1994. Biosynthesis of p-hydroxyphenylbutan-2-one
in raspberry fruits and tissue cultures. Phytochemistry 35, 623–628. https://doi.org/
10.1016/S0031-9422(00)90575-2.
Bouallagui, Z., Sayadi, S., 2018. Bioconversion of p -tyrosol into hydroxytyrosol under
bench-scale fermentation. Biomed Res. Int. 2018, 7390751 https://doi.org/10.1155/
2018/7390751.
Braga, A., Faria, N., 2020. Bioprocess optimization for the production of aromatic
compounds with metabolically engineered hosts: Recent developments and future
challenges. Front. Bioeng. Biotechnol. 8, 96. https://doi.org/10.3389/
fbioe.2020.00096.
Brochado, A.R., Matos, C., Møller, B.L., Hansen, J., Mortensen, U.H., Patil, K.R., 2010.
Improved vanillin production in baker’s yeast through in silico design. Microb. Cell
Fact. 9 (1), 84. https://doi.org/10.1186/1475-2859-9-84.
Bych, K., Mikˇ
s, M.H., Johanson, T., Hederos, M.J., Vigsnæs, L.K., Becker, P., 2019.
Production of HMOs using microbial hosts — from cell engineering to large scale
production. Curr. Opin. Biotechnol. 56, 130–137. https://doi:10.1016/j.copbio.20
18.11.003.
Cao, L., Liang, D., Hao, P., Song, Q., Xue, E., Caiyin, Q., Cheng, Z., Qiao, J., 2018. The
increase of O-acetylation and N-deacetylation in cell wall promotes acid resistance
and nisin production through improving cell wall integrity in Lactococcus lactis.
J. Ind. Microbiol. Biotechnol. 45, 813–825. https://doi.org/10.1007/s10295-018-
2052-2.
Carlson-S, S. J., 2020. Agency Response Letter GRAS Notice No. GRN 000876.
Hydroxytyrosol. Available online: https://www.fda.gov/media/134955/download
(Accessed on 23 November 2020).
Carly, F., Fickers, P., 2018. Erythritol production by yeasts: a snapshot of current
knowledge. Yeast 35 (7), 455–463.
Chaitali, M., Kapadi, M., Suraishkumar, G.K., Gudi, R.D., 2008. Productivity
improvement in xanthan gum fermentation using multiple substrate optimization.
Biotechnol. Prog. 19 (4), 1190–1198.
Ch´
avez-B´
ejar, M.I., Balderas-Hernandez, V.E., Guti´
errez-Alejandre, A., Martinez, A.,
Bolívar, F., Gosset, G., 2013. Metabolic engineering of Escherichia coli to optimize
melanin synthesis from glucose. Microb. Cell Fact. 12, 1–12. https://doi.org/
10.1186/1475-2859-12-108.
Chen, Y., Xiao, W., Wang, Y., Liu, H., Li, X., Yuan, Y., 2016. Lycopene overproduction in
Saccharomyces cerevisiae through combining pathway engineering with host
engineering. Microb. Cell Fact. 15, 1–13. https://doi.org/10.1186/s12934-016-
0509-4.
Chen, W., Yao, J., Meng, J., Han, W., Tao, Y., Chen, Y., Guo, Y., Shi, G., He, Y., Jin, J.M.,
Tang, S.Y., 2019. Promiscuous enzymatic activity-aided multiple-pathway network
design for metabolic ux rearrangement in hydroxytyrosol biosynthesis. Nat.
Commun. 10, 1–12. https://doi.org/10.1038/s41467-019-08781-2.
Cheng, S., Liu, X., Jiang, G., Wu, J., Zhang, J.L., Lei, D., Yuan, Y.J., Qiao, J., Zhao, G.R.,
2019. Orthogonal engineering of biosynthetic pathway for efcient production of
limonene in Saccharomyces cerevisiae. ACS Synth. Biol. 8, 968–975. https://doi.org/
10.1021/acssynbio.9b00135.
Connor, K. O., 2019. GRAS Notice No. GRN 000876, Hydroxytyrosol, Available online:
https://www.fda.gov/media/134474/download (Accessed on 23 November 2020).
Contreras-Llano, L.E., Guerrero-Rubio, M.A., Lozada-Ramírez, J.D., García-Carmona, F.,
Gandía-Herrero, F., 2019. First betalain-producing bacteria break the exclusive
presence of the pigments in the Plant Kingdom. MBio 10, 1–12. https://doi.org/
10.1128/mBio.00345-19.
Cumbers, J., 2020. Synthetic biology startups raised $3 billion in the rst half of 2020,
Available online: https://www.forbes.com/sites/johncumbers/2020/09/09/synthe
tic-biology-startups-raised-30-billion-in-the-first-half-of-2020/?sh=2a6462481265
(Accessed on 23 November 2020).
da Silva, J.A., Cardoso, L.G., de Jesus Assis, D., Gomes, G.V.P., Oliveira, M.B.P.P., de
Souza, C.O., Druzian, J.I., 2018. Xanthan gum production by Xanthomonas campestris
pv. campestris IBSBF 1866 and 1867 from lignocellulosic agroindustrial wastes. Appl.
Biochem. Biotechnol. 186, 750–763. https://doi.org/10.1007/s12010-018-2765-8.
da Silva, D.V.T., dos Santos Bai˜
ao, D., de Oliveira Silva, F., Alves, G., Perrone, D., Del
Aguila, E.M., Flosi Paschoalin, V.M., 2019. Betanin, a natural food additive:
Stability, bioavailability, antioxidant and preservative ability assessments. Molecules
24 (3), 458. https://doi.org/10.3390/molecules24030458.
Dai, X., Gao, G., Wu, M., Wei, W., Qu, J., Li, G., Ma, T., 2019. Construction and
application of a Xanthomonas campestris CGMCC15155 strain that produces white
xanthan gum. Microbiologyopen 8, e00631. https://doi.org/10.1002/mbo3.631.
Del Toro-S´
anchez, L., S´
anchez, S., Ortiz, M.A., Villanueva, S., Lugo-Cervantes, E., 2006.
Generation of aroma compounds from Ditaxis heterantha by Saccharomyces cerevisiae.
Appl. Microbiol. Biotechnol. 72, 155–162. https://doi.org/10.1007/s00253-005-
0244-y.
Deng, J., Chen, C., Gu, Y., Lv, X., Liu, Y., Li, J., Ledesma-Amaro, R., Du, G., Liu, L., 2019.
Creating an in vivo bifunctional gene expression circuit through an aptamer-based
regulatory mechanism for dynamic metabolic engineering in Bacillus subtilis. Metab.
Eng. 55, 179–190. https://doi.org/10.1016/j.ymben.2019.07.008.
Dinh, C.V., Prather, K.L.J., 2019. Development of an autonomous and bifunctional
quorum-sensing circuit for metabolic ux control in engineered Escherichia coli. Proc.
Natl. Acad. Sci. U. S. A. 116, 25562–25568. https://doi.org/10.1073/
pnas.1911144116.
Dinh, C.V., Chen, X., Prather, K.L.J., 2020. Development of a quorum-sensing based
circuit for control of coculture population composition in a naringenin production
system. ACS Synth. Biol. 9, 590–597. https://doi.org/10.1021/acssynbio.9b00451.
Diretto, G., Ahrazem, O., Rubio-Moraga, ´
A., Fiore, A., Sevi, F., Argando˜
na, J., G´
omez-
G´
omez, L., 2019. UGT709G1: a novel uridine diphosphate glycosyltransferase
involved in the biosynthesis of picrocrocin, the precursor of safranal in saffron
(Crocus sativus). New Phytol. 224, 725–740. https://doi.org/10.1111/nph.16079.
Doshi, R., Nguyen, T., Chang, G., 2013. Transporter-mediated biofuel secretion. Proc.
Natl. Acad. Sci. U. S. A. 110, 7642–7647. https://doi.org/10.1073/
pnas.1301358110.
Dudley, Q.M., Nash, C.J., Jewett, M.C., 2019. Cell-free biosynthesis of limonene using
enzyme-enriched Escherichia coli lysates. Synth. Biol. 4 (1) ysz003.
Dudley, Q.M., Karim, A.S., Nash, C.J., Jewett, M.C., 2020. In vitro prototyping of
limonene biosynthesis using cell-free protein synthesis. Metab. Eng. 61, 251–260.
https://doi.org/10.1016/j.ymben.2020.05.006.
EFSA ANS Panel (EFSA Panel on Food Addtives and Nutrient Sources added to Food),
2015. Scientic opinion on the safety of the extension of use of thaumatin (E 957).
EFSA Journal 13 (11), 4290.
Fact.MR, 2020, Hydroxytyrosol market slammed by slow demand during Covid-19
outbreak; Olive leaf to attain signicant momentum: Fact.MR, 2020, Available
online: https://apnews.com/press-release/accesswire/consumer-products-and-ser
vices-pharmaceutical-manufacturing-health-care-industry-dairy-product-manufact
uring-health-420bd0cc2206ef2b455f9a1d46035501 (Accessed on 23 November
2020).
FDA, 2020. Overview of food ingredients, additives & colors. Available online: https
://www.fda.gov/food/food-ingredients-packaging/overview-food-ingredients-add
itives-colors. (Accessed on 23 November 2020).
Fior Markets, 2019, Global Vitamin B2 (Riboavin) Market Growth 2019-2024,
Available online: https://www.ormarkets.com/report/global-vitamin-b2-riboa
vin-market-growth-2019-2024-374891.html (Accessed on 23 November 2020).
Fleige, C., Meyer, F., Steinbüchel, A., 2016. Metabolic engineering of the actinomycete
Amycolatopsis sp. strain ATCC 39116 towards enhanced production of natural
vanillin. Appl. Environ. Microbiol. 82, 3410–3419. https://doi.org/10.1128/
AEM.00802-16.
Forney, C.F., Song, J., 2017. Flavors and aromas: chemistry and biological functions. In:
Fruit and vegetable phytochemicals, pp. 515–540.
Fraser, R., Davis, S.C., Brown, P.O., 2017. Secretion of heme-containing polypeptides.
Patent US20170342132A1.
Fraser, R.Z., Shitut, M., Agrawal, P., Mendes, O., Klapholz, S., 2018. Safety evaluation of
soy leghemoglobin protein preparation derived from Pichia pastoris, Intended for use
as a avor catalyst in plant-based meat. Int. J. Toxicol. 37, 241–262. https://doi.org/
10.1177/1091581818766318.
Freitas-D¨
orr, B.C., Machado, C.O., Pinheiro, A.C., Fernandes, A.B., D¨
orr, F.A., Pinto, E.,
Lopes-Ferreira, M., Abdellah, M., S´
a, J., Russo, L.C., Forti, F.L., Gonçalves, L.C.P.,
Bastos, E.L., 2020. A metal-free blue chromophore derived from plant pigments. Sci.
Adv. 6, 1–8. https://doi.org/10.1126/sciadv.aaz0421.
Frusciante, S., Diretto, G., Bruno, M., Ferrante, P., Pietrella, M., Prado-Cabrero, A.,
Rubio-Moraga, A., Beyer, P., Gomez-Gomez, L., Al-Babili, S., Giuliano, G., 2014.
Novel carotenoid cleavage dioxygenase catalyzes the rst dedicated step in saffron
crocin biosynthesis. Proc. Natl. Acad. Sci. U. S. A. 111, 12246–12251. https://doi.
org/10.1073/pnas.1404629111.
Galaev, I., 2015. Recovery of steviol glycosides. Patent WO2015/014959.
Gao, S., Lyu, Y., Zeng, W., Du, G., Zhou, J., Chen, J., 2020. Efcient biosynthesis of (2 S)-
naringenin from p-coumaric acid in Saccharomyces cerevisiae. J. Agric. Food Chem.
68, 1015–1021. https://doi.org/10.1021/acs.jafc.9b05218.
Ge, Y.Y., Zhang, J.R., Corke, H., Gan, R.Y., 2020. Screening and spontaneous mutation of
pickle-derived Lactobacillus plantarum with overproduction of riboavin, related
mechanism, and food application. Foods. 9 (1), 88. https://doi.org/10.3390/
foods9010088.
Global Market Insights, 2020, Glutamic acid and monosodium glutamate (MSG) market
size, potential, industry outlook, regional analysis, application development,
competitive landscape & forecast, 2020 – 2026, Available online: https://www.gmi
nsights.com/industry-analysis/glutamic-acid-and-monosodium-glutamate-msg-ma
rket (Accessed on 23 November 2020).
Grand View Research, 2020, Curcumin market size, share & trends analysis report by
application (pharmaceutical, food, cosmetics), by region (North America, Europe,
Asia Pacic, Central & South America, Middle East & Africa), and segment forecasts,
2020 – 2027, Available online: https://www.grandviewresearch.com/industry-ana
lysis/turmeric-extract-curcumin-market (Accessed on 23 November 2020).
Grewal, P.S., Modavi, C., Russ, Z.N., Harris, N.C., Dueber, J.E., 2018. Bioproduction of a
betalain color palette in Saccharomyces cerevisiae. Metab. Eng. 45, 180–188. https://
doi.org/10.1016/j.ymben.2017.12.008.
Guerrero-Rubio, M.A., L´
opez-Llorca, R., Henarejos-Escudero, P., García-Carmona, F.,
Gandía-Herrero, F., 2019. Scaled-up biotechnological production of individual
betalains in a microbial system. Microb. Biotechnol. 12, 993–1002. https://doi.org/
10.1111/1751-7915.13452.
Gurkok, S., Cekmecelioglu, D., Ogel, Z.B., 2011. Optimization of culture conditions for
Aspergillus sojae expressing an Aspergillus fumigatus
α
-galactosidase. Bioresour.
Technol. 102, 4925–4929. https://doi.org/10.1016/j.biortech.2011.01.036.
Guti´
errez-Preciado, A., Torres, A.G., Merino, E., Bonomi, H.R., Goldbaum, F.A., García-
Angulo, V.A., 2015. Extensive identication of bacterial riboavin transporters and
their distribution across bacterial species. PLoS One 10, 1–17. https://doi.org/
10.1371/journal.pone.0126124.
L. Sun et al.
Biotechnology Advances 47 (2021) 107694
17
Halmschlag, B., Steurer, X., Putri, S.P., Fukusaki, E., Blank, L.M., 2019. Tailor-made poly-
γ-glutamic acid production. Metab. Eng. 55, 239–248. https://doi.org/10.1016/j.
ymben.2019.07.009.
Han, L., Usher, S., Sandgrind, S., Hassall, K., Sayanova, O., Michaelson, L.V., Haslam, R.
P., Napier, J.A., 2020. High level accumulation of EPA and DHA in eld-grown
transgenic Camelina – a multi-territory evaluation of TAG accumulation and
heterogeneity. Plant Biotechnol. J. 1–12. https://doi.org/10.1111/pbi.13385.
Hansen, E.H., Møller, B.L., Kock, G.R., Bünner, C.M., Kristensen, C., Jensen, O.R.,
Okkels, F.T., Olsen, C.E., Motawia, M.S., Hansen, J., 2009. De novo biosynthesis of
vanillin in ssion yeast (Schizosaccharomyces pombe) and baker’s yeast
(Saccharomyces cerevisiae). Appl. Environ. Microbiol. 75, 2765–2774. https://doi.
org/10.1128/AEM.02681-08.
Hao, P., Liang, D., Cao, L., Qiao, B., Wu, H., Caiyin, Q., Zhu, H., Qiao, J., 2017.
Promoting acid resistance and nisin yield of Lactococcus lactis F44 by genetically
increasing D-Asp amidation level inside cell wall. Appl. Microbiol. Biotechnol. 101,
6137–6153. https://doi.org/10.1007/s00253-017-8365-7.
Hassen, I., Casabianca, H., Hosni, K., 2015. Biological activities of the natural antioxidant
oleuropein: Exceeding the expectation - A mini-review. J. Funct. Foods. 18, 926–940.
https://doi.org/10.1016/j.jff.2014.09.001.
Healey, R.D., Lebhar, H., Hornung, S., Thordarson, P., Marquis, C.P., 2017. An improved
process for the production of highly puried recombinant thaumatin tagged-
variants. Food Chem. 237, 825–832. https://doi.org/10.1016/j.
foodchem.2017.06.018.
Heefner, D., Weaver, C.A., Yarus, M.J., Burdzinski, L.A., Gyure, D.C., Foster, E.W., 1988.
Riboavin producing strains of microorganisms, method for selecting, and method
for fermentation. Patent WO 88/09822.
Hegar, B., Wibowo, Y., Basrowi, R.W., Ranuh, R.G., Sudarmo, S.M., Munasir, Z.,
Atthiyah, A.F., Widodo, A.D., Supriatmo, Kadim, Suryawan, A., Diana, N.R.,
Manoppo, C., Vandenplas, Y., 2019. The role of two human milk oligosaccharides,
2’-fucosyllactose and lacto-N-neotetraose, in infant nutrition. Pediatr. Gastroenterol.
Hepatol. Nutr 22, 330–340. https://doi.org/10.5223/pghn.2019.22.4.330.
Hemberger, S., Pedrolli, D.B., Stolz, J., Vogl, C., Lehmann, M., Mack, M., 2011. RibM
from Streptomyces davawensis is a riboavin/roseoavin transporter and may be
useful for the optimization of riboavin production strains. BMC Biotechnol. 11,
119. https://doi.org/10.1186/1472-6750-11-119.
Hern´
andez-Almanza, A., Monta˜
nez, J., Martínez, G., Aguilar-Jim´
enez, A., Contreras-
Esquivel, J.C., Aguilar, C.N., 2016. Lycopene: Progress in microbial production.
Trends Food Sci. Technol. 56, 142–148. https://doi.org/10.1016/j.tifs.2016.08.013.
Hirasawa, T., Wachi, M., 2016. Glutamate fermentation-2: mechanism of l-glutamate
overproduction in Corynebacterium glutamicum. Adv. Biochem. Eng. Biotechnol. 159,
57–72.
Hoeven, V.D.R.A.M., Galaev, I., Boer, V.M., 2015. Diterpene production. In: Patent
WO2015/007748.
Hollands, K., Baron, C.M., Gibson, K.J., Kelly, K.J., Krasley, E.A., Laffend, L.A.,
Lauchli, R.M., Maggio-Hall, L.A., Nelson, M.J., Prasad, J.C., Ren, Y., Rice, B.A.,
Rice, G.H., Rothman, S.C., 2019. Engineering two species of yeast as cell factories for
2′-fucosyllactose. Metab. Eng. 52, 232–242. https://doi.org/10.1016/j.
ymben.2018.12.005.
Huang, J., Du, Y., Xu, G., Zhang, H., Zhu, F., Huang, L., Xu, Z., 2011. High yield and cost-
effective production of poly(γ-glutamic acid) with Bacillus subtilis. Eng. Life Sci. 11,
291–297. https://doi.org/10.1002/elsc.201000133.
Incha, M.R., Thompson, M.G., Blake-Hedges, J.M., Liu, Y., Pearson, A.N., Schmidt, M.,
Gin, J.W., Petzold, C.J., Deutschbauer, A.M., Keasling, J.D., 2020. Leveraging host
metabolism for bisdemethoxycurcumin production in Pseudomonas putida. Metab.
Eng. Commun. 10, e00119. https://doi.org/10.1016/j.mec.2019.e00119.
Ismail, I., Hwang, Y.-H., Joo, S.-T., 2020. Meat analog as future food: a review. J. Anim.
Sci. Technol. 62 (2), 111–120. https://doi.org/10.5187/jast.2020.62.2.111.
Jiang, L., Liu, Y., Yan, G., Cui, Y., Cheng, Q., Zhang, Z., Meng, Q., Teng, L., Ren, X., 2015.
Aeration and fermentation strategies on nisin production. Biotechnol. Lett. 37,
2039–2045. https://doi.org/10.1007/s10529-015-1886-1.
Jin, Y., He, X., Andoh-Kumi, K., Fraser, R.Z., Lu, M., Goodman, R.E., 2018. Evaluating
potential risks of food allergy and toxicity of soy leghemoglobin expressed in Pichia
pastoris. Mol. Nutr. Food Res. 62 (1), 1700297 https://doi.org/10.1002/
mnfr.201700297.
Jindal, N., Singh Khattar, J., 2018. Microbial polysaccharides in food industry. In:
Biopolymers for food design. Elsevier Inc, pp. 95–123. https://doi.org/10.1016/
B978-0-12-811449-0.00004-9.
Jongedijk, E., Cankar, K., Buchhaupt, M., Schrader, J., Bouwmeester, H., Beekwilder, J.,
2016. Biotechnological production of limonene in microorganisms. Appl. Microbiol.
Biotechnol. 100, 2927–2938. https://doi.org/10.1007/s00253-016-7337-7.
Joseph, J.A., Akkermans, S., Nimmegeers, P., Van Impe, J.F.M., 2019. Bioproduction of
the recombinant SWEET protein thaumatin: Current state of the art and perspectives.
Front. Microbiol. 10, 1–19. https://doi.org/10.3389/fmicb.2019.00695.
Ju, L.-K., Zhao, S., 1993. Xanthan fermentations in water/oil dispersions. Biotechnol.
Tech. 7 (7), 463–468.
Jung, S.M., Chin, Y.W., Lee, Y.G., Seo, J.H., 2019. Enhanced production of 2’-
fucosyllactose from fucose by elimination of rhamnose isomerase and arabinose
isomerase in engineered Escherichia coli. Biotechnol. Bioeng. 116, 2412–2417.
https://doi.org/10.1002/bit.27019.
Kallscheuer, N., 2018. Engineered microorganisms for the production of food additives
approved by the European Union-A systematic analysis. Front. Microbiol. 9, 1–20.
https://doi.org/10.3389/fmicb.2018.01746.
Katrolia, P., Rajashekhara, E., Yan, Q., Jiang, Z., 2014. Biotechnological potential of
microbial
α
-galactosidases. Crit. Rev. Biotechnol. 34, 307–317. https://doi.org/
10.3109/07388551.2013.794124.
Katsuyama, Y., Hirose, Y., Funa, N., Ohnishi, Y., Horinouchi, S., 2010. Precursor-directed
biosynthesis of curcumin analogs in escherichia coli. Biosci. Biotechnol. Biochem.
74, 641–645. https://doi.org/10.1271/bbb.90866.
Kaur, B., Chakraborty, D., 2013. Biotechnological and molecular approaches for vanillin
production: A review. Appl. Biochem. Biotechnol. 169 (4), 1353–1372. https://doi.
org/10.1007/s12010-012-0066-1.
Kawasaki, H., Martinac, B., 2020. Mechanosensitive channels of Corynebacterium
glutamicum functioning as exporters of L-glutamate and other valuable metabolites.
Curr. Opin. Chem. Biol. 59, 77–83. https://doi.org/10.1016/j.cbpa.2020.05.005.
Keefe, D. M., 2016. Agency response letter GRAS notice No. GRN 000600.
Hydroxytyrosol. Available online: https://www.fda.gov/food/gras-notice-inventory
/agency-response-letter-gras-notice-no-grn-000600 (Accessed on 23 November
2020).
Khelissa, S., Chihib, N.E., Gharsallaoui, A., 2020. Conditions of nisin production by
Lactococcus lactis subsp. lactis and its main uses as a food preservative. Arch.
Microbiol. https://doi.org/10.1007/s00203-020-02054-z.
Kim, E.J., Cha, M.N., Kim, B.G., Ahn, J.H., 2017. Production of curcuminoids in
engineered Escherichia coli. J. Microbiol. Biotechnol. 27, 975–982. https://doi.org/
10.4014/jmb.1701.01030.
Kimura, E., 2003. Metabolic engineering of glutamate production. Adv. Biochem. Eng.
Biotechnol. 79, 37–57. https://doi.org/10.1007/3-540-45989-8_2.
Koeduka, T., Watanabe, B., Suzuki, S., Hiratake, J., Mano, J., Yazaki, K., 2011.
Characterization of raspberry ketone/zingerone synthase, catalyzing the alpha, beta-
hydrogenation of phenylbutenones in raspberry fruits. Biochem. Biophys. Res.
Commun. 412, 104–108. https://doi.org/10.1016/j.bbrc.2011.07.052.
Kongklom, N., Shi, Z., Chisti, Y., Sirisansaneeyakul, S., 2017. Enhanced production of
poly-γ-glutamic acid by Bacillus licheniformis TISTR 1010 with environmental
controls. Appl. Biochem. Biotechnol. 182, 990–999. https://doi.org/10.1007/
s12010-016-2376-1.
Koopman, F., Beekwilder, J., Crimi, B., van Houwelingen, A., Hall, R.D., Bosch, D., van
Maris, A.J.A., Pronk, J.T., Daran, J.M., 2012. De novo production of the avonoid
naringenin in engineered Saccharomyces cerevisiae. Microb. Cell Fact. 11, 1–15.
https://doi.org/10.1186/1475-2859-11-155.
Kunjapur, A.M., Tarasova, Y., Prather, K.L.J., 2014. Synthesis and accumulation of
aromatic aldehydes in an engineered strain of Escherichia coli. J. Am. Chem. Soc.
136, 11644–11654. https://doi.org/10.1021/ja506664a.
Kuppuswami, G.M., 2014. Fermentation (industrial): production of xanthan gum. In:
Encyclopedia of Food Microbiology: Second Edition, Second Edi. Elsevier,
pp. 816–821. https://doi.org/10.1016/B978-0-12-384730-0.00110-5.
Lee, K.-H., Park, Y.-H., Han, J.-K., Park, J.-H., Lee, K.-H., Choi, H., 2007. Microorganism
for producing riboavin and method for producing riboavin using the same. Patent
US7166456B2.
Lee, D., Lloyd, N.D.R., Pretorius, I.S., Borneman, A.R., 2016. Heterologous production of
raspberry ketone in the wine yeast Saccharomyces cerevisiae via pathway engineering
and synthetic enzyme fusion. Microb. Cell Fact. 15, 1–7. https://doi.org/10.1186/
s12934-016-0446-2.
Lee, A., Choi, K.H., Yoon, D., Kim, S., Cha, J., 2017. Characterization of a thermostable
glycoside hydrolase family 36
α
-galactosidase from Caldicellulosiruptor bescii.
J. Biosci. Bioeng. 124, 289–295. https://doi.org/10.1016/j.jbiosc.2017.04.011.
Lee, J.M., Kim, J.H., Kim, K.W., Lee, B.J., Kim, D.G., Kim, Y.O., Lee, J.H., Kong, I.S.,
2018. Physicochemical properties, production, and biological functionality of poly-
γ-D-glutamic acid with constant molecular weight from halotolerant Bacillus sp. SJ-
10. Int. J. Biol. Macromol. 108, 598–607. https://doi.org/10.1016/j.
ijbiomac.2017.12.055.
Li, H., Alper, H.S., 2020. Producing biochemicals in Yarrowia lipolytica from xylose
through a strain mating approach. Biotechnol. J. 15 (2), e1900304 https://doi.org/
10.1002/biot.201900304.
Li, P., Li, T., Zeng, Y., Li, X., Jiang, X., Wang, Y., Xie, T., Zhang, Y., 2016. Biosynthesis of
xanthan gum by Xanthomonas campestris LRELP-1 using kitchen waste as the sole
substrate. Carbohydr. Polym. 151, 684–691. https://doi.org/10.1016/j.
carbpol.2016.06.017.
Li, X., Chen, Z., Wu, Y., Yan, Y., Sun, X., Yuan, Q., 2018a. Establishing an articial
pathway for efcient biosynthesis of hydroxytyrosol. ACS Synth. Biol. 7, 647–654.
https://doi.org/10.1021/acssynbio.7b00385.
Li, Z., Meng, T., Ling, X., Li, J., Zheng, C., Shi, Y., Chen, Z., Li, Zhenqi, Li, Q., Lu, Y.,
He, N., 2018b. Overexpression of Malonyl-CoA: ACP transacylase in Schizochytrium
sp. to improve polyunsaturated fatty acid production. J. Agric. Food Chem. 66,
5382–5391. https://doi.org/10.1021/acs.jafc.8b01026.
Li, Q., Ray, C.S., Callow, N.V., Loman, A.A., Islam, S.M.M., Ju, L.K., 2020a. Aspergillus
niger production of pectinase and
α
-galactosidase for enzymatic soy processing.
Enzyme Microb. Technol. 134, 109476 https://doi.org/10.1016/j.
enzmictec.2019.109476.
Li, Z., Li, F., Cai, L., Chen, Z., Qin, L., Gao, X.D., 2020b. One-pot multienzyme synthesis
of rare ketoses from glycerol. J. Agric. Food Chem. 68, 1347–1353. https://doi.org/
10.1021/acs.jafc.9b06748.
Liang, C., Zhang, X., Wu, J., Mu, S., Wu, Z., Jin, J.M., Tang, S.Y., 2020. Dynamic control
of toxic natural product biosynthesis by an articial regulatory circuit. Metab. Eng.
57, 239–246. https://doi.org/10.1016/j.ymben.2019.12.002.
Lim, B.C., Kim, H.J., Oh, D.K., 2007. High production of D-tagatose by the addition of
boric acid. Biotechnol. Prog. 23, 824–828. https://doi.org/10.1021/bp070056y.
Lin, Z., Xu, Z., Li, Y., Wang, Z., Chen, T., Zhao, X., 2014. Metabolic engineering of
Escherichia coli for the production of riboavin. Microb. Cell Fact. https://doi.org/
10.1186/s12934-014-0104-5.
Liu, C.Q., Chen, Q.H., Tang, B., Ruan, H., He, G.Q., 2007. Response surface methodology
for optimizing the fermentation medium of alpha-galactosidase in solid-state
L. Sun et al.
Biotechnology Advances 47 (2021) 107694
18
fermentation. Lett. Appl. Microbiol. 45, 206–212. https://doi.org/10.1111/j.1472-
765X.2007.02173.x.
Liu, L., Martínez, J.L., Liu, Z., Petranovic, D., Nielsen, J., 2014. Balanced globin protein
expression and heme biosynthesis improve production of human hemoglobin in
Saccharomyces cerevisiae. Metab. Eng. 21, 9–16. https://doi.org/10.1016/j.
ymben.2013.10.010.
Liu, X., Yu, X., Lv, J., Xu, J., Xia, J., Wu, Z., Zheng, T., Deng, Y., 2017. A cost-effective
process for the coproduction of erythritol and lipase with Yarrowia lipolytica M53
from waste cooking oil. Food Bioprod. Process. 103, 86–94. https://doi.org/
10.1016/j.fbp.2017.03.002.
Liu, J., Kwak, S., Pathanibul, P., Lee, J., Yu, S., Yun, E.J., Jin, Y-S., 2018a. Biosynthesis of
a functional human milk oligosaccharide, 2′-Fucosyllactose, and L-fucose, using
engineered Saccharomyces cerevisiae. ACS Synth. Biol. 7, 2529–2536. https://doi
:10.1021/acssynbio.8b00134.
Liu, Y., Wang, Z., Liu, M., Zhou, L., Sha, Y., 2018b. Bioproduction of low-pigment
xanthan gum by a cell-wall decient mutant of Xanthomonas campestris. Prep.
Biochem. Biotechnol. 48 (10), 940–945. https://doi.org/10.1080/
10826068.2018.1525561.
Liu, J.J., Zhang, G.C., Kwak, S., Oh, E.J., Yun, E.J., Chomvong, K., Cate, J.H.D., Jin, Y.S.,
2019. Overcoming the thermodynamic equilibrium of an isomerization reaction
through oxidoreductive reactions for biotransformation. Nat. Commun. 10, 1–8.
https://doi.org/10.1038/s41467-019-09288-6.
Liu, S., Hu, W., Wang, Z., Chen, T., 2020. Production of riboavin and related cofactors
by biotechnological processes. Microb. Cell Fact. 19, 1–16. https://doi.org/10.1186/
s12934-020-01302-7.
Luo, Z., Guo, Y., Liu, J., Qiu, H., Zhao, M., Zou, W., Li, S., 2016. Microbial synthesis of
poly-γ-glutamic acid: Current progress, challenges, and future perspectives.
Biotechnol. Biofuels 9, 1–12. https://doi.org/10.1186/s13068-016-0537-7.
Luziatelli, F., Brunetti, L., Ficca, A.G., Ruzzi, M., 2019. Maximizing the efciency of
vanillin production by biocatalyst enhancement and process optimization. Front.
Bioeng. Biotechnol. 7, 1–14. https://doi.org/10.3389/fbioe.2019.00279.
Ma, Q., Zhang, Q., Xu, Q., Zhang, C., Li, Y., Fan, X., Xie, X., Chen, N., 2017. Systems
metabolic engineering strategies for the production of amino acids. Synth. Syst.
Biotechnol. 2, 87–96. https://doi.org/10.1016/j.synbio.2017.07.003.
Ma, T., Shi, B., Ye, Z., Li, X., Liu, M., Chen, Y., Xia, J., Nielsen, J., Deng, Z., Liu, T., 2019.
Lipid engineering combined with systematic metabolic engineering of Saccharomyces
cerevisiae for high-yield production of lycopene. Metab. Eng. 52, 134–142. https://
doi.org/10.1016/j.ymben.2018.11.009.
Market Study Report, 2020, Global isoamyl acetate market 2020 by manufacturers,
regions, type and application, forecast to 2025, Available online: https://www.mar
ketstudyreport.com/reports/global-isoamyl-acetate-market-2020-by-manufact
urers-regions-type-and-application-forecast-to-2025 (Accessed on 23 November
2020).
Market Watch, 2020a, Global limonene market size top manufacturers entry, global
industry analysis, market share, growth, trends, showing impressive growth by 2026,
Available online: https://www.marketwatch.com/press-release/global-limonene-
market-size-top-manufacturers-entry-global-industry-analysis-market-share-gr
owth-trends-showing-impressive-growth-by-2026-2020-10-09 (Accessed on 23
November 2020).
Market Watch, 2020b, Thaumatin market 2020: Top countries data, market size,
denation, brief analysis of global industry with forecast growth by 2024, Available
online: https://www.marketwatch.com/press-release/thaumatin-market-2020-top-
countries-data-market-size-denation-brief-analysis-of-global-industry-with-forecas
t-growth-by-2024-2020-08-05 (Accessed on 23 November 2020).
Markets and Markets, 2016, Low intensity sweeteners market by type (xylitol, tagatose,
allulose, trehalose, & isomaltuolose), application, & by geography - global trends &
forecasts to 2020, Available online: https://www.marketsandmarkets.com/Market-
Reports/low-intensity-sweeteners-market-4777320.html (Accessed on 23 November
2020).
Markets and Markets, 2019a, Ketones market by application (supplements, food &
beverage, cosmetics & personal care), supplement type (ketone salts, ketone esters,
ketone oils, raspberry ketones), form and region – global forecast to 2025, Available
online: https://www.marketsandmarkets.com/Market-Reports/ketone-market
-123702593.html (Accessed on 23 November 2020).
Markets and Markets, 2019b, Omega-3 market by type (DHA, EPA, and ALA), application
(dietary supplements, functional foods & beverages, pharmaceuticals, infant
formula, and pet food & feed), source (marine and plant), and region - global
forecasts to 2025, Available online: https://www.marketsandmarkets.com/Ma
rket-Reports/omega-3-omega-6-227.html (Accessed on 23 November 2020).
Markets and Markets, 2020a, Lycopene market by source (synthetic and natural),
application (dietary supplements, food, pharmaceuticals, and personal care
products), form (beadlets, oil suspension, emulsiers, and powder), property, and
region - global forecast to 2025, Available online: https://www.marketsandmarkets.
com/Market-Reports/lycopene-market-104131737.html (Accessed on 23 November
2020).
Markets and Markets, 2020b, Nisin market by application (meat, poultry & seafood
products, dairy products, beverages, bakery & confectionery products, canned &
frozen food products, and other applications) and region - global trends and forecast
to 2025, Available online: https://www.marketsandmarkets.com/Market-Repor
ts/nisin-market-29041412.html (Accessed on 23 November 2020).
Mart˘
au, G.A., Coman, V., Vodnar, D.C., 2020. Recent advances in the biotechnological
production of erythritol and mannitol. Crit. Rev. Biotechnol. 40 (5), 608–622.
https://doi.org/10.1080/07388551.2020.1751057.
Martins, N., Roriz, C.L., Morales, P., Barros, L., Ferreira, I.C.F.R., 2017. Coloring
attributes of betalains: A key emphasis on stability and future applications. Food
Funct. 8, 1357–1372. https://doi.org/10.1039/c7fo00144d.
Martins, F.C.O.L., Sentanin, M.A., De Souza, D., 2019. Analytical methods in food
additives determination: Compounds with functional applications. Food Chem. 272,
732–750. https://doi.org/10.1016/j.foodchem.2018.08.060.
Masuda, T., Ide, N., Ohta, K., Kitabatake, N., 2010. High-yield secretion of the
recombinant sweet-tasting protein thaumatin I. Food Sci. Technol. Res. 16, 585–592.
https://doi.org/10.3136/fstr.16.585.
Mbonyiryivuze, A., Nuru, Y., Diop Ngom, B., Mwakikunga, B., Mokhotjwa Dhlamini, S.,
Park, E., Maaza, M., 2015. Morphological and chemical composition
characterization of commercial sepia melanin. Am. J. Nanomater 3, 22–27. https://
doi.org/10.12691/ajn-3-1-3.
Mejía-Caballero, A., Anda, R., Hern´
andez-Ch´
avez, G., Rogg, S., Martinez, A., Bolívar, F.,
Casta˜
no, V.M., Gosset, G., 2016. Biosynthesis of catechol melanin from glycerol
employing metabolically engineered Escherichia coli. Microb. Cell Fact. 15, 1–8.
https://doi.org/10.1186/s12934-016-0561-0.
Mikkelsen, M.D., Hansen, J., Simon, E., Brianza, F., Semmler, A., Olsson, K., Carlsen, S.,
Düring, L., Ouspensku, A., Hicks, P., 2014. Methods for improved production of
Rebaudioside D and Rebaudioside M. Patent WO2014/122227 A9.
Milke, L., Aschenbrenner, J., Marienhagen, J., Kallscheuer, N., 2018. Production of plant-
derived polyphenols in microorganisms: current state and perspectives. Appl.
Microbiol. Biotechnol. 102, 1575–1585. https://doi.org/10.1007/s00253-018-8747-
5.
Milke, L., Mutz, M., Marienhagen, J., 2020. Synthesis of the character impact compound
raspberry ketone and additional avoring phenylbutanoids of biotechnological
interest with Corynebacterium glutamicum. Microb. Cell Fact. 19, 1–12. https://doi.
org/10.1186/s12934-020-01351-y.
Miro´
nczuk, A.M., Dobrowolski, A., Rakicka, M., Rywi´
nska, A., Rymowicz, W., 2015.
Newly isolated mutant of Yarrowia lipolytica MK1 as a proper host for efcient
erythritol biosynthesis from glycerol. Process Biochem. 50 (1), 61–68. https://doi.
org/10.1016/j.procbio.2014.10.020.
Mohsin, A., Zhang, K., Hu, J., Salim-ur-Rehman, Tariq, Zaman, W.Q., Khan, I.M.,
Zhuang, Y., Guo, M., 2018. Optimized biosynthesis of xanthan via effective
valorization of orange peels using response surface methodology: A kinetic model
approach. Carbohydr. Polym. 181, 793–800. https://doi.org/10.1016/j.
carbpol.2017.11.076.
Moralejo, F.J., Watson, A.J., Jeenes, D.J., Archer, D.B., Martín, J.F., 2001. A dened level
of protein disulde isomerase expression is required for optimal secretion of
thaumatin by Aspergillus awamori. Mol. Genet. Genomics 266, 246–253. https://doi.
org/10.1007/s004380100550.
Mordor Intelligence LLP, 2019, Vanillin Market - Growth, Trends, And Forecast (2019-
2024), Available online: https://www.businesswire.com/news/home/20191121005
621/en/Global-Vanillin-Industry-Report-2019-2024-Key-Players-are-Solvay-Evolva
-Camlin-Fine-Sciences-Lesaffre-Ennloys-and-Prinova-Group—ResearchAndMarkets.
com (Accessed on 23 November 2020).
Naikwadi, D.R., Naikwadi, D.R., Ravi, K., Ravi, K., Singh, A.S., Singh, A.S., Advani, J.H.,
Biradar, A.V., Biradar, A.V., 2020. Gram-scale synthesis of avoring ketones in one
pot via alkylation-decarboxylation on benzylic carbon using a commercial solid acid
catalyst. ACS Omega 5, 14291–14296. https://doi.org/10.1021/acsomega.0c00416.
Ni, J., Tao, F., Du, H., Xu, P., 2015. Mimicking a natural pathway for de novo
biosynthesis: Natural vanillin production from accessible carbon sources. Sci. Rep. 5,
13670 https://doi.org/10.1038/srep13670.
Ni, J., Gao, Y.Y., Tao, F., Liu, H.Y., Xu, P., 2018a. Temperature-directed biocatalysis for
the sustainable production of aromatic aldehydes or alcohols. Angew. Chemie - Int.
Ed. 57 (5), 1214–1217. https://doi.org/10.1002/anie.201710793.
Ni, J., Wu, Y.T., Tao, F., Peng, Y., Xu, P., 2018b. A coenzyme-free biocatalyst for the
value-added utilization of lignin-derived aromatics. J. Am. Chem. Soc. 140,
16001–16005. https://doi.org/10.1021/jacs.8b08177.
Ogunleye, A., Bhat, A., Irorere, V.U., Hill, D., Williams, C., Radecka, I., 2015. Poly-
γ-glutamic acid: Production, properties and applications. Microbiol. (United
Kingdom) 161, 1–17. https://doi.org/10.1099/mic.0.081448-0.
Olsson, K., Carlsen, S., Semmler, A., Sim´
on, E., Mikkelsen, M.D., Møller, B.L., 2016.
Microbial production of next-generation stevia sweeteners. Microb. Cell Fact. 15,
1–14. https://doi.org/10.1186/s12934-016-0609-1.
¨
Ozel, B., S
¸ims
¸ek, ¨
O., Akçelik, M., Saris, P.E.J., 2018. Innovative approaches to nisin
production. Appl. Microbiol. Biotechnol. 102, 6299–6307. https://doi.org/10.1007/
s00253-018-9098-y.
Palmer, C.M., Alper, H.S., 2019. Expanding the chemical palette of industrial microbes:
Metabolic engineering for type III PKS-derived polyketides. Biotechnol. J. 14 (1),
e1700463 https://doi.org/10.1002/biot.201700463.
Palmer, C.M., Miller, K.K., Nguyen, A., Alper, H.S., 2020. Engineering 4-coumaroyl-CoA
derived polyketide production in Yarrowia lipolytica through a β-oxidation mediated
strategy. Metab. Eng. 57, 174–181. https://doi.org/10.1016/j.ymben.2019.11.006.
Palms, B., 2017. Evolva’s 21st century brewing. Available online: https://www.bio.or
g/sites/default/les/legacy/bioorg/docs/1030AM-Benoit%20Palms.pdf (Accessed
on 23 November 2020).
Pang, B., Pang, B., Pang, B., Chen, Y., Chen, Y., Gan, F., Gan, F., Gan, F., Yan, C., Jin, L.,
Gin, J.W., Gin, J.W., Petzold, C.J., Petzold, C.J., Keasling, J.D., 2020. Investigation of
indigoidine synthetase reveals a conserved active-site base residue of nonribosomal
peptide synthetase oxidases. J. Am. Chem. Soc. 142, 10931–10935. https://doi.org/
10.1021/jacs.0c04328.
Park, Y.C., Oh, E.J., Jo, J.H., Jin, Y.S., Seo, J.H., 2016. Recent advances in biological
production of sugar alcohols. Curr. Opin. Biotechnol. 37, 105–113. https://doi.org/
10.1016/j.copbio.2015.11.006.
Parschat, K., Schreiber, S., Wartenberg, D., Engels, B., Jennewein, S.M., 2020. High-titer
de novo biosynthesis of the predominant human milk oligosaccharide 2′
fucosyllactose from sucrose in Escherichia coli. ACS Synth 9 (10), 2784–2796.
L. Sun et al.
Biotechnology Advances 47 (2021) 107694
19
Patel, Y., Schneider, J.C., Ielpi, L., Ielmini, M.V., 2008. High viscosity xanthan polymer
preparations, Patent US7439044B2.
Pavan, M.E., L´
opez, N.I., Pettinari, M.J., 2020. Melanin biosynthesis in bacteria,
regulation and production perspectives. Appl. Microbiol. Biotechnol. 104,
1357–1370. https://doi.org/10.1007/s00253-019-10245-y.
Pombeiro-Sponchiado, S.R., Sousa, G.S., Andrade, J.C.R., Lisboa, H.F., Gonçalves, R.C.R.,
2017. Production of melanin pigment by fungi and its biotechnological applications.
In: Melanin, pp. 47–75. https://doi.org/10.5772/67375.
Prakash, I., Markosyan, A., Bunders, C., 2014. Development of next generation Stevia
sweetener: Rebaudioside M. Foods 3, 162–175. https://doi.org/10.3390/
foods3010162.
Prasad, S., Roy, I., 2017. Converting enzymes into tools of industrial importance. Recent
Pat. Biotechnol. 12, 33–56. https://doi.org/10.2174/
1872208311666170612113303.
Qiu, X., Xu, P., Zhao, X., Du, G., Zhang, J., Li, J., 2020. Combining genetically-encoded
biosensors with high throughput strain screening to maximize erythritol production
in Yarrowia lipolytica. Metab. Eng. 60, 66–76. https://doi.org/10.1016/j.
ymben.2020.03.006.
Raghavan, S., Hansen, J., Sonkar, S., Kumar, S., Panchapagesa, M., Hansen, E.H.,
Hansen, K.R., Kumar, K., 2014. Methods and materials for recombinant production
of saffron compounds. Patent US20140248668A1.
Rahman, D.Y., Sarian, F.D., van Wijk, A., Martinez-Garcia, M., van der Maarel, M.J.E.C.,
2017. Thermostable phycocyanin from the red microalga Cyanidioschyzon merolae, a
new natural blue food colorant. J. Appl. Phycol. 29 (3), 1233–1239. https://doi.org/
10.1007/s10811-016-1007-0.
Rakicka, M., Lazar, Z., Rywi´
nska, A., Rymowicz, W., 2016. Efcient utilization of inulin
and glycerol as fermentation substrates in erythritol and citric acid production using
Yarrowia lipolytica expressing inulinase. Chem. Pap. 70, 1452–1459. https://doi.org/
10.1515/chempap-2016-0085.
Ranveer, R.C., 2018. Lycopene: A natural red pigment, Natural and artiial avoring
agents and food dyes: Handbook of Food Bioengineering. Elsevier Inc. https://doi.
org/10.1016/C2016-0-00380-7.
Ren, Y., Liu, S., Jin, G., Yang, X., Zhou, Y.J., 2020. Microbial production of limonene and
its derivatives: Achievements and perspectives. Biotechnol. Adv. 44, 107628 https://
doi.org/10.1016/j.biotechadv.2020.107628.
Reports Express, 2020a, Global safranal market report 2020, Available online: https
://reportsexpress.com/safranal-market-report/ (Accessed on 23 November 2020).
Reports Express, 2020b, Global isobutyl acetate market report 2020, Available online:
https://reportsexpress.com/isobutyl-acetate-market-report/ (Accessed on 23
November 2020).
Revuelta, J.L., Ledesma-Amaro, R., Jim´
enez, A., 2016. Industrial production of vitamin
B2 by microbial fermentation. In: Industrial biotechnology of vitamins, biopigments,
and antioxidants. Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany,
pp. 15–40. https://doi.org/10.1002/9783527681754.ch2.
Ribera, J., Panzarasa, G., Stobbe, A., Osypova, A., Rupper, P., Klose, D., Schwarze, F.W.
M.R., 2019. Scalable biosynthesis of melanin by the Basidiomycete Armillaria
cepistipes. J. Agric. Food Chem. 67, 132–139. https://doi.org/10.1021/acs.
jafc.8b05071.
Rodrigues, J.L., Prather, K.L.J., Kluskens, L.D., Rodrigues, L.R., 2015. Heterologous
production of curcuminoids. Microbiol. Mol. Biol. Rev. 79, 39–60. https://doi.org/
10.1128/mmbr.00031-14.
Rodrigues, J.L., Gomes, D., Rodrigues, L.R., 2020. A combinatorial approach to optimize
the production of curcuminoids from tyrosine in Escherichia coli. Front. Bioeng.
Biotechnol. 8, 1–15. https://doi.org/10.3389/fbioe.2020.00059.
Rodriguez, G.M., Tashiro, Y., Atsumi, S., 2014. Expanding ester biosynthesis in
Escherichia coli. Nat. Chem. Biol. 10 (4), 259–265. https://doi.org/10.1038/
nchembio.1476.
Rolf, J., Julsing, M.K., Rosenthal, K., Lütz, S., 2020. A gram-scale limonene production
process with engineered Escherichia coli. Molecules 25, 1–12. https://doi.org/
10.3390/molecules25081881.
Ronˇ
cevi´
c, Z., Grahovac, J., Dodi´
c, S., Vuˇ
curovi´
c, D., Dodi´
c, J., 2019. Utilisation of winery
wastewater for xanthan production in stirred tank bioreactor: Bioprocess modelling
and optimisation. Food Bioprod. Process. 117, 113–125. https://doi:10.1016/j.
fbp.2019.06.019.
Sahm, H., Antranikian, G., Stahmann, K.-P., Takors, R., 2013. Riboavin (vitamin B2).
In: Sahm, H., Antranikian, G., Stahmann, K.-P., Takors, R. (Eds.), Industrielle
Mikrobiologie. Springer-Verlag, Berlin-Heidelberg, pp. 132–140. https://doi.org/
10.1007/978-3-8274-3040-3.
S´
anchez, C., Bra˜
na, A.F., M´
endez, C., Salas, J.A., 2006. Reevaluation of the violacein
biosynthetic pathway and its relationship to indolocarbazole biosynthesis.
ChemBioChem 7, 1231–1240. https://doi.org/10.1002/cbic.200600029.
Sano, C., 2009. History of glutamate production. Am. J. Clin. Nutr. 90 (3), 728S–732S.
https://doi.org/10.3945/ajcn.2009.27462F.
Santos, C.N.S., Koffas, M., Stephanopoulos, G., 2011. Optimization of a heterologous
pathway for the production of avonoids from glucose. Metab. Eng. 13, 392–400.
https://doi.org/10.1016/j.ymben.2011.02.002.
Satoh, Y., Tajima, K., Munekata, M., Keasling, J.D., Lee, T.S., 2012. Engineering of l-
tyrosine oxidation in Escherichia coli and microbial production of hydroxytyrosol.
Metab. Eng. 14, 603–610. https://doi.org/10.1016/j.ymben.2012.08.002.
Schempp, F.M., Drummond, L., Buchhaupt, M., Schrader, J., 2018. Microbial cell
factories for the production of terpenoid avor and fragrance compounds. J. Agric.
Food Chem. 66, 2247–2258. https://doi.org/10.1021/acs.jafc.7b00473.
Schwechheimer, S.K., Park, E.Y., Revuelta, J.L., Becker, J., Wittmann, C., 2016.
Biotechnology of riboavin. Appl. Microbiol. Biotechnol. 100, 2107–2119. https://
doi.org/10.1007/s00253-015-7256-z.
Sen, T., Barrow, C.J., Deshmukh, S.K., 2019. Microbial pigments in the food
industry—challenges and the way forward. Front. Nutr. 6, 1–14. https://doi.org/
10.3389/fnut.2019.00007.
Seo, H., Nicely, P.N., Trinh, C.T., 2020. Endogenous carbohydrate esterases of Clostridium
thermocellum are identied and disrupted for enhanced isobutyl acetate production
from cellulose. Biotechnol. Bioeng. 117, 2223–2236. https://doi.org/10.1002/
bit.27360.
Sevgili, A., Erkmen, O., 2019. Improved lycopene production from different substrates by
mated fermentation of Blakeslea trispora. Foods 8 (4), 120. https://doi.org/10.3390/
foods8040120.
Seydametova, E., Yu, J., Shin, J., Park, Yoonjung, Kim, C., Kim, H., Yu, S.H.,
Park, Yongcheol, Kweon, D.H., 2019. Search for bacterial
α
1,2-fucosyltransferases
for whole-cell biosynthesis of 2′-fucosyllactose in recombinant Escherichia coli.
Microbiol. Res. 222, 35–42. https://doi.org/10.1016/j.micres.2019.02.009.
Sha, Y., Zhang, Y., Qiu, Y., Xu, Z., Li, S., Feng, X., Wang, M., Xu, H., 2019. Efcient
Biosynthesis of low-molecular-weight poly-γ-glutamic acid by stable overexpression
of PgdS hydrolase in Bacillus amyloliquefaciens NB. J. Agric. Food Chem. 67,
282–290. https://doi.org/10.1021/acs.jafc.8b05485.
Shankar, S., Hoyt, M.A., 2019a. Expression constructs and methods of genetically
engineering methylotrophic yeast, Patent US10273492B2.
Shankar, S., Hoyt, M.A., 2019b. Expression constructs and methods of genetically
engineering methylotrophic yeast, Patent US10273492B2.
Shi, B., Ma, T., Ye, Z., Li, X., Huang, Y., Zhou, Z., Ding, Y., Deng, Z., Liu, T., 2019.
Systematic metabolic engineering of Saccharomyces cerevisiae for lycopene
overproduction. J. Agric. Food Chem. 67, 11148–11157. https://doi.org/10.1021/
acs.jafc.9b04519.
Sigurdson, G.T., Tang, P., Giusti, M.M., 2017. Natural colorants: Food colorants from
natural sources. Annu. Rev. Food Sci. Technol. 8, 261–280. https://doi.org/
10.1146/annurev-food-030216-025923.
Singh, O.V., 2017. Bio-pigmentation and biotechnological implementations. John Wiley
& Sons, Inc. https://doi.org/10.1002/9781119166191 (Chapter 1).
Siripong, W., Wolf, P., Kusumoputri, T.P., Downes, J.J., Kocharin, K., Tanapongpipat, S.,
Runguphan, W., 2018. Metabolic engineering of Pichia pastoris for production of
isobutanol and isobutyl acetate. Biotechnol. Biofuels 11, 1–16. https://doi.org/
10.1186/s13068-017-1003-x.
Soleymanpour, Z., Nikzad, M., Talebnia, F., Niknezhad, V., 2018. Xanthan gum
production from acid hydrolyzed broomcorn stem as a sole carbon source by
Xanthomonas campestris. 3 Biotech 8, 1–12. https://doi.org/10.1007/s13205-018-
1322-z.
Sun, L., Alper, H.S., 2020. Non-conventional hosts for the production of fuels and
chemicals. Curr. Opin. Chem. Biol. 59, 15–22. https://doi.org/10.1016/j.
cbpa.2020.03.004.
Sun, H., Zhao, D., Xiong, B., Zhang, C., Bi, C., 2016. Engineering Corynebacterium
glutamicum for violacein hyper production. Microb. Cell Fact. 15, 1–9. https://doi.
org/10.1186/s12934-016-0545-0.
Tai, Y.S., Xiong, M., Zhang, K., 2015. Engineered biosynthesis of medium-chain esters in
Escherichia coli. Metab. Eng. 27, 20–28. https://doi.org/10.1016/j.
ymben.2014.10.004.
Tan, H., Chen, X., Liang, N., Chen, R., Chen, J., Hu, C., Li, Qi, Li, Qing, Pei, W., Xiao, W.,
Yuan, Y., Chen, W., Zhang, L., 2019. Transcriptome analysis reveals novel enzymes
for apo-carotenoid biosynthesis in saffron and allows construction of a pathway for
crocetin synthesis in yeast. J. Exp. Bot. 70, 4819–4833. https://doi.org/10.1093/
jxb/erz211.
Tocher, D.R., Betancor, M.B., Sprague, M., Olsen, R.E., Napier, J.A., 2019. Omega-3 long-
chain polyunsaturated fatty acids, EPA and DHA: Bridging the gap between supply
and demand. Nutrients 11, 1–20. https://doi.org/10.3390/nu11010089.
van Berlo, D., Wallinga, A.E., van Acker, F.A., Delsing, D.J., 2018. Safety assessment of
biotechnologically produced 2′-Fucosyllactose, a novel food additive. Food Chem.
Toxicol. 118, 84–93. https://doi.org/10.1016/j.fct.2018.04.049.
Venil, C.K., Aruldass, C.A., Abd Halim, M.H., Khasim, A.R., Zakaria, Z.A., Ahmad, W.A.,
2015. Spray drying of violet pigment from Chromobacterium violaceum UTM 5 and its
application in food model systems. Int. Biodeterior. Biodegrad. 102, 324–329.
https://doi.org/10.1016/j.ibiod.2015.02.006.
Wagner, J.M., Liu, L., Yuan, S.F., Venkataraman, M.V., Abate, A.R., Alper, H.S., 2018.
A comparative analysis of single cell and droplet-based FACS for improving
production phenotypes: Riboavin overproduction in Yarrowia lipolytica. Metab.
Eng. 47, 346–356. https://doi.org/10.1016/j.ymben.2018.04.015.
Wang, Z., Wu, J., Gao, M.J., Zhu, L., Zhan, X.B., 2017a. High production of xanthan gum
by a glycerol-tolerant strain Xanthomonas campestris WXLB-006. Prep. Biochem.
Biotechnol. 47 (5), 468–472. https://doi.org/10.1080/10826068.2017.1292288.
Wang, Z., Wu, J., Zhu, L., Zhan, X., 2017b. Characterization of xanthan gum produced
from glycerol by a mutant strain Xanthomonas campestris CCTCC M2015714.
Carbohydr. Polym. 157, 521–526. https://doi.org/10.1016/j.carbpol.2016.10.033.
Wang, Y., Cao, G., Xu, D., Fan, L., Wu, X., Ni, X., Zhao, S., Zheng, P., Sun, J., Ma, Y.,
2018. A novel Corynebacterium glutamicum L-glutamate exporter. Appl. Environ.
Microbiol. 84 (6) https://doi.org/10.1128/AEM.02691-17 e02691-17.
Wang, C., Zheng, P., Chen, P., 2019a. Construction of synthetic pathways for raspberry
ketone production in engineered Escherichia coli. Appl. Microbiol. Biotechnol. 103
(9), 3715–3725. https://doi.org/10.1007/s00253-019-09748-5.
Wang, F., Bi, Y., Diao, J., Lv, M., Cui, J., Chen, L., Zhang, W., 2019b. Metabolic
engineering to enhance biosynthesis of both docosahexaenoic acid and odd-chain
fatty acids in Schizochytrium sp. S31. Biotechnol. Biofuels 12, 1–14. https://doi.org/
10.1186/s13068-019-1484-x.
Wang, W., He, P., Zhao, D., Ye, L., Dai, L., Zhang, X., Sun, Y., Zheng, J., Bi, C., 2019c.
Construction of Escherichia coli cell factories for crocin biosynthesis. Microb. Cell
Fact. 18 (1), 120. https://doi.org/10.1186/s12934-019-1166-1.
L. Sun et al.
Biotechnology Advances 47 (2021) 107694
20
Wang, Z., Tschirhart, T., Schultzhaus, Z., Kelly, E.E., Chen, A., Oh, E., Nag, O., Glaser, E.
R., Kim, E., Lloyd, P.F., Charles, P.T., Li, W., Leary, D., Compton, J., Phillips, D.A.,
Dhinojwala, A., Payne, G.F., Vora, G.J., 2020. Melanin produced by the fast-growing
marine bacterium Vibrio natriegens through heterologous biosynthesis:
Characterization and application. Appl. Environ. Microbiol. 86, 1–18. https://doi.
org/10.1128/AEM.02749-19.
Watson, E., 2017. Special report: navigating the vanilla avors market. Available online:
https://www.foodnavigator-usa.com/Article/2017/10/21/Navigating-the-vanilla-
vanillin-market-with-Solvay (Accessed on 23 November 2020).
Watson, E., 2019. Available online: https://www.foodnavigator-usa.com/Article/201
9/08/07/Amyris-talks-steviol-glycosides-at-IFT-Our-Reb-M-has-the-highest-purity-o
n-the-market-today (Accessed on 23 November 2020).
Wehrs, M., Prahl, J.P., Moon, J., Li, Y., Tanjore, D., Keasling, J.D., Pray, T.,
Mukhopadhyay, A., 2018. Production efciency of the bacterial non-ribosomal
peptide indigoidine relies on the respiratory metabolic state in S. cerevisiae. Microb.
Cell Fact. 17, 1–11. https://doi.org/10.1186/s12934-018-1045-1.
Wehrs, M., Gladden, J.M., Liu, Y., Platz, L., Prahl, J.P., Moon, J., Papa, G., Sundstrom, E.,
Geiselman, G.M., Tanjore, D., Keasling, J.D., Pray, T.R., Simmons, B.A.,
Mukhopadhyay, A., 2019. Sustainable bioproduction of the blue pigment
indigoidine: Expanding the range of heterologous products in: R. toruloides to include
non-ribosomal peptides. Green Chem. 21, 3394–3406. https://doi.org/10.1039/
c9gc00920e.
Wen, J., Bao, J., 2019. Engineering Corynebacterium glutamicum triggers glutamic acid
accumulation in biotin-rich corn stover hydrolysate. Biotechnol. Biofuels 12, 1–11.
https://doi.org/10.1186/s13068-019-1428-5.
Willrodt, C., David, C., Cornelissen, S., Bühler, B., Julsing, M.K., Schmid, A., 2014.
Engineering the productivity of recombinant Escherichia coli for limonene formation
from glycerol in minimal media. Biotechnol. J. 9 (8), 1000–1012. https://doi:10.
1002/biot.201400023.
Wong, L., Engel, J., Jin, E., Holdridge, B., Xu, P., 2017. YaliBricks, a versatile genetic
toolkit for streamlined and rapid pathway engineering in Yarrowia lipolytica. Metab.
Eng. Commun. 5, 68–77. https://doi.org/10.1016/j.meteno.2017.09.001.
Wu, J., Cheng, S., Cao, J., Qiao, J., Zhao, G.R., 2019a. Systematic optimization of
limonene production in engineered Escherichia coli. J. Agric. Food Chem. 67,
7087–7097. https://doi.org/10.1021/acs.jafc.9b01427.
Wu, M., Qu, J., Tian, X., Zhao, X., Shen, Y., Shi, Z., Chen, P., Li, G., Ma, T., 2019b. Tailor-
made polysaccharides containing uniformly distributed repeating units based on the
xanthan gum skeleton. Int. J. Biol. Macromol. 131, 646–653. https://doi.org/
10.1016/j.ijbiomac.2019.03.130.
Wu, H., Xue, E., Zhi, N., Song, Q., Tian, K., Caiyin, Q., Yuan, L., Qiao, J., 2020. D-
methionine and D-phenylalanine improve Lactococcus lactis F44 acid resistance and
nisin yield by governing cell wall remodeling. Appl. Environ. Microbiol. 86 (9)
https://doi.org/10.1128/AEM.02981-19 e02981–19.
Xu, H., Qin, Y., Huang, Z., Liu, Z., 2014. Characterization and site-directed mutagenesis
of an
α
-galactosidase from the deep-sea bacterium Bacillus megaterium. Enzyme
Microb. Technol. 56, 46–52. https://doi.org/10.1016/j.enzmictec.2014.01.004.
Xu, F., Gage, D., Zhan, J., 2015. Efcient production of indigoidine in Escherichia coli.
J. Ind. Microbiol. Biotechnol. 42, 1149–1155. https://doi.org/10.1007/s10295-015-
1642-5.
Xu, J., Xu, X., Xu, Q., Zhang, Z., Jiang, L., Huang, H., 2018a. Efcient production of
lycopene by engineered E. coli strains harboring different types of plasmids.
Bioprocess Biosyst. Eng. 41, 489–499. https://doi.org/10.1007/s00449-017-1883-y.
Xu, Y., Wang, Y. hui, Liu, T. qi, Zhang, Hui, Zhang, He, Li, J., 2018b. The GlaA signal
peptide substantially increases the expression and secretion of
α
-galactosidase in
Aspergillus niger. Biotechnol. Lett. 40, 949–955. https://doi.org/10.1007/s10529-
018-2540-5.
Xue, Z., Sharpe, P.L., Hong, S.P., Yadav, N.S., Xie, D., Short, D.R., Damude, H.G.,
Rupert, R.A., Seip, J.E., Wang, J., Pollak, D.W., Bostick, M.W., Bosak, M.D.,
Macool, D.J., Hollerbach, D.H., Zhang, H., Arcilla, D.M., Bledsoe, S.A., Croker, K.,
McCord, E.F., Tyreus, B.D., Jackson, E.N., Zhu, Q., 2013. Production of omega-3
eicosapentaenoic acid by metabolic engineering of Yarrowia lipolytica. Nat.
Biotechnol. 31, 734–740. https://doi.org/10.1038/nbt.2622.
Yang, C., Jiang, P., Xiao, S., Zhang, C., Lou, K., Xing, X.H., 2011. Fed-batch fermentation
of recombinant Citrobacter freundii with expression of a violacein-synthesizing gene
cluster for efcient violacein production from glycerol. Biochem. Eng. J. 57, 55–62.
https://doi.org/10.1016/j.bej.2011.08.008.
Yao, J., He, Y., Su, N., Bharath, S.R., Tao, Y., Jin, J.M., Chen, W., Song, H., Tang, S.Y.,
2020a. Developing a highly efcient hydroxytyrosol whole-cell catalyst by de-
bottlenecking rate-limiting steps. Nat. Commun. 11, 1–12. https://doi.org/10.1038/
s41467-020-14918-5.
Yao, X., Lv, Y., Yu, H., Cao, H., Wang, L., Wen, B., Gu, T., Wang, F., Sun, L., Xin, F.,
2020b. Site-directed mutagenesis of coenzyme-independent carotenoid oxygenase
CSO2 to enhance the enzymatic synthesis of vanillin. Appl. Microbiol. Biotechnol.
104 (9), 3897–3907. https://doi.org/10.1007/s00253-020-10433-1.
Yoon, S.H., Lee, E.G., Das, A., Lee, S.H., Li, C., Ryu, H.K., Choi, M.S., Seo, W.T., Kim, S.
W., 2007. Enhanced vanillin production from recombinant E. coli using NTG
mutagenesis and adsorbent resin. Biotechnol. Prog. 23, 1143–1148. https://doi.org/
10.1021/bp070153r.
Yu, D., Xu, F., Valiente, J., Wang, S., Zhan, J., 2013. An indigoidine biosynthetic gene
cluster from Streptomyces chromofuscus ATCC 49982 contains an unusual IndB
homologue. J. Ind. Microbiol. Biotechnol. 40, 159–168. https://doi.org/10.1007/
s10295-012-1207-9.
Yu, S., Liu, J.J., Yun, E.J., Kwak, S., Kim, K.H., Jin, Y.S., 2018. Production of a human
milk oligosaccharide 2’-fucosyllactose by metabolically engineered Saccharomyces
cerevisiae. Microb. Cell Fact. 17, 1–10. https://doi.org/10.1186/s12934-018-0947-2.
Yuan, J., Mishra, P., Ching, C.B., 2016. Metabolically engineered Saccharomyces
cerevisiae for branched-chain ester productions. J. Biotechnol. 239, 90–97. https://
doi.org/10.1016/j.jbiotec.2016.10.013.
Yue, J., Wang, R., Ma, X., Liu, Jiayi, Lu, X., Balaso Thakar, S., An, N., Liu, Jia, Xia, E., Liu,
Y., 2020. Full-length transcriptome sequencing provides insights into the evolution
of apocarotenoid biosynthesis in Crocus sativus. Comput. Struct. Biotechnol. J. 18,
774–783. doi:https://doi.org/10.1016/j.csbj.2020.03.022.
Zhang, D., Guan, D., Liang, J., Guo, C., Xie, X., Zhang, C., Xu, Q., Chen, N., 2014.
Reducing lactate secretion by ldhA deletion in L-glutamate- producing strain
Corynebacterium glutamicum GDK-9. Brazilian J. Microbiol. 45, 1477–1483. https://
doi.org/10.1590/S1517-83822014000400044.
Zhang, J., Caiyin, Q., Feng, W., Zhao, X., Qiao, B., Zhao, G., Qiao, J., 2016. Enhance nisin
yield via improving acid-tolerant capability of Lactococcus lactis F44. Sci. Rep. 6,
1–12. https://doi.org/10.1038/srep27973.
Zhang, W., Du, F., Tian, G., Zhao, Y., Wang, H., Ng, T.B., 2018. Characterization of an
acidic
α
-galactosidase from hemp (Cannabis sativa L.) seeds and its application in
removal of rafnose family oligosaccharides (RFOs). Acta Biochim. Pol. 65 (3),
383–389. https://doi.org/10.18388/abp.2017_1535.
Zhang, X.K., Nie, M.Y., Chen, J., Wei, L.J., Hua, Q., 2019. Multicopy integrants of crt
genes and co-expression of AMP deaminase improve lycopene production in
Yarrowia lipolytica. J. Biotechnol. 289, 46–54. https://doi.org/10.1016/j.
jbiotec.2018.11.009.
Zhao, L.Q., Sun, Z.H., Zheng, P., Zhu, L.L., 2005. Biotransformation of isoeugenol to
vanillin by a novel strain of Bacillus fusiformis. Biotechnol. Lett. 27, 1505–1509.
https://doi.org/10.1007/s10529-005-1466-x.
Zhao, Ruili, Zhao, Rui, Tu, Y., Zhang, X., Deng, L., Chen, X., 2018a. A novel
α
-galactosidase from the thermophilic probiotic Bacillus coagulans with remarkable
protease-resistance and high hydrolytic activity. PLoS One 13, 1–16. https://doi.
org/10.1371/journal.pone.0197067.
Zhao, X.R., Choi, K.R., Lee, S.Y., 2018b. Metabolic engineering of Escherichia coli for
secretory production of free haem. Nat. Catal. 1, 720–728. https://doi.org/10.1038/
s41929-018-0126-1.
Zhao, X., Zhou, J., Du, G., Chen, J., 2020. Recent advances in the microbial synthesis of
hemoglobin. Trends Biotechnol. 7 s0167-7799(20)30226-2. doi:10.1016/j.
tibtech.2020.08.004.
Zhou, Y., Fang, M.Y., Li, G., Zhang, C., Xing, X.H., 2018. Enhanced production of crude
violacein from glucose in Escherichia coli by overexpression of rate-limiting key
enzyme(S) involved in violacein biosynthesis. Appl. Biochem. Biotechnol. 186,
909–916. https://doi.org/10.1007/s12010-018-2787-2.
Zuo, Z.Q., Xue, Q., Zhou, J., Zhao, D.H., Han, J., Xiang, H., 2018. Engineering Haloferax
mediterranei as an efcient platform for high level production of lycopene. Front.
Microbiol. 9, 1–13. https://doi.org/10.3389/fmicb.2018.02893.
L. Sun et al.
