Saccharomyces cerevisiae strains for second-generation ethanol production: from academic exploration to industrial implementation

Abstract

The recent start-up of several full-scale ‘second generation’ ethanol plants marks a major milestone in the development of Saccharomyces cerevisiae strains for fermentation of lignocellulosic hydrolysates of agricultural residues and energy crops. After discussing the challenges that these novel industrial contexts impose on yeast strains, this mini-review describes key metabolic engineering strategies that have been developed to address these challenges. Additionally, it outlines how proof-of-concept studies, often developed in academic settings, can be used for the development of robust strain platforms that meet requirements for industrial application. Fermentation performance of current, engineered industrial S. cerevisiae strains is no longer a bottleneck in efforts to achieve the projected outputs of the first large-scale second-generation ethanol plants. Academic and industrial yeast research will continue to strengthen the economic value position of second-generation ethanol production by further improving fermentation kinetics, product yield and cellular robustness under process conditions.

Full text

Saccharomyces cerevisiae strains for second-generation ethanol production: from
academic exploration to industrial implementation
Mickel L.A. Jansen1, Jasmine M. Bracher2, Ioannis Papapetridis2, Maarten D. Verhoeven2, Hans de
Bruijn1, Paul P. de Waal1, Antonius J.A. van Maris2, †, Paul Klaassen1 and Jack T. Pronk2,*
1DSM Biotechnology Centre, Alexander Fleminglaan 1, 2613 AX Delft, The Netherlands
2Department of Biotechnology, Delft University of Technology, Van der Maasweg 9, 2629 HZ
Delft, The Netherlands
†Current address: Division of Industrial Biotechnology, School of Biotechnology, KTH Royal
Institute of Technology, AlbaNova University Center, SE 106 91, Stockholm, Sweden.
Review manuscript for publication in FEMS Yeast Research
*Corresponding author: Jack Pronk, e-mail j.t.pronk@tudelft.nl; telephone +31 15 2782416

ABSTRACT
The recent start-up of several full-scale ‘second generation’ ethanol plants marks a major
milestone in the development of Saccharomyces cerevisiae strains for fermentation of
lignocellulosic hydrolysates of agricultural residues and energy crops. After discussing the
challenges that these novel industrial contexts impose on yeast strains, this mini-review
describes key metabolic engineering strategies that have been developed to address these
challenges. Additionally, it outlines how proof-of-concept studies, often developed in academic
settings, can be used for the development of robust strain platforms that meet requirements for
industrial application. Fermentation performance of current, engineered industrial S. cerevisiae
strains is no longer a bottleneck in efforts to achieve the projected outputs of the first large-scale
second-generation ethanol plants. Academic and industrial yeast research will continue to
strengthen the economic value position of second-generation ethanol production by further
improving fermentation kinetics, product yield and cellular robustness under process
conditions.
Key words: biofuels, metabolic engineering, strain improvement, industrial fermentation, yeast
biotechnology, pentose fermentation, biomass hydrolysates

INTRODUCTION
Alcoholic fermentation is a key catabolic process in most yeasts and in many fermentative
bacteria, which concentrates the heat of combustion of carbohydrates into two thirds of their
carbon atoms ((CH2O)n → ⅓n C2H6O + ⅓n CO2). Its product, ethanol, has been used as an
automotive fuel for over a century (Bernton et al. 1982). With an estimated global production of
100 Mton (Renewable Fuels Association 2016), ethanol is the largest-volume product in
industrial biotechnology. Its production is, currently, mainly based on fermentation of cane
sugar or hydrolysed corn starch with the yeast Saccharomyces cerevisiae. Such ‘first generation’
bioethanol processes are characterized by high ethanol yields on fermentable sugars (> 90 % of
the theoretical maximum yield of 0.51 g ethanol·(g hexose sugar)-1), ethanol titers of up to 21 %
(w/w) and volumetric productivities of 2 to 3 kg·m-3·h-1 (Della-Bianca et al. 2013; Lopes et al.
2016; Thomas and Ingledew 1992).
Over the past two decades, a large international effort, involving researchers in
academia, research institutes and industry, aimed to access abundantly available agricultural
and forestry residues, as well as fast-growing energy crops, as alternative feedstocks for fuel
ethanol production (Rude and Schirmer 2009). Incentives for this effort, whose relative impact
depends on geographical location and varies over time, include reduction of the carbon footprint
of ethanol production (Otero et al. 2007), prevention of competition with food production for
arable land (Nordhoff 2007; Tenenbaum 2008), energy security in fossil-fuel importing
countries (Farrell et al. 2006) and development of rural economies (Kleinschmidt 2007).
Techno-economic forecasts of low-carbon scenarios for global energy supply almost invariably
include liquid biofuels as a significant contributor (Yan et al. 2010). Moreover, successful
implementation of economically and environmentally sustainable ‘second generation’
bioethanol processes can pave the way for similar processes to produce other biofuels and
commodity chemicals (Pereira et al. 2015).
In contrast to starch, a plant storage carbohydrate that can be easily hydrolysed, the
major carbohydrate polymers in lignocellulosic plant biomass (cellulose, hemicellulose and, in

some cases, pectin) contribute to the structure and durability of stalks, leaves and roots (Hahn-
Hägerdal et al. 2006). Consistent with these natural functions and with their chemical diversity
and complexity, mobilization of these polymers by naturally occurring cellulose-degrading
microorganisms requires complex arrays of hydrolytic enzymes (Lynd et al. 2002; Van den Brink
and de Vries 2011).
The second-generation ethanol processes that are now coming on line at demonstration
and full commercial scale (Table 1) are mostly based on fermentation of lignocellulosic biomass
hydrolysates by engineered strains of S. cerevisiae. While this yeast has a strong track record in
first-generation bioethanol production and its amenability to genetic modification is excellent, S.
cerevisiae cannot hydrolyse cellulose or hemicellulose. Therefore, in conventional process
configurations for second-generation bioethanol production, the fermentation step is preceded
by chemical/physical pretreatment and enzyme-catalysed hydrolysis by cocktails of fungal
hydrolases, which can either be produced on- or off site (Figure 1, (Sims-Borre 2010).
Alternative process configurations, including simultaneous saccharification and fermentation
(SSF) and consolidated bioprocessing (CBP) by yeast cells expressing heterologous hydrolases
are intensively investigated (Den Haan et al. 2015; Olson et al. 2012). However, the high
temperature optima of fungal enzymes and low productivity of heterologously expressed
hydrolases in S. cerevisiae have so far precluded large-scale implementation of these alternative
strategies for lignocellulosic ethanol production (Den Haan et al. 2015; Vohra et al. 2014). This
mini-review will, therefore, focus on the development of yeast strains for conventional process
designs.
Over the past decade, the authors have collaborated in developing metabolic engineering
concepts for fermentation of lignocellulosic hydrolysates with engineered S. cerevisiae strains
and in implementing these in advanced industrial strain platforms. Based on their joint
academic-industrial vantage point, this paper reviews key conceptual developments and
challenges in the development and industrial implementation of S. cerevisiae strains for second
generation bioethanol production processes.

FERMENTING LIGNOCELLULOSIC HYDROLYSATES: CHALLENGES FOR YEAST STRAIN
DEVELOPMENT
A wide range of agricultural and forestry residues, as well as energy crops, are being considered
as feedstocks for bioethanol production (Khoo 2015). Full-scale and demonstration plants using
raw materials such as corn stover, sugar-cane bagasse, wheat straw and switchgrass are now in
operation (Table 1). These lignocellulosic feedstocks have different chemical compositions,
which further depend on factors such as seasonal variation, weather and climate, crop maturity
and storage conditions (Kenney et al. 2013). Despite this variability, common features of
feedstock composition and biomass-deconstruction methods generate several generic
challenges that have to be addressed in the development of yeast strains for second-generation
bioethanol production.
Pentose fermentation
For large-volume products such as ethanol, maximizing the product yield on feedstock and,
therefore, efficient conversion of all potentially available substrate molecules in the feedstock is
of paramount economic importance (Lin and Tanaka 2006). In addition to readily fermentable
hexoses such as glucose and mannose, lignocellulosic biomass contains substantial amounts of
D-xylose and L-arabinose. These pentoses, derived from hemicellulose and pectin polymers in
plant biomass, cannot be fermented by wild-type S. cerevisiae strains. D-xylose and L-arabinose
typically account for 10 to 25 % and 2 to 3 %, respectively, of the carbohydrate content of
lignocellulosic feedstocks (Lynd 1996). However, in some feedstocks, such as corn fiber
hydrolysates and sugar beet pulp, the L-arabinose content can be up to ten-fold higher
(Grohmann and Bothast 1994; Grohmann and Bothast 1997). Early studies already identified
metabolic engineering of S. cerevisiae for efficient, complete pentose fermentation as key
prerequisite for its application in second-generation ethanol production (Bruinenberg et al.
1983; Hahn-Hägerdal et al. 2001; Kötter et al. 1990; Sedlak and Ho 2001).

Acetic acid inhibition
Since hemicellulose is acetylated (Van Hazendonk et al. 1996), its complete hydrolysis inevitably
results in the release of acetic acid. Bacterial contamination during biomass storage,
pretreatment and/or fermentation may further increase the acetic acid concentrations to which
yeasts are exposed in the fermentation process. First-generation bioethanol processes are
typically run at pH values of 4 to 5 to counter contamination with lactic acid bacteria (Beckner et
al. 2011). At these low pH values, undissociated acetic acid (pKa = 4.76) easily diffuses across
the yeast plasma membrane. In the near-neutral pH environment of the yeast cytosol, the acid
readily dissociates and releases a proton, which forces cells to expend ATP for proton export via
the plasma-membrane ATPase to prevent cytosolic acidification (Axe and Bailey 1995;
Pampulha and Loureiro-Dias 2000; Verduyn et al. 1992). The accompanying accumulation of the
acetate anion in the cytosol can cause additional toxicity effects (Palmqvist and Hahn-Hägerdal
2000b; Russel 1992; Ullah et al. 2013). Acetic acid concentrations in some lignocellulosic
hydrolysates exceed 5 g·l-1, which can cause strong inhibition of anaerobic growth and sugar
fermentation by S. cerevisiae (Taherzadeh et al. 1997). Acetic acid tolerance at low culture pH is
therefore a key target in yeast strain development for second-generation ethanol production.
Inhibitors formed during biomass deconstruction
In biomass deconstruction, a trade-off exists between the key objective to release all
fermentable sugars at minimal process costs and the need to minimize generation and release of
compounds that compromise yeast performance. Biomass deconstruction generally
encompasses three steps: (i) size reduction to increase surface area and reduce degree of
polymerization, (ii) thermal pretreatment, often at low pH and high pressure, to disrupt the
crystalline structure of cellulose while already (partly) solubilizing hemicellulose and/or lignin
and (iii) hydrolysis with cocktails of fungal cellulases and hemicellulases to release fermentable
sugars (Hendriks and Zeeman 2009; Narron et al. 2016; Silveira et al. 2015). Several inhibitors
of yeast performance are generated in chemical reactions that occur during biomass

deconstruction and, especially, in high-temperature pretreatment. 5-Hydroxymethyl-2-
furaldehyde (HMF) and 2-furaldehyde (furfural) are formed when hexoses and pentoses,
respectively, are exposed to high temperature and low pH (Dunlop 1948; Palmqvist and Hahn-
Hägerdal 2000b; Ulbricht et al. 1984). These furan derivatives inhibit yeast glycolysis, alcoholic
fermentation and TCA cycle (Banerjee et al. 1981; Modig et al. 2002; Sárvári Horváth et al. 2003)
while, additionally, depleting intracellular pools of NAD(P)H and ATP (Almeida et al. 2007).
Their further degradation, during biomass deconstruction, yields formic acid and levulinic acid
(Dunlop 1948; Ulbricht et al. 1984), whose inhibitory effects overlap with those of acetic acid
(Palmqvist and Hahn-Hägerdal 2000b). Inhibitor profiles of hydrolysates depend on biomass
structure and composition as well as on the type and intensity of the biomass deconstruction
method used (Almeida et al. 2007; Kumar et al. 2009). During pressurized pretreatment at
temperatures above 160 °C, phenolic inhibitors are generated by partial degradation of lignin.
This diverse class of inhibitors includes aldehydes, ketones, alcohols and aromatic acids
(Almeida et al. 2007). Ferulic acid, a phenolic compound that is an integral part of the lignin
fraction of herbaceous plants (Klinke et al. 2002; Lawther et al. 1996) is a potent inhibitor of S.
cerevisiae fermentations (Larsson et al. 2000). The impact of phenolic inhibitors on membrane
integrity and other cellular functions depends on the identity and position of functional groups
and carbon-carbon double bonds (Adeboye et al. 2014).
Concentrations of inorganic salts in hydrolysates vary depending on the feedstock used
(Klinke et al. 2004). Moreover, high salt concentrations in hydrolysates can originate from pH
adjustments during pretreatment (Jönsson et al. 2013). Salt- and osmotolerance can therefore be
important additional requirements in yeast strain development (Casey et al. 2013).
The inhibitors in lignocellulosic hydrolysates do not always act independently but can
exhibit complex synergistic effects, both with each other and with ethanol (Liu et al. 2004;
Palmqvist and Hahn-Hägerdal 2000b; Taherzadeh et al. 1999), while their impact can also be
modulated by the presence of water-insoluble solids (Koppram et al. 2016). Furthermore, their
absolute and relative impact can change over time due to variations in feedstock composition,

process modifications, or malfunctions in biomass deconstruction. While process adaptations to
detoxify hydrolysates have been intensively studied (Canilha et al. 2012; Jönsson et al. 2013;
Palmqvist and Hahn-Hägerdal 2000a; Sivers et al. 1994), the required additional unit operations
typically result in a loss of fermentable sugar and are generally considered to be too expensive
and complicated. Therefore, as research on optimization of biomass deconstruction processes
continues, tolerance to the chemical environments generated by current methods is a key design
criterion for yeast strain development.
YEAST STRAIN DEVELOPMENT FOR SECOND-GENERATION ETHANOL PRODUCTION: KEY
CONCEPTS
For almost three decades, yeast metabolic engineers have vigorously explored strategies to
address the challenges outlined above. This quest benefited from rapid technological
development in genomics, genome editing, evolutionary engineering and protein engineering.
Box 1 lists key technologies and examples of their application in research on yeast strain
development for second-generation ethanol production.
Xylose fermentation
Efficiently linking D-xylose metabolism to glycolysis requires two key modifications of the S.
cerevisiae metabolic network (Figure 2) (Jeffries and Jin 2004; Van Maris et al. 2007):
introduction of a heterologous pathway that converts D-xylose into D-xylulose and,
simultaneously, alleviation of the limited capacity of the native S. cerevisiae xylulokinase and
non-oxidative pentose-phosphate pathway (PPP). Two strategies for converting D-xylose into D-
xylulose have been implemented in S. cerevisiae: (i) simultaneous expression of heterologous
xylose reductase (XR) and xylitol dehydrogenase (XDH) and (ii) expression of a heterologous
xylose isomerase (XI).
The first S. cerevisiae strains engineered for xylose utilization were based on expression
of XR and XDH from the xylose-metabolising yeast Scheffersomyces stipitis (Kötter and Ciriacy

1993). Due to the non-matching redox-cofactor preferences of these enzymes, these strains
produced large amounts of the by-product D-xylitol (Hahn-Hägerdal et al. 2001; Jeffries 2006;
Kötter and Ciriacy 1993). Modification of these cofactor preferences by protein engineering
resulted in reduced xylitol formation under laboratory conditions (Runquist et al. 2010a;
Watanabe et al. 2007b). A much lower xylitol formation by XR/XDH-based strains in
lignocellulosic hydrolysates was attributed to NADH-dependent reduction of furfural, which may
contribute to in situ detoxification of this inhibitor (Karhumaa et al. 2007; Katahira et al. 2006;
Moniruzzaman et al. 1997; Sedlak and Ho 2003; Wahlbom and Hahn–Hägerdal 2002). A
potential drawback of XR/XDH-based strains for application in large-scale anaerobic processes
is that, even after prolonged laboratory evolution, their anaerobic growth rates are very low
(Sonderegger and Sauer 2003).
Combined expression of a fungal XI (Harhangi et al. 2003) and overexpression of the
native S. cerevisiae genes encoding xylulokinase and non-oxidative PPP enzymes enabled
anaerobic growth of a laboratory strain on D-xylose. In anaerobic cultures of this strain, in which
the aldose-reductase encoding GRE3 gene was deleted to eliminate xylitol formation, ethanol
yields on D-xylose were the same as on glucose (Kuyper et al. 2005). This metabolic engineering
strategy, complemented with laboratory evolution under anaerobic conditions, has been
successfully reproduced in different S. cerevisiae genetic backgrounds and/or with different XI
genes (Brat et al. 2009; Dun 2012; Ha et al. 2011; Hector et al. 2013; Hou et al. 2016b; Madhavan
et al. 2009).
Laboratory evolution (Box 1) for faster D-xylose fermentation and analysis of evolved
strains identified high-level expression of XI as a major contributing factor (Demeke et al. 2015;
Hou et al. 2016a; Zhou et al. 2012). Multi-copy introduction of XI expression cassettes,
optimization of their codon usage, and mutagenesis of their coding sequences have contributed
to higher D-xylose fermentation rates (Brat et al. 2009; Crook et al. 2016; Lee et al. 2012).
Whole-genome sequencing of evolved D-xylose-fast-fermenting strains expressing Piromyces XI
identified mutations affecting intracellular homeostasis of Mn2+, a preferred metal ion for this XI

(Verhoeven et al. 2017). Other mutations affected stress-response regulators and, thereby,
increased expression of yeast chaperonins that assisted functional expression of XI (Hou et al.
2016a). Consistent with this observation, co-expression of the Escherichia coli GroEL and GroES
chaperonins enabled in vivo activity of E. coli XI in S. cerevisiae (Xia et al. 2016). A positive effect
of mutations in the PHO13 phosphatase gene on xylose fermentation rates in XI- and XR/XDH-
based strains has been attributed to transcriptional upregulation of PPP-related genes by an as
yet unknown mechanism (Bamba et al. 2016; Ni et al. 2007; Van Vleet et al. 2008; Xu et al. 2016).
Additionally, Pho13 has been implicated in dephosphorylation of the PPP intermediate
sedoheptulose-7-phosphate (Xu et al. 2016). For other mutations in evolved strains, e.g. in genes
involved in iron-sulfur cluster assembly and in the MAP-kinase signaling pathway (dos Santos et
al. 2016; Sato et al. 2016), the mechanisms by which they affect D-xylose metabolism remain to
be identified.
Arabinose fermentation
The metabolic engineering strategy for constructing L-arabinose-fermenting S. cerevisiae is
based on heterologous expression of a bacterial pathway for conversion of L-arabinose into
xylulose-5-phosphate, involving L-arabinose isomerase (AraA), L-ribulokinase (AraB) and L-
ribulose-5-phosphate-4-epimerase (AraD) (Lee et al. 1986). Together with the non-oxidative
PPP and glycolysis, these reactions enable redox-cofactor-balanced alcoholic fermentation of L-
arabinose (Figure 2).
Combined expression of Bacillus subtilis or B. licheniformis araA and E. coli araBD
(Becker and Boles 2003; Bettiga et al. 2008; Wiedemann and Boles 2008) allowed aerobic
growth of S. cerevisiae on L-arabinose. Anaerobic growth of S. cerevisiae on arabinose was first
achieved by expressing the Lactobacillus plantarum araA, B and D genes in an XI-based xylose-
fermenting strain that already overexpressed the enzymes of the non-oxidative PPP (Figure 2),
followed by evolutionary engineering under anaerobic conditions (Wisselink et al. 2007).
Increased expression levels of GAL2, which encodes a galactose transporter that also transports

L-arabinose (Kou et al. 1970), was essential for L-arabinose fermentation (Becker and Boles
2003; Subtil and Boles 2011; Subtil and Boles 2012; Wisselink et al. 2010). Increased expression
of the transaldolase and transketolase isoenzymes Nqm1 and Tkl2 contributed to an increased
rate of arabinose fermentation in strains evolved for fast arabinose fermentation (Wisselink et
al. 2010). The set of arabinose isomerase genes that can be functionally expressed in S. cerevisiae
was recently expanded by coexpression of E. coli araA with the groEL and groES chaperonins (Xia
et al. 2016).
Engineering of sugar transport and mixed-substrate fermentation
In early S. cerevisiae strains engineered for pentose fermentation, uptake of D-xylose and L-
arabinose exclusively relied on their native hexose transporters. While several of the 18 S.
cerevisiae Hxt transporters (Hxt1-17 and Gal2) transport D-xylose, their Km values for this
pentose are one to two orders of magnitude higher than for glucose (Farwick et al. 2014;
Hamacher et al. 2002; Lee et al. 2002; Reifenberger et al. 1997; Saloheimo et al. 2007). High-
affinity glucose transporters, which are only expressed at low glucose concentrations (Diderich
et al. 1999), display a lower Km for D-xylose than low-affinity glucose transporters (Hamacher et
al. 2002; Lee et al. 2002). The galactose transporter Gal2, which also catalyses high-affinity
glucose transport (Reifenberger et al. 1997) also has a much higher Km for L-arabinose than for
glucose (Subtil and Boles 2011; Subtil and Boles 2012).
The higher affinities of Hxt transporters for glucose, combined with the transcriptional
repression of Gal2 (Horak et al. 2002; Horak and Wolf 1997) and other high-affinity Hxt
transporters (Diderich et al. 1999; Sedlak and Ho 2004) at high glucose concentrations,
contribute to a sequential use of glucose and pentoses during mixed-substrate cultivation of
engineered strains that depend on Hxt-mediated pentose uptake. Furthermore, the high Km
values of Hxt transporters for pentoses cause a deceleration of sugar fermentation during the
pentose-fermentation phase. This ‘tailing’ effect is augmented by accumulation of ethanol and by
the reduced inhibitor tolerance of S. cerevisiae at low sugar fermentation rates (Ask et al. 2013;

Bellissimi et al. 2009; Demeke et al. 2013b). Intensive efforts have been made to generate yeast
strains that can either co-consume hexoses and pentose sugars or sequentially consume all
sugars in hydrolysates in an economically acceptable time frame (Kim et al. 2012; Moysés et al.
2016).
Evolutionary engineering experiments played a major role in accelerating mixed-sugar
utilization by engineered pentose-fermenting strains (Kuyper et al. 2005b; Sanchez et al. 2010;
Sonderegger and Sauer 2003; Wisselink et al. 2009; Zhou et al. 2012). Repeated batch cultivation
on a sugar mixture can favour selection of mutants that rapidly ferment one of the sugars, while
showing deteriorated fermentation kinetics with other sugars in the mixture. In practice, such
trade-off scenarios can increase rather than decrease the time required for complete conversion
of sugar mixtures (Wisselink et al. 2009). A modified strategy for repeated batch cultivation,
designed to equally distribute the number of generations of selective growth on each of the
individual substrates in a mixture, enabled acceleration of the anaerobic conversion of glucose-
xylose-arabinose mixtures by an engineered S. cerevisiae strain (Wisselink et al. 2009).
Recently constructed glucose-phosphorylation-negative, pentose-fermenting S. cerevisiae
strains enabled evolutionary engineering experiments for in vivo directed evolution of Hxt
variants that supported growth on D-xylose or L-arabinose in the presence of high glucose
concentrations (Farwick et al. 2014; Nijland et al. 2014; Shin et al. 2015a; Wisselink et al. 2015).
Several of the evolved HXT alleles were confirmed to encode transporters whose D-xylose-
transport kinetics were substantially less sensitive to glucose inhibition (Farwick et al. 2014;
Nijland et al. 2014; Shin et al. 2015a; Wisselink et al. 2015). Remarkably, independent
evolutionary engineering studies aimed at selecting glucose-insensitive D-xylose and L-arabinose
Hxt transporters yielded single-amino-acid substitutions at the exact corresponding positions in
Hxt7(N370), Gal2 (N376), and in a chimera of Hxt3 and Hxt6 (N367) (Farwick et al. 2014;
Nijland et al. 2014; Wisselink et al. 2015). Additional Hxt variants with improved relative
affinities for pentoses and glucose were obtained by in vitro directed evolution and knowledge-
based protein engineering (Farwick et al. 2014; Reznicek et al. 2015) (Box 1).

Low-, moderate- and high-affinity pentose transporters from pentose-metabolizing
filamentous fungi or non-Saccharomyces yeasts, have been functionally expressed in S. cerevisiae
(Colabardini et al. 2014; Du et al. 2010; Ferreira et al. 2013; Katahira et al. 2008; Knoshaug et al.
2015; Leandro et al. 2006; Li et al. 2015; Reis et al. 2016; Runquist et al. 2010b; Subtil and Boles
2011; Weierstall et al. 1999; Young et al. 2012). Expression and/or activity of several of these
transporters were further improved by directed evolution (Li et al. 2016b; Li et al. 2015; Young
et al. 2012) or evolutionary engineering (Moysés et al. 2016; Wang et al. 2016). Such high-
affinity transporters may be suited to ‘mop up’ low concentrations of pentoses towards the end
of a fermentation process. Since high-affinity sugar transporters are typically proton
symporters, care should be taken to avoid scenarios in which their simultaneous expression
with Hxt-like transporters, which mediate facilitated diffusion, causes futile cycles and
negatively affects inhibitor tolerance.
Inhibitor tolerance
Yeast enzymes involved in detoxification of specific inhibitors provide logical targets for
metabolic engineering. For example, overexpression of native NAD(P)+-dependent alcohol
dehydrogenases stimulates conversion of furfural and HMF to the less toxic alcohols
furanmethanol and furan-2,5-dimethanol, respectively (Almeida et al. 2009; Jeppsson et al.
2003; Lewis Liu et al. 2008). Similarly, combined overexpression of the aldehyde dehydrogenase
Ald5, the decarboxylase Pad1 and the alcohol acetyltransferases Atf1 and Atf2 increased
resistance to several phenolic inhibitors (Adeboye et al. 2017).
Genome-wide expression studies have revealed intricate, strain- and context-dependent
stress-response networks as major key contributors to inhibitor tolerance (Abbott et al. 2007;
Almeida et al. 2007; Guo and Olsson 2014; Li and Yuan 2010; Liu 2011; Mira et al. 2010; Ullah et
al. 2013). An in-depth transcriptome analysis implicated SFP1 and ACE2, which encode
transcriptional regulators involved in ribosomal biogenesis and septum destruction after
cytokinesis, respectively, in the phenotype of an acetic-acid and furfural-tolerant strain. Indeed,

overexpression of these transcriptional regulators significantly enhanced ethanol productivity in
the presence of these inhibitors (Chen et al. 2016).
Whole-genome resequencing of tolerant strains derived from evolutionary engineering,
mutagenesis and/or genome shuffling has yielded strains with increased tolerance whose causal
mutations could be identified (Almario et al. 2013; Demeke et al. 2013a; González-Ramos et al.
2016; Pinel et al. 2015; Thompson et al. 2016). Physiological and evolutionary engineering
experiments demonstrated the importance of high sugar fermentation rates for acetic acid
tolerance (Bellissimi et al. 2009; Wright et al. 2011). When the acetic-acid concentration in
anaerobic, xylose-grown continuous cultures was continually increased over time, evolving
cultures acquired the ability to grow at acetic-acid concentrations that prevented growth of the
non-evolved S. cerevisiae strain. However, after growth in the absence of acetic acid, full
expression of their increased tolerance required pre-exposure to a lower acetic-acid
concentration. This observation indicated that the acquired tolerance was inducible rather than
constitutive (Wright et al. 2011). Constitutive tolerance to acetic acid was shown to reflect the
fraction of yeast populations able to initiate growth upon exposure to acetic acid stress
(Swinnen et al. 2014). Based on this observation, an evolutionary engineering strategy that
involved alternating batch cultivation cycles in the presence and absence of acetic acid was
successfully applied to select for constitutive acetic acid tolerance (González-Ramos et al. 2016).
Exploration of the natural diversity of inhibitor tolerance among S. cerevisiae strains
(Favaro et al. 2013; Field et al. 2015; Wimalasena et al. 2014) is increasingly used to identify
genes and alleles that contribute to tolerance. In particular, combination of whole genome
sequencing and classical genetics is a powerful approach to identify relevant genomic loci, genes
and even nucleotides (Liti and Louis 2012) (Quantitative Trait Loci (QTL) analysis, see Box 1).
For example, Meijnen et al. (2016) used whole-genome sequencing of pooled tolerant and
sensitive segregants from crosses between a highly acetic-acid tolerant S. cerevisiae strain and a
reference strain to identify mutations in five genes that contributed to tolerance.

Reduction of acetic acid to ethanol: converting an inhibitor into a co-substrate
Even small improvements of the product yield on feedstock can substantially improve the
economics of biotechnological processes for manufacturing large-volume products such as
ethanol (Nielsen et al. 2013; Van Maris et al. 2006a). In industrial, anaerobic ethanol production
processes, a significant amount of sugar is converted into the byproduct glycerol (Nissen et al.
2000). Glycerol formation, catalyzed by the two isoforms of glycerol-3-phosphate
dehydrogenase (Gpd1 and Gpd2) and of glycerol-3-phosphate phosphatase (Gpp1 and Gpp2), is
required during anaerobic growth of S. cerevisiae for reoxidation of NADH generated in
biosynthetic reactions (Björkqvist et al. 1997; Van Dijken and Scheffers 1986). Metabolic
engineering strategies to diminish glycerol formation focused on modification of intracellular
redox reactions (Guo et al. 2011; Nissen et al. 2000) or modulation of GPD1 and GPD2 expression
(Hubmann et al. 2011). Replacement of GPD1 and GPD2 with a heterologous gene encoding an
acetylating acetaldehyde dehydrogenase (A-ALD) and supplementation of acetic acid eliminated
glycerol formation in anaerobic S. cerevisiae cultures (Guadalupe-Medina et al. 2010). By
enabling NADH-dependent reduction of acetic acid to ethanol (Figure 2), this strategy resulted
in a significant increase in the final ethanol yield, while consuming acetic acid. This engineering
strategy has recently been extended by altering the redox-cofactor specificities of alcohol
dehydrogenase (Henningsen et al. 2015) and 6-phosphogluconate dehydrogenase (Papapetridis
et al. 2016). These further interventions increased the availability of cytosolic NADH for acetate
reduction and should, upon implementation in industrial strains, further improve in situ
detoxification of acetic acid. The A-ALD strategy was also shown to decrease xylitol formation in
XR/XDH-based xylose-fermenting engineered strains by reoxidation of excess NADH formed in
the XDH reaction (Wei et al. 2013; Zhang et al. 2016a).
DEVELOPMENT OF INDUSTRIAL YEAST STRAINS AND PROCESSES
Much of the research discussed in the preceding paragraphs was based on laboratory yeast
strains, grown in synthetic media whose composition can be different from that of industrial

lignocellulosic hydrolysates. Table 2 provides examples of ethanol yields and biomass-specific
conversion rates that have been obtained with engineered S. cerevisiae strains in synthetic
media.
While data on the performance of current industrial strains on industrial feedstocks are
proprietary, many scientific publications describe the fermentation of hydrolysates by D-xylose-
fermenting strains (either XI or XR-XDH-based, but so far without arabinose pathways). These
studies cover a wide variety of feedstocks, biomass deconstruction and fermentation strategies
(batch, fed-batch, SSF), aeration regimes and nutritional supplementations (e.g. yeast extract,
peptone, low-cost industrial supplements, trace elements, nitrogen sources). However, with few
exceptions, these data are restricted to final ethanol yields and titers, and do not include
quantitative information of the biomass-specific conversion rates (qxylose, qethanol, expressed in
g·(g biomass)-1·h-1 that are essential for strain comparison and process design. Table 3
summarizes results studies on fermentation of biomass hydrolysates that include or enable
calculation of biomass-specific conversion rates and ethanol yields.
Despite the heterogeneity of the studies included in Tables 2 and 3, the available data
clearly illustrate that, while even ‘academic’ strain platforms can exhibit high ethanol yields in
hydrolysates, conversion rates under these conditions are much lower than in synthetic media.
Improving kinetics and robustness in industrial hydrolysates is therefore the single most
important objective in industrial yeast strain development platforms.
In the authors’ experience, aspects such as spatial and temporal heterogeneity,
hydrostatic pressure and CO2 concentrations, which are highly important for down-scaling
aerobic industrial fermentation processes (Noorman 2011), do not represent substantial
challenges in down-scaling second-generation ethanol processes. Provided that anaerobic
conditions can be maintained, strain performance can therefore be adequately assessed in small-
scale systems. Access to hydrolysates whose composition and concentration are fully
representative for the target industrial substrate(s) may be necessary for strain development.
This requirement is not a trivial one due to feedstock variability, the plethora of pretreatment

options and the limited scalability and continuous innovation in biomass deconstruction (Knoll
et al. 2013; Li et al. 2016a).
Due to the complex, multigene nature of inhibitor tolerance, screening of natural and
industrial S. cerevisiae strains is a logical first step in the development of industrial strain
platforms. The power of this approach is illustrated by the Brazilian first-generation bioethanol
strain PE-2. Stable maintenance of this strain in non-aseptically operated industrial reactors,
over many production campaigns (Basso et al. 2008), was attributed to its innate tolerance to
the sulfuric-acid washing steps that are employed between fermentation cycles to combat
bacterial contamination (Della-Bianca et al. 2014). In contrast to most laboratory strains, robust
industrial strains of S. cerevisiae are heterozygous diploids or polyploids which, additionally, are
prone to whole-chromosome or segmental aneuploidy (Gorter De Vries et al. 2017; Zhang et al.
2016b). Acquiring high-quality, well annotated genome sequences (Box 1) of these complex
genomes is an important prerequisite for interpreting the results of strain improvement
campaigns and for targeted genetic modification.
Episomal expression vectors carrying auxotrophic marker genes, which are commonly
used in academic research, do not allow for stable replication and selection, respectively, in
complex industrial media (Hahn-Hägerdal et al. 2007; Karim et al. 2013; Pronk 2002). Instead,
industrial strain development requires chromosomal integration of expression cassettes. Even
basic academic designs of xylose- and arabinose-fermenting strains encompass the introduction
of 10-12 different expression cassettes (Wisselink et al. 2010; Wisselink et al. 2007), some of
which need to be present in multiple copies (e.g. for high-level expression of XI genes (Demeke
et al. 2015; Verhoeven et al. 2017; Wang et al. 2014; Zhou et al. 2012)). Additional genetic
modifications, on multiple chromosomes in the case of diploid or polyploid strains, are required
to reduce by-product formation, improve inhibitor tolerance and/or improve product yields.
Genetic modification of complex industrial yeast genomes has now been strongly accelerated by
novel, CRISPR-based genome editing tools (Box 1).

Non-targeted strategies for strain improvement (Box 1) including mutagenesis with
chemical mutagens or irradiation, evolutionary engineering, recursive breeding and/or genome
shuffling remain essential for industrial strain improvement. Down-scaling, automation and
integration with high-throughput screening of the resulting strains in hydrolysates strongly
increases the success rates of these approaches (e.g. for ethanol tolerance, (Snoek et al. 2015)).
In non-targeted strain improvement campaigns, it is important to maintain selective pressure on
all relevant aspects of strain performance, to avoid trade-offs between, for example,
fermentation kinetics with different sugars (glucose, xylose and arabinose), and/or inhibitor
tolerance (Demeke et al. 2013a; Smith et al. 2014; Wisselink et al. 2009).
Even when kinetics of yeast growth and fermentation in hydrolysates are suboptimal
(Table 2) due to the impact of inhibitors and/or strain characteristics, industrial fermentation
processes need to achieve complete sugar conversion within acceptable time limits (typically 72
h or less). This can be accomplished by increasing the initial yeast biomass densities, which, in
second generation processes, are typically 2- to 8-fold higher than the initial concentrations of
0.125-0.25 g·l-1 that are used in first-generation processes without biomass recycling (Jacques et
al. 2003). Several second-generation bioethanol plants therefore include on-site bioreactors for
cost-effective generation of the required yeast biomass. Precultivation in the presence of mild
concentrations of inhibitors can prime yeast cells for improved performance upon exposure to
stressful conditions (Alkasrawi et al. 2006; Nielsen et al. 2015; Sànchez i Nogué et al. 2013).
Especially when biomass propagation uses non-lignocellulosic feedstocks (Narendranath and
Lewis 2013; Steiner 2008) and/or is operated aerobically to maximize biomass yields, yeast
strain development must take into account the need to maintain pentose- fermentation kinetics
and inhibitor tolerance during biomass propagation.
FULL-SCALE IMPLEMENTATION: STATUS AND CHALLENGES
Vigorous lab-scale optimization of each of the unit operations in yeast-based ethanol production
from lignocellulosic feedstocks enabled the design, construction and operation of processes at

pilot scale. Recently, several industrial parties started or announced the first commercial-scale
cellulosic ethanol plants, most of which rely on yeast for the fermentation step (Table 1). Actual
cellulosic ethanol production volumes in the United States of America, derived from registered
RIN (Renewable Identification Numbers) credits (United States Environmental Protection
Agency 2017), indicate an increase in recent years (Figure 3). However, based on these
numbers and estimates for plants elsewhere in the world, the global production volume of
cellulosic ethanol is still below 1 % of that of first-generation processes. This places actual
production volumes years behind earlier projections (Lane 2015) and indicates that currently
installed commercial-scale plants still operate below their nominal capacity. For obvious
reasons, industrial parties cannot always be fully transparent on factors that impede
acceleration and intensification of cellulosic ethanol production. However, presentations at
conferences and trade fairs enable a few general observations. Many aspects of full-scale plants
can be assessed prior to commercialization by carefully down-scaling all process steps. Such
down-scaling is crucial for optimal process development and equipment design (sizing, layout,
mixing requirements, scheduling etc. (Noorman 2011; Villadsen and Noorman 2015; Wang et al.
2015b). As indicated above, most aspects of the performance of engineered yeast strains in full-
scale plants can be, and indeed have been, adequately predicted from such lab-scale studies.
Other aspects, such as impacts of seasonal and regional variation of plant biomass and other in-
process streams, are more difficult to predict. Additionally, continued optimization of upstream
unit operations in commercial-scale plants requires continual ‘tuning’ of yeast strain
characteristics to address impacts on the fermentation process.
An aspect that may have been underestimated in down-scaled experiments is bacterial
contamination. Yield losses caused by contamination with lactic acid bacteria (LAB) is a well-
known problem in first-generation bioethanol production (Beckner et al. 2011; Bischoff et al.
2009). The longer pretreatment and fermentation times in current cellulosic ethanol processes,
caused by inhibitors in the hydrolysates, allow LAB more time to compete with the engineered
yeast strains than in first-generation processes. Moreover, concentrations of ethanol, a potent

inhibitor of LAB, are typically lower in second generation processes (Albers et al. 2011). While
requiring constant attention, bacterial contamination is a manageable problem that can be
addressed with currently available technology and without insurmountable additional costs.
Strict attention for hygiene aspects in all aspects of plant design and operation, e.g. by avoiding
dead legs, implementing full drainability and robust cleaning-in-place (CIP) procedures, is
crucial in this respect. For example, installing appropriate valves and filters should be an integral
part of plant design and be combined with measures to minimize survival and propagation of
bacterial contaminants that do make it into the process. As a last and sometimes inevitable
resort, antibacterial compounds can be used to minimize bacterial load and impact (Muthaiyan
et al. 2011).
An important factor that appears to have escaped attention in most small-scale studies is
that the agricultural residues entering a factory contain an abundance of non-plant solids. Rocks,
sand and metal particles coming off agricultural fields and/or equipment can rapidly damage
and erode expensive equipment (Figure 4). In pilot- and commercial-scale plants, clogging of
pipes and reactors during biomass handling and pretreatment remains a point of attention.
These challenges, which can result in significant down-time of plants, can either be addressed by
elimination of high-density solids during harvesting and storage of the biomass or by installing
extra unit operations in factories. For example, Beta Renewables installed a biomass washing
step at their Crescentino plant (Lane 2014). While these engineering solutions cannot be easily
down-scaled and retrofitting of existing processes may be complicated and expensive, they are
technologically surmountable.
OUTLOOK
Second-generation bioethanol plants are complex, multi-step biorefineries for conversion of
crude and variable feedstocks. Just as high-efficiency petrochemical refineries did not appear
overnight, optimizing the performance of the current frontrunner plants requires significant
process engineering efforts. As remaining challenges in biomass processing and deconstruction

are conquered, yeast-based processes for second-generation biofuels should soon leave the
demonstration phase, become fully economically viable, and expand production volume. Such an
expansion will generate new incentives for improving conversion yields, while reducing carbon
footprints and overall costs. For example, the stillage fraction that remains after distillation is
currently considered a waste stream and treated by anaerobic digestion. As proposed for first-
generation processes (Clomburg and Gonzalez 2013), options may be explored to convert
stillage fractions from second-generation plants into biogas or higher value products.
The yeast technology developed for conversion of second-generation feedstocks can also
be applied to improve ethanol yields of first-generation bioethanol production processes and
plants. For example, in current first-generation ethanol processes, corn fiber is separated from
whole stillage as “wet-distillers’ grains”, mixed with the concentrated stillage liquid fraction
(CDS, “condensed distillers’ solubles’’) and dried to yield DDGS (“dried distillers’ grains with
solubles”), which is sold as cattle feed (Jacques et al. 2003; Kim et al. 2008). Processes that
enable conversion of this fiber-based side stream, which is more easily hydrolysed than other
cellulosic feedstocks, in the context of existing first-generation bioethanol facilities, are referred
to as ‘Gen 1.5’ technology. Several Gen 1.5 processes are currently being implemented
commercially and have the potential to increase the ethanol yield per bushel of corn by
approximately 10 % (Lane 2016b; D3MAX 2017; ICM 2017).
Metabolic engineering strategies to further improve yeast performance in second
generation bioethanol processes are already being explored. For example, the option is
investigated to implement the strategies discussed above in non-Saccharomyces yeasts with
industrially interesting properties, such as high-temperature and low-pH tolerance strains
(Goshima et al. 2013; Radecka et al. 2015; Ryabova et al. 2003; Yuan et al. 2012). Other research
focuses on the improvement of these characteristics in S. cerevisiae (Caspeta et al. 2014; Fletcher
et al. 2017). Furthermore, as production volume increases, the economic relevance of the
conversion of minor, potentially fermentable substrates such as uronic acids and deoxysugars
into ethanol (Van Maris et al. 2006b) will increase. Co-feeding of additional, low-value carbon

sources can be explored as a strategy to further increase ethanol yield. For example, glycerol,
derived from fermentation stills or biodiesel manufacturing (Yang et al. 2012) is considered as a
potential co-substrate. Significant rates of glycerol utilization have already been achieved in S.
cerevisiae strains by simultaneously (over-) expressing glycerol dehydrogenase (GCY1),
dihydroxyacetone kinase (DAK1) and a heterologous glycerol transporter (Yu et al. 2010). These
glycerol conversion pathways can be combined with the engineered pathways for acetic acid
reduction discussed above to further optimize ethanol yields and process robustness (De Bont
et al. 2012; Klaassen and Hartman 2014).
Consolidated bio-processing (CBP), i.e., the full integration of pretreatment, hydrolysis
and fermentation towards ethanol in a single microbial process step, remains a ‘holy grail’ in
lignocellulosic ethanol production. Engineered starch-hydrolysing S. cerevisiae strains are
already applied in first-generation processes (Kumar and Singh 2016). The first important steps
towards efficient cellulose and xylan hydrolysis by S. cerevisiae have been made by functional
expression of heterologous polysaccharide hydrolases (Den Haan et al. 2015; Olson et al. 2012).
The resulting engineered strains often produce significant amounts of di- and/or trisaccharides
(Katahira et al. 2004; Lee et al. 2009; La Grange et al. 2001). The ability to ferment cellobiose has
been successfully introduced into S. cerevisiae by combined expression of a heterologous
cellobiose transporter and β-glucosidase (Galazka et al. 2010, Hu et al. 2016).
Our confidence in yeast-based processes notwithstanding, it is relevant to look beyond
yeasts. Fast progress is made in engineering thermophilic and cellulolytic bacteria for efficient
ethanol production. High-temperature fermentation processes require less cooling and reduce
contamination risks (Scully and Orlygsson 2015). If, moreover, thermophilic CBP can integrate a
simple mechanical pretreatment with biomass deconstruction and fermentation by a single
organism (Lynd et al. 2005; Olson et al. 2012), while matching the robustness of yeasts under
industrial conditions, it could develop into a highly interesting approach for second-generation
ethanol production.

Technological and scientific progress aside, development of yeast platforms for
lignocellulosic ethanol production has provided a generation of academic and industrial
researchers with a challenging common goal. We hope that this mini-review not only informs
readers about scientific and technological progress in this field, but also conveys our genuine
conviction that combining and integrating academic and industrial research efforts (Pronk et al.
2015) is a stimulating, positively challenging way towards sustainable innovation.
Funding
Our joint research on second generation ethanol production is performed within the BE-Basic
R&D Program (http://www.be-basic.org/), which is financially supported by an EOS Long Term
grant from the Dutch Ministry of Economic Affairs, Agriculture and Innovation (EL&I). The PhD
project of IP is funded by DSM Bio-based Products & Services B.V. (Delft, The Netherlands).
Acknowledgements
We gratefully acknowledge our current and former colleagues and students at DSM and TU Delft
for their contributions to our research collaboration. We thank Jim Lane from BiofuelsDigest and
POET-DSM Advanced Biofuels for their kind permission to reproduce the photographs shown in
Figure 4 and in the Graphical Abstract.
References
Abbott DA, Knijnenburg TA, De Poorter LMI et al. Generic and specific transcriptional responses to
different weak organic acids in anaerobic chemostat cultures of Saccharomyces cerevisiae.
FEMS Yeast Res 2007;7:819-33.
Adeboye PT, Bettiga M, Olsson L. The chemical nature of phenolic compounds determines their
toxicity and induces distinct physiological responses in Saccharomyces cerevisiae in
lignocellulose hydrolysates. AMB Express 2014;4:46.
Adeboye PT, Bettiga M, Olsson L. ALD5, PAD1, ATF1 and ATF2 facilitate the catabolism of coniferyl
aldehyde, ferulic acid and p-coumaric acid in Saccharomyces cerevisiae. Sci Rep
2017;7:42635.
Albers E, Johansson E, Franzén CJ et al. Selective suppression of bacterial contaminants by process
conditions during lignocellulose based yeast fermentations. Biotechnol Biofuels 2011;4:59.
Alkasrawi M, Rudolf A, Lidén G et al. Influence of strain and cultivation procedure on the
performance of simultaneous saccharification and fermentation of steam pretreated spruce.
Enzyme Microb Technol 2006;38:279-86.

Almario MP, Reyes LH, Kao KC. Evolutionary engineering of Saccharomyces cerevisiae for enhanced
tolerance to hydrolysates of lignocellulosic biomass. Biotechnol Bioeng 2013;110:2616-23.
Almeida JRM, Bertilsson M, Hahn-Hägerdal B et al. Carbon fluxes of xylose-consuming Saccharomyces
cerevisiae strains are affected differently by NADH and NADPH usage in HMF reduction. Appl
Microbiol Biotechnol 2009;84:751-61.
Almeida JRM, Modig T, Petersson A et al. Increased tolerance and conversion of inhibitors in
lignocellulosic hydrolysates by Saccharomyces cerevisiae. J Chem Technol Biotechnol
2007;82:340-9.
Ask M, Bettiga M, Duraiswamy VR et al. Pulsed addition of HMF and furfural to batch-grown xylose-
utilizing Saccharomyces cerevisiae results in different physiological responses in glucose and
xylose consumption phase. Biotechnol Biofuels 2013;6:181.
Axe DD, Bailey JE. Transport of lactate and acetate through the energized cytoplasmic membrane of
Escherichia coli. Biotechnol Bioeng 1995;47:8-19.
Bailey J. Toward a science of metabolic engineering. Science 1991;252:1668-75.
Bamba T, Hasunuma T, Kondo A. Disruption of PHO13 improves ethanol production via the xylose
isomerase pathway. Amb Express 2016;6:4.
Banerjee N, Bhatnagar R, Viswanathan L. Inhibition of glycolysis by furfural in Saccharomyces
cerevisiae. Appl Microbiol Biotechnol 1981;11:226-8.
Basso LC, De Amorim HV, De Oliveira AJ et al. Yeast selection for fuel ethanol production in Brazil.
FEMS Yeast Res 2008;8:1155-63.
Becker J, Boles E. A modified Saccharomyces cerevisiae strain that consumes L-arabinose and
produces ethanol. Appl Environ Microbiol 2003;69:4144-50.
Beckner M, Ivey ML, Phister TG. Microbial contamination of fuel ethanol fermentations. Lett Appl
Microbiol 2011;53:387-94.
Bellissimi E, Van Dijken JP, Pronk JT et al. Effects of acetic acid on the kinetics of xylose fermentation
by an engineered, xylose‐isomerase‐based Saccharomyces cerevisiae strain. FEMS Yeast Res
2009;9:358-64.
Bernton H, Kovarik B, Sklar S. The forbidden fuel: Power alcohol in the twentieth century. New York:
Caroline House Pubns, 1982.
Bettiga M, Hahn-Hägerdal B, Gorwa-Grauslund MF. Comparing the xylose reductase/xylitol
dehydrogenase and xylose isomerase pathways in arabinose and xylose fermenting
Saccharomyces cerevisiae strains. Biotechnol Biofuels 2008;1:1.
Lane J. Editor's Sketchbook: Beta Renewables cellulosic ethanol project, Crescentino, Italy. Biofuels
Digest 2014. http://www.biofuelsdigest.com/bdigest/2014/09/25/editors-sketchbook-beta-
renewables-cellulosic-ethanol-project-crescentino-italy/ (24 March 2017, date last accessed).
Lane J. Cellulosic ethanol: Where are the gallons? Answers for your questions. Biofuels Digest 2015.
http://www.biofuelsdigest.com/bdigest/2015/07/01/cellulosic-ethanol-where-are-the-
gallons-answers-for-your-questions/ (24 March 2017, date last accessed).
Lane J. Abengoa’s Hugoton cellulosic ethanol project goes on the Block. Biofuels Digest 2016a.
http://www.biofuelsdigest.com/bdigest/2016/07/18/abengoas-hugoton-cellulosic-ethanol-
project-goes-on-the-block/ (24 March 2017, date last accessed).
Lane J. What’s After Gen 1? The Digest’s 2016 Multi-Slide Guide to ICM’s Gen 1.5 cellulosic
technology. Biofuels Digest 2016b. http://www.biofuelsdigest.com/bdigest/2016/12/14/
whats-after-gen-1-the-digests-2016-multi-slide-guide-to-icms-gen-1-5-cellulosic-technology/
(24 March 2017, date last accessed).
Lane J. Getting it right: The digest’s multi-slide guide to Iogen. Biofuels Digest 2016c.
http://www.biofuelsdigest.com/bdigest/2016/05/30/getting-it-right-the-digests-multi-slide-
guide-to-iogen/ (24 March 2017, date last accessed).
Bischoff KM, Liu S, Leathers TD et al. Modeling bacterial contamination of fuel ethanol fermentation.
Biotechnol Bioeng 2009;103:117-22.

Björkqvist S, Ansell R, Adler L et al. Physiological response to anaerobicity of glycerol-3-phosphate
dehydrogenase mutants of Saccharomyces cerevisiae. Appl Environ Microbiol 1997;63:128-
32.
Brat D, Boles E, Wiedemann B. Functional expression of a bacterial xylose isomerase in
Saccharomyces cerevisiae. Appl Environ Microbiol 2009;75:2304-11.
Bruinenberg PM, Bot PH, Dijken JP et al. The role of redox balances in the anaerobic fermentation of
xylose by yeasts. Appl Microbiol Biotechnol 1983;18:287-92.
Canilha L, Chandel AK, Suzane Dos Santos Milessi T et al. Bioconversion of sugarcane biomass into
ethanol: An overview about composition, pretreatment methods, detoxification of
hydrolysates, enzymatic saccharification, and ethanol fermentation. J Biomed Biotechnol
2012, DOI Review.
Casey E, Mosier NS, Adamec J et al. Effect of salts on the co-fermentation of glucose and xylose by a
genetically engineered strain of Saccharomyces cerevisiae. Biotechnol Biofuels 2013;6:83.
Caspeta L, Chen Y, Ghiaci P et al. Altered sterol composition renders yeast thermotolerant. Science
2014;346:75-8.
Chen Y, Sheng J, Jiang T et al. Transcriptional profiling reveals molecular basis and novel genetic
targets for improved resistance to multiple fermentation inhibitors in Saccharomyces
cerevisiae. Biotechnol Biofuels 2016;9.
Clomburg JM, Gonzalez R. Anaerobic fermentation of glycerol: a platform for renewable fuels and
chemicals. Trends Biotechnol 2013;31:20-8.
Colabardini AC, Ries LNA, Brown NA et al. Functional characterization of a xylose transporter in
Aspergillus nidulans. Biotechnol Biofuels 2014;7:1.
Costa CE, Romaní A, Cunha JT et al. Integrated approach for selecting efficient Saccharomyces
cerevisiae for industrial lignocellulosic fermentations: Importance of yeast chassis linked to
process conditions. Bioresour Technol 2017;227:24-34.
Crook N, Abatemarco J, Sun J et al. In vivo continuous evolution of genes and pathways in yeast. Nat
Commun 2016;7.
D3MAX. Advantages of D3MAX. 2017. https://www.d3maxllc.com/ (24 March 2017, date last
accessed).
De Bont JAM, Teunissen AWRH, Klaassen P et al. Yeast strains engineered to produce ethanol from
acetic acid and glycerol. US Patent 20150176032 A1. 2012.
Della-Bianca BE, Basso TO, Stambuk BU et al. What do we know about the yeast strains from the
Brazilian fuel ethanol industry? Appl Microbiol Biotechnol 2013;97:979-91.
Della-Bianca BE, de Hulster E, Pronk JT et al. Physiology of the fuel ethanol strain Saccharomyces
cerevisiae PE-2 at low pH indicates a context-dependent performance relevant for industrial
applications. FEMS Yeast Res 2014;14:1196-205.
Demeke MM, Dietz H, Li Y et al. Development of a D-xylose fermenting and inhibitor tolerant
industrial Saccharomyces cerevisiae strain with high performance in lignocellulose
hydrolysates using metabolic and evolutionary engineering. Biotechnol Biofuels 2013a;6:89.
Demeke MM, Dumortier F, Li Y et al. Combining inhibitor tolerance and D-xylose fermentation in
industrial Saccharomyces cerevisiae for efficient lignocellulose-based bioethanol production.
Biotechnol Biofuels 2013b;6:120.
Demeke MM, Foulquié-Moreno MR, Dumortier F et al. Rapid evolution of recombinant
Saccharomyces cerevisiae for xylose fermentation through formation of extra-chromosomal
circular DNA. PLoS Genet 2015;11:e1005010.
Den Haan R, Van Rensburg E, Rose SH et al. Progress and challenges in the engineering of non-
cellulolytic microorganisms for consolidated bioprocessing. Curr Opin Biotechnol 2015;33:32-
8.
DiCarlo JE, Norville JE, Mali P et al. Genome engineering in Saccharomyces cerevisiae using CRISPR-
Cas systems. Nucleic Acids Res 2013:gkt135.
Diderich JA, Schepper M, van Hoek P et al. Glucose uptake kinetics and transcription of HXT genes in
chemostat cultures of Saccharomyces cerevisiae. J Biol Chem 1999;274:15350-9.

Dos Santos LV, Carazzolle MF, Nagamatsu ST et al. Unraveling the genetic basis of xylose
consumption in engineered Saccharomyces cerevisiae strains. Sci Rep 2016;6.
Du J, Li S, Zhao H. Discovery and characterization of novel d-xylose-specific transporters from
Neurospora crassa and Pichia stipitis. Mol Biosyst 2010;6:2150-6.
Dun B, Wang, Z., Ye, K., Zhang, B., Li, G., Lu, M. Functional expression of Arabidopsis thaliana xylose
isomerase in Saccharomyces cerevisiae. Xinjiang Agric Sci 2012;49:681-6.
Dunlop AP. Furfural Formation and Behavior. Ind Eng Chem 1948;40:204-9.
Ethanol Producer Magazine. U.S. Ethanol Plants. 2017.
http://www.ethanolproducer.com/plants/listplants/US/All/Cellulosic/ (14 February 2017,
date last accessed).
Farrell AE, Plevin RJ, Turner BT et al. Ethanol can contribute to energy and environmental goals.
Science 2006;311:506.
Farwick A, Bruder S, Schadeweg V et al. Engineering of yeast hexose transporters to transport D-
xylose without inhibition by D-glucose. Proc Natl Acad Sci USA 2014;111:5159-64.
Favaro L, Basaglia M, Trento A et al. Exploring grape marc as trove for new thermotolerant and
inhibitor-tolerant Saccharomyces cerevisiae strains for second-generation bioethanol
production. Biotechnol Biofuels 2013;6:168.
Ferreira D, Nobre A, Silva ML et al. XYLH encodes a xylose/H+ symporter from the highly related yeast
species Debaryomyces fabryi and Debaryomyces hansenii. FEMS Yeast Res 2013;13:585-96.
Field SJ, Ryden P, Wilson D et al. Identification of furfural resistant strains of Saccharomyces
cerevisiae and Saccharomyces paradoxus from a collection of environmental and industrial
isolates. Biotechnol Biofuels 2015;8.
Fletcher E, Feizi A, Bisschops MMM et al. Evolutionary engineering reveals divergent paths when
yeast is adapted to different acidic environments. Metab Eng 2017;39:19-28.
Fonseca C, Olofsson K, Ferreira C et al. The glucose/xylose facilitator Gxf1 from Candida intermedia
expressed in a xylose-fermenting industrial strain of Saccharomyces cerevisiae increases
xylose uptake in SSCF of wheat straw. Enzyme Microb Technol 2011;48:518-25.
Galazka JM, Tian C, Beeson WT et al. Cellodextrin transport in yeast for improved biofuel production.
Science 2010;330:84-6.
Garcia Sanchez R, Karhumaa K, Fonseca C et al. Improved xylose and arabinose utilization by an
industrial recombinant Saccharomyces cerevisiae strain using evolutionary engineering.
Biotechnol Biofuels 2010;3:13.
González-Ramos D, Gorter de Vries AR, Grijseels SS et al. A new laboratory evolution approach to
select for constitutive acetic acid tolerance in Saccharomyces cerevisiae and identification of
causal mutations. Biotechnol Biofuels 2016;9:1.
Gorter De Vries AR, Pronk JT, Daran JM. Industrial relevance of chromosomal copy number variation
in Saccharomyces yeasts. Appl Environ Microbiol 2017;In press.
Goshima T, Tsuji M, Inoue H et al. Bioethanol production from lignocellulosic biomass by a novel
Kluyveromyces marxianus strain. Biosci Biotechnol Biochem 2013;77:1505-10.
Grohmann K, Bothast R. Pectin-rich residues generated by processing of citrus fruits, apples, and
sugar beets.Enzymatic Conversion of Biomass for Fuels Production 566: ACS Publications,
1994,pp 372-90.
Grohmann K, Bothast RJ. Saccharification of corn fibre by combined treatment with dilute sulphuric
acid and enzymes. Process Biochem 1997;32:405-15.
Guadalupe-Medina V, Almering MJH, van Maris AJA et al. Elimination of glycerol production in
anaerobic cultures of a Saccharomyces cerevisiae strain engineered to use acetic acid as an
electron acceptor. Appl Environ Microbiol 2010;76:190-5.
Guo Z, Olsson L. Physiological response of Saccharomyces cerevisiae to weak acids present in
lignocellulosic hydrolysate. FEMS Yeast Res 2014;14:1234-48.
Guo Z, Zhang L, Ding Z et al. Minimization of glycerol synthesis in industrial ethanol yeast without
influencing its fermentation performance. Metab Eng 2011;13:49-59.

Ha S, Galazka JM, Rin Kim S et al. Engineered Saccharomyces cerevisiae capable of simultaneous
cellobiose and xylose fermentation. Proc Natl Acad Sci USA 2011;108:504-9.
Hahn-Hägerdal B, Galbe M, Gorwa-Grauslund MF et al. Bio-ethanol – the fuel of tomorrow from the
residues of today. Trends Biotechnol 2006;24:549-56.
Hahn-Hägerdal B, Karhumaa K, Fonseca C et al. Towards industrial pentose-fermenting yeast strains.
Appl Microbiol Biotechnol 2007;74:937-53.
Hahn-Hägerdal B, Wahlbom CF, Gárdonyi M et al. Metabolic engineering of Saccharomyces cerevisiae
for xylose utilization.Metab Eng,73: Springer, 2001,53-84.
Hamacher T, Becker J, Gárdonyi M et al. Characterization of the xylose-transporting properties of
yeast hexose transporters and their influence on xylose utilization. Microbiology
2002;148:2783-8.
Harhangi HR, Akhmanova AS, Emmens R et al. Xylose metabolism in the anaerobic fungus Piromyces
sp. strain E2 follows the bacterial pathway. Arch Microbiol 2003;180:134-41.
Hector RE, Dien BS, Cotta MA et al. Growth and fermentation of D-xylose by Saccharomyces
cerevisiae expressing a novel D-xylose isomerase originating from the bacterium Prevotella
ruminicola TC2-24. Biotechnol Biofuels 2013;6:84.
Hendriks ATWM, Zeeman G. Pretreatments to enhance the digestibility of lignocellulosic biomass.
Bioresour Technol 2009;100:10-8.
Henningsen BM, Hon S, Covalla SF et al. Increasing anaerobic acetate consumption and ethanol yields
in Saccharomyces cerevisiae with NADPH-specific alcohol dehydrogenase. Appl Environ
Microbiol 2015;81:8108-17.
Horak J, Regelmann J, Wolf DH. Two distinct proteolytic systems responsible for glucose-induced
degradation of fructose-1,6-bisphosphatase and the Gal2p transporter in the yeast
Saccharomyces cerevisiae share the same protein components of the glucose signaling
pathway. J Biol Chem 2002;277:8248-54.
Horak J, Wolf DH. Catabolite inactivation of the galactose transporter in the yeast Saccharomyces
cerevisiae: ubiquitination, endocytosis, and degradation in the vacuole. J Bacteriol
1997;179:1541-9.
Hou J, Jiao C, Peng B et al. Mutation of a regulator Ask10p improves xylose isomerase activity
through up-regulation of molecular chaperones in Saccharomyces cerevisiae. Metab Eng
2016a.
Hou J, Shen Y, Jiao C et al. Characterization and evolution of xylose isomerase screened from the
bovine rumen metagenome in Saccharomyces cerevisiae. J Biosci Bioeng 2016b;121:160-5.
Hu ML, Zha J, He LW et al. Enhanced bioconversion of cellobiose by industrial Saccharomyces
cerevisiae used for cellulose utilization. Front Microbiol 2016;7.
Hubmann G, Guillouet S, Nevoigt E. Gpd1 and Gpd2 fine-tuning for sustainable reduction of glycerol
formation in Saccharomyces cerevisiae. Appl Environ Microbiol 2011;77:5857-67.
Hubmann G, Mathé L, Foulquié-Moreno MR et al. Identification of multiple interacting alleles
conferring low glycerol and high ethanol yield in Saccharomyces cerevisiae ethanolic
fermentation. Biotechnol Biofuels 2013;6:87.
ICM. Generation 1.5: Grain Fiber to Cellulosic Ethanol Technology. 2012.
http://icminc.com/products/generation-1-5.html (24 March 2017, date last accessed).
Ilmén M, den Haan R, Brevnova E et al. High level secretion of cellobiohydrolases by Saccharomyces
cerevisiae. Biotechnol Biofuels 2011;4:30.
Jacques KA, Lyons TP, Kelsall DR. The alcohol textbook: a reference for the beverage, fuel and
industrial alcohol industries: Nottingham University Press, 2003.
Jeffries TW. Engineering yeasts for xylose metabolism. Curr Opin Biotechnol 2006;17:320-6.
Jeffries TW, Jin Y. Metabolic engineering for improved fermentation of pentoses by yeasts. Appl
Microbiol Biotechnol 2004;63:495-509.
Jeppsson M, Johansson B, Jensen PR et al. The level of glucose-6-phosphate dehydrogenase activity
strongly influences xylose fermentation and inhibitor sensitivity in recombinant
Saccharomyces cerevisiae strains. Yeast 2003;20:1263-72.

Jönsson LJ, Alriksson B, Nilvebrant NO. Bioconversion of lignocellulose: inhibitors and detoxification.
Biotechnol Biofuels 2013;6:16.
Karhumaa K, Sanchez RG, Hahn-Hägerdal B et al. Comparison of the xylose reductase-xylitol
dehydrogenase and the xylose isomerase pathways for xylose fermentation by recombinant
Saccharomyces cerevisiae. Microb Cell Fact 2007;6:5.
Karim AS, Curran KA, Alper HS. Characterization of plasmid burden and copy number in
Saccharomyces cerevisiae for optimization of metabolic engineering applications. FEMS Yeast
Res 2013;13:107-16.
Katahira S, Fujita Y, Mizuike A et al. Construction of a xylan-fermenting yeast strain through codisplay
of xylanolytic enzymes on the surface of xylose-utilizing Saccharomyces cerevisiae cells. Appl
Environ Microbiol 2004;70:5407-14.
Katahira S, Ito M, Takema H et al. Improvement of ethanol productivity during xylose and glucose co-
fermentation by xylose-assimilating S. cerevisiae via expression of glucose transporter Sut1.
Enzyme Microb Technol 2008;43:115-9.
Katahira S, Mizuike A, Fukuda H et al. Ethanol fermentation from lignocellulosic hydrolysate by a
recombinant xylose-and cellooligosaccharide-assimilating yeast strain. Appl Microbiol
Biotechnol 2006;72:1136-43.
Kenney KL, Smith WA, Gresham GL et al. Understanding biomass feedstock variability. Biofuels
2013;4:111-27.
Khoo HH. Review of bio-conversion pathways of lignocellulose-to-ethanol: Sustainability assessment
based on land footprint projections. Renew Sustainable Energy Rev 2015;46:100-19.
Kim SR, Ha S-J, Wei N et al. Simultaneous co-fermentation of mixed sugars: a promising strategy for
producing cellulosic ethanol. Trends Biotechnol 2012;30:274-82.
Kim SR, Skerker JM, Kang W et al. Rational and evolutionary engineering approaches uncover a small
set of genetic changes efficient for rapid xylose fermentation in Saccharomyces cerevisiae.
PLOS ONE 2013;8:e57048.
Kim Y, Mosier NS, Hendrickson R et al. Composition of corn dry-grind ethanol by-products: DDGS, wet
cake, and thin stillage. Bioresour Technol 2008;99:5165-76.
Klaassen P, Hartman WWA. Glycerol and acetic acid converting yeast cells with improved acetic acid
conversion. US Patent 20160208291 A1. 2014.
Kleinschmidt J. Biofueling rural development: making the case for linking biofuel production to rural
revitalization. Carsey Institute 2007. (24 March 2017, date last accessed).
Klinke HB, Ahring BK, Schmidt AS et al. Characterization of degradation products from alkaline wet
oxidation of wheat straw. Bioresour Technol 2002;82:15-26.
Klinke HB, Thomsen AB, Ahring BK. Inhibition of ethanol-producing yeast and bacteria by degradation
products produced during pre-treatment of biomass. Appl Microbiol Biotechnol 2004;66:10-
26.
Knoll JE, Anderson WF, Richard EP et al. Harvest date effects on biomass quality and ethanol yield of
new energycane (Saccharum hyb.) genotypes in the Southeast USA. Biomass Bioenergy
2013;56:147-56.
Knoshaug EP, Vidgren V, Magalhães F et al. Novel transporters from Kluyveromyces marxianus and
Pichia guilliermondii expressed in Saccharomyces cerevisiae enable growth on l‐arabinose
and d‐xylose. Yeast 2015;32:615-28.
Koppram R, Albers E, Olsson L. Evolutionary engineering strategies to enhance tolerance of xylose
utilizing recombinant yeast to inhibitors derived from spruce biomass. Biotechnol Biofuels
2012;5:32.
Koppram R, Mapelli V, Albers E et al. The presence of pretreated lignocellulosic solids from birch
during Saccharomyces cerevisiae fermentations leads to increased tolerance to inhibitors – a
proteomic study of the effects. PLOS ONE 2016;11:e0148635.
Kötter P, Amore R, Hollenberg CP et al. Isolation and characterization of the Pichia stipitis xylitol
dehydrogenase gene, XYL2, and construction of a xylose-utilizing Saccharomyces cerevisiae
transformant. Curr Genet 1990;18:493-500.

Kötter P, Ciriacy M. Xylose fermentation by Saccharomyces cerevisiae. Appl Microbiol Biotechnol
1993;38:776-83.
Kou S, Christensen MS, Cirillo VP. Galactose transport in Saccharomyces cerevisiae II. Characteristics
of galactose uptake and exchange in galactokinaseless cells. J Bacteriol 1970;103:671-8.
Krahulec S, Klimacek M, Nidetzky B. Engineering of a matched pair of xylose reductase and xylitol
dehydrogenase for xylose fermentation by Saccharomyces cerevisiae. Biotechnol J
2009;4:684-94.
Kumar D, Singh V. Dry-grind processing using amylase corn and superior yeast to reduce the
exogenous enzyme requirements in bioethanol production. Biotechnol Biofuels 2016;9:228.
Kumar R, Mago G, Balan V et al. Physical and chemical characterizations of corn stover and poplar
solids resulting from leading pretreatment technologies. Bioresour Technol 2009;100:3948-
62.
Kuyper M, Hartog MM, Toirkens MJ et al. Metabolic engineering of a xylose‐isomerase‐expressing
Saccharomyces cerevisiae strain for rapid anaerobic xylose fermentation. FEMS Yeast Res
2005a;5:399-409.
Kuyper M, Toirkens MJ, Diderich JA et al. Evolutionary engineering of mixed‐sugar utilization by a
xylose‐fermenting Saccharomyces cerevisiae strain. FEMS Yeast Res 2005b;5:925-34.
Kuyper M, Winkler AA, van Dijken JP et al. Minimal metabolic engineering of Saccharomyces
cerevisiae for efficient anaerobic xylose fermentation: a proof of principle. FEMS Yeast Res
2006;4:655-64.
La Grange DC, Pretorius IS, Claeyssens M et al. Degradation of xylan to d-xylose by recombinant
Saccharomyces cerevisiae coexpressing the Aspergillus niger β-xylosidase (xlnD) and the
Trichoderma reesei xylanase II (xyn2) genes. Appl Environ Microbiol 2001;67:5512-9.
Larsson S, Quintana-Sainz A, Reimann A et al. Influence of lignocellulose-derived aromatic
compounds on oxygen-limited growth and ethanolic fermentation by Saccharomyces
cerevisiae. Appl Biochem Biotechnol 2000;84-86:617-32.
Lawther JM, Sun R, Banks WB. Fractional characterization of alkali-labile lignin and alkali-insoluble
lignin from wheat straw. Ind Crops Prod 1996;5:291-300.
Leandro MJ, Gonçalves P, Spencer-Martins I. Two glucose/xylose transporter genes from the yeast
Candida intermedia: first molecular characterization of a yeast xylose–H+ symporter. Biochem
J 2006;395:543-9.
Lee JH, Heo SY, Lee JW et al. Thermostability and xylan-hydrolyzing property of endoxylanase
expressed in yeast Saccharomyces cerevisiae. Biotechnol Bioprocess Eng 2009;14:639-44.
Lee N, Gielow W, Martin R et al. The organization of the araBAD operon of Escherichia coli. Gene
1986;47:231-44.
Lee SM, Jellison T, Alper HS. Directed Evolution of xylose isomerase for improved xylose catabolism
and fermentation in the yeast Saccharomyces cerevisiae. Appl Environ Microbiol
2012;78:5708-16.
Lee SM, Jellison T, Alper HS. Systematic and evolutionary engineering of a xylose isomerase-based
pathway in Saccharomyces cerevisiae for efficient conversion yields. Biotechnol Biofuels
2014;7:122.
Lee W, Kim M, Ryu Y et al. Kinetic studies on glucose and xylose transport in Saccharomyces
cerevisiae. Appl Microbiol Biotechnol 2002;60:186-91.
Lewis Liu Z, Moon J, Andersh BJ et al. Multiple gene-mediated NAD(P)H-dependent aldehyde
reduction is a mechanism of in situ detoxification of furfural and 5-hydroxymethylfurfural by
Saccharomyces cerevisiae. Appl Microbiol Biotechnol 2008;81:743-53.
Li BZ, Yuan YJ. Transcriptome shifts in response to furfural and acetic acid in Saccharomyces
cerevisiae. Appl Microbiol Biotechnol 2010;86:1915-24.
Li C, Aston JE, Lacey JA et al. Impact of feedstock quality and variation on biochemical and
thermochemical conversion. Renewable and Sustainable Energy Reviews 2016a;65:525-36.
Li H, Schmitz O, Alper HS. Enabling glucose/xylose co-transport in yeast through the directed
evolution of a sugar transporter. Appl Microbiol Biotechnol 2016b;100:10215-23.

Li H, Shen Y, Wu M et al. Engineering a wild-type diploid Saccharomyces cerevisiae strain for second-
generation bioethanol production. Bioresour Bioprocess 2016c;3:51.
Li J, Xu J, Cai P et al. Functional analysis of two l-arabinose transporters from filamentous fungi
reveals promising characteristics for improved pentose utilization in Saccharomyces
cerevisiae. Appl Environ Microbiol 2015;81:4062-70.
Lin Y, Tanaka S. Ethanol fermentation from biomass resources: current state and prospects. Appl
Microbiol Biotechnol 2006;69:627-42.
Liti G, Louis EJ. Advances in quantitative trait analysis in yeast. PLoS Genet 2012;8:e1002912.
Liu ZL. Molecular mechanisms of yeast tolerance and in situ detoxification of lignocellulose
hydrolysates. Appl Microbiol Biotechnol 2011;90:809-25.
Liu ZL, Slininger PJ, Dien BS et al. Adaptive response of yeasts to furfural and 5-hydroxymethylfurfural
and new chemical evidence for HMF conversion to 2,5-bis-hydroxymethylfuran. J Ind
Microbiol Biotechnol 2004;31:345-52.
Lopes ML, de Lima Paulillo SCdL, Godoy A et al. Ethanol production in Brazil: a bridge between
science and industry. Braz J Microbiol 2016;47:64-76.
Lynd LR. Overview and evaluation of fuel ethanol from cellulosic biomass: technology, economics,
the environment, and policy. Annu Rev Energy Env 1996;21:403-65.
Lynd LR, van Zyl WH, McBride JE et al. Consolidated bioprocessing of cellulosic biomass: an update.
Curr Opin Biotechnol 2005;16:577-83.
Lynd LR, Weimer PJ, van Zyl WH et al. Microbial cellulose utilization: fundamentals and
biotechnology. Microbiol Mol Biol Rev 2002;66:506-77.
Madhavan A, Tamalampudi S, Ushida K et al. Xylose isomerase from polycentric fungus Orpinomyces:
gene sequencing, cloning, and expression in Saccharomyces cerevisiae for bioconversion of
xylose to ethanol. Appl Microbiol Biotechnol 2009;82:1067-78.
Mans R, van Rossum HM, Wijsman M et al. CRISPR/Cas9: a molecular Swiss army knife for
simultaneous introduction of multiple genetic modifications in Saccharomyces cerevisiae.
FEMS Yeast Res 2015;15::fov004.
Marcheschi RJ, Gronenberg LS, Liao JC. Protein engineering for metabolic engineering: current and
next-generation tools. Biotechnol J 2013;8:545-55.
Meijnen JP, Randazzo P, Foulquié-Moreno MR et al. Polygenic analysis and targeted improvement of
the complex trait of high acetic acid tolerance in the yeast Saccharomyces cerevisiae.
Biotechnol Biofuels 2016;9:1.
Mira NP, Palma M, Guerreiro JF et al. Genome-wide identification of Saccharomyces cerevisiae genes
required for tolerance to acetic acid. Microb Cell Fact 2010;9:79.
Modig T, Lidén G, Taherzadeh MJ. Inhibition effects of furfural on alcohol dehydrogenase, aldehyde
dehydrogenase and pyruvate dehydrogenase. Biochem J 2002;363:769-76.
Moniruzzaman M, Dien B, Skory C et al. Fermentation of corn fibre sugars by an engineered xylose
utilizing Saccharomyces yeast strain. World J Microbiol Biotechnol 1997;13:341-6.
Moon J, Liu ZL. Engineered NADH-dependent GRE2 from Saccharomyces cerevisiae by directed
enzyme evolution enhances HMF reduction using additional cofactor NADPH. Enzyme Microb
Technol 2012;50:115-20.
Moysés DN, Reis VCB, de Almeida JRMM, Lidia Maria Pepe de et al. Xylose Fermentation by
Saccharomyces cerevisiae: Challenges and Prospects. Int J Mol Sci 2016;17:207.
Muthaiyan A, Limayem A, Ricke S. Antimicrobial strategies for limiting bacterial contaminants in fuel
bioethanol fermentations. Prog Energy Combust Sci 2011;37:351-70.
Narendranath NV, Lewis SM. Systems and methods for yeast propagation. US Patent 9034631 B2.
2013.
Narron RH, Kim H, Chang HM et al. Biomass pretreatments capable of enabling lignin valorization in a
biorefinery process. Curr Opin Biotechnol 2016;38:39-46.
Ni H, Laplaza JM, Jeffries TW. Transposon mutagenesis to improve the growth of recombinant
Saccharomyces cerevisiae on D-xylose. Appl Environ Microbiol 2007;73:2061-6.

Nielsen F, Tomás-Pejó E, Olsson L et al. Short-term adaptation during propagation improves the
performance of xylose-fermenting Saccharomyces cerevisiae in simultaneous saccharification
and co-fermentation. Biotechnol Biofuels 2015;8:1.
Nielsen J, Larsson C, van Maris A et al. Metabolic engineering of yeast for production of fuels and
chemicals. Curr Opin Biotechnol 2013;24:398-404.
Nijland JG, Shin HY, de Jong RM et al. Engineering of an endogenous hexose transporter into a
specific D-xylose transporter facilitates glucose-xylose co-consumption in Saccharomyces
cerevisiae. Biotechnol Biofuels 2014;7:168.
Nijland JG, Vos E, Shin HY et al. Improving pentose fermentation by preventing ubiquitination of
hexose transporters in Saccharomyces cerevisiae. Biotechnol Biofuels 2016;9:158.
Nissen TL, Kielland-Brandt MC, Nielsen J et al. Optimization of ethanol production in Saccharomyces
cerevisiae by metabolic engineering of the ammonium assimilation. Metab Eng 2000;2:69-77.
Noorman H. An industrial perspective on bioreactor scale-down: What we can learn from combined
large-scale bioprocess and model fluid studies. Biotechnol J 2011;6:934-43.
Nordhoff S. Editorial: Food vs fuel – the role of biotechnology. Biotechnol J 2007;2:1451.
Olson DG, McBride JE, Joe Shaw A et al. Recent progress in consolidated bioprocessing. Curr Opin
Biotechnol 2012;23:396-405.
Ota M, Sakuragi H, Morisaka H et al. Display of Clostridium cellulovorans xylose isomerase on the cell
surface of Saccharomyces cerevisiae and its direct application to xylose fermentation.
Biotechnol Prog 2013;29:346-51.
Otero JM, Panagiotou G, Olsson L. Fueling industrial biotechnology growth with bioethanol. In:
Olsson L (ed.) Biofuels Springer Berlin Heidelberg, 2007.
Oud B, van Maris AJ, Daran J-M et al. Genome-wide analytical approaches for reverse metabolic
engineering of industrially relevant phenotypes in yeast. FEMS Yeast Res 2012;12:183-96.
Palmqvist E, Hahn-Hägerdal B. Fermentation of lignocellulosic hydrolysates. I: inhibition and
detoxification. Bioresour Technol 2000a;74:17-24.
Palmqvist E, Hahn-Hägerdal B. Fermentation of lignocellulosic hydrolysates. II: inhibitors and
mechanisms of inhibition. Bioresour Technol 2000b;74:25-33.
Pampulha ME, Loureiro-Dias MC. Energetics of the effect of acetic acid on growth of Saccharomyces
cerevisiae. FEMS Microbiol Lett 2000;184:69-72.
Papapetridis I, van Dijk M, Dobbe AP et al. Improving ethanol yield in acetate-reducing
Saccharomyces cerevisiae by cofactor engineering of 6-phosphogluconate dehydrogenase
and deletion of ALD6. Microb Cell Fact 2016;15:67.
Pereira LG, Dias MOS, Mariano AP et al. Economic and environmental assessment of n-butanol
production in an integrated first and second generation sugarcane biorefinery: Fermentative
versus catalytic routes. Appl Energy 2015;160:120-31.
Petschacher B, Leitgeb S, Kavanagh Kathryn L et al. The coenzyme specificity of Candida tenuis xylose
reductase (AKR2B5) explored by site-directed mutagenesis and X-ray crystallography.
Biochem J 2005;385:75-83.
Petschacher B, Nidetzky B. Altering the coenzyme preference of xylose reductase to favor utilization
of NADH enhances ethanol yield from xylose in a metabolically engineered strain of
Saccharomyces cerevisiae. Microb Cell Fact 2008;7:9.
Pinel D, Colatriano D, Jiang H et al. Deconstructing the genetic basis of spent sulphite liquor tolerance
using deep sequencing of genome-shuffled yeast. Biotechnol Biofuels 2015;8:53.
Pronk JT. Auxotrophic yeast strains in fundamental and applied research. Appl Environ Microbiol
2002;68:2095-100.
Pronk JT, Lee SY, Lievense J et al. How to set up collaborations between academia and industrial
biotech companies. Nat Biotech 2015;33:237-40.
Radecka D, Mukherjee V, Mateo RQ et al. Looking beyond Saccharomyces: the potential of non-
conventional yeast species for desirable traits in bioethanol fermentation. FEMS Yeast Res
2015;15:fov053.

Reifenberger E, Boles E, Ciriacy M. Kinetic characterization of individual hexose transporters of
Saccharomyces cerevisiae and their relation to the triggering mechanisms of glucose
repression. Eur J Biochem 1997;245:324-33.
Reis TF, Lima PBA, Parachin NS et al. Identification and characterization of putative xylose and
cellobiose transporters in Aspergillus nidulans. Biotechnol Biofuels 2016;9:204.
Renewable Fuels Association. World fuel ethanol production. 2016.
http://ethanolrfa.org/resources/industry/statistics/ (24 March 2017, date last accessed).
Reznicek O, Facey SJ, de Waal PP et al. Improved xylose uptake in Saccharomyces cerevisiae due to
directed evolution of galactose permease Gal2 for sugar co-consumption. J Appl Microbiol
2015;119:99-111.
Roca C, Nielsen J, Olsson L. Metabolic engineering of ammonium assimilation in xylose-fermenting
Saccharomyces cerevisiae improves ethanol production. Appl Environ Microbiol
2003;69:4732-6.
Rude MA, Schirmer A. New microbial fuels: a biotech perspective. Curr Opin Microbiol 2009;12:274-
81.
Runquist D, Hahn-Hägerdal B, Bettiga M. Increased ethanol productivity in xylose-utilizing
Saccharomyces cerevisiae via a randomly mutagenized xylose reductase. Appl Environ
Microbiol 2010a;76:7796-802.
Runquist D, Hahn-Hägerdal B, Rådström P. Comparison of heterologous xylose transporters in
recombinant Saccharomyces cerevisiae. Biotechnol Biofuels 2010b;3:1.
Russel JB. Another explanation for the toxicity of fermentation acids at low pH: anion accumulation
versus uncoupling. J Appl Microbiol 1992;73:363-70.
Ryabova OB, Chmil OM, Sibirny AA. Xylose and cellobiose fermentation to ethanol by the
thermotolerant methylotrophic yeast Hansenula polymorpha. FEMS Yeast Res 2003;4:157-
64.
Sadie CJ, Rose SH, den Haan R et al. Co-expression of a cellobiose phosphorylase and lactose
permease enables intracellular cellobiose utilisation by Saccharomyces cerevisiae. Appl
Microbiol Biotechnol 2011;90:1373-80.
Saloheimo A, Rauta J, Stasyk V et al. Xylose transport studies with xylose-utilizing Saccharomyces
cerevisiae strains expressing heterologous and homologous permeases. Appl Microbiol
Biotechnol 2007;74:1041-52.
Sànchez i Nogué V, Narayanan V, Gorwa-Grauslund MF. Short-term adaptation improves the
fermentation performance of Saccharomyces cerevisiae in the presence of acetic acid at low
pH. Appl Microbiol Biotechnol 2013;97:7517-25.
Sanchez RG, Karhumaa K, Fonseca C et al. Improved xylose and arabinose utilization by an industrial
recombinant Saccharomyces cerevisiae strain using evolutionary engineering. Biotechnol
Biofuels 2010;3:1.
Sander JD, Joung JK. CRISPR-Cas systems for editing, regulating and targeting genomes. Nat Biotech
2014;32:347-55.
Sárvári Horváth I, Franzén CJ, Taherzadeh MJ et al. Effects of furfural on the respiratory metabolism
of Saccharomyces cerevisiae in glucose-limited chemostats. Appl Environ Microbiol
2003;69:4076-86.
Sato TK, Tremaine M, Parreiras LS et al. Directed evolution reveals unexpected epistatic interactions
that alter metabolic regulation and enable anaerobic xylose use by Saccharomyces cerevisiae.
PLoS Genet 2016;12:e1006372.
Sauer U. Evolutionary engineering of industrially important microbial phenotypes. Metabolic
Engineering Springer Berlin Heidelberg, 2001,129-69.
Scully S, Orlygsson J. Recent advances in second generation ethanol production by thermophilic
bacteria. Energies 2015;8:1.
Sedlak M, Ho NW. Expression of E. coli araBAD operon encoding enzymes for metabolizing L-
arabinose in Saccharomyces cerevisiae. Enzyme Microb Technol 2001;28:16-24.

Sedlak M, Ho NW. Production of ethanol from cellulosic biomass hydrolysates using genetically
engineered Saccharomyces yeast capable of cofermenting glucose and xylose. Appl Biochem
Biotechnol 2004;114:403-16.
Sedlak M, Ho NW. Characterization of the effectiveness of hexose transporters for transporting
xylose during glucose and xylose co‐fermentation by a recombinant Saccharomyces yeast.
Yeast 2004;21:671-84.
Shi S, Liang Y, Zhang MM et al. A highly efficient single-step, markerless strategy for multi-copy
chromosomal integration of large biochemical pathways in Saccharomyces cerevisiae. Metab
Eng 2016;33:19-27.
Shin H, Nijland J, de Waal P et al. An engineered cryptic Hxt11 sugar transporter facilitates glucose-
xylose co-consumption in Saccharomyces cerevisiae. Biotechnol Biofuels 2015a;8:176.
Shin HY, Nijland JG, de Waal PP et al. An engineered cryptic Hxt11 sugar transporter facilitates
glucose–xylose co-consumption in Saccharomyces cerevisiae. Biotechnol Biofuels
2015b;8:176.
Silveira MHL, Morais ARC, Da Costa Lopes AM et al. Current pretreatment technologies for the
development of cellulosic ethanol and biorefineries. Chem Sus Chem 2015;8:3366-90.
Sims-Borre P. The economics of enzyme production. Ethanol Producer Magazine 2010.
http://www.ethanolproducer.com/articles/7048/ (24 March 2017, date last accessed).
Sinha H, Nicholson BP, Steinmetz LM et al. Complex genetic interactions in a quantitative trait locus.
PLoS Genet 2006;2:e13.
Sivers MV, Zacchi G, Olsson L et al. Cost analysis of ethanol production from willow using
recombinant Escherichia coli. Biotechnol Prog 1994;10:555-60.
Smith J, van Rensburg E, Görgens JF. Simultaneously improving xylose fermentation and tolerance to
lignocellulosic inhibitors through evolutionary engineering of recombinant Saccharomyces
cerevisiae harbouring xylose isomerase. BMC Biotechnol 2014;14:41.
Snoek T, Nicolino MP, van den Bremt S et al. Large-scale robot-assisted genome shuffling yields
industrial Saccharomyces cerevisiae yeasts with increased ethanol tolerance. Biotechnol
Biofuels 2015;8:32.
Sonderegger M, Sauer U. Evolutionary engineering of Saccharomyces cerevisiae for anaerobic growth
on xylose. Appl Environ Microbiol 2003;69:1990-8.
Steiner G. Use of ethanol plant by-products for yeast propagation. US Patent 8183022 B2. 2008.
Stovicek V, Borodina I, Forster J. CRISPR–Cas system enables fast and simple genome editing of
industrial Saccharomyces cerevisiae strains. Metab Eng Commun 2015;2:13-22.
Subtil T, Boles E. Improving L-arabinose utilization of pentose fermenting Saccharomyces cerevisiae
cells by heterologous expression of L-arabinose transporting sugar transporters. Biotechnol
Biofuels 2011;4:38.
Subtil T, Boles E. Competition between pentoses and glucose during uptake and catabolism in
recombinant Saccharomyces cerevisiae. Biotechnol Biofuels 2012;5:1.
Swinnen S, Fernández-Niño M, González-Ramos D et al. The fraction of cells that resume growth after
acetic acid addition is a strain-dependent parameter of acetic acid tolerance in
Saccharomyces cerevisiae. FEMS Yeast Res 2014;14:642-53.
Swinnen S, Schaerlaekens K, Pais T et al. Identification of novel causative genes determining the
complex trait of high ethanol tolerance in yeast using pooled-segregant whole-genome
sequence analysis. Genome Res 2012;22:975-84.
Taherzadeh MJ, Eklund R, Gustafsson L et al. Characterization and fermentation of dilute-acid
hydrolyzates from wood. Ind Eng Chem Res 1997;36:4659-65.
Taherzadeh MJ, Gustafsson L, Niklasson C et al. Conversion of furfural in aerobic and anaerobic batch
fermentation of glucose by Saccharomyces cerevisiae. J Biosci Bioeng 1999;87:169-74.
Tantirungkij M, Nakashima N, Seki T et al. Construction of xylose-assimilating Saccharomyces
cerevisiae. J Ferment Bioeng 1993;75:83-8.
Tenenbaum DJ. Food vs. fuel: Diversion of crops could cause more hunger. Environ Health Perspect
2008;116:A254-A7.

Thomas K, Ingledew W. Production of 21%(v/v) ethanol by fermentation of very high gravity (VHG)
wheat mashes. J Ind Microbiol Biotechnol 1992;10:61-8.
Thompson OA, Hawkins GM, Gorsich SW et al. Phenotypic characterization and comparative
transcriptomics of evolved Saccharomyces cerevisiae strains with improved tolerance to
lignocellulosic derived inhibitors. Biotechnol Biofuels 2016;9.
Tsai C-S, Kong II, Lesmana A et al. Rapid and marker-free refactoring of xylose-fermenting yeast
strains with Cas9/CRISPR. Biotechnol Bioeng 2015;112:2406-11.
Ulbricht R, Northup S, Thomas J. A review of 5-hydroxymethylfurfural (HMF) in parenteral solutions.
Fundam Appl Toxicol 1984;4:843-53.
Ullah A, Chandrasekaran G, Brul S et al. Yeast adaptation to weak acids prevents futile energy
expenditure. Front Microbiol 2013;4.
UNCTAD. Second generation biofuel markets: state of play, trade and developing country
perspectives. United Nations Conference on Trade and Development 2016.
United States Environmental Protection Agency. Public data for the Renewable Fuel Standard 2017.
https://www.epa.gov/fuels-registration-reporting-and-compliance-help/public-data-
renewable-fuel-standard (4 Jan 2017, date last accessed).
Van den Brink J, de Vries RP. Fungal enzyme sets for plant polysaccharide degradation. Appl Microbiol
Biotechnol 2011;91:1477.
Van Dijken JP, Scheffers WA. Redox balances in the metabolism of sugars by yeasts. FEMS Microbiol
Lett 1986;32:199-224.
Van Hazendonk JM, Reinerik EJM, de Waard P et al. Structural analysis of acetylated hemicellulose
polysaccharides from fibre flax (Linum usitatissimum L.). Carbohydr Res 1996;291:141-54.
Van Maris AJA, Abbott DA, Bellissimi E et al. Alcoholic fermentation of carbon sources in biomass
hydrolysates by Saccharomyces cerevisiae: current status. Antonie Van Leeuwenhoek
2006a;90:391-418.
Van Maris AJA, Winkler AA, Kuyper M et al. Development of efficient xylose fermentation in
Saccharomyces cerevisiae: xylose isomerase as a key component. In: Biofuels: Springer, 2007.
Van Maris AJA, Abbott DA, Bellissimi E et al. Alcoholic fermentation of carbon sources in biomass
hydrolysates by Saccharomyces cerevisiae: current status. Antonie Van Leeuwenhoek
2006b;90:391-418.
Van Rossum HM, Kozak BU, Niemeijer MS et al. Requirements for carnitine shuttle-mediated
translocation of mitochondrial acetyl moieties to the yeast cytosol. mBio 2016;7.
Van Vleet JH, Jeffries TW, Olsson L. Deleting the para-nitrophenyl phosphatase (pNPPase), PHO13, in
recombinant Saccharomyces cerevisiae improves growth and ethanol production on D-
xylose. Metab Eng 2008;10:360-9.
Verduyn C, Postma E, Scheffers WA et al. Effect of benzoic acid on metabolic fluxes in yeasts: A
continuous-culture study on the regulation of respiration and alcoholic fermentation. Yeast
1992;8:501-17.
Verhoeven MD, Lee M, Kamoen L et al. Mutations in PMR1 stimulate xylose isomerase activity and
anaerobic growth on xylose of engineered Saccharomyces cerevisiae by influencing
manganese homeostasis. Sci Rep 2017; In press.
Villadsen J, Noorman H. Scale-Up and Scale-Down. In: Villadsen J (ed.) Fundamental Bioengineering
Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, 2016.
Voegele E. Shell files bid to purchase Abengoa’s cellulosic ethanol plant. Biomass Magazine 2016.
http://biomassmagazine.com/articles/13792/ (24 March 2017, date last accessed).
Vohra M, Manwar J, Manmode R et al. Bioethanol production: Feedstock and current technologies.
Journal of Environmental Chemical Engineering 2014;2:573-84.
Wahlbom CF, Hahn–Hägerdal B. Furfural, 5‐hydroxymethyl furfural, and acetoin act as external
electron acceptors during anaerobic fermentation of xylose in recombinant Saccharomyces
cerevisiae. Biotechnol Bioeng 2002;78:172-8.
Wang BL, Ghaderi A, Zhou H et al. Microfluidic high-throughput culturing of single cells for selection
based on extracellular metabolite production or consumption. Nat Biotechnol 2014;32:473-8.

Wang C, Bao X, Li Y et al. Cloning and characterization of heterologous transporters in Saccharomyces
cerevisiae and identification of important amino acids for xylose utilization. Metab Eng
2015a;30:79-88.
Wang G, Tang W, Xia J et al. Integration of microbial kinetics and fluid dynamics toward model-driven
scale-up of industrial bioprocesses. Eng Life Sci 2015b;15:20-9.
Wang M, Yu C, Zhao H. Directed evolution of xylose specific transporters to facilitate glucose‐xylose
co‐utilization. Biotechnol Bioeng 2016;113:484-91.
Watanabe S, Abu Saleh A, Pack SP et al. Ethanol production from xylose by recombinant
Saccharomyces cerevisiae expressing protein-engineered NADH-preferring xylose reductase
from Pichia stipitis. Microbiology 2007a;153:3044-54.
Watanabe S, Kodaki T, Makino K. Complete reversal of coenzyme specificity of xylitol dehydrogenase
and increase of thermostability by the introduction of structural zinc. J Biol Chem
2005;280:10340-9.
Watanabe S, Saleh AA, Pack SP et al. Ethanol production from xylose by recombinant Saccharomyces
cerevisiae expressing protein-engineered NADH-preferring xylose reductase from Pichia
stipitis. Microbiology 2007b;153:3044-54.
Wei N, Quarterman J, Kim SR et al. Enhanced biofuel production through coupled acetic acid and
xylose consumption by engineered yeast. Nat Commun 2013;4.
Weierstall T, Hollenberg CP, Boles E. Cloning and characterization of three genes (SUT1–3) encoding
glucose transporters of the yeast Pichia stipitis. Mol Microbiol 1999;31:871-83.
Wiedemann B, Boles E. Codon-optimized bacterial genes improve L-arabinose fermentation in
recombinant Saccharomyces cerevisiae. Appl Environ Microbiol 2008;74:2043-50.
Wilkening S, Lin G, Fritsch ES et al. An evaluation of high-throughput approaches to QTL mapping in
Saccharomyces cerevisiae. Genetics 2014;196:853-65.
Wimalasena TT, Greetham D, Marvin ME et al. Phenotypic characterisation of Saccharomyces spp.
yeast for tolerance to stresses encountered during fermentation of lignocellulosic residues to
produce bioethanol. Microb Cell Fact 2014;13:47.
Wisselink HW, Cipollina C, Oud B et al. Metabolome, transcriptome and metabolic flux analysis of
arabinose fermentation by engineered Saccharomyces cerevisiae. Metab Eng 2010;12:537-
51.
Wisselink HW, Toirkens MJ, del Rosario Franco Berriel M et al. Engineering of Saccharomyces
cerevisiae for efficient anaerobic alcoholic fermentation of L-arabinose. Appl Environ
Microbiol 2007;73:4881-91.
Wisselink HW, Toirkens MJ, Wu Q et al. Novel evolutionary engineering approach for accelerated
utilization of glucose, xylose, and arabinose mixtures by engineered Saccharomyces
cerevisiae strains. Appl Environ Microbiol 2009;75:907-14.
Wisselink HW, Van Maris AJA, Pronk JT et al. Polypeptides with permease activity. US Patent 9034608
B2. 2015.
Wright J, Bellissimi E, de Hulster E et al. Batch and continuous culture‐based selection strategies for
acetic acid tolerance in xylose‐fermenting Saccharomyces cerevisiae. FEMS Yeast Res
2011;11:299-306.
Xia PF, Zhang GC, Liu JJ et al. GroE chaperonins assisted functional expression of bacterial enzymes in
Saccharomyces cerevisiae. Biotechnol Bioeng 2016.
Xu H, Kim S, Sorek H et al. PHO13 deletion-induced transcriptional activation prevents sedoheptulose
accumulation during xylose metabolism in engineered Saccharomyces cerevisiae. Metab Eng
2016;34:88-96.
Yan X, Inderwildi OR, King DA. Biofuels and synthetic fuels in the US and China: A review of Well-to-
Wheel energy use and greenhouse gas emissions with the impact of land-use change. Energy
Environ Sci 2010;3:190-7.
Yang F, Hanna MA, Sun R. Value-added uses for crude glycerol-a byproduct of biodiesel production.
Biotechnol Biofuels 2012;5:13.

Young EM, Comer AD, Huang H et al. A molecular transporter engineering approach to improving
xylose catabolism in Saccharomyces cerevisiae. Metab Eng 2012;14:401-11.
Young EM, Tong A, Bui H et al. Rewiring yeast sugar transporter preference through modifying a
conserved protein motif. Proc Natl Acad Sci USA 2014;111:131-6.
Yu KO, Kim SW, Han SO. Engineering of glycerol utilization pathway for ethanol production by
Saccharomyces cerevisiae. Bioresour Technol 2010;101:4157-61.
Yuan WJ, Chang BL, Ren JG et al. Consolidated bioprocessing strategy for ethanol production from
Jerusalem artichoke tubers by Kluyveromyces marxianus under high gravity conditions. J Appl
Microbiol 2012;112:38-44.
Zhang GC, Kong II, Wei N et al. Optimization of an acetate reduction pathway for producing cellulosic
ethanol by engineered yeast. Biotechnol Bioeng 2016a;113:2587-96.
Zhang K, Zhang L-J, Fang Y-H et al. Genomic structural variation contributes to phenotypic change of
industrial bioethanol yeast Saccharomyces cerevisiae. FEMS Yeast Res 2016b;16:fov118.
Zhou H, Cheng J-s, Wang BL et al. Xylose isomerase overexpression along with engineering of the
pentose phosphate pathway and evolutionary engineering enable rapid xylose utilization and
ethanol production by Saccharomyces cerevisiae. Metab Eng 2012;14:611-22.
Figure 1. Schematic process-flow diagram for ethanol production from lignocellulose, based on
physically separated processes for pretreatment, hydrolysis and fermentation, combined with
on-site cultivation of filamentous fungi for production of cellulolytic enzymes and on-site
propagation of engineered pentose-fermenting yeast strains.

Figure 2. Key strategies for engineering carbon and redox metabolism in S. cerevisiae strains for
alcoholic fermentation of lignocellulosic feedstocks. Colours indicate the following pathways and
processes: Black: native S. cerevisiae enzymes of glycolysis and alcoholic fermentation;
Magenta: native enzymes of the non-oxidative pentose-phosphate pathway (PPP),
overexpressed in pentose-fermenting strains; Red: conversion of D-xylose into D-xylulose-5-
phosphate by heterologous expression of a xylose isomerase (XI) or combined expression of
heterologous xylose reductase (XR) and xylitol dehydrogenase (XDH), together with the
overexpression of (native) xylulokinase (Xks1); Green: conversion of L-arabinose into D-
xylulose-5-phosphate by heterologous expression of a bacteria AraA/AraB/AraD pathway; Blue:
expression of a heterologous acetylating acetaldehyde dehydrogenase (A-ALD) for reduction of
acetic acid to ethanol; Grey: native glycerol pathway.

Figure 3. Annual production volumes of cellulosic ethanol in the USA from 2010 until November
2016. Numbers are based on RIN D code 3 RIN (Renewable Identification Number) credits
generated (accounted as cellulosic ethanol, United States Environmental Protection Agency
2017).
A
B
Figure 4. Problems not encountered in shake flask cultures: non-yeast-related challenges in
large-scale processing of lignocellulosic biomass. A. Small rocks collected from corn stover
(picture courtesy of POET-DSM Liberty). B. Example of severely eroded equipment (picture
courtesy of Iogen Corporation (Lane 2016c)).

Table 1. Overview of operational commercial-scale (demonstration) plants for second-
generation bioethanol production. Data for US and Canada reflect status in May 2017 (source:
Ethanol Producer Magazine 2017), data for other countries (source: UNCTAD 2016) reflect
status in 2016.
Company/Plant
Country
(State)
Feedstock
Capacity
ML·y-1
DuPont Cellulosic Ethanol LLC - Nevada
USA (IA)
Corn stover
113.6
Poet-DSM Advanced Biofuels LLC - Project
Liberty1
USA (IA)
Corn cobs/corn stover
75.7
Quad County Cellulosic Ethanol Plant
USA (IA)
Corn fiber
7.6
Fiberight Demonstration Plant
US (VA)
Waste stream
1.9
ICM Inc. Pilot integrated Cellulosic Biorefinery
US (MO)
Biomass crops
1.2
American Process Inc. – Thomaston Biorefinery
USA (GA)
Other
1.1
ZeaChem Inc. – Demonstration plant
US (OR)
Biomass crops
1.0
Enerkem Alberta Biofuels LP
Canada (AB)
Sorted municipal solid waste
38
Enerkem Inc.-Westbury
Canada (QC)
Woody biomass
5.0
Iogen Corporation
Canada (ON)
Crop residue
2.0
Woodlands Biofuels Inc. – Demonstration plant
Canada (ON)
Woody biomass
2.0
GranBio
Brazil
Bagasse
82.4
Raizen
Brazil
Sugarcane bagasse/straw
40.3
Longlive Bio-technology Co. Ltd. – commercial
demo
China
Corn cobs
63.4
Mussi Chemtex / Beta Renewables
Italy
Arundo donax, rice straw, wheat
straw
75
Borregaard Industries AS – ChemCell Ethanol
Norway
Wood pulping residues
20
1 With expansion capacity to 94.6 ML per year

Jansen et al. - 40 -
40
Table 2. Ethanol yields (YE/S, g ethanol·(g sugar)-1) and biomass-specific rates of xylose and/or arabinose consumption and ethanol production
(qxylose, qarabinose and qethanol, respectively, g·(g biomass)-1·h-1) in cultures of S. cerevisiae strains engineered for pentose fermentation, grown in
synthetic media. Asterisks (*) indicate values estimated from graphs in the cited reference.
S. cerevisiae
strain
Pentose fermentation
strategy
Key genetic modifications
Fermentation conditions
YE/S
g·g-1
qethanol
g·g-1·h-1
qxylose
g·g·h-1
qarabinose
g·g·h-1
Reference
TMB3400
XR/XDH
(S. stipitis XYL1, XYL2)
SsXYL1, SsXYL2 + XKS1↑, random mutagenesis
Anaerobic batch (bioreactor),
5 % xylose
0.33
0.04
0.13
-
(Karhumaa et
al. 2007)
GLBRCY87
XR/XDH
(S. stipitis XYL1, XYL2)
SsXYL1, SsXYL2, SsXYL3, evolved on xylose and
hydrolysate inhibitors
Semi-anaerobic batch (flask)
5 % glucose and 5% xylose
0.34*
0.036*
0.13
-
(Sato et al.
2016)
SR8
XR/XDH
(S. stipitis XYL1, XYL2)
SsXYL1,Ss XYL2, Ss XYL3, ald6Δ, evolved on
xylose
Anaerobic batch (reactor),
4 % xylose
0.39
0.25
0.64
-
(Wei et al.
2013)
TMB3421
XR/XDH
(S. stipitis XYL1, XYL2)
S. stipitis XYL1N272D/P275Q, XYL2 + XKS1↑ TAL1↑
TKL1↑ RPE1↑ RKI1↑ gre3Δ, evolved on xylose
Anaerobic batch (reactor),
6 % xylose
0.35
0.20
0.57
-
(Runquist et al.
2010)
RWB 217
XI
(Piromyces XylA)
Piromyces XylA + XKS1↑ TAL1↑ TKL1↑ RPE1↑
RKI1↑ , gre3Δ
Anaerobic batch (reactor),
2 % xylose
0.43
0.46
1.06
-
(Kuyper et al.
2005a)
RWB 218
XI
(Piromyces XylA)
Derived from RWB 217 after evolution on
glucose/xylose mixtures
Anaerobic batch (reactor)
2 % xylose
0.41
0.49
1.2
-
(Kuyper et al.
2005b)
H131-A3-
ALCS
XI
(Piromyces XylA)
XylA, Xyl3, XKS1↑ TAL1↑ TKL1↑ RPE1↑ RKI1↑ ,
gre3Δ, evolved on xylose
Anaerobic batch (reactor),
4 % xylose
0.43
0.76
1.9
-
(Zhou et al.
2012)
IMS0010
XI/AraABD
(Piromyces XylA,
L. plantarum AraA, B,D
XylA; XKS1↑ TAL1↑ TKL1↑ RPE1↑ RKI1↑ AraT,
AraA, AraB, AraD, evolved on glucose, xylose,
arabinose mixtures
Anaerobic batch (reactor),
3 % glucose, 1.5 % xylose and
1.5 % arabinose
0.43
-
0.35
0.53
(Wisselink et
al. 2009)
GS1.11-26
XI/AraABD
(Piromyces XylA,
L. plantarum AraA, B,D,.
K. lactis ARAT).
XylA, XKS1↑ TAL1↑ TKL1↑ RPE1↑ RKI1↑ XylA
HXT7↑ KlAraT, AraA, AraB, AraD, TAL2↑ TKL2↑,
several rounds of mutagenesis and evolution on
xylose
Semi-anaerobic batch (flask),
synthetic medium, 3.5 %
xylose
0.46
0.48
1.1
-
(Demeke et al.
2013a)
Piromyces: XylA.
L. plantarum:
AraA, AraB, AraD.

Jansen et al. - 41 -
41
Table 3. Ethanol yields on consumed sugar (YE/S, g ethanol·(g sugar)-1) and biomass-specific rates of glucose and xylose consumption and ethanol
production (qglucose, qxylose and qethanol, respectively, g·(g biomass)-1·h-1) in cultures of S. cerevisiae strains engineered for pentose fermentation, grown
in lignocellulosic hydrolysates. Asterisks (*) indicate specific conversion rates estimated from graphs in the cited reference; daggers (†) indicate crude
estimates of biomass-specific rates calculated based on the assumption that biomass concentrations did not change after inoculation, these estimates
probably overestimate actual biomass-specific conversion rates.
1Abbreviations of supplements: YE, yeast extract; YP, yeast extract and peptone; YNB, Yeast Nitrogen Base.
S. cerevisiae
strain
Description
Feedstock, pretreatment
conditions, hydrolysate sugar
composition3
Fermentation
conditions, added
nutrients1
YE/S
g·g-1
qglucose
g·g·h-1
qethanol
g·g·h-1
qxylose
g·g·h-1
Reference
TMB3400
XR/XDH
S. stipitis XYL1 and XYL2; XKS1↑
Spruce, two-step dilute acid
hydrolysis, 1.6 % glucose, 0.4 %
xylose, 1 % mannose, 1 %
galactose,
Anaerobic batch
(flasks), (NH4)2HPO4
NaH2PO4 MgSO4, YE
0.41
0.021
0.005
0.005
(Karhumaa et
al. 2007)
GLBRCY87
XR/XDH
S. stipitis XYL1, XYL2 and XYL3
evolved on xylose and hydrolysate
inhibitors
Corn Stover, ammonia fiber
expansion, , 8 % glucose, 3.8 %
xylose.
Semi-anaerobic batch
(flasks), pH 5.5, Urea,
YNB
0.28
1.4*
0.27*
0.04
(Sato et al.
2016)
GLBRCY87
XR/XDH
S. stipitis XYL1, XYL2 and XYL3
evolved on xylose and hydrolysate
inhibitors
Switchgrass, ammonia fiber
expansion, 6.1 % glucose, 3.9 %
xylose.
Semi-anaerobic batch
(flasks), Urea, YNB
0.35
1.65*
0.28*
0.07
(Sato et al.
2016)
MEC1122
XR/XDH, industrial host strain
S. stipitis XYL1(N272D/P275Q) and
XYL2, XKS1↑ TAL1↑
Corn cobs, autohydrolysis (202
°C), liquid fraction acid-treated.
0.3 % glucose, 2.6 % xylose.
Oxygen limited batch
(flasks), cheese whey,
urea, YE, K2O5S2
0.3
-
0.12†,*
0.25†
(Costa et al.
2017)
RWB 218
XI
Piromyces XylA, XKS1↑ TAL1↑
TKL1↑ RPE1↑ RKI1↑, gre3Δ, evolved
on glucose/xylose mixed substrate
Wheat straw hydrolysate, steam
explosion, 5 % glucose, 2 % xylose
Anaerobic batch
(reactor), (NH4)2PO4
0.47
1.58†
1.0†
0.32†
(Van Maris et
al. 2007)
GS1.11-26
XI, AraABD
Piromyces XylA, XKS1↑ TAL1↑
TKL1↑ RPE1↑ RKI1↑ HXT7↑AraT,
AraA, AraB, AraD, TAL2↑ TKL2↑,
several rounds of mutagenesis and
evolution on xylose
Spruce (no hydrolysis), acid pre-
treated, 6.2 % glucose, 1.8 %
xylose, 1 % mannose
Semi-anaerobic batch
(flasks), YNB,
(NH4)2SO4, amino
acids added,
0.43
2.46†
0.3†
0.11†
(Demeke et al.
2013a)
XH7
Multiple integrations of
RuXylA; XKS1↑ TAL1↑ TKL1↑ RPE1↑
RKI1↑ pho13Δ gre3Δ, evolved on
xylose
Corn stover, steam explosion, 6.2
% glucose, 1.8 % xylose
Semi-anaerobic batch
(flasks), urea
0.39
0.14
0.080
0.096
(Li et al.
2016c)
LF1
Selection mutant of XH7 further
evolved on xylose and hydrolysates
with MGT transporter introduced
Corn stover, steam explosion,
8.7% glucose, 3.9% xylose
Semi-anaerobic batch
(flasks), urea
0.41
0.57
0.34
0.23
(Li et al.
2016c)
Box 1. Overview of key technologies used for development of Saccharomyces cerevisiae strains for second-generation bioethanol production and
examples of their application.
Metabolic engineering
Application of recombinant-DNA techniques for
the improvement of catalytic and regulatory
processes in living cells, to improve and extend
their applications in industry (Bailey 1991).
Metabolic engineering of pentose-fermenting strains commenced with the functional expression of pathways for XR/XDH- (Kötter and
Ciriacy 1993; Tantirungkij et al. 1993) or XI-based (Kuyper et al. 2005a) xylose utilization and pathways for isomerase-based arabinose
utilization (Becker and Boles 2003; Wisselink et al. 2007). Further research focused on improvement of pathway capacity (Kuyper et al.
2006; Wiedemann and Boles 2008), engineering of sugar transport (Fonseca et al. 2011; Subtil and Boles 2011; Nijland et al. 2014;
Nijland et al. 2016), redox engineering to decrease byproduct formation and increase ethanol yield (Guadalupe-Medina et al. 2010;
Henningsen et al. 2015; Papapetridis et al. 2016; Roca et al. 2003; Sonderegger and Sauer 2003; Watanabe et al. 2005; Wei et al. 2013; Yu
et al. 2010; Zhang et al. 2016) and expression of alternative pathway enzymes (Brat et al. 2009; Ota et al. 2013). Expression of
heterologous hydrolases provided the first steps towards consolidated bioprocessing (den Haan et al. 2015; Ha et al. 2011; Ilmén et al.
2011; Sadie et al. 2011).
Evolutionary engineering
Application of laboratory evolution to select for
industrially relevant traits (Sauer 2001). Also
known as adaptive laboratory evolution (ALE).
Evolutionary engineering in repeated-batch and chemostat cultures has been intensively utilized to improve growth and fermentation
kinetics on pentoses (e.g., (Demeke et al. 2013a; Garcia Sanchez et al. 2010; Kim et al. 2013; Kuyper et al. 2005b; Lee et al. 2014;
Sonderegger and Sauer 2003; Wisselink et al. 2009; Zhou et al. 2012) and inhibitor tolerance (Almario et al. 2013; González-Ramos et al.
2016; Koppram et al. 2012; Smith et al. 2014; Wright et al. 2011).
Whole genome (re)sequencing
Determination of the entire DNA sequence of an
organism.
Availability of a high-quality reference genome sequence is essential for experimental design in metabolic engineering. When genomes of
strains that have been obtained by non-targeted approaches (e.g. evolutionary engineering or mutagenesis) are (re)sequenced, the
relevance of identified mutations can subsequently be tested by their reintroduction in naïve strains, non-evolved strains and/or by
classical genetics (reverse engineering; (Oud et al. 2012)). This approach has been successfully applied to identify mutations contributing

Jansen et al. - 42 -
42
to fast pentose fermentation (dos Santos et al. 2016; Hou et al. 2016a; Nijland et al. 2014) and inhibitor tolerance (e.g., (González-Ramos
et al. 2016; Pinel et al. 2015).
Quantitative trait loci (QTL) analysis
QTL identifies alleles that contribute to (complex)
phenotypes based on their meiotic co-segregation
with a trait of interest (Liti and Louis 2012;
Wilkening et al. 2014). In contrast to whole-
genome (re)sequencing alone, QTL analysis can
identify epistatic interactions.
QTL analysis currently enables resolution to gene or even nucleotide level (Swinnen et al. 2012). QTL analysis has been used to identify
alleles contributing to high-temperature tolerance (Sinha et al. 2006), ethanol tolerance (Swinnen et al. 2012) and improved ethanol-to-
glycerol product ratios (Hubmann et al. 2013). The requirement of QTL analysis for mating limits its applicability in aneuploidy and/or
poorly sporulating industrial S. cerevisiae strains.
Protein engineering
Modification of the amino acid sequences of
proteins with the aim to improve their catalytic
properties, regulation and/or stability in
industrial contexts (Marcheschi et al. 2013).
Protein engineering has been used to improve the pentose-uptake kinetics, reduce the glucose sensitivity and improve the stability of
yeast hexose transporters (e.g., (Farwick et al. 2014; Li et al. 2016b; Wang et al. 2015a; Farwick et al. 2014; Nijland et al. 2016; Reznicek
et al. 2015; Shin et al. 2015; Young et al. 2014)). The approach has been utilized to improve the redox cofactor specificity of XR and/or
XDH to decrease xylitol formation (Krahulec et al. 2009; Petschacher et al. 2005; Petschacher and Nidetzky 2008; Watanabe et al. 2007;
Watanabe et al. 2005). Directed evolution of xylose isomerase yielded XI variants with increased enzymatic activity (Lee et al. 2012).
Directed evolution of native yeast dehydrogenases has yielded strains with increased HMF tolerance (Moon and Liu 2012).
Genome editing
Where ‘classical’ genetic engineering encompass
iterative, one-by-one introduction of genetic
modifications, genome editing techniques enable
simultaneous introduction of multiple (types of)
modifications at different genomic loci (Sander
and Joung 2014).
The combination of CRISPR-Cas9-based genome editing (DiCarlo et al. 2013; Mans et al. 2015) with in vivo assembly of DNA fragments
has enabled the one-step introduction of all genetic modifications needed to enable S. cerevisiae to ferment xylose (Shi et al. 2016; Tsai et
al. 2015; Verhoeven et al. 2017). Recent developments have enabled the application of the system in industrial backgrounds (Stovicek et
al. 2015). CRISPR-Cas9 has been used in reverse engineering studies to rapidly introduce multiple single-nucleotide mutations observed
in evolutionary engineering experiments in naïve strains (e.g., (van Rossum et al. 2016)).