Editorial: Development and Application of Clostridia as Microbial Cell-Factories for Biofuels and Biochemicals Production

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Editorial: Development and
Application of Clostridia as Microbial
Cell-Factories for Biofuels and
Biochemicals Production
Hongxin Fu
1
* and Shang-Tian Yang
2
*
1
Guangdong Key Laboratory of Fermentation and Enzyme Engineering, School of Biology and Biological Engineering, South
China University of Technology, Guangzhou, China,
2
William G. Lowrie Department of Chemical and Biomolecular Engineering,
The Ohio State University, Columbus, OH, United States
Keywords: clostridia, butanol, butyric acid, isobutanol, 1,3-butanediol (1,3-BDO), biofilm, short- and medium-chain
ester, metabolic engineering
Editorial on the Research Topic
Development and Application of Clostridia as Microbial Cell-factories for Biofuels and
Biochemicals Production
Clostridia are Gram-positive, spore-forming, obligate anaerobic bacteria with versatile substrate
utilization and metabolite production capabilities (Xue et al., 2017). The exploitation of clostridia for
large-scale production of commodity chemicals can be dated back to 100 years ago with
solventogenic clostridia in acetone-butanol-ethanol (ABE) fermentation, which had fallen out of
favor since the establishment of more economical petrochemical processes (Cheng et al., 2019a).
However, the necessity of sustainable development has renewed interest in the production of biofuels
and biochemicals from abundant renewable biomass. In recent years, several Clostridium species,
including conventional solventogenic clostridia such as C. acetobutylicum and C. beijerinckii,
cellulolytic clostridia like C. thermocellum,C. cellulolyticum and C. cellulovorans (Yang et al.,
2015), acetogens such as C. formicoaceticum (Bao et al., 2019) and C. carboxidivorans (Cheng et al.,
2019b), and acidogens including C. tyrobutyricum (Li et al., 2019;Bao et al., 2020;Fu et al., 2021)and
C. kluyveri, have garnered immense interests in the field of industrial biotechnology for their abilities
to produce various chemicals and biofuels from low-cost agricultural residues and industrial wastes.
The high toxicity of fermentative products (such as butanol and butyrate) is a drawback limiting
Clostridia for industrial applications. Random chemical mutagenesis aiming at increasing butanol
efflux capacity was used to improve butanol tolerance of C. beijerinckii (Vasylkivska et al.). Mutant
strains obtained by different approaches behaved differently in terms of efflux pump substrates
(butanol, ethanol, ethidium bromide and antibiotics) tolerance and metabolites production. The best
mutant obtained with ethidium bromide for mutagenesis and selection showed a 127% improvement
in butanol tolerance. The genomes of various mutant strains were sequenced and analyzed, and the
results indicated that the improved butanol tolerance was attributed to mutations in genes related to
stress responses (chemotaxis, autolysis or changes in cell membrane structure) and efflux pump
regulators. This study confirmed the importance of efflux in butanol stress and provided new gene
targets for rational strain engineering. On another hand, Gao et al. screened 5 C. acetobutylicum
mutants through carbon ion beam irradiation, which had advantages of excellent biological
effectiveness and dose conformity over traditional mutation methods. The mutant Y217 showed
enhanced butanol tolerance and production of 13.67 g/L (vs. 9.77 g/L for the control), which was
attributed to its ability to maintain cell membrane integrity and permeability under butanol stress. As
strain mutagenesis and screening is a time-consuming process, Cao et al. developed and verified non-
Edited and reviewed by:
Jean Marie François,
Institut Biotechnologique de Toulouse
(INSA), France
*Correspondence:
Hongxin Fu
hongxinfu@scut.edu.cn
Shang-Tian Yang
yang.15@osu.edu
Specialty section:
This article was submitted to
Synthetic Biology,
a section of the journal
Frontiers in Bioengineering and
Biotechnology
Received: 08 December 2021
Accepted: 13 December 2021
Published: 11 January 2022
Citation:
Fu H and Yang S-T (2022) Editorial:
Development and Application of
Clostridia as Microbial Cell-Factories
for Biofuels and
Biochemicals Production.
Front. Bioeng. Biotechnol. 9:831135.
doi: 10.3389/fbioe.2021.831135
Frontiers in Bioengineering and Biotechnology | www.frontiersin.org January 2022 | Volume 9 | Article 8311351
EDITORIAL
published: 11 January 2022
doi: 10.3389/fbioe.2021.831135

structured mathematical models which could reflect strain
growth, glucose consumption, and butyric acid production of
12
C
6+
irradiation-mutation strain of C. tyrobutyricum.
Although clostridia possess the ability to ferment a broad
range of substrates, carbon catabolite repression (CCR), which
usually happens when mixed sugars (glucose and non-glucose
substrates) are present in the fermentation medium, is limiting
their uses of lignocellulosic biomass hydrolysates as substrates
(Fu et al., 2017). Although catabolite control protein A (CcpA)
knockout resulted in simultaneous utilization of glucose and
xylose in C. acetobutylicum, some negative influences were
observed due to the multiple roles of CcpA (Wu et al., 2015).
To solve this problem, Ujor et al. explored a ribozyme-mediated
approach to downregulate CcpA in C. beijerinckii. The expression
of CcpA-/DisA (encoding DNA integrity scanning protein A)-
specific M1 RNA-based ribozyme led to obvious decreases in
CcpA/DisA mRNA levels and modest increase in mixed sugars
utilization and ABE production compared to the control. This
study demonstrated that DisA played an important role in
regulating solvent production and the feasibility of using
ribozyme-mediated approach for gene knockdown in C.
beijerinckii.
Isobutanol is an important platform chemical widely used in
food, chemical, biofuel and pharmaceutical industries. The
market of bio-based isobutanol reached $1 billion in 2019 and
is expected to rise significantly in the near future. Weitz et al.
introduced two different isobutanol synthesis pathways,
ketoisovalerate ferredoxin oxidoreductase (Kor) and
ketoisovalerate decarboxylase (Kivd), into two acetogenic
bacteria, Clostridium ljungdahlii and Acetobacterium woodii,
and evaluated their effects on isobutanol production under
both heterotrophic and autotrophic conditions. When syngas
was used as the substrate, the engineered C. ljungdahlii produced
0.4 and 1 mM isobutanol without and with ketoisovalerate
addition via the Kivd pathway, which was better than the
results (0 and 0.2 mM isobutanol) obtained via the Kor
pathway. To identify the bottlenecks of autotrophic isobutanol
production, syngas-based batch cultivation together with
metabolic profiling and flux balance analysis were performed
for various C. ljungdahlii strains (Hermann et al.), and the results
indicated that further work could be focused on improving the
activities of key enzymes and changing their coenzyme specificity
or supply. The chirally pure (R)-1,3-butanediol (BDO) is used
for fragrances, insecticides and as precursor molecules for penem
and carbapenem antibiotic synthesis. Grosse-Honebrink et al.
expressed acetoacetyl-CoA reductase gene phaB from
Cupriavidus necator in C. saccharoperbutylacetonicum and
optimized the heterologous pathway at transcriptional
(promoters and gene expression methods optimization),
translational (codon optimization), enzyme (point mutations),
and population (medium optimization) levels for (R)-1,3-BDO
production from glucose. The optimized mutant produced 1.8 g/
L (R)-1,3-BDO, a 217% increase compared to the control. A
higher concentration could be achieved by further optimizing the
fermentation process.
C. acetobutylicum has long been and extensively used in
industrial production of acetone, butanol, and ethanol in ABE
fermentation, but little attention has been paid to its biofilm.
Zhang et al. summarized the cell physiological changes,
extracellular matrix components, production advantages,
influencing factors and regulatory genes of C. acetobutylicum
biofilm, which provided valuable insights into its molecular basis
useful for developing efficient biofilm processes. Clostridia can
produce a variety of organic acids and alcohols, and thus are
promising whole cell biocatalysts for short- and medium-
chain esters. Wang et al. reviewed the advances in ester
production by Clostridia including in vitro lipase catalysis and
in vivo acyltransferase reaction. In addition, the potential of
several Clostridia and clostridial consortia which can utilize
cheap substrates such as industrial waste gas and lignocellulose
for bio-ester production and the recent development of
synthetic biology in clostridial chassis development were also
discussed. Furthermore, cellulolytic Clostridia in a constructed
cellulolytic microflora played an important role in anaerobic co-
digestion of pig manure and rice straw for methane production
(Zhong et al.).
In summary, the original research and review papers
published in this Research Topic provide valuable information
on the selection and construction of robust and novel Clostridium
strains for biofuels and biochemicals production from renewable
resources, which will contribute to the further development of
Clostridia as microbial cell-factories for industrial applications.
AUTHOR CONTRIBUTIONS
HF initiated the Research Topic and wrote the draft. S-TY revised
and approved the final version for publication.
ACKNOWLEDGMENTS
We thank all authors, reviewers, topic editors, and editorial staff
at Frontiers who contributed to this Research Topic.
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Frontiers in Bioengineering and Biotechnology | www.frontiersin.org January 2022 | Volume 9 | Article 8311352
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Conflict of Interest: The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be construed as a
potential conflict of interest.
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