Metabolic engineering of Clostridium acetobutylicum for butyric acid production with high butyric acid selectivity

Jang, Yu‐Sin; Im, Jung Ae; Choi, So Young; Lee, Jung Im; Lee, Sang Yup

doi:10.1016/j.ymben.2014.03.004

Cited by 91 publications

(56 citation statements)

References 41 publications

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“…It was reported that most butyrate-producing bacteria preferred glucose as their substrates [49], and the enzymes related to butyrate production were mainly transferases of acetyl-coenzyme and butyrate kinase. Over 50% of butyrate-producing bacteria, like Clostridium sensu stricto and Brochothrix , have both enzymes, which means that they could produce butyrate through the transformation of intracellular acetate [50]. However, some butyrate-producing bacteria could only generate butyrate by using extracellular acetate because of the absence of butyrate kinase.…”

Section: Resultsmentioning

confidence: 99%

Novel insight into the relationship between organic substrate composition and volatile fatty acids distribution in acidogenic co-fermentation

Zhang

et al. 2017

Biotechnol Biofuels

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BackgroundCo-fermentation is an attractive technology for improving volatile fatty acids (VFAs) production by treatment of solid organic wastes. However, it remains unclear how the composition of different organic matters in solid waste influences the VFAs distribution, microbial community structure, and metabolic pathway during acidogenic co-fermentation. In this study, different organic wastes were added into waste activated sludge (WAS) as co-fermentation substrates to explore the impact of organic matter composition on VFAs pattern and the microbiological mechanism .ResultsAcetate was the most dominant VFA produced in all fermentation groups, making up 41.3–57.6% of the total VFAs produced during acidogenic co-fermentation under alkaline condition. With the increased addition of potato peel waste, the concentrations of propionate and valerate decreased dramatically, while ethanol and butyrate concentrations increased. The addition of food waste caused gradual decreases of valerate and propionate, but ethanol increased and butyrate was relatively stable. Some inconsistency was observed between hydrolysis efficiency and acidification efficiency. Our results revealed that starch was mainly responsible for butyrate and ethanol formation, while lipids and protein favored the synthesis of valerate and propionate. Microbial community analysis by high-throughput sequencing showed that Firmicutes had the highest relative abundance at phylum level in all fermentation groups. With 75% potato peel waste or 75% food waste addition to WAS, Bacilli (72.2%) and Clostridia (56.2%) were the dominant respective classes. In fermentation using only potato peel waste, the Bacilli content was 64.1%, while the Clostridia content was 53.6% in the food-only waste fermentation.ConclusionsAcetate was always the dominant product in acidogenic co-fermentation, regardless of the substrate composition. The addition of carbon-rich substrates significantly enhanced butyrate and ethanol accumulation, while protein-rich substrate substantially benefited propionate and valerate generation. Potato peel waste substantially favored the enrichment of Bacilli, while food waste dramatically increased Clostridia content in the sludge.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-017-0821-1) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

Novel insight into the relationship between organic substrate composition and volatile fatty acids distribution in acidogenic co-fermentation

Zhang

et al. 2017

Biotechnol Biofuels

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“…More recently, several metabolic engineering and systems metabolic engineering strategies of Clostridium have been developed for the production of diverse chemicals including butyric acid, ethanol, isopropanol, butanol, 1,3‐propanediol, 2,3‐butanediol, isobutanol, and hydrogen (Fig. ) . For the details in metabolic engineering tools and strategies, historical strain development, carbohydrate utilization, consolidated bioprocesses, and recovery processes on butanol fermentation, several review articles are available …”

Section: Introductionmentioning

confidence: 99%

Metabolic engineering of clostridia for the production of chemicals

Cho

Jang

Moon

et al. 2014

Biofuels Bioprod Bioref

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There have recently been signifi cant advances in bio-based production of chemicals from renewable resources. Microorganisms belonging to the genus Clostridium have been considered as one of the promising hosts for the production of desired chemicals of wide industrial use. Clostridium strains have capability to utilize diverse carbon sources, including C5 and C6 substrates, which thus allows production of chemicals from inexpensive and abundant biomass such as corn stover, straw, and woody waste. In addition, Clostridium strains naturally produce various chemicals, such as acetic acid, butyric acid, ethanol, isopropanol, butanol, 1,3-propanediol, 2,3-butanediol, and acetone. Recently, several important strategies for the metabolic engineering of Clostridium have been developed not only for the enhanced production of these natural products and but also for the production of non-natural isobutanol production. Here, we review the strategies employed for the development of metabolically engineered Clostridium strains for the production of such chemicals and provide future perspectives.

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“…C. acetobutylicum is a promising platform strain for the production of important chemicals and biofuels (Cho et al, ; Tracy, ; Tracy et al, ). Recent studies have focused on metabolic engineering of C. acetobutylicum using mobile group II introns to knock out genes of interest in strain development (Jang et al, , ; Lehmann and Lutke‐Eversloh, ). While triple, quadruple, and quintuple gene knockout C. acetobutylicum strains have been developed using the Targetron method, the curing process required for constructing quadruple and quintuple mutants is still quite complicated and labor intensive.…”

Section: Discussionmentioning

confidence: 99%

“…Thus, extensive studies have been conducted to develop genetic and metabolic engineering tools for this strain. Several notable tools that have been developed in C. acetobutylicum include shuttle vectors to overexpress genes (Heap et al, 2009;Lee et al, 1992;Mermelstein et al, 1992), antisense RNA (asRNA) to downregulate gene expression (Desai and Papoutsakis, 1999;Tummala et al, 2003), expression reporters (Girbal et al, 2003;Tummala et al, 1999), inducible promoters (Girbal et al, 2003;Hartman et al, 2011), repressor systems (Girbal et al, 2003), and gene knockout methods based on homologous recombination Liu et al, 2006;Liyanage et al, 2001;Sillers et al, 2008;Tracy et al, 2011) and mobile group II introns (Heap et al, 2007;Jang et al, 2012aJang et al, , 2014Shao et al, 2007;Wang et al, 2013). For the fine control of metabolic flux, it is essential to develop a tool for efficiently knocking down gene expression in C. acetobutylicum.…”

Section: Introductionmentioning

confidence: 99%

Efficient gene knockdown in Clostridium acetobutylicum by synthetic small regulatory RNAs

Cho

Lee

2016

Biotech & Bioengineering

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Clostridium is considered a promising microbial host for the production of valuable industrial chemicals. However, Clostridium is notorious for the difficulty of genetic manipulations, and consequently metabolic engineering. Thus, much effort has been exerted to develop novel tools for genetic and metabolic engineering of Clostridium strains. Here, we report the development of a synthetic small regulatory RNA (sRNA)-based system for controlled gene expression in Clostridium acetobutylicum, consisting of a target recognition site, MicC sRNA scaffold, and an RNA chaperone Hfq. To examine the functional operation of sRNA system in C. acetobutylicum, expression control was first examined with the Evoglow fluorescent protein as a model protein. Initially, a C. acetobutylicum protein annotated as Hfq was combined with the synthetic sRNA based on the Escherichia coli MicC scaffold to knockdown Evoglow expression. However, C. acetobutylicum Hfq did not bind to E. coli MicC, while MicC scaffold-based synthetic sRNA itself was able to knockdown the expression of Evoglow. When E. coli hfq gene was introduced, the knockdown efficiency assessed by measuring fluorescence intensity, could be much enhanced. Then, this E. coli MicC scaffold-Hfq system was used to knock down adhE1 gene expression in C. acetobutylicum. Knocking down the adhE1 gene expression using the synthetic sRNA led to a 40% decrease in butanol production (2.5 g/L), compared to that (4.5 g/L) produced by the wild-type strain harboring an empty vector. The sRNA system was further extended to knock down the pta gene expression in the buk mutant C. acetobutylicum strain PJC4BK for enhanced butanol production. The PJC4BK (pPta-Hfq ) strain, which has the pta gene expression knocked down, was able to produce 16.9 g/L of butanol, which is higher than that (14.9 g/L) produced by the PJC4BK strain, mainly due to reduced acetic acid production. Fed-batch culture of PJC4BK (pPta-Hfq ) strain coupled with in situ gas stripping produced 105.5 g of total solvents (70.7 g butanol, 20.5 g acetone, and 14.3 g ethanol), demonstrating that the sRNA-based engineered C. acetobutylicum strain can be cultured without instability. The synthetic sRNA system reported in this study will be useful for more efficient development of engineered C. acetobutylicum strains capable of producing valuable chemicals and fuels. Biotechnol. Bioeng. 2017;114: 374-383. © 2016 Wiley Periodicals, Inc.

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Metabolic engineering of Clostridium acetobutylicum for butyric acid production with high butyric acid selectivity

Cited by 91 publications

References 41 publications

Novel insight into the relationship between organic substrate composition and volatile fatty acids distribution in acidogenic co-fermentation

Novel insight into the relationship between organic substrate composition and volatile fatty acids distribution in acidogenic co-fermentation

Metabolic engineering of clostridia for the production of chemicals

Efficient gene knockdown in Clostridium acetobutylicum by synthetic small regulatory RNAs

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