Enhanced sucrose fermentation by introduction of heterologous sucrose transporter and invertase into
            Clostridium beijerinckii
            for acetone–butanol–ethanol production

A heterologous pathway for sucrose transport and metabolism was introduced into Clostridium beijerinckii to improve sucrose use for n -butanol production. The combined expression of StSUT1 , encoding a sucrose transporter from potato ( Solanum tuberosum ), and SUC2 , encoding a sucrose invertase from Saccharomyces cerevisiae , remarkably enhanced n -butanol production. With sucrose, sugarcane molasses and sugarcane juice as substrates, the C. beijerinckii strain harbouring StSUT1 and SUC2 increased acetone–butanol–ethanol production by 38.7%, 22.3% and 52.8%, respectively, compared with the wild-type strain. This is the first report to demonstrate enhanced sucrose fermentation due to the heterologous expression of a sucrose transporter and invertase in Clostridium . The metabolic engineering strategy used in this study can be widely applied in other microorganisms to enhance the production of high-value compounds from sucrose-based biomass.

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Milling byproducts are an economically viable substrate for butanol production using clostridial ABE fermentation

Applied Microbiology and Biotechnology ◽

10.1007/s00253-020-10882-8 ◽

2020 ◽

Vol 104 (20) ◽

pp. 8679-8689

Author(s):

Nils Thieme ◽

Johanna C. Panitz ◽

Claudia Held ◽

Birgit Lewandowski ◽

Wolfgang H. Schwarz ◽

...

Keyword(s):

Clostridium Beijerinckii ◽

Abe Fermentation ◽

Liquid Fuels ◽

Butanol Production ◽

Enzymatic Pretreatment ◽

Profitability Analysis ◽

Key Points ◽

Wide Range ◽

Wheat Middlings ◽

Acetone Butanol Ethanol

Abstract Butanol is a platform chemical that is utilized in a wide range of industrial products and is considered a suitable replacement or additive to liquid fuels. So far, it is mainly produced through petrochemical routes. Alternative production routes, for example through biorefinery, are under investigation but are currently not at a market competitive level. Possible alternatives, such as acetone-butanol-ethanol (ABE) fermentation by solventogenic clostridia are not market-ready to this day either, because of their low butanol titer and the high costs of feedstocks. Here, we analyzed wheat middlings and wheat red dog, two wheat milling byproducts available in large quantities, as substrates for clostridial ABE fermentation. We could identify ten strains that exhibited good butanol yields on wheat red dog. Two of the best ABE producing strains, Clostridium beijerinckii NCIMB 8052 and Clostridium diolis DSM 15410, were used to optimize a laboratory-scale fermentation process. In addition, enzymatic pretreatment of both milling byproducts significantly enhanced ABE production rates of the strains C. beijerinckii NCIMB 8052 and C. diolis DSM 15410. Finally, a profitability analysis was performed for small- to mid-scale ABE fermentation plants that utilize enzymatically pretreated wheat red dog as substrate. The estimations show that such a plant could be commercially successful. Key points • Wheat milling byproducts are suitable substrates for clostridial ABE fermentation. • Enzymatic pretreatment of wheat red dog and middlings increases ABE yield. • ABE fermentation plants using wheat red dog as substrate are economically viable.

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Butanol production from the effluent of hydrogen fermentation

Water Science & Technology ◽

10.2166/wst.2011.365 ◽

2011 ◽

Vol 63 (6) ◽

pp. 1236-1240 ◽

Cited By ~ 7

Author(s):

W. H. Chen ◽

S. Y. Chen ◽

S. J. Chao ◽

Z. C. Jian

Keyword(s):

Consumption Rate ◽

Clostridium Beijerinckii ◽

Abe Fermentation ◽

Acetate Buffer ◽

Microbial Populations ◽

Butanol Production ◽

Practical Application ◽

Carbohydrate Consumption ◽

Acetone Butanol Ethanol ◽

Acetate Butyrate

The purpose of the study was to recover butanol from the effluent of the hydrogen-producing bioreactor containing acetate, butyrate, and carbohydrate. The butanol production by Clostridium beijerinckii NRRL B592 was evaluated under both unsterilized and sterilized conditions for examining the potential of butanol production for the practical application. Sucrose of 10 g/L and butyrate of 2 g/L coupled with acetate buffer were used to mimic the effluent. Sucrose was completely consumed in the both unsterilized and sterilized conditions during acetone-butanol-ethanol (ABE) fermentation. However, the results illustrate that the carbohydrate consumption rate in the unsterilized condition was higher than that in the sterilized condition. The maximum butanol concentrations of 3,500 and 3,750 mg/L were achieved in the sterilized and unsterilized conditions, respectively. Meanwhile, it was found that the acetate and the butyrate concentrations of 600 and 1,500 mg/L, and 300 and 1,000 mg/L were ingested to yield butanol in the sterilized condition and in the unsterilized condition, respectively. The results concluded that high levels of acetate and butyrate could eliminate the interference of other microbial populations, resulting in the enrichment of C. beijerinckii NRRL B592 in the fermentor. The butanol production by C. beijerinckii NRRL B592 could be, therefore, produced from the effluent of the hydrogen-producing bioreactor. It promised that the microbial butanol production is one of attractive bioprocesses to recover energy from wastes.

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A Convenient Fluorescence-Based Assay for the Detection of Sucrose Transport and the Introduction of a Sucrose Transporter from Potato into Clostridium Strains

Molecules ◽

10.3390/molecules24193495 ◽

2019 ◽

Vol 24 (19) ◽

pp. 3495

Author(s):

Zhikai Zhang ◽

Lihua Lin ◽

Hongchi Tang ◽

Shaowei Zeng ◽

Yuan Guo ◽

...

Keyword(s):

Heterologous Expression ◽

Sucrose Transporter ◽

Sucrose Transport ◽

Radioactive Materials ◽

Traditional Methods ◽

Isotope Labelling ◽

Transport Assay ◽

Sucrose Fermentation ◽

Technological Platform ◽

High Level

A convenient and effective sucrose transport assay for Clostridium strains is needed. Traditional methods, such as 14C-sucrose isotope labelling, use radioactive materials and are not convenient for many laboratories. Here, a sucrose transporter from potato was introduced into Clostridium, and a fluorescence assay based on esculin was used for the analysis of sucrose transport in Clostridium strains. This showed that the heterologously expressed potato sucrose transporter is functional in Clostridium. Recombinant engineering of high-level sucrose transport would aid sucrose fermentation in Clostridium strains. The assay described herein provides an important technological platform for studying sucrose transporter function following heterologous expression in Clostridium.

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Enhanced Acetone-Butanol-Ethanol Production by Clostridium beijerinckii Using Biochar

10.13031/aim.201900256 ◽

2019 ◽

Cited By ~ 1

Author(s):

Xiao Sun ◽

Hasan K. Atiyeh ◽

Yinka A. Adesanya ◽

Hailin Zhang ◽

Christopher Okonkwo ◽

...

Keyword(s):

Ethanol Production ◽

Clostridium Beijerinckii ◽

Acetone Butanol Ethanol

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Efficient acetone–butanol–ethanol production by Clostridium beijerinckii from sugar beet pulp

Bioresource Technology ◽

10.1016/j.biortech.2015.04.082 ◽

2015 ◽

Vol 190 ◽

pp. 332-338 ◽

Cited By ~ 32

Author(s):

Carolina Bellido ◽

Celia Infante ◽

Mónica Coca ◽

Gerardo González-Benito ◽

Susana Lucas ◽

...

Keyword(s):

Sugar Beet ◽

Ethanol Production ◽

Clostridium Beijerinckii ◽

Sugar Beet Pulp ◽

Beet Pulp ◽

Acetone Butanol Ethanol

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Enhanced acetone/butanol/ethanol production by Clostridium beijerinckii IB4 using pH control strategy

Process Biochemistry ◽

10.1016/j.procbio.2014.04.017 ◽

2014 ◽

Vol 49 (8) ◽

pp. 1238-1244 ◽

Cited By ~ 31

Author(s):

Min Jiang ◽

Jia-nan Chen ◽

Ai-yong He ◽

Hao Wu ◽

Xiang-ping Kong ◽

...

Keyword(s):

Ethanol Production ◽

Control Strategy ◽

Ph Control ◽

Clostridium Beijerinckii ◽

Acetone Butanol Ethanol

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The significance of aspartate on NAD(H) biosynthesis and ABE fermentation in Clostridium acetobutylicum ATCC 824

AMB Express ◽

10.1186/s13568-019-0874-6 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 3

Author(s):

Zhengping Liao ◽

Xitong Yang ◽

Hongxin Fu ◽

Jufang Wang

Keyword(s):

Clostridium Acetobutylicum ◽

Wild Type Strain ◽

De Novo ◽

Reference Value ◽

Butanol Production ◽

Wild Type ◽

De Novo Synthesis ◽

Fermentation Performance ◽

Engineering Strategy ◽

Clostridium Acetobutylicum Atcc 824

Abstract The co-factor NADH plays an important role in butanol biosynthesis. In this study, we found that aspartate could effectively improve the butanol production of Clostridium acetobutylicum ATCC 824. Further study showed that aspartate could be used as the precursor of NADH de novo synthesis in C. acetobutylicum ATCC 824. When 2 g/L aspartate was added, the transcription levels of essential genes (nadA, nadB and nadC) for NADH de novo synthesis were significantly higher than that of without aspartate addition. The levels of intracellular NAD+, NADH, total NAD(H) and the ratio of NADH/NAD+ were also significantly increased, which were 63.9 ± 8.0%, 85.0 ± %, 77.7 ± 8.0% and 12.7 ± 2.9% higher than those of without aspartate addition, respectively. Furthermore, the butanol production was improved by overexpressing the NADH de novo synthesis genes, and the fermentation performance could be further enhanced by strengthening the VB1 biosynthesis and NADH de novo synthesis pathway simultaneously. As a result, the butanol titer of the engineered strain 824(thiCGE–nadC) reached 13.96 ± 0.11 g/L, 7.2 ± 0.4%, 18.1 ± 0.1%, 34.1 ± 0.1% higher than that of 824(thiCGE), 824(nadC) and the wild type strain, respectively. This study has a reference value for the NADH related researches of other microbes, and the engineering strategy used in this study provides a new idea for construction of efficient fuel-producing strains.

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