Skip to Main Content
 

Global Search Box

 
 
 
 

Files

File List


Teng Bao Dissertation.pdf (7.34 MB)

ETD Abstract Container

Abstract Header

Metabolic engineering of Clostridium cellulovorans for selective n-butanol production from cellulose

Bao, Teng
http://rave.ohiolink.edu/etdc/view?acc_num=osu1574686346943506

Abstract Details

2019, Doctor of Philosophy, Ohio State University, Chemical Engineering.
n-Butanol as a promising alternative biofuel has gained high interest. Especially, compared with methanol and ethanol, n-butanol presents superior fuel properties and can be used as an ideal substitute of gasoline because of its high energy density, low water solubility, and low vapor pressure. Unfortunately, bio-butanol production through the conventional acetone-butanol-ethanol (ABE) fermentation process is not economically feasible because of the co-production of other metabolites and the use of expensive food-based feedstock. Lignocellulosic biomass, an abundant, cheap, and renewable source, is a desired feedstock for biofuels production. Conventional biorefinery of lignocellulosic biomass requires different operations, which is not cost-effective due to the complex process and high equipment capital requirement. Currently, several process strategies have been developed for cellulosic butanol production. Among them, consolidated bioprocessing (CBP) combines enzyme production, biomass hydrolysis, and sugar fermentation into one step, which greatly simplifies the process and dramatically reduces the equipment investment. Clostridium cellulovorans is a strictly anaerobic, cellulolytic bacterium among the most interesting candidates for CBP of lignocellulosic biomass. Recently, a bifunctional aldehyde/alcohol dehydrogenase gene adhE2 from Clostridium acetobutylicum has been overexpressed in C. cellulovorans for n-butanol production from cellulose. However, this recombinant strain produced n-butanol from microcrystalline cellulose at a low titer and yield insufficient for industrial application. In addition, there are two type II restriction modification (RM) systems in C. cellulovorans and currently available plasmids would be digested by C. cellulovorans cell extract even after in vivo methylation, making it difficult to transfer and express genes in C. cellulovorans for further metabolic engineering. Therefore, efficient transformation for developing better recombinant C. cellulovorans for butanol production is urgently required. In this study, the RM system of C. cellulovorans was analyzed and a new Clostridium shuttle plasmid pYL001 without Cce743I and Cce743II restriction sites was constructed to overcome the transformation barrier in C. cellulovorans. The new plasmid with improved post-electroporation procedure significantly improved the transformation efficiency and allowed the use of unmethylated DNA for metabolic engineering of C. cellulovorans. Then a synthetic butanol formation pathway was introduced in C. cellulovorans by overexpressing three different aldehyde/alcohol dehydrogenases gene combinations (acidogenesis: adhE2-bdhB, solventogenesis: adhE1-bdhB, and alcohologenesis: adhE2) from C. acetobutylicum. Due to different substrate and cofactor specificities, overexpressing different ADH and BDH in C. cellulovorans resulted in different amounts of alcohols production. Among them, C. cellulovorans adhE2 with the highest aldehyde/alcohol dehydrogenase activities produced the highest n-butanol titer of ~4 g/L with a high yield of ~0.22 g/g. Finally, modular metabolic engineering strategies, including strengthening the butyryl-CoA biosynthesis and improving intracellular NADH availability, were applied to redistribute the carbon flux towards butanol production in C. cellulovorans. Heterologous thiolase (thlA) and 3-hydroxybutyryl-CoA dehydrogenase (hbd) genes from C. acetobutylicum and Clostridium tyrobutyricum, respectively, were overexpressed in C. cellulovorans adhE2 to increase the flux from C2 to C4 metabolites. In addition, ferredoxin-NAD(P)+ oxidoreductase (fnr) from C. acetobutylicum, which can regenerate the intracellular NAD(P)H and thus increase alcohols formation, was also overexpressed. In batch fermentation with adding methyl viologen (MV) as an electron carrier, the recombinant C. cellulovorans adhE2-fnrCA-thlACA-hbdCT was able to direct the carbon flux towards n-butanol biosynthesis, leading to a high n-butanol titer of 5.56 g/L with a high yield of 0.34 g/g cellulose, which were the highest ever obtained for butanol production from cellulose in a mono-culture fermentation. These results indicated that C. cellulovorans is a promising CBP platform host for bio-butanol production from lignocellulosic biomass.
Shang-Tian Yang (Advisor)
Charles Bell (Committee Member)
Jeffrey Chalmers (Committee Member)
Eduardo Reátegui (Committee Member)
224 p.
Chemical Engineering
Biofuel, Clostridium Cellulovorans, Metabolic engineering

Recommended Citations

Citations

  • Bao, T. (2019). Metabolic engineering of Clostridium cellulovorans for selective n-butanol production from cellulose [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1574686346943506

    APA Style (7th edition)

  • Bao, Teng. Metabolic engineering of Clostridium cellulovorans for selective n-butanol production from cellulose. 2019. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1574686346943506.

    MLA Style (8th edition)

  • Bao, Teng. "Metabolic engineering of Clostridium cellulovorans for selective n-butanol production from cellulose." Doctoral dissertation, Ohio State University, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=osu1574686346943506

    Chicago Manual of Style (17th edition)

osu1574686346943506
392
© 2019, some rights reserved.Creative Commons License Metabolic engineering of Clostridium cellulovorans for selective n-butanol production from cellulose by Teng Bao is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. Based on a work at etd.ohiolink.edu.
This open access ETD is published by The Ohio State University and OhioLINK.