Metabolic engineering of cellulolytic Clostridium cellulovorans for biofuel production directly from cellulosic biomass

Bibliographic Details
Main Author:	Yang, Xiaorui
Language:	English
Published:	The Ohio State University / OhioLINK 2014
Subjects:	Biology
Online Access:	http://rave.ohiolink.edu/etdc/view?acc_num=osu1404229128

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spelling	ndltd-OhioLink-oai-etd.ohiolink.edu-osu14042291282021-08-03T06:25:31Z Metabolic engineering of cellulolytic Clostridium cellulovorans for biofuel production directly from cellulosic biomass Yang, Xiaorui Biology With worldwide energy crisis, biofuel has been proposed as a renewable, sustainable and environmentally-friendly fuel to replace petroleum fuel or be used as fuel additive. Currently, ethanol fuel from corn takes the major market share of biofuel. With many advantageous properties, n-butanol is considered to be a more advanced fuel than ethanol. To reduce the feedstock cost as well as avoid competing with human food, the second-generation biofuel is being developed to produce ethanol and butanol fuels from sustainable and low-cost lignocellulosic biomass, pretreated with chemical and biological hydrolysis processes prior to fermentation. Recently, consolidated bioprocessing (CBP) has been proposed to produce the third-generation biofuel directly from lignocellulosic biomass, combining cellulase production, cellulose hydrolysis, and fermentation into a single-step process. CBP can greatly simplify the process and reduce the cost. Clostridium cellulovorans, a cellulolytic acid-producing anaerobe, was chosen to be the host of metabolic engineering for biofuel production directly from cellulosic biomass. To introduce butanol synthesis pathway into C. cellulovorans, its transformation method was developed at first. Understanding its restriction-modification (RM) systems, as well as establishment of methylation system were shown to be the key factors for successful transformation of C. cellulovorans. Particularly, two RM systems, Cce743I and Cce743II were determined with the details of their restriction specificities and methylation specificities. An in vivo methylation system, expressing M. Cce743I and M. Cce743II in E. coli, was then established to protect plasmids from being degraded. With the proper methylated plasmids, an engineered strain of C. cellulovorans, producing n-butanol and ethanol directly from cellulosic biomass by expressing an aldehyde/alcohol dehydrogenase 2 (adhE2), was obtained. Basic fermentation studies with this engineered C. cellulovorans were then performed, including medium optimization, comparison of fermentation bioreactors, and comparison of wild-type and engineered C. cellulovorans. The results showed the fermentation performance and metabolic flux distributions were greatly affected by these factors. To further enhance biofuel production, methyl viologen, an artificial electron carrier was added into the fermentation broth to increase NADH availability, since n-butanol and ethanol production was usually limited by NADH availability. The result showed that biofuel production was increased sharply, and acids production was inhibited greatly by the addition of optimal concentration of methyl viologen from the beginning of fermentation with glucose, cellobiose, or cellulose as the carbon substrates. In summary, C. cellulovorans was metabolically engineered to produce n-butanol and ethanol directly from cellulosic biomass, with the development of its transformation method for the first time. In addition, the engineered C. cellulovorans could produce 1.6 g/L n-butanol directly from cellulose, which is the highest, compared to other wild-type and engineered cellulolytic strains. This project provided a promising platform for the production of biofuel and other value-added products directly from lignocellulosic biomass. 2014-10-07 English text The Ohio State University / OhioLINK http://rave.ohiolink.edu/etdc/view?acc_num=osu1404229128 http://rave.ohiolink.edu/etdc/view?acc_num=osu1404229128 unrestricted This thesis or dissertation is protected by copyright: all rights reserved. It may not be copied or redistributed beyond the terms of applicable copyright laws.
collection	NDLTD
language	English
sources	NDLTD
topic	Biology
spellingShingle	Biology Yang, Xiaorui Metabolic engineering of cellulolytic Clostridium cellulovorans for biofuel production directly from cellulosic biomass
author	Yang, Xiaorui
author_facet	Yang, Xiaorui
author_sort	Yang, Xiaorui
title	Metabolic engineering of cellulolytic Clostridium cellulovorans for biofuel production directly from cellulosic biomass
title_short	Metabolic engineering of cellulolytic Clostridium cellulovorans for biofuel production directly from cellulosic biomass
title_full	Metabolic engineering of cellulolytic Clostridium cellulovorans for biofuel production directly from cellulosic biomass
title_fullStr	Metabolic engineering of cellulolytic Clostridium cellulovorans for biofuel production directly from cellulosic biomass
title_full_unstemmed	Metabolic engineering of cellulolytic Clostridium cellulovorans for biofuel production directly from cellulosic biomass
title_sort	metabolic engineering of cellulolytic clostridium cellulovorans for biofuel production directly from cellulosic biomass
publisher	The Ohio State University / OhioLINK
publishDate	2014
url	http://rave.ohiolink.edu/etdc/view?acc_num=osu1404229128
work_keys_str_mv	AT yangxiaorui metabolicengineeringofcellulolyticclostridiumcellulovoransforbiofuelproductiondirectlyfromcellulosicbiomass
_version_	1719436535790567424

Metabolic engineering of cellulolytic Clostridium cellulovorans for biofuel production directly from cellulosic biomass

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