Abstract
Large-scale production of lignocellulosic biofuel is a potential solution to sustainably meet global energy needs. One-step consolidated bioprocessing (CBP) is a potentially advantageous approach for the production of biofuels, but requires an organism capable of hydrolyzing biomass to sugars and fermenting the sugars to ethanol at commercially viable titers and yields. Clostridium thermocellum, a thermophilic anaerobe, can ferment cellulosic biomass to ethanol and organic acids, but low yield, low titer, and ethanol sensitivity remain barriers to industrial production. Here, we deleted the hypoxanthine phosphoribosyltransferase gene in ethanol tolerant strain of C. thermocellum adhE*(EA) in order to allow use of previously developed gene deletion tools, then deleted lactate dehydrogenase (ldh) to redirect carbon flux towards ethanol. Upon deletion of ldh, the adhE*(EA) Δldh strain produced 30% more ethanol than wild type on minimal medium. The adhE*(EA) Δldh strain retained tolerance to 5% v/v ethanol, resulting in an ethanol tolerant platform strain of C. thermocellum for future metabolic engineering efforts.
Highlights
A major challenge of this century is to develop sustainable technology for production of fuels and chemicals independent of fossil fuels
Primer set (P3) amplified the 3200 bp region of the wild type locus, while amplification from alcohol dehydrogenase (adhE)*(EA) Dldh resulted in a 2200 bp fragment, confirming gene deletion
The mechanism of ethanol tolerance in C. thermocellum adhE*(EA) appears to be related to correcting an imbalance between NADH-NADPH cofactors, perhaps with an overabundance of NADPH being detrimental to growth in the presence of added ethanol [7]
Summary
A major challenge of this century is to develop sustainable technology for production of fuels and chemicals independent of fossil fuels. Lignocellulosic biomass is an abundant resource [1] that has potential to be used as a feedstock for the production of fuels and chemicals. Consolidated Bioprocessing (CBP) [2,3] is a promising approach that could help make cellulosic fuel production economical; no natural organisms are known that can both hydrolyze cellulosic biomass and produce a liquid fuel at high yield and titer under industrially relevant conditions. Unlike highly ethanologenic microbes such as Saccharomyces cerevisiae and Zymomonas mobilis, wild type strains are only able to tolerate low levels of ethanol Increased tolerance would allow higher titer and more economic product recovery. Recent studies have aimed to understand the mechanisms by which C. thermocellum is able to evolve tolerance to higher levels of ethanol [5,6]. An ethanol tolerant strain of C. thermocellum ATCC 27405 was developed to tolerate ethanol concentration up to 5% v/v [6]
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