Abstract
Clostridium thermocellum is a promising candidate for ethanol production from cellulosic biomass, but requires metabolic engineering to improve ethanol yield. A key gene in the ethanol production pathway is the bifunctional aldehyde and alcohol dehydrogenase, adhE. To explore the effects of overexpressing wild-type, mutant, and exogenous adhEs, we developed a new expression plasmid, pDGO144, that exhibited improved transformation efficiency and better gene expression than its predecessor, pDGO-66. This new expression plasmid will allow for many other metabolic engineering and basic research efforts in C. thermocellum. As proof of concept, we used this plasmid to express 12 different adhE genes (both wild type and mutant) from several organisms. Ethanol production varied between clones immediately after transformation, but tended to converge to a single value after several rounds of serial transfer. The previously described mutant C. thermocellum D494G adhE gave the best ethanol production, which is consistent with previously published results.
Highlights
Clostridium thermocellum is a good candidate for producing biofuels from cellulosic biomass via consolidated bioprocessing (Olson et al, 2012)
A recent report describing isobutanol production in C. thermocellum documented the spontaneous integration of plasmid DNA onto the chromosome (Lin et al, 2015)
The improved expression plasmid, pDGO144, more reliably resulted in high levels of adhE expression, whereas with pDGO-66, we saw in most cases that adhE expression was non-existent (i.e., equivalent to the negative control, parent adhE deletion strain, LL1111, Fig. 2 (A))
Summary
Clostridium thermocellum is a good candidate for producing biofuels from cellulosic biomass via consolidated bioprocessing (Olson et al, 2012). This microorganism is among the most effective described at solubilizing lignocellulose (Lynd et al, 2002), and ferments glucose and glucan oligomers to organic acids, hydrogen, and ethanol. One example related to metabolic engineering is the expression of the Thermoanaerobacterium saccharolyticum pyruvate kinase in C. thermocellum (Deng et al, 2013) Another example is the complementing of adhE activity in C. thermocellum adhE deletion strain (Lo et al, 2015; Zheng et al, 2015). Gene expression was achieved via targeted recombination of the gene of interest onto the chromosome, a process that takes several weeks under ideal conditions (Olson and Lynd, 2012a)
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