Genetic engineering of Clostridium thermocellum DSM1313 for enhanced ethanol production

Abstract

BackgroundThe twin problem of shortage in fossil fuel and increase in environmental pollution can be partly addressed by blending of ethanol with transport fuel. Increasing the ethanol production for this purpose without affecting the food security of the countries would require the use of cellulosic plant materials as substrate. Clostridium thermocellum is an anaerobic thermophilic bacterium with cellulolytic property and the ability to produce ethanol. But its application as biocatalyst for ethanol production is limited because pyruvate ferredoxin oxidoreductase, which diverts pyruvate to ethanol production pathway, has low affinity to the substrate. Therefore, the present study was undertaken to genetically modify C. thermocellum for enhancing its ethanol production capacity by transferring pyruvate carboxylase (pdc) and alcohol dehydrogenase (adh) genes of the homoethanol pathway from Zymomonas mobilis.ResultsThe pdc and adh genes from Z. mobilis were cloned in pNW33N, and transformed to Clostridium thermocellum DSM 1313 by electroporation to generate recombinant CTH-pdc, CTH-adh and CTH-pdc-adh strains that carried heterologous pdc, adh, and both genes, respectively. The plasmids were stably maintained in the recombinant strains. Though both pdc and adh were functional in C. thermocellum, the presence of adh severely limited the growth of the recombinant strains, irrespective of the presence or absence of the pdc gene. The recombinant CTH-pdc strain showed two-fold increase in pyruvate carboxylase activity and ethanol production when compared with the wild type strain.ConclusionsPyruvate decarboxylase gene of the homoethanol pathway from Z mobilis was functional in recombinant C. thermocellum strain and enhanced its ability to produced ethanol. Strain improvement and bioprocess optimizations may further increase the ethanol production from this recombinant strain.

Single mutation at a highly conserved region of chloramphenicol acetyltransferase enables isobutyl acetate production directly from cellulose by Clostridium thermocellum at elevated temperatures

“…Unfortunately, the AATs known to date are isolated from mesophilic yeasts or plants [9][10][11][12], and none of them has been reported to be active at a temperature above 50 °C. To tackle this problem, we chose CATs to investigate their potential functions as a thermostable AAT, because some thermostable CATs have been successfully used as a selection marker in thermophiles [17][18][19][20][21] and others have been shown to perform the acetylation for not only chloramphenicol but various alcohols like AATs [7,[22][23][24][25] (Fig. 1a, Additional file 1: Figure S1A).…”

Single mutation at a highly conserved region of chloramphenicol acetyltransferase enables thermophilic isobutyl acetate production directly from cellulose by Clostridium thermocellum

“…To tackle this problem, we chose CATs to investigate their potential functions as a thermostable AAT because some thermophilic CATs have been successfully used as a selection marker in thermophiles [13–17] and others have been shown to perform the acetylation for not only chloramphenicol but various alcohols like AATs [18–21] [7] (Figure 1A, S1A). As a proof-of-study, we investigated CAT Sa , classified as Type A-9, from the plasmid pNW33N for a broad range of alcohol substrates as it has been widely used for genetic engineering in C. thermocellum at elevated temperature (≥ 50°C) [13–15].…”

CRISPR interference (CRISPRi) as transcriptional repression tool for Hungateiclostridium thermocellum DSM 1313

“…Two main strategies have been followed. On one hand, competing pathways for carbon flux are disrupted (Argyros et al , ; Biswas et al , ; Papanek et al , ; Kannuchamy et al , ). In another approach, competing pathways for electron flux are eliminated (Biswas et al , ; Rydzak et al , ; Lo et al , ).…”