The toxicity of n-butanol in microbial fermentations limits its formation. The stress response of Clostridium acetobutylicum involves various stress proteins and therefore, over-expression of genes encoding stress proteins constitutes an option to improve solvent tolerance. Over-expression of groESL, grpE and htpG, significantly improved butanol tolerance of C. acetobutylicum. Whereas the wild type and vector control strain did not survive 2 % (v/v) butanol for 2 h, the recombinant strains showed 45 % (groESL), 25 % (grpE) and 56 % (htpG), respectively, of the initial c.f.u. after 2 h of butanol exposure. As previously, over-expression of groESL led to higher butanol production rates, but the novel strains over-expressing grpE or htpG produced only 51 and 68 %, respectively, of the wild type butanol concentrations after 72 h clearly differentiating butanol tolerance and production. Not only butanol tolerance but also the adaptation to butanol in successive stress experiments was significantly facilitated by increased levels of GroESL, GrpE and HtpG. Re-transformation and sequence analyses of the plasmids confirmed that not the plasmids, but the host cells evolved to a more robust phenotype.
Biosynthetic thiolases catalyze the condensation of two molecules acetyl-CoA to acetoacetyl-CoA and represent key enzymes for carbon-carbon bond forming metabolic pathways. An important biotechnological example of such a pathway is the clostridial n-butanol production, comprising various natural constraints that limit titer, yield, and productivity. In this study, the thiolase of Clostridium acetobutylicum, the model organism for solventogenic clostridia, was specifically engineered for reduced sensitivity towards its physiological inhibitor coenzyme A (CoA-SH). A high-throughput screening assay in 96-well microtiter plates was developed employing Escherichia coli as host cells for expression of a mutant thiolase gene library. Screening of this library resulted in the identification of a thiolase derivative with significantly increased activity in the presence of free CoA-SH. This optimized thiolase comprised three amino acid substitutions (R133G, H156N, G222V) and its gene was expressed in C. acetobutylicum ATCC 824 to assess the effect of reduced CoA-SH sensitivity on solvent production. In addition to a clearly delayed ethanol and acetone formation, the ethanol and butanol titers were increased by 46% and 18%, respectively, while the final acetone concentrations were similar to the vector control strain. These results demonstrate that thiolase engineering constitutes a suitable methodology applicable to improve clostridial butanol production, but other biosynthetic pathways involving thiolase-mediated carbon flux limitations might also be subjected to this new metabolic engineering approach.
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