Engineering and chromosomal integration of a functional butanol pathway in Escherichia coli for butanol fermentation

Advisors: Shengde Zhou.Committee members: Neil Blackstone; Stuart Hill; Gabriel Holbrook; Rangaswamy Meganathan.A butanol fermentation pathway was engineered in Escherichia coli through cloning and chromosomal integration of the butanol genes (thiL, CA_C2873, hbd, crt, bcd, etfA, etfB, adhe1, adhe2, bdhAB) from Clostridium acetobutylicum ATCC824. Initially, a minimal set of butanol pathway genes (thiL, hbd, crt, bcd, etfAB, adhe2) was transcriptionally fused with a pflBp6 promoter and were integrated into the chromosome of E. coli EG10, a strain lacking endogenous competing fermentation pathways, resulting in strain EG60. Anaerobic fermentations showed that EG60 was able to produce 100 muM butanol, suggesting that the butanol pathway genes were effectively expressed for butanol production. Although the titer needs a 2-3 order of magnitude improvement for industrial relevance, a detectable level of butanol produced by EG60 proves, for the first time in principle, that a functional butanol pathway can be established in E. coli via chromosomal integration of butanol genes. The butanol pathway in EG60 was then improved through a stepwise integration of the redundant butanol pathway genes: 1) integration of a second clostridial thiolase (CA_C2873) gene, resulting in strain EG72 which produced 210 muM butanol, achieving a 2-fold improvement in the butanol titer; 2) integration of a redundant alcohol dehydrogenase gene (adhe1) into EG72, resulting in strain EG80 (adhe1, adhe2) which produced 360 muM butanol, a 3.5-fold increase in butanol production over that of EG60; 3) integration of the butanol dehydrogenase genes (bdhAB), resulting in strains EG78 (bdhAB, adhe2) and EG79 (bdhAB, adhe1, adhe2), achieving an increased butanol to ethanol ratio under the same fermentation conditions. These results indicate that along with the minimum set of butanol genes (thiL, hbd, crt, bcd, etfAB, adhe2) an "optimized" butanol pathway should include CA_C2873, bdhAB, and possibly adhe1.Ph.D. (Doctor of Philosophy