Sunday, May 3, 2009
2-54
Riboflavin production during fermentation of genetically modified biobutanol-producing Clostridium acetobutylicum
Xianpeng Cai, Department of Biochemistry & Cell Biology, MS 140, Rice University, 6100 Main Street, Houston, TX 77005-1892 and George Bennett, Biochemistry and Cell Biology, Rice University, 6100 Main St., Houston, TX 77005-1892.
Solvent producing Clostridia are well known for their capacity to use a wide variety of renewable biomass and agricultural waste materials for biobutanol production, which make the process economic and friendly to the environment. To explore the possibility of co-production of a high-value product during biobutanol production, the C. acetobutylicum riboflavin operon ribGBAH was over-expressed in C. acetobutylicum on an E. coli-Clostridium shuttle vector J-ribGBAH; riboflavin was successfully produced in both E. coli and C. acetobutylicum using this vector. The Clostridium acetobutylicum purine pathway was engineered by over-expression of the Clostridium purF gene, which encodes the rate-limiting enzyme PRPP-aminotransferase. The function of the shuttle vector J-purF was verified by its ability to complement an E. coli purF mutation. However, co-production of riboflavin with biobutanol by use of the overexpression plasmid J-ribGBAH-purF was not significantly improved because of the strict regulation of the purine pathway. Rational mutation of the purF gene by replacement of a few amino acid codons was made to yield plasmid J-purFC. This construct also was verified by complementation of the E. coli purF mutation. In E. coli, co-expression of ribGBAH and purFC improved riboflavin production by more than 30%. Function of the co-expression of ribGBAH and purFC in C. acetobutylicum was also examined.