Doubling the catabolic reducing power (NADH) output of Escherichia coli fermentation for production of reduced products.

Homofermentative production of reduced products requires additional reducing power output (NADH) from glucose catabolism. Anaerobic expression of the pyruvate dehydrogenase complex (PDH, encoded by aceEF-lpd, a normal aerobic operon) is able to provide the additional NADH required for production of reduced products in Escherichia coli fermentation. The multiple promoters (pflBp(1-7)) of pyruvate formate lyase (pflB) were evaluated for anaerobic expression of the aceEF-lpd operon. Four chromosomal constructs, pflBp(1-7)-aceEF-lpd, pflBp(1-6)-aceEF-lpd, pflBp(6,7)-aceEF-lpd, and pflBp6-aceEF-lpd efficiently expressed the PDH complex in anaerobically grown cells. Doubling the reducing power output was achieved when glucose was oxidized to acetyl-CoA through glycolysis and pyruvate oxidation by the anaerobically expressed PDH complex (glucose -->2 acetyl-CoA + 4 NADH). This additional reducing power output can be used for production of reduced products in anaerobic E. coli fermentation.