Engineering of Bacillus subtilis for Enhanced Total Synthesis of Folic Acid

Abstract

ABSTRACT We investigated whether the yield of the B vitamin folic acid could be elevated in Bacillus subtilis . Strategies for increasing the folic acid yield were investigated by employing computer-aided flux analysis and mutation. Controlling the activity of the enzyme pyruvate kinase by placing it under inducible control was one strategy devised to elevate yield while insuring that a rapid growth rate results. Other single mutation strategies included amplifying the expression of the genes in the folate operon and overexpressing the Escherichia coli aroH gene, which encodes 2-dehydro-3-deoxyphosphoheptonate aldolase. The latter could conceivably elevate the abundance of the folic acid precursor, para -aminobenzoic acid. Strains that combined two or more mutations were also constructed. Overall, a strain possessing inducible pyruvate kinase, overexpressed aroH , and increased transcription and translation of genes from the folic operon exhibited the best yield. The yield was eightfold higher than that displayed by the parent B. subtilis 168 strain.