Engineering of Bacillus subtilis for enhanced total synthesis of folic acid

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.

FIG. 1.

Folic pathway schematic showing the flow of metabolites and the genes that encode the enzymes involved. Metabolite abbreviations not found in the text are Phe (phenylalanine), Tyr (tyrosine), Trp (tryptophan), and GTP. The transketolase-catalyzed reaction that forms E4P is omitted on the figure for brevity.

FIG. 2.

Folic acid biosynthetic and regulatory genes in B. subtilis from the National Center for Biotechnology Information website (http://www.ncbi.nih.gov). The major folic operon located at 7° encodes 6 genes (pabB, pabA, pabC, sul, folA, and folK) required for folate synthesis. GTP cyclohydrolase I (mtrA) and trp RNA-binding attenuation protein (mtrB) are located on an operon at 203.7°. The last two enzymes for folate synthesis, folC and dfrA, are located at 244.8° and 196.1°. The dashed arrow highlights the transcriptional and translational control of TRAP on the folic operon. Binding of TRAP to folic operon-derived mRNA terminates the translation of the DNA (2.1-kb mRNA would be the major product) and translation of the mRNA as well. Disruption of mtrB increases the expression level of 5.9-kb mRNA.

FIG. 3.

One output from Metabologica that shows a metabolite trafficking pattern that is predicted by linear programming to confer the highest carbon yield for folic acid production. Flux values shown correspond to mmol/h g cell (basis growth rate is 0.4 h⁻¹). The maximized yield solutions also correspond to minimized pyruvate kinase flux (r₅). Metabolite abbreviations not found in the text are GAP (glyceraldehyde-3-phosphate), S7P (sedoheptulose-7-phosphate), PGP (1,3-biphosphoglycerate), OAA (oxaloacetate), Succ (succinate), aa (amino acid), na (nucleic acids), and c (carbon dioxide).

FIG. 4.

Phase planes for folic acid production. (a) Folic acid formation rate (Fig. 3, r₁) versus PYK flux (r₅); (b) folic acid formation rate (r₁) versus HMP flux (r₃); (c) folic acid formation rate (r₁) versus transketolase flux converting R5P and X5P to GAP and S7P (r₁₆); and (d) folic acid formation rate (r₁) versus transketolase flux converting E4P and X5P to F6P and G3P (r₃₆).

FIG. 5.

How folic acid yield (white bar) and doubling time (gray bar) depend on IPTG concentration in strain BSZT0402 [trpC2, pyk(Ind)], which possesses IPTG-inducible pyruvate kinase. The parent strain's (B. subtilis 168) folic acid yield and doubling time are included for comparison.