Evaluation of Carbon and Electron Flow in Lactobacillus brevis as a Potential Host for Heterologous 1-Butanol Biosynthesis

Abstract

Heterofermentative lactic acid bacterium Lactobacillus brevis may be considered as a promising host for heterologous butanol synthesis because of tolerance to butanol and ability to ferment pentose and hexose sugars from wood hydro- lysates that are cheap and renewable carbohydrate source. Carbon and electron flow was evaluated in two L. brevis strains in order to assess metabolic potential of these bacteria for heterologous butanol synthesis. Conditions required for generation of acetyl-CoA and NADH which are necessary for butanol biosynthesis have been determined. Key en- zymes controlling direction of metabolic fluxes in L. brevis in various redox conditions were defined. In anaerobic glu- cose fermentation, the carbon flow through acetyl-CoA is regulated by aldehyde dehydrogenase ALDH possessing low NADH affinity to NADH and activity ( K m = 200 µM, Vmax= 0.03 U/mg of total cell protein). Aerobically, the NADH- NADH oxidase NOX ( K m = 25 µM, Vmax = 1.7 U/mg) efficiently competes with ALDH for NADH that results in forma- tion of acetate instead of acetyl-CoA. In general, external electron acceptors (oxygen, fructose) and pentoses decrease NADH availability for native ethanol and recombinant butanol enzymes and therefore reduce carbon flux through ace- tyl-CoA. Pyruvate metabolism was studied in order to reveal redirection possibilities of competitive carbon fluxes to- wards butanol synthesis. The study provides a basis for the rational development of L. brevis strains producing butanol from wood hydrolysate.