High Yields of 2,3-Butanediol and Mannitol in Lactococcus lactis through Engineering of NAD+Cofactor Recycling

Abstract

ABSTRACTManipulation of NADH-dependent steps, and particularly disruption of thelas-located lactate dehydrogenase (ldh) gene inLactococcus lactis, is common to engineering strategies envisaging the accumulation of reduced end products other than lactate. Reverse transcription-PCR experiments revealed that three out of the four genes assigned to lactate dehydrogenase in the genome ofL. lactis, i.e., theldh,ldhB, andldhXgenes, were expressed in the parental strain MG1363. Given that genetic redundancy is often a major cause of metabolic instability in engineered strains, we set out to develop a genetically stable lactococcal host tuned for the production of reduced compounds. Therefore, theldhBandldhXgenes were sequentially deleted inL. lactisFI10089, a strain with a deletion of theldhgene. The single, double, and triple mutants, FI10089, FI10089ΔldhB, and FI10089ΔldhBΔldhX, showed similar growth profiles and displayed mixed-acid fermentation, ethanol being the main reduced end product. Hence, the alcohol dehydrogenase-encoding gene, theadhEgene, was inactivated in FI10089, but the resulting strain reverted to homolactic fermentation due to induction of theldhBgene. The three lactate dehydrogenase-deficient mutants were selected as a background for the production of mannitol and 2,3-butanediol. Pathways for the biosynthesis of these compounds were overexpressed under the control of a nisin promoter, and the constructs were analyzed with respect to growth parameters and product yields under anaerobiosis. Glucose was efficiently channeled to mannitol (maximal yield, 42%) or to 2,3-butanediol (maximal yield, 67%). The theoretical yield for 2,3-butanediol was achieved. We show that FI10089ΔldhBis a valuable basis for engineering strategies aiming at the production of reduced compounds.