초록
<P><B>ABSTRACT</B><P> Manipulation of NADH-dependent steps, and particularly disruption of the <I>las</I> -located lactate dehydrogenase ( <I>ldh</I> ) gene in Lactococcus 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 of L. lactis , i.e., the <I>ldh</I> , <I>ldhB</I> , and <I>ldhX</I> genes, 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, the <I>ldhB</I> and <I>ldhX</I> genes were sequentially deleted in L. lactis FI10089, a strain with a deletion of the <I>ldh</I> gene. The single, double, and triple mutants, FI10089, FI10089Δ <I>ldhB</I> , and FI10089Δ <I>ldhB</I> Δ <I>ldhX</I> , showed similar growth profiles and displayed mixed-acid fermentation, ethanol being the main reduced end product. Hence, the alcohol dehydrogenase-encoding gene, the <I>adhE</I> gene, was inactivated in FI10089, but the resulting strain reverted to homolactic fermentation due to induction of the <I>ldhB</I> gene. 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 FI10089Δ <I>ldhB</I> is a valuable basis for engineering strategies aiming at the production of reduced compounds. </P></P>