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首页> 外文期刊>Applied Microbiology >High Yields of 2,3-Butanediol and Mannitol in Lactococcus lactis through Engineering of NAD+ Cofactor Recycling
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High Yields of 2,3-Butanediol and Mannitol in Lactococcus lactis through Engineering of NAD+ Cofactor Recycling

机译：通过NAD +辅因子循环工程可提高乳酸乳球菌中2,3-丁二醇和甘露醇的产量

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摘要

Manipulation of NADH-dependent steps, and particularly disruption of the las -located lactate dehydrogenase ( ldh ) 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 ldh , ldhB , and ldhX 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 ldhB and ldhX genes were sequentially deleted in L. lactis FI10089, a strain with a deletion of the ldh gene. The single, double, and triple mutants, FI10089, FI10089Δ ldhB , and FI10089Δ ldhB Δ ldhX , showed similar growth profiles and displayed mixed-acid fermentation, ethanol being the main reduced end product. Hence, the alcohol dehydrogenase-encoding gene, the adhE gene, was inactivated in FI10089, but the resulting strain reverted to homolactic fermentation due to induction of the ldhB 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 ldhB is a valuable basis for engineering strategies aiming at the production of reduced compounds.
机译：操纵NADH依赖性步骤，尤其是破坏乳酸乳球菌中位于las的乳酸脱氢酶（ldh）基因，对于设想除乳酸以外的还原终产物的积累的工程策略来说是很常见的。逆转录-PCR实验表明，在乳酸乳球菌基因组中分配给乳酸脱氢酶的四个基因中的三个，即ldh，ldhB和ldhX基因在亲本菌株MG1363中表达。鉴于遗传冗余通常是工程菌株代谢不稳定的主要原因，因此我们着手开发一种遗传稳定的乳球菌宿主，该宿主可针对还原化合物的生产进行调整。因此，ldhB和ldhX基因在乳酸乳球菌FI10089（具有ldh基因缺失的菌株）中顺序缺失。单突变体，双突变体和三突变体FI10089，FI10089ΔldhB和FI10089ΔldhBΔldhX表现出相似的生长曲线并显示出混合酸发酵，乙醇是主要的还原终产物。因此，醇脱氢酶编码基因adhE基因在FI10089中失活，但是由于ldhB基因的诱导，所得菌株恢复到纯乳酸发酵。选择了三个乳酸脱氢酶缺陷型突变体作为甘露醇和2,3-丁二醇生产的背景。在乳链菌肽启动子的控制下，这些化合物的生物合成途径被过表达，并且在厌氧条件下分析了构建体的生长参数和产物产量。葡萄糖被有效地引导至甘露醇（最大产率，42％）或2，3-丁二醇（最大产率，67％）。获得了2，3-丁二醇的理论产率。我们表明，FI10089ΔldhB是旨在生产还原化合物的工程策略的宝贵基础。

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