D-lactic acid is a chiral three-carbon organic acid that can improve the thermostability of polylactic acid. Here, we systematically engineered Saccharomyces cerevisiae to produce D-lactic acid from glucose, a renewable carbon source, at near theoretical yield. Specifically, we screened D-lactate dehydrogenase (DLDH) variants from lactic acid bacteria in three different genera and identified the Leuconostoc pseudomesenteroides variant (LpDLDH) as having the highest activity in yeast. We then screened single-gene deletions to minimize the production of the side products ethanol and glycerol as well as prevent the conversion of D-lactic acid back to pyruvate. Based on the results of the DLDH screening and the single-gene deletions, we created a strain called ASc-d789M which overexpresses LpDLDH and contains deletions in glycerol pathway genes GPD1 and GPD2 and lactate dehydrogenase gene DLD1, as well as downregulation of ethanol pathway gene ADH1 using the L-methionine repressible promoter to minimize impact on growth. ASc-d789M produces D-lactic acid at a titer of 17.09 g/L in shake-flasks (yield of 0.89 g/g glucose consumed or 89% of the theoretical yield). Fed-batch fermentation resulted in D-lactic acid titer of 40.03 g/L (yield of 0.81 g/g glucose consumed). Altogether, our work represents progress towards efficient microbial production of D-lactic acid.

中文翻译：

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酿酒酵母系统工程用于生产 D-乳酸，接近理论产率

D-乳酸是一种手性三碳有机酸，可以提高聚乳酸的热稳定性。在这里，我们系统地设计了酿酒酵母，以接近理论产量从葡萄糖（一种可再生碳源）生产 D-乳酸。具体来说，我们从三个不同属的乳酸菌中筛选出 D-乳酸脱氢酶 (DLDH) 变体，并确定假肠系膜明串珠菌变体 (LpDLDH) 在酵母中具有最高活性。然后，我们筛选了单基因缺失，以尽量减少副产物乙醇和甘油的产生，并防止 D-乳酸转化回丙酮酸。根据 DLDH 筛选结果和单基因缺失，我们创建了一个名为 ASc-d789M 的菌株，它过度表达 LpDLDH，并包含甘油途径基因 GPD1 和 GPD2 以及乳酸脱氢酶基因 DLD1 的缺失，以及使用 L-蛋氨酸可抑制启动子下调乙醇途径基因 ADH1 以尽量减少对生长的影响。ASc-d789M 在摇瓶中以 17.09 g/L 的滴度产生 D-乳酸（消耗的葡萄糖产量为 0.89 g/g 或理论产量的 89%）。补料分批发酵导致 D-乳酸滴度为 40.03 g/L（消耗的葡萄糖产量为 0.81 g/g）。总之，我们的工作代表了高效微生物生产 D-乳酸的进展。ASc-d789M 在摇瓶中以 17.09 g/L 的滴度产生 D-乳酸（消耗的葡萄糖产量为 0.89 g/g 或理论产量的 89%）。补料分批发酵导致 D-乳酸滴度为 40.03 g/L（消耗的葡萄糖产量为 0.81 g/g）。总之，我们的工作代表了高效微生物生产 D-乳酸的进展。ASc-d789M 在摇瓶中以 17.09 g/L 的滴度产生 D-乳酸（消耗的葡萄糖产量为 0.89 g/g 或理论产量的 89%）。补料分批发酵导致 D-乳酸滴度为 40.03 g/L（消耗的葡萄糖产量为 0.81 g/g）。总之，我们的工作代表了高效微生物生产 D-乳酸的进展。