Lactic acid (LA) is chemically synthesized or fermentatively produced using glucose as substrate, mainly using lactic acid bacteria. Polylactic acid is used as a biodegradable bioplastic for packaging materials, medical materials, and filaments for 3D printers. In this study, we aimed to construct a LA-tolerant yeast to reduce the neutralization cost in LA production. The pHLA2-51 strain was obtained through a previously developed genome evolution strategy, and transcriptome analysis revealed the gene expression profile of the mutant yeast. Furthermore, the expression of the genes associated with glycolysis and the LA synthesis pathway in the LA-tolerant yeast was comprehensively and randomly modified to construct a D-LA-producing, LA-tolerant yeast. In detail, DNA fragments expressing thirteen genes, HXT7, HXK2, PGI1, PFK1, PFK2, FBA1, TPI1, TDH3, PGK1, GPM1, ENO2, and PYK2, and D-lactate dehydrogenase (D-LDH) from Leuconostoc mesenteroides were randomly integrated into the genomic DNA in the LA-tolerant yeast. The resultant engineered yeast produced about 33.9 g/L of D-LA from 100 g/L glucose without neutralizing agents in a non-neutralized condition and 52.2 g/L of D-LA from 100 g/L glucose with 20 g/L CaCO3 in a semi-neutralized condition. Our research provides valuable insights into non-neutralized fermentative production of LA. KEY POINTS: • Lactic acid (LA) tolerance of yeast was improved by genome evolution. • The transcription levels of 751 genes were changed under LA stress. • Rapid LA production with semi-neutralization was achieved by modifying glycolysis. • A versatile yeast strain construction method based on the CRISPR system was proposed.

中文翻译：

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通过使用CRISPR-Cas介导的基因组进化构建乳酸耐受的酿酒酵母，以高效生产D-乳酸。

乳酸（LA）是使用葡萄糖为底物，主要使用乳酸菌化学合成或发酵生产的。聚乳酸被用作包装材料，医疗材料和3D打印机的细丝的可生物降解的生物塑料。在这项研究中，我们旨在构建耐LA的酵母，以降低LA生产中的中和成本。通过先前开发的基因组进化策略获得了pHLA2-51菌株，转录组分析揭示了该突变酵母的基因表达谱。此外，在耐LA的酵母中与糖酵解和LA合成途径相关的基因的表达被全面随机地修饰以构建产生D-LA的耐LA的酵母。详细来说，表达13个基因的DNA片段HXT7，HXK2，PGI1，PFK1，PFK2，FBA1，TPI1，TDH3，PGs1，GPM1，ENO2和PYK2，以及肠膜间苯二酚的D-乳酸脱氢酶（D-LDH）随机整合到耐LA酵母中的基因组DNA中。所得的工程酵母可在无中和条件下，从无中和剂的100 g / L葡萄糖中产生约33.9 g / L D-LA，从100 g / L葡萄糖中含20 g / L CaCO3产生52.2 g / L D-LA在半中和状态下。我们的研究为LA的非中和发酵生产提供了宝贵的见识。要点：•基因组进化提高了酵母对乳酸（LA）的耐受性。•在LA胁迫下751个基因的转录水平发生了变化。•通过修饰糖酵解，实现半中和的快速LA生产。•提出了一种基于CRISPR系统的通用酵母菌株构建方法。Mesenteroides的D-乳酸脱氢酶（D-LDH）和D-LDH被随机整合到耐LA的酵母的基因组DNA中。所得的工程酵母可在无中和条件下，从无中和剂的100 g / L葡萄糖中产生约33.9 g / L D-LA，从100 g / L葡萄糖中含20 g / L CaCO3产生52.2 g / L D-LA在半中和状态下。我们的研究为LA的非中和发酵生产提供了宝贵的见识。要点：•基因组进化提高了酵母对乳酸（LA）的耐受性。•在LA胁迫下751个基因的转录水平发生了变化。•通过修饰糖酵解，实现半中和的快速LA生产。•提出了一种基于CRISPR系统的通用酵母菌株构建方法。Mesenteroides的D-乳酸脱氢酶（D-LDH）和D-LDH被随机整合到耐LA的酵母的基因组DNA中。所得的工程酵母可在无中和条件下，从无中和剂的100 g / L葡萄糖中产生约33.9 g / L D-LA，从100 g / L葡萄糖中含20 g / L CaCO3产生52.2 g / L D-LA在半中和的条件下。我们的研究为LA的非中和发酵生产提供了宝贵的见识。要点：•基因组进化提高了酵母对乳酸（LA）的耐受性。•在LA胁迫下751个基因的转录水平发生了变化。•通过修饰糖酵解，可以半中和地快速生产LA。•提出了一种基于CRISPR系统的通用酵母菌株构建方法。