Domesticating a food spoilage yeast into an organic acid‐tolerant metabolic engineering host: Lactic acid production by engineered Zygosaccharomyces bailii,Biotechnology and Bioengineering

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Domesticating a food spoilage yeast into an organic acid‐tolerant metabolic engineering host: Lactic acid production by engineered Zygosaccharomyces bailii
Biotechnology and Bioengineering ( IF 3.5 ) Pub Date : 2020-10-08 , DOI: 10.1002/bit.27576
Nurzhan Kuanyshev _{1,

2} , Christopher V Rao _{2,

3} , Bruce Dien _{2,

4} , Yong-Su Jin _{1,

2,

5}

Affiliation

Lactic acid represents an important class of commodity chemicals, which can be produced by microbial cell factories. However, due to the toxicity of lactic acid at lower pH, microbial production requires the usage of neutralizing agents to maintain neutral pH. Zygosaccharomyces bailii, a food spoilage yeast, can grow under the presence of organic acids used as food preservatives. This unique trait of the yeast might be useful for producing lactic acid. With the goal of domesticating the organic acid‐tolerant yeast as a metabolic engineering host, seven Z. bailii strains were screened in a minimal medium with 10 g/L of acetic, or 60 g/L of lactic acid at pH 3. The Z. bailii NRRL Y7239 strain was selected as the most robust strain to be engineered for lactic acid production. By applying a PAN‐ARS‐based CRISPR‐Cas9 system consisting of a transfer RNA promoter and NAT selection, we demonstrated the targeted deletion of ADE2 and site‐specific integration of Rhizopus oryzae ldhA coding for lactate dehydrogenase into the PDC1 locus. The resulting pdc1::ldhA strain produced 35 g/L of lactic acid without ethanol production. This study demonstrates the feasibility of the CRISPR‐Cas9 system in Z. bailii, which can be applied for a fundamental study of the species.

中文翻译：

将食品腐败酵母驯化为耐有机酸代谢工程宿主：通过工程化接合酵母菌生产乳酸

乳酸是一类重要的商品化学品，可由微生物细胞工厂生产。然而，由于乳酸在较低 pH 值下的毒性，微生物生产需要使用中和剂来维持中性 pH 值。Zygosaccharomyces Baiii是一种食品腐败酵母，可以在用作食品防腐剂的有机酸存在下生长。酵母的这种独特特性可能有助于生产乳酸。与驯化有机酸耐受酵母作为代谢工程宿主的目标，七Z.拜耳接合酵母菌株在基本培养基中在pH 3.筛选用10克/乙酸L，或60克/升乳酸ž . 百利NRRL Y7239 菌株被选为最强大的菌株，用于生产乳酸。通过应用由转移 RNA 启动子和 NAT 选择组成的基于 PAN-ARS 的 CRISPR-Cas9 系统，我们证明了ADE2的靶向缺失和编码乳酸脱氢酶的米根霉 ldhA位点特异性整合到PDC1基因座中。所得的pdc1::ldhA菌株产生 35 g/L 的乳酸，但不产生乙醇。该研究证明了 CRISPR-Cas9 系统在Z. Baili 中的可行性，可用于该物种的基础研究。

更新日期：2020-10-08

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