Highly Multiplexed CRISPRi Repression of Respiratory Functions Enhances Mitochondrial Localized Ethyl Acetate Biosynthesis in Kluyveromyces marxianus,ACS Synthetic Biology

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Highly Multiplexed CRISPRi Repression of Respiratory Functions Enhances Mitochondrial Localized Ethyl Acetate Biosynthesis in Kluyveromyces marxianus
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2018-10-24 00:00:00 , DOI: 10.1021/acssynbio.8b00331
Ann-Kathrin Löbs ₁ , Cory Schwartz ₁ , Sarah Thorwall ₁ , Ian Wheeldon _{1,

2}

Affiliation

The emergence of CRISPR-Cas9 for targeted genome editing and regulation has enabled the manipulation of desired traits and enhanced strain development of nonmodel microorganisms. The natural capacity of the yeast Kluyveromyces marxianus to produce volatile esters at high rate and at elevated temperatures make it a potentially valuable production platform for industrial biotechnology. Here, we identify the native localization of ethyl acetate biosynthesis in K. marxianus and use this information to develop a multiplexed CRISPRi system for redirecting carbon flux along central metabolic pathways, increasing ethyl acetate productivity. First, we identified the primary pathways of precursor and acetate ester biosynthesis. A genetic knockout screen revealed that the alcohol acetyltransferase Eat1 is the critical enzyme for ethyl, isoamyl, and phenylethyl acetate production. Truncation studies revealed that high ester biosynthesis is contingent on Eat1 mitochondrial localization. As ethyl acetate is formed from the condensation of ethanol and acetyl-CoA, we modulated expression of the TCA cycle and electron transport chain genes using a highly multiplexed CRISPRi approach. The simultaneous knockdown of ACO2b, SDH2, RIP1, and MSS51 resulted in a 3.8-fold increase in ethyl acetate productivity over the already high natural capacity. This work demonstrates that multiplexed CRISPRi regulation of central carbon flux, supported by a fundamental understanding of pathway biochemistry, is a potent strategy for metabolic engineering in nonconventional microorganisms.

中文翻译：

呼吸功能的高度多重CRISPRi抑制增强线粒体马克斯克鲁维酵母的线粒体定位乙酸乙酯生物合成。

用于靶向基因组编辑和调控的CRISPR-Cas9的出现使所需特性的操纵和非模型微生物的菌株开发得以增强。马尔克斯克鲁维酵母酵母以高速率和高温产生挥发性酯的天然能力使其成为工业生物技术的潜在有价值的生产平台。在这里，我们确定了K. marxianus中的乙酸乙酯生物合成的本地化本地化并利用这些信息开发出一个多重CRISPRi系统，用于沿着中央代谢途径重定向碳通量，从而提高乙酸乙酯的生产率。首先，我们确定了前体和乙酸酯生物合成的主要途径。基因敲除筛选表明，醇乙酰基转移酶Eat1是乙基，异戊基和苯乙基乙酸酯生产的关键酶。截断研究表明，高酯生物合成取决于Eat1线粒体的定位。由于乙酸乙酯是由乙醇和乙酰辅酶A的缩合形成的，因此我们使用高度复用的CRISPRi方法调节了TCA循环和电子传输链基因的表达。同时击倒ACO2b，SDH2，RIP1和MSS51使乙酸乙酯的生产率比原本就很高的天然能力提高了3.8倍。这项工作表明，通过对路径生物化学的基本理解，对中心碳通量的多重CRISPRi调节是一种在非常规微生物中进行代谢工程的有效策略。

更新日期：2018-10-24

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