Highly Multiplexed CRISPRi Repression of Respiratory Functions Enhances Mitochondrial Localized Ethyl Acetate Biosynthesis in Kluyveromyces marxianus
Highly Multiplexed CRISPRi Repression of Respiratory Functions Enhances Mitochondrial Localized Ethyl Acetate Biosynthesis in Kluyveromyces marxianus
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DOI：
10.1021/acssynbio.8b00331
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发表时间：
2018-11-01
影响因子：
4.7
通讯作者：
Wheeldon, Ian
中科院分区：
文献类型：
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作者：
Lobs, Ann-Kathrin;Schwartz, Cory;Wheeldon, Ian
用于靶向基因组编辑和调控的CRISPR-Cas9的出现使得能够操纵所需的性状并增强非模型微生物的菌株开发。马克斯克鲁维酵母（Kluyveromycesmarxianus）在高温下以高速率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，我们确定了乙酸乙酯生物合成的天然定位在K。marxianus，并使用该信息开发多路CRISPRi系统，用于沿沿着中心代谢途径重定向碳通量，提高乙酸乙酯生产率。首先，我们确定了前体和乙酸酯生物合成的主要途径。基因敲除筛选揭示了醇乙酰转移酶Eatl是乙酸乙酯、乙酸异戊酯和乙酸苯乙酯生产的关键酶。截断研究表明，高酯的生物合成是偶然的Eatl线粒体定位。由于乙酸乙酯是由乙醇和乙酰辅酶A缩合形成的，我们使用高度多重的CRISPRi方法调节TCA循环和电子传递链基因的表达。同时敲除ACO 2b、SDH 2、RIP 1和MSSSI导致乙酸乙酯生产率比已经很高的天然能力增加3.8倍。这项工作表明，在对途径生物化学的基本理解的支持下，中心碳通量的多重CRISPRi调节是非常规微生物代谢工程的有效策略。
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 Eatl is the critical enzyme for ethyl, isoamyl, and phenylethyl acetate production. Truncation studies revealed that high ester biosynthesis is contingent on Eatl 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 MSSSI 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.