Löbs Ann-Kathrin, Schwartz Cory, Thorwall Sarah, Wheeldon Ian
Chemical and Environmental Engineering , University of California Riverside , Riverside , California 92521 , United States.
Center for Industrial Biotechnology, Bourns College of Engineering , University of California Riverside , Riverside , California 92521 , United States.
ACS Synth Biol. 2018 Nov 16;7(11):2647-2655. doi: 10.1021/acssynbio.8b00331. Epub 2018 Nov 5.
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.
用于靶向基因组编辑和调控的CRISPR-Cas9技术的出现，使得对非模式微生物的理想性状进行操纵以及加强菌株开发成为可能。马克斯克鲁维酵母天然具有在高温下高速率生产挥发性酯类的能力，这使其成为工业生物技术领域一个潜在的重要生产平台。在此，我们确定了马克斯克鲁维酵母中乙酸乙酯生物合成的天然定位，并利用这一信息开发了一种多重CRISPR干扰系统，用于重定向中心代谢途径中的碳通量，提高乙酸乙酯的生产力。首先，我们确定了前体和乙酸酯生物合成的主要途径。一项基因敲除筛选表明，醇乙酰转移酶Eat1是乙酸乙酯、乙酸异戊酯和乙酸苯乙酯生产的关键酶。截短研究表明，高酯生物合成取决于Eat1在线粒体中的定位。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CRISPR干扰方法调节三羧酸循环和电子传递链基因的表达。同时敲低ACO2b、SDH2、RIP1和MSS51，使得乙酸乙酯的生产力在原本就很高的天然能力基础上提高了3.8倍。这项工作表明，在对途径生物化学有基本了解的支持下，对中心碳通量进行多重CRISPR干扰调控，是非常规微生物代谢工程的一种有效策略。
