Joint BioEnergy Institute, 5885 Hollis Street, Emeryville, CA 94608, USA; Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
Joint BioEnergy Institute, 5885 Hollis Street, Emeryville, CA 94608, USA; Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA; Environmental Genomics and Systems Biology, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
Metab Eng. 2021 Mar;64:154-166. doi: 10.1016/j.ymben.2021.02.002. Epub 2021 Feb 10.
Isoprenol (3-methyl-3-butene-1-ol) is a valuable drop-in biofuel and an important precursor of several commodity chemicals. Synthetic microbial systems using the heterologous mevalonate pathway have recently been developed for the production of isoprenol in Escherichia coli, and a significant yield and titer improvement has been achieved through a decade of research. Saccharomyces cerevisiae has been widely used in the biotechnology industry for isoprenoid production, but there has been no good example of isoprenol production reported in this host. In this study, we engineered the budding yeast S. cerevisiae for improved biosynthesis of isoprenol. The strain engineered with the mevalonate pathway achieved isoprenol production at the titer of 36.02 ± 0.92 mg/L in the flask. The IPP (isopentenyl diphosphate)-bypass pathway, which has shown more efficient isoprenol production by avoiding the accumulation of the toxic intermediate in E. coli, was also constructed in S. cerevisiae and improved the isoprenol titer by 2-fold. We further engineered the strains by deleting a promiscuous endogenous kinase that could divert the pathway flux away from the isoprenol production and improved the titer to 130.52 ± 8.01 mg/L. Finally, we identified a pathway bottleneck using metabolomics analysis and overexpressed a promiscuous alkaline phosphatase to relieve this bottleneck. The combined efforts resulted in the titer improvement to 383.1 ± 31.62 mg/L in the flask. This is the highest isoprenol titer up to date in S. cerevisiae and this work provides the key strategies to engineer yeast as an industrial platform for isoprenol production.
异戊烯醇（3-甲基-3-丁烯-1-醇）是一种有价值的可替代生物燃料，也是几种商品化学品的重要前体。最近，利用异源甲羟戊酸途径构建了合成微生物系统，用于在大肠杆菌中生产异戊烯醇，经过十年的研究，产量和浓度都有了显著提高。酿酒酵母在生物技术工业中被广泛用于异戊二烯的生产，但在该宿主中尚未有生产异戊烯醇的良好实例报道。在这项研究中，我们对 budding yeast S. cerevisiae 进行了工程改造，以提高其异戊烯醇的生物合成能力。工程菌株通过构建甲羟戊酸途径，在摇瓶中达到了 36.02±0.92mg/L 的异戊烯醇产量。IPP（异戊烯二磷酸）旁路途径通过避免在大肠杆菌中积累有毒中间体，显示出更高效的异戊烯醇生产能力，该途径也在 S. cerevisiae 中构建，并将异戊烯醇浓度提高了 2 倍。我们进一步通过敲除一种能够将途径通量从异戊烯醇生产中转移的杂泛内源性激酶来工程化这些菌株，将浓度提高到 130.52±8.01mg/L。最后，我们通过代谢组学分析确定了一个途径瓶颈，并过表达了一种杂泛的碱性磷酸酶来缓解这个瓶颈。综合努力使浓度提高到摇瓶中的 383.1±31.62mg/L。这是迄今为止 S. cerevisiae 中异戊烯醇的最高浓度，这项工作为工程化酵母作为工业平台生产异戊烯醇提供了关键策略。
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