Mitochondrial Compartmentalization Confers Specificity to the 2-Ketoacid Recursive Pathway: Increasing Isopentanol Production in Saccharomyces cerevisiae
Mitochondrial Compartmentalization Confers Specificity to the 2-Ketoacid Recursive Pathway: Increasing Isopentanol Production in Saccharomyces cerevisiae
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DOI：
10.1021/acssynbio.9b00420
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发表时间：
2020-03-20
影响因子：
4.7
通讯作者：
Avalos, Jose L.
中科院分区：
文献类型：
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作者：
Hammer, Sarah K.;Zhang, Yanfei;Avalos, Jose L.
递归延伸途径产生碳链长度随着每个迭代循环而增加的化合物。特别令人感兴趣的是来自递归延伸的2-酮酸，其作为被称为支链高级醇（BCHAs）的一类有价值的高级生物燃料的前体。蛋白质工程已被用于增加完成的迭代延伸循环的数量，但长链2-酮酸的特异性生产仍然难以实现。在这里，我们表明，线粒体区室化是一种有效的策略，以增加递归途径的特异性，有利于长链的产品。使用2-酮酸延伸作为概念的证明，我们表明，三个延伸酶-LEU 4，LEU 1，和LEU 2-在异丁醇生产菌株的线粒体中的过表达导致异戊醇与异丁醇的产物比相对于在胞质溶胶中过表达相同的延伸酶增加2.3倍，相对于野生型酶表达增加31倍。减少中间体的损失使我们能够进一步将异戊醇产量提高到1.24 +/- 0.06 g/L。在该菌株中，异戊醇占所产生的总BCHAs的86%，同时实现了酿酒酵母所报道的最高异戊醇滴度。将延伸酶定位在线粒体中使得能够开发其中异戊醇构成BCHA生产的多达93%的菌株。这项工作建立了线粒体区室化作为一种新的方法，有利于高滴度和产品特异性的较大的产品从递归途径。
Recursive elongation pathways produce compounds of increasing carbon-chain length with each iterative cycle. Of particular interest are 2-ketoacids derived from recursive elongation, which serve as precursors to a valuable class of advanced biofuels known as branched-chain higher alcohols (BCHAs). Protein engineering has been used to increase the number of iterative elongation cycles completed, yet specific production of longer-chain 2-ketoacids remains difficult to achieve. Here, we show that mitochondrial compartmentalization is an effective strategy to increase specificity of recursive pathways to favor longer-chain products. Using 2-ketoacid elongation as a proof of concept, we show that overexpression of the three elongation enzymes-LEU4, LEU1, and LEU2-in mitochondria of an isobutanol production strain results in a 2.3-fold increase in the isopentanol to isobutanol product ratio relative to overexpressing the same elongation enzymes in the cytosol, and a 31-fold increase relative to wild-type enzyme expression. Reducing the loss of intermediates allows us to further boost isopentanol production to 1.24 +/- 0.06 g/L of isopentanol. In this strain, isopentanol accounts for 86% of the total BCHAs produced, while achieving the highest isopentanol titer reported for Saccharomyces cerevisiae. Localizing the elongation enzymes in mitochondria enables the development of strains in which isopentanol constitutes as much as 93% of BCHA production. This work establishes mitochondrial compartmentalization as a new approach to favor high titers and product specificities of larger products from recursive pathways.