Directed Evolution of Xylose Isomerase for Improved Xylose Catabolism and Fermentation in the Yeast Saccharomyces cerevisiae
Directed Evolution of Xylose Isomerase for Improved Xylose Catabolism and Fermentation in the Yeast Saccharomyces cerevisiae
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DOI：
10.1128/aem.01419-12
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发表时间：
2012-08-01
影响因子：
4.4
通讯作者：
Alper, Hal S.
中科院分区：
文献类型：
--
作者：
Lee, Sun-Mi;Jellison, Taylor;Alper, Hal S.
高功能木糖异构酶途径在酿酒酵母中的异源表达将对乙醇产率具有显著优势，因为该途径绕过了在传统使用的氧化还原酶途径中发现的辅因子要求。然而，几乎所有的报道木糖异构酶为基础的途径在S。与氧化还原酶途径相比，酿酒酵母的乙醇生产率差、木糖消耗率低、细胞生长差，此外，通常需要适应性菌株进化。在这里，我们报告的Piromyces属木糖异构酶（xylA编码）用于酵母的定向进化。在三轮诱变和基于生长的筛选之后，我们分离出含有六个突变（E15D、E114G、E129D、T142S、A177T和V433I）的变体，其表现出酶活性增加77%。当在含有gre3敲除和tall和XKS 1过表达的最低限度工程化酵母宿主中表达时，表达该突变酶的菌株将其需氧生长速率提高了61倍，乙醇生产和木糖消耗速率提高了近8倍。此外，与野生型酶不同，突变酶使这些酵母在限氧发酵条件下能够生产乙醇。在微需氧条件下，表达突变木糖异构酶的菌株的乙醇生产率显著高于先前报道的含有木糖异构酶途径的酵母的值，并且也与含有氧化还原酶途径的菌株的乙醇生产率相当。因此，这项研究显示了发展木糖异构酶途径以更有效地利用木糖的潜力。
The heterologous expression of a highly functional xylose isomerase pathway in Saccharomyces cerevisiae would have significant advantages for ethanol yield, since the pathway bypasses cofactor requirements found in the traditionally used oxidoreductase pathways. However, nearly all reported xylose isomerase-based pathways in S. cerevisiae suffer from poor ethanol productivity, low xylose consumption rates, and poor cell growth compared with an oxidoreductase pathway and, additionally, often require adaptive strain evolution. Here, we report on the directed evolution of the Piromyces sp. xylose isomerase (encoded by xylA) for use in yeast. After three rounds of mutagenesis and growth-based screening, we isolated a variant containing six mutations (E15D, E114G, E129D, T142S, A177T, and V433I) that exhibited a 77% increase in enzymatic activity. When expressed in a minimally engineered yeast host containing a gre3 knockout and tall and XKS1 overexpression, the strain expressing this mutant enzyme improved its aerobic growth rate by 61-fold and both ethanol production and xylose consumption rates by nearly 8-fold. Moreover, the mutant enzyme enabled ethanol production by these yeasts under oxygen-limited fermentation conditions, unlike the wild-type enzyme. Under microaerobic conditions, the ethanol production rates of the strain expressing the mutant xylose isomerase were considerably higher than previously reported values for yeast harboring a xylose isomerase pathway and were also comparable to those of the strains harboring an oxidoreductase pathway. Consequently, this study shows the potential to evolve a xylose isomerase pathway for more efficient xylose utilization.