Improvement of xylose uptake and ethanol production in recombinant Saccharomyces cerevisiae through an inverse metabolic engineering approach
Improvement of xylose uptake and ethanol production in recombinant Saccharomyces cerevisiae through an inverse metabolic engineering approach
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DOI：
10.1128/aem.71.12.8249-8256.2005
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发表时间：
2005-12-01
影响因子：
4.4
通讯作者：
Stephanopoulos, G
中科院分区：
文献类型：
--
作者：
Jin, YS;Alper, H;Stephanopoulos, G
我们使用逆代谢工程的方法，以确定基因的目标，以改善重组酿酒酵母中的木糖同化。具体来说，我们从树干毕赤酵母中构建了一个基因组片段文库，并将其导入重组S。酿酒酵母表达XYL 1和XYL 2。通过对文库的连续传代富集，鉴定了16个转化子，并证实其在木糖上具有较高的生长速率。从这些转化体分离的16个质粒的测序显示，大多数插入片段（16个中的10个）含有XYL 3基因，从而证实了先前的发现，即XYL 3是增加木糖同化的共有靶标。在顺序搜索基因靶点后，我们在XYL 1 XYL 2 XYL 3背景中重复互补富集过程，并鉴定了15个快速生长的转化体，所有这些转化体都含有相同的质粒。该质粒含有一个开放阅读框（ORF），基于与S的高度同源性命名为PsTAL 1。酿酒酵母TAL 1。为了进一步研究新鉴定的PsTAL 1 ORF是否负责增强生长表型，我们构建了在组成型启动子控制下含有PsTAL 1 ORF的表达盒，并将其转化到S.表达XYL 1、ATL 2和XYL 3的酿酒酵母重组体。得到的重组菌株表现出100%的生长速率增加和70%的乙醇产量增加（0.033对0.019 g乙醇/g细胞（.）h）与亲本菌株相比对木糖的影响。有趣的是，与ScTAL 1基因的过表达不同，当细胞在葡萄糖上生长时，PsTAL 1的过表达不会引起生长抑制。这些结果表明，PsTAL 1是重组S.啤酒。
We used an inverse metabolic engineering approach to identify gene targets for improved xylose assimilation in recombinant Saccharomyces cerevisiae. Specifically, we created a genomic fragment library from Pichia stipitis and introduced it into recombinant S. cerevisiae expressing XYL1 and XYL2. Through serial subculturing enrichment of the transformant library, 16 transformants were identified and confirmed to have a higher growth rate on xylose. Sequencing of the 16 plasmids isolated from these transformants revealed that the majority of the inserts (10 of 16) contained the XYL3 gene, thus confirming the previous finding that XYL3 is the consensus target for increasing xylose assimilation. Following a sequential search for gene targets, we repeated the complementation enrichment process in a XYL1 XYL2 XYL3 background and identified 15 fast-growing transformants, all of which harbored the same plasmid. This plasmid contained an open reading frame (ORF) designated PsTAL1 based on a high level of homology with S. cerevisiae TAL1. To further investigate whether the newly identified PsTAL1 ORF is responsible for the enhanced-growth phenotype, we constructed an expression cassette containing the PsTAL1 ORF under the control of a constitutive promoter and transformed it into an S. cerevisiae recombinant expressing XYL1, ATL2, and XYL3. The resulting recombinant strain exhibited a 100% increase in the growth rate and a 70% increase in ethanol production (0.033 versus 0.019 g ethanol/g cells (.) h) on xylose compared to the parental strain. Interestingly, overexpression of PsTAL1 did not cause growth inhibition when cells were grown on glucose, unlike overexpression of the ScTAL1 gene. These results suggest that PsTAL1 is a better gene target for engineering of the pentose phosphate pathway in recombinant S. cerevisiae.