Verdezyne, Carlsbad, CA 92010, USA.
J Ind Microbiol Biotechnol. 2012 Nov;39(11):1597-604. doi: 10.1007/s10295-012-1169-y. Epub 2012 Aug 5.
We have integrated and coordinately expressed in Saccharomyces cerevisiae a xylose isomerase and cellobiose phosphorylase from Ruminococcus flavefaciens that enables fermentation of glucose, xylose, and cellobiose under completely anaerobic conditions. The native xylose isomerase was active in cell-free extracts from yeast transformants containing a single integrated copy of the gene. We improved the activity of the enzyme and its affinity for xylose by modifications to the 5'-end of the gene, site-directed mutagenesis, and codon optimization. The improved enzyme, designated RfCO*, demonstrated a 4.8-fold increase in activity compared to the native xylose isomerase, with a K(m) for xylose of 66.7 mM and a specific activity of 1.41 μmol/min/mg. In comparison, the native xylose isomerase was found to have a K(m) for xylose of 117.1 mM and a specific activity of 0.29 μmol/min/mg. The coordinate over-expression of RfCO* along with cellobiose phosphorylase, cellobiose transporters, the endogenous genes GAL2 and XKS1, and disruption of the native PHO13 and GRE3 genes allowed the fermentation of glucose, xylose, and cellobiose under completely anaerobic conditions. Interestingly, this strain was unable to utilize xylose or cellobiose as a sole carbon source for growth under anaerobic conditions, thus minimizing yield loss to biomass formation and maximizing ethanol yield during their fermentation.
我们在酿酒酵母中整合并协调表达了来自瘤胃球菌的木糖异构酶和纤维二糖磷酸酶，使酵母在完全厌氧条件下发酵葡萄糖、木糖和纤维二糖。天然木糖异构酶在含有单个整合基因拷贝的酵母转化体的无细胞提取物中具有活性。我们通过基因 5'端的修饰、定点突变和密码子优化来提高酶的活性及其对木糖的亲和力。改良后的酶命名为 RfCO*，与天然木糖异构酶相比，其活性提高了 4.8 倍，对木糖的 K(m)值为 66.7 mM，比活为 1.41 μmol/min/mg。相比之下，天然木糖异构酶对木糖的 K(m)值为 117.1 mM，比活为 0.29 μmol/min/mg。RfCO*与纤维二糖磷酸酶、纤维二糖转运蛋白、内源性基因 GAL2 和 XKS1 的协调过表达，以及天然 PHO13 和 GRE3 基因的破坏，使得酵母在完全厌氧条件下能够发酵葡萄糖、木糖和纤维二糖。有趣的是，该菌株在厌氧条件下无法将木糖或纤维二糖作为唯一碳源用于生长，从而最大限度地减少生物质形成的产量损失，并在发酵过程中最大限度地提高乙醇产量。
