Central Laboratories for Frontier Technology, KIRIN Holdings Co., Ltd., 1-13-5 Fukuura Kanazawa-ku, Yokohama, Kanagawa 236-0004, Japan.
J Biosci Bioeng. 2013 May;115(5):532-9. doi: 10.1016/j.jbiosc.2012.12.001. Epub 2013 Jan 5.
We previously reported the construction of a recombinant Candida utilis strain expressing mXYL1, XYL2 and XYL3, which encode mutated Candida shehatae xylose reductase K275R/N277D, C. shehatae xylitol dehydrogenase and Pichia stipitis xylulokinase to produce ethanol from xylose. However, its productivity was low. In this study, to breed a strain with higher productivity of ethanol from xylose, we used a cocktail multicopy integration method to attain optimized gene dosage of the three enzymes. Gene expression cassettes of the xylose-metabolizing enzymes were simultaneously integrated into C. utilis chromosomes in one step. Measurement of integrated gene copy number and xylose fermentability in all of the resulting integrant strains revealed that the copy number ratio of XYL2/mXYL1 in strains with higher ethanol yield was higher than that in strains with lower ethanol yield, whereas the copy number ratio of mXYL1/XYL3 was lower in strains with higher ethanol yield. The resultant strain CIS35, which was found to be the best producer of ethanol from xylose produced 29.2 g/L of ethanol, yielding 0.402 g ethanol/g xylose. This result provides that C. utilis may be a good candidate as a host for ethanol production from xylose.
我们之前曾报道过构建一株能够表达 mXYL1、XYL2 和 XYL3 的重组 Candida utilis 菌株，这三种酶分别编码突变型 Candida shehatae 木糖还原酶 K275R/N277D、C. shehatae 木糖醇脱氢酶和 Pichia stipitis 木酮糖激酶，可将木糖转化为乙醇。然而，其生产效率较低。在这项研究中，为了培育出一种能够从木糖生产出更高产量乙醇的菌株，我们使用鸡尾酒多拷贝整合方法来获得这三种酶的最佳基因剂量。木糖代谢酶的基因表达盒一步同时整合到 C. utilis 染色体中。对所有整合子菌株的整合基因拷贝数和木糖发酵能力进行测量，结果显示，在乙醇产量较高的菌株中，XYL2/mXYL1 的基因拷贝数比值高于乙醇产量较低的菌株，而 mXYL1/XYL3 的基因拷贝数比值在乙醇产量较高的菌株中较低。结果发现，从木糖生产乙醇能力最强的菌株 CIS35 可生产 29.2 g/L 的乙醇，得率为 0.402 g 乙醇/g 木糖。这一结果表明，C. utilis 可能是一种从木糖生产乙醇的良好宿主。
