产甘油假丝酵母甘油分解代谢CgGCY1、CgGCY2和CgDAK基因的克隆、敲除及其功能  

Cloning, deletion, and function of genes involved in the glycerol catabolism pathway of Candida glycerinogenes

作　　者：秦丽娟 高瑛 陆信曜[2] 宗红[2] 诸葛斌 方慧英[1,2] 宋健 

机构地区：[1]江南大学糖化学与生物技术教育部重点实验室,无锡214122 [2]江南大学生物工程学院,工业生物技术教育部重点实验室,工业微生物研究中心,无锡214122 [3]江南大学化学与材料工程学院,无锡214122

出　　处：《应用与环境生物学报》2017年第6期999-1005,共7页Chinese Journal of Applied and Environmental Biology

基　　金：国家自然科学基金项目(31570052,31601456);江苏省自然科学基金项目(BK20140138);111项目(111-2-06)资助~~

摘　　要：产甘油假丝酵母(Candida glycerinogenes)能够利用甘油大量生长菌体而无有机酸、醇等代谢物积累,是潜在的优良宿主细胞.为了解C.glycerinogenes甘油分解代谢途径,成功克隆得到二羟基丙酮(DHA)途径的编码基因Cg GCY1、Cg GCY2和Cg DAK.利用"Ura-Blaster"敲除盒分别构建的缺失突变菌Cggcy1?/gcy2?和Cgdak?均不能在甘油培养基中生长.q RT-PCR及酶活测定结果显示,与葡萄糖培养相比,甘油培养下细胞通过强化糖异生、HMP途径积累生物量,下调EMP途径和副产物合成关键酶表达以弱化有机酸、醇的合成,同时上调TCA循环以补偿EMP途径下调带来的能量和还原力不足,使得生物量提高24.5%而不积累有机酸、醇等代谢物.以甘油为共底物进行木糖发酵,木糖醇产量和转化率达到39.4 g/L和89%,与葡萄糖为共底物相比分别提高了79%和32.8%.本研究表明C.glycerinogenes甘油分解代谢仅依赖于DHA途径,以甘油为共底物更有利于木糖醇的合成和转化;结果可为代谢改造C.glycerinogenes以甘油为共底物合成高附加值化合物打下基础.Candida glycerinogenes is a potentially valuable cell factory that can utilize glycerol as a carbon source to generate high-grade biomass without accumulation of organic acids and alcohol. To understand glycerol catabolism in C. glycerinogenes, the key genes involved in the dihydroxyacetone （DHA） pathway were cloned. The deficient mutants Cggcy1？/gcy2？ and Cgdak？ were unable to grow on the glycerol-based medium, indicating that glycerol catabolism in C. glycerinogenes is solely dependent on the DHA pathway. qRT-PCR and enzyme activity assays showed that when cells were cultivated with glycerol as the sole carbon source, the gluconeogenesis and pentose phosphate （HMP） pathways were upregulated to generate the cell skeleton, the Embden-Meyerhof （EMP） and by-product synthesis pathways were downregulated to reduce the production of organic acids and alcohol, and the tricarboxylic acid （TCA） cycle was upregulated to compensate for the loss of reducing power and energy resulting from the reduced EMP pathway activity. These changes increased biomass production by 25% without accumulation of organic acids and alcohol. When cells were cultivated with glycerol and xylose as co-substrates, the titer and conversion rate of xylitol were 39.4 g/L and 89%, respectively. In comparison with cells cultivated with xylose and glucose as co-substrates, this titer and conversion were 1.79 and 1.32-fold higher, respectively. These results suggest that the use of glycerol as a co-substrate can promote the titer and conversion rate of xylitol. This study provides a valuable insight into the production of high-value chemicals by engineered C. glycerinogenes using glycerol as a co-substrate.

关 键 词：产甘油假丝酵母 甘油分解代谢 甘油脱氢酶 二羟丙酮激酶 

分 类 号：Q78[生物学—分子生物学] Q939.9