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[1]秦丽娟,高瑛,陆信曜,等.产甘油假丝酵母甘油分解代谢CgGCY1、gGCY2和CgDAK基因的克隆、敲除及其功能[J].应用与环境生物学报,2017,23(06):999-1005.[doi:10.3724/SP.J.1145.2017.01017]
　QIN Lijuan,GAO Ying,et al.Cloning, deletion, and function of genes involved in the glycerol catabolism pathway of Candida glycerinogenes[J].Chinese Journal of Applied & Environmental Biology,2017,23(06):999-1005.[doi:10.3724/SP.J.1145.2017.01017]
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产甘油假丝酵母甘油分解代谢CgGCY1、gGCY2和CgDAK基因的克隆、敲除及其功能()
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《应用与环境生物学报》[ISSN:1006-687X/CN:51-1482/Q]

卷:
23卷
期数:
2017年06期
页码:
999-1005
栏目:
微生物资源发掘与生物合成专栏论文
出版日期:
2017-12-25

文章信息/Info

Title:
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;  宗红;  诸葛斌;  方慧英;  宋健
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
Author(s):
QIN Lijuan1;  2;  GAO Ying2;  LU Xinyao2;  ZONG Hong2;  ZHUGE Bin1;  2**;  FANG Huiying1;  2 & SONG Jian3
1 Key Laboratory of Carbohydrate Chemisty and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China 2 Key Laboratory of Industrial Biotechnology, Ministry of Education, Laboratory of Industrial Microorganisms, School of Biotechnology, Jiangnan University, Wuxi 214122, China 3 School of Chemical and Material Engineering, Jiangnan University, Wuxi 214122, China
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
Keywords:
Candida glycerinogenes;  catabolism of glycerol;  glycerol dehydrogenase;  dihydroxyacetone kinase
分类号:
Q786 : Q939.97
DOI:
10.3724/SP.J.1145.2017.01017
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CgGCY1、CgGCY2和CgDAK. 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. qRT-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. （图8 表3 参21）
Abstract:
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.

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更新日期/Last Update: 2017-12-25