热带假丝酵母高效利用甘油研究

Studies on Efficient Utilization of Glycerol of Candida tropicalis 1798

目的：热带假丝酵母以油脂为底物发酵时会产生副产物甘油,研究对热带假丝酵母gk基因进行过表达,将副产物甘油转化为能量,提高油脂转化利用效率。方法：以热带假丝酵母Candida tropicalis 1798中的甘油激酶（gk）为研究对象,利用PCR技术获得同源臂基因gkpR,通过一步法无缝克隆将同源臂和G418抗性基因（kanr）连接至pPICzαA载体,同时将解脂假丝酵母Candida lipolytica 1457中的启动子基因pGAP无缝连接至载体中的gkpR,构成质粒pPICzαA-gkp,并电转化至C.tropicalis 1798感受态细胞中,通过一次同源单交换,将启动子pGK替换为pGAP。结果：经过G418抗性筛选和PCR鉴定,成功获得pGAP基因替换菌株C.tropicalis 1798-gkPr;发酵验证结果显示,启动子基因替换C.tropicalis 1798在以甘油为底物培养时重组菌OD600值比野生型菌株高46.4%,重组菌培养基中甘油剩余量比野生菌降低56.1%,表明启动子替换能促进C.tropicalis1798对甘油的吸收利用。此外,以油脂为底物进行发酵实验时还发现重组菌产长链二元酸的量比野生菌提高32.7%。结论：通过启动子替换手段构建的重组菌C.tropicalis 1798-gkPr,提高了热带假丝酵母对油脂组分中甘油成分的利用效率。

Candida tropicalis1798 can produce glycerol when ferment grease. C. tropicalis1798 efficient use of glycerol and it will provides energy for fermentation and improve the utilization of grease by genetically modified. A related glycerol metabolism gene gk in C. tropicalis 1798 was intended to replace the promoter gene about 500bp DNA fragment gkpR in gk gene was cloned by using PCR, gkpR and a G418 resistance gene （kant） was connected to vector will be connected to the of pPICzaA by One Step Cloning Kit ,the promoter gene of pGAP from C. lipolytica 1457 gkpR in recombinant vector by One Step Cloning Kit. The recombinant plasmid pPICzaAgkp was transformed into C. tropicalis 1798 competent cells. Through a single homologous exchange, the promoter pGK was replaced by pGAP. After G418 resistance experiments, PCR determination, the pGAP promoter replacement C. tropicalis 1798-gkPr was obtained successfully;The verification results offer mentation shows, The analysis founded that the OD~ of C. tropicalis 1798-gkPr was 46.4% higher than that of C. tropicalis 1798 and the glycerol content of C. tropicalis 1798-gkPr was accounted for just 56.1% when it was cultured for 12 hours with glycerin as substrate. It revealed that the replacement of promoter pGAP gene affected utilization of glycerol in C. tropicalis 1798-gkPr. Another analysis also founded that the long-chain dicarboxylic acid production of C. tropicalis 1798-gkPr was 32.7% higher than that of C. tropicalis 1798 when it was cultured for 6 days with oil as substrate. Conclusion： A C. tropicalis 1798 strain with the replaced promote gene was successfully constructed and it increased the utilization of C. tropicalis 1798 for glycerol constituents in the grease component.