热带假丝酵母高效利用甘油研究
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摘要
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 500 bp DNA fragment gkpR in gk gene was cloned by using PCR,gkpR and a G418 resistance gene( kanr)was connected to vector of pPICzαA by One Step Cloning Kit,the promoter gene of p GAP from C. lipolytica 1457 will be connected to the gkpR in recombinant vector by One Step Cloning Kit. The recombinant plasmid pPICzαAgkp was transformed into C. tropicalis 1798 competent cells. Through a single homologous exchange,the promoter p GK was replaced by pGAP. After G418 resistance experiments, PCR determination, the p AP promoter replacement C. tropicalis 1798-gk Pr was obtained successfully; The verification results offer mentation shows,The analysis founded that the OD_(600) 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.
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 500 bp DNA fragment gkpR in gk gene was cloned by using PCR,gkpR and a G418 resistance gene( kanr)was connected to vector of pPICzαA by One Step Cloning Kit,the promoter gene of p GAP from C. lipolytica 1457 will be connected to the gkpR in recombinant vector by One Step Cloning Kit. The recombinant plasmid pPICzαAgkp was transformed into C. tropicalis 1798 competent cells. Through a single homologous exchange,the promoter p GK was replaced by pGAP. After G418 resistance experiments, PCR determination, the p AP promoter replacement C. tropicalis 1798-gk Pr was obtained successfully; The verification results offer mentation shows,The analysis founded that the OD_(600) 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.
引文
[1]A dedayo M R,Ajiboye E A,Akintunde J K,et al.Single cell proteins:As nutritional enhancer.Advances in Applied Science Research.,2011,2(5):396-409.
[2]刘继东,付灿,王同阳.热带假丝酵母在木薯淀粉酒精废液治理中的应用研究.中国酿造,2009,208(7):121-123.Liu J D,Fu C,Wang T Y.Application of Candida tropicallis in the treatment of cassava-starch-alcohol waste water.China Brewing,2009,208(7):121-123.
[3]Gao Y R,Li D P,Liu Y.Production of single cell protein from soy molasses using Candida tropicalis.Ann Microbiol,2011,62(3):1165-1172.
[4]刘祖同.长链二元酸发酵的研究.微生物学通报,1979,19(1):71-75.Liu Z T.Study on long-chain dicarboxylic acid fermentation.Microbiology Bulletin,1979,19(1):71-75.
[5]桂秋芬,姚嘉旻,蒋洋松,等.利用热带假丝酵母发酵生产长链二元酸的研究进展.化学与生物工程,2014,31(1):17-22.Gui Q F,Yao J M,Jiang Y S,et al.Advances in production of long-chain dicarboxylic acid by fermentation of tropical candida.Chemistry&Bioengineering,2014,31(1):17-22.
[6]Blandin G,Ozier-Kalogeropoulos O,Wincker P,et al.Genomic exploration of the hemiascomycetous yeasts:16.Candida tropicalis.FEBS Letters,2000,487(1):91-94.
[7]诸葛健,王正祥.产甘油假丝酵母甘油代谢关键酶的研究.微生物学报,1999,39(1):91-93.Zhu G J,Wang Z X.Study on key enzymes of glycerol metabolism of candida glycerol.Acta Microbiological Sinica,1999,39(1):91-93.
[8]Gerhard M.Biochemical Pathways.3nded.Germany:Boehringer Mannheim Press,1996.
[9]郭雪娜,诸葛斌,诸葛健.甘油代谢中甘油激酶的研究进展.微生物学报,2002,42(4):510-513.Guo X N,Zhu Ge B,Zhu Ge J.Research progress on the glycerol kinase.Acta Microbiological Sinica,2002,42(4):510-513.
[10]谢涛,方慧英,诸葛健,等.渗透压对产甘油假丝酵母甘油合成与胞内磷积累的影响.中国生物工程杂志,2009,29(4):61-66.Xie T,Fang H Y,Zhu G J,et al.Effect of osmotic pressure on glycerol production and intracellular phosphorus accumulation in Candida.China Biotechnology,2009,29(4):61-66.
[11]王正祥,诸葛健,方慧英.耐高渗压高产甘油的假丝酵母新种一产甘油假丝酵母.微生物学报,1999,39(l):68-74.Wang Z X,Zhuge J,Fang H Y,et al.A new breviscapus of candida glycerin with high osmotic pressure and high yield glycerol.Acta Microbiological Sinica.1999,39(l):68-74.
[12]Shen W,Wang Z X,Rao Z M,et al.A genetic transformationsystem based on trp1 complementation in Candida glycerinogenes.World Journal of Microbiology and Biotechnology,2011,27(4):1005-1008.
[13]奥斯博F M,金士顿R E.精编分子生物学实验指南.北京:科学出版社,1998.Osbo F M,Kingston R E.A Guide to Molecular Biology Experiment.Beijing:Science Press,1998.
[14]邢竹青,王彦宁,刘兆贤,等.Lactobacillus kefiranofaciens乳糖酶基因克隆及在毕赤酵母中表达.中国酿造,2016,35(1):10-13.Xing Z Q,Wang Y N,Liu Z X,et a.Cloning of Lactobacillus kefiranofaciensβ-galactosidase and expression in Pichia pastoris.China Brewing,2016,35(1):10-13.
[15]Wolff A M,Arnaul J.Cloning of glyceraldehyde-3-phosphate dehydrogenase-encoding genes in Mucor circinelloides(Syn.racemosus)and use of the gpdl promoter for pecombinant protein production.Fungal Genetics and Biology,2002,35(1):21-29.
[16]Schmitt E K,Kempken R,Kuck U.Functional analysis of promoter sequences of cephalosporin C biosynthesis genes from Acremonium chrysogenum:specific DNA-protein interactions and characterization of the transcription factor PACC.Mol Genet Genomics,2001,265(3):508-510.
[17]刘桂明,赵智,张英姿,等.谷氨酸棒杆菌10147基因组中启动子活性片段的克隆与分析.微生物学报,2009,49(7):972-977.Liu G M,Zhao Z,Zhang Y Z,et al.Cloning and analysis of promoter fragment of Corynebacterium glutamicum 10147 genome.Acta Microbiological Sinica,2009,49(7):972-977.
[18]耿宏伟,侯红燕,王丕武,等.p GAP-毕赤酵母表达系统的研究进展.食品工程技术,2011,29(1):159-161.Gong H W,Hou H Y,Wang P W,et al.Research progress of p GAP-pichia pastoris expression system.Food Engineering Technology,2011,29(1):159-161.
[19]Xu Y,Tu Z.Application and progress of filamentous fungi gene targeting.J Food Sci Biotechnol,2007,26(1):120-126.
[20]Xiang Z,Chen X Z,Zhang L H,et al.Development of a genetic transformation system for Candida tropicalis based on a reusable selection marker of URA3 gene.Hereditas,2014,36(10):1053-1061.
[21]Xue K J,Liu B,Chang S H,et al.A high efficient method to knockout target gene by two-step homologous recombination in Pichia pastoris.Lett Biotechnol,2010,21(5):650-654.
[2]刘继东,付灿,王同阳.热带假丝酵母在木薯淀粉酒精废液治理中的应用研究.中国酿造,2009,208(7):121-123.Liu J D,Fu C,Wang T Y.Application of Candida tropicallis in the treatment of cassava-starch-alcohol waste water.China Brewing,2009,208(7):121-123.
[3]Gao Y R,Li D P,Liu Y.Production of single cell protein from soy molasses using Candida tropicalis.Ann Microbiol,2011,62(3):1165-1172.
[4]刘祖同.长链二元酸发酵的研究.微生物学通报,1979,19(1):71-75.Liu Z T.Study on long-chain dicarboxylic acid fermentation.Microbiology Bulletin,1979,19(1):71-75.
[5]桂秋芬,姚嘉旻,蒋洋松,等.利用热带假丝酵母发酵生产长链二元酸的研究进展.化学与生物工程,2014,31(1):17-22.Gui Q F,Yao J M,Jiang Y S,et al.Advances in production of long-chain dicarboxylic acid by fermentation of tropical candida.Chemistry&Bioengineering,2014,31(1):17-22.
[6]Blandin G,Ozier-Kalogeropoulos O,Wincker P,et al.Genomic exploration of the hemiascomycetous yeasts:16.Candida tropicalis.FEBS Letters,2000,487(1):91-94.
[7]诸葛健,王正祥.产甘油假丝酵母甘油代谢关键酶的研究.微生物学报,1999,39(1):91-93.Zhu G J,Wang Z X.Study on key enzymes of glycerol metabolism of candida glycerol.Acta Microbiological Sinica,1999,39(1):91-93.
[8]Gerhard M.Biochemical Pathways.3nded.Germany:Boehringer Mannheim Press,1996.
[9]郭雪娜,诸葛斌,诸葛健.甘油代谢中甘油激酶的研究进展.微生物学报,2002,42(4):510-513.Guo X N,Zhu Ge B,Zhu Ge J.Research progress on the glycerol kinase.Acta Microbiological Sinica,2002,42(4):510-513.
[10]谢涛,方慧英,诸葛健,等.渗透压对产甘油假丝酵母甘油合成与胞内磷积累的影响.中国生物工程杂志,2009,29(4):61-66.Xie T,Fang H Y,Zhu G J,et al.Effect of osmotic pressure on glycerol production and intracellular phosphorus accumulation in Candida.China Biotechnology,2009,29(4):61-66.
[11]王正祥,诸葛健,方慧英.耐高渗压高产甘油的假丝酵母新种一产甘油假丝酵母.微生物学报,1999,39(l):68-74.Wang Z X,Zhuge J,Fang H Y,et al.A new breviscapus of candida glycerin with high osmotic pressure and high yield glycerol.Acta Microbiological Sinica.1999,39(l):68-74.
[12]Shen W,Wang Z X,Rao Z M,et al.A genetic transformationsystem based on trp1 complementation in Candida glycerinogenes.World Journal of Microbiology and Biotechnology,2011,27(4):1005-1008.
[13]奥斯博F M,金士顿R E.精编分子生物学实验指南.北京:科学出版社,1998.Osbo F M,Kingston R E.A Guide to Molecular Biology Experiment.Beijing:Science Press,1998.
[14]邢竹青,王彦宁,刘兆贤,等.Lactobacillus kefiranofaciens乳糖酶基因克隆及在毕赤酵母中表达.中国酿造,2016,35(1):10-13.Xing Z Q,Wang Y N,Liu Z X,et a.Cloning of Lactobacillus kefiranofaciensβ-galactosidase and expression in Pichia pastoris.China Brewing,2016,35(1):10-13.
[15]Wolff A M,Arnaul J.Cloning of glyceraldehyde-3-phosphate dehydrogenase-encoding genes in Mucor circinelloides(Syn.racemosus)and use of the gpdl promoter for pecombinant protein production.Fungal Genetics and Biology,2002,35(1):21-29.
[16]Schmitt E K,Kempken R,Kuck U.Functional analysis of promoter sequences of cephalosporin C biosynthesis genes from Acremonium chrysogenum:specific DNA-protein interactions and characterization of the transcription factor PACC.Mol Genet Genomics,2001,265(3):508-510.
[17]刘桂明,赵智,张英姿,等.谷氨酸棒杆菌10147基因组中启动子活性片段的克隆与分析.微生物学报,2009,49(7):972-977.Liu G M,Zhao Z,Zhang Y Z,et al.Cloning and analysis of promoter fragment of Corynebacterium glutamicum 10147 genome.Acta Microbiological Sinica,2009,49(7):972-977.
[18]耿宏伟,侯红燕,王丕武,等.p GAP-毕赤酵母表达系统的研究进展.食品工程技术,2011,29(1):159-161.Gong H W,Hou H Y,Wang P W,et al.Research progress of p GAP-pichia pastoris expression system.Food Engineering Technology,2011,29(1):159-161.
[19]Xu Y,Tu Z.Application and progress of filamentous fungi gene targeting.J Food Sci Biotechnol,2007,26(1):120-126.
[20]Xiang Z,Chen X Z,Zhang L H,et al.Development of a genetic transformation system for Candida tropicalis based on a reusable selection marker of URA3 gene.Hereditas,2014,36(10):1053-1061.
[21]Xue K J,Liu B,Chang S H,et al.A high efficient method to knockout target gene by two-step homologous recombination in Pichia pastoris.Lett Biotechnol,2010,21(5):650-654.