解脂耶氏酵母(Yarrowia lipolytica)ATCC30162脂肪酶基因Yllip1和Yllip2的结构及表达特征
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摘要
以目前报道油脂产量最高的解脂耶氏酵母菌株(Yarrowia lipolytica)ATCC 30162为对象,采用逆转录PCR扩增到脂肪酶编码基因Yllip1和Yllip2,编码产物分别为816和549个氨基酸。保守结构域预测表明,Yllip1包含Patatin类磷脂酶和功能未知的DUF3336结构域,而Yllip2包含lipase_3类脂肪酶结构域,且这两个蛋白都具有1～4个跨膜区域。与不同物种来源的脂肪酶同源蛋白的多序列比对表明Yllip1和Yllip2分别包含8和6个保守区域,这些生物信息学分析表明这两个来源于解脂耶氏酵母的脂肪酶作用底物可能分别为细胞内膜磷脂和酰基甘油酯。荧光定量PCR分析表明:培养基中添加油酸在短期内(6 h)诱导了这两个脂肪酶基因Yllip1和Yllip2的显著上调表达,表明它们可能参与了酵母分解利用油酸的生化过程。
Reverse transcription PCR was conducted to amplify two lipase genes from the yeast strain Yarrowia lipolytica ATCC 30162,which was reported to produce highest lipid yield till now. The sizes of the encoding products of these two lipase genes,Yllip1 and Yllip2,are 816 and 549 amino acids,respectively. Prediction of conserved domain suggests that Yllip1 contains a Patatin - like phospholipase and a DUF3336 conserved domain with unknown function,whereas Yllip2 contains a lipase_3 class domain. Both of these two lipase proteins have 1～4 transmembrane regions.Multiple sequence alignment of Yllip1,Yllip2 and their homologues shows that they contain 8 and 6 conserved regions,respectively. These bioinformatics analyses suggest that these two lipase proteins from the yeast Yarrowia lipolytica ATCC 30162 may have different substrates with Yllip1 using intracellular membrane phospholipids as substrate while Yl-lip2 using acyl glycerides as substrate. Quantitative RT-PCR analysis shows that supplementation of oleic acid in the media within 6 h induces the up-regulation of both Yllip1 and Yllip2,suggesting that these two lipases possibly participate in the biochemical process of the utilization of oleic acid.
Reverse transcription PCR was conducted to amplify two lipase genes from the yeast strain Yarrowia lipolytica ATCC 30162,which was reported to produce highest lipid yield till now. The sizes of the encoding products of these two lipase genes,Yllip1 and Yllip2,are 816 and 549 amino acids,respectively. Prediction of conserved domain suggests that Yllip1 contains a Patatin - like phospholipase and a DUF3336 conserved domain with unknown function,whereas Yllip2 contains a lipase_3 class domain. Both of these two lipase proteins have 1～4 transmembrane regions.Multiple sequence alignment of Yllip1,Yllip2 and their homologues shows that they contain 8 and 6 conserved regions,respectively. These bioinformatics analyses suggest that these two lipase proteins from the yeast Yarrowia lipolytica ATCC 30162 may have different substrates with Yllip1 using intracellular membrane phospholipids as substrate while Yl-lip2 using acyl glycerides as substrate. Quantitative RT-PCR analysis shows that supplementation of oleic acid in the media within 6 h induces the up-regulation of both Yllip1 and Yllip2,suggesting that these two lipases possibly participate in the biochemical process of the utilization of oleic acid.
引文
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[15] Beckerich J M, Boisrame-Baudevin A, Gaillardin C.Yarrowia lipolytica:a model organism for protein secre-tion studies[J]. International Microbiology, 1998, 1(2):123-130.
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[17] Mileto D, Brocca S, Lotti M, et al. Characterization of the Candida rugosa lipase system and overexpression of the Lip1 isoenzyme in a non-conventional yeast[J].Chemistry and Physics of Lipids, 1998, 93(1-2):47-55.
[18] Pignede G, Wang H, Fudalej F, et al. Characterization of an extracellular lipase encoded by LIP2 in Yarrowia lipolytica[J]. Journal of Bacteriology, 2000, 182(10):2802-2810.
[19] Kamoun J, SchuéM, Messaoud W, et al. Biochemical characterization of Yarrowia lipolytica LIP8, a secret-ed lipase with a cleavable C-terminal region[J]. Bio-chim Biophys Acta, 2015, 1851(2):129-140.
[2] Zang W, Yan B, Meng Y H, et al. Study on the pro-duction of ferulic acid by hydrolysis of oryzanol by lipo-polysaccharide Lip2 lipase[J]. Food Science and Tech-nology, 2014,35(22):189-192.张宸,严勃,孟永宏,等.解脂耶氏酵母Lip2脂肪酶水解谷维素产生阿魏酸的研究[J].食品工业科技, 2014,35(22):189-192.
[3] Xiao C L. The twilight and application of microbial li-pase[J]. Journal of Microbiology,1997, 17(4):56-59.肖春玲.微生物脂肪酶的硏光与应用[J].微生物学杂志,1997, 17(4):56-59.
[4] de Azeredo L A, Gomes P M, Sant′Anna G L Jr, et al. Production and regulation of lipase activity from Pen-icillium restrictum in submerged and solid-state fermen-tations[J]. Curr Microbiol, 2007, 54(5):361-365.
[5] Thakur S. Lipases, its sources, properties and applica-tions:a review[J]. Int J Sci Eng Res, 2012, 3:1-29.
[6] Ciafardini G, Zullo BA, Iride A. Lipase production by yeasts from extra virgin olive oil[J]. Food Microbiol,2006, 23:60-67.
[7] Potumarthi P, Subhakar C, Vanajakshi J, et al. Effect of aeration and agitation regimes on lipase production by newly isolated Rhodotorula mucilaginosa-MTCC 8737in stirred tank reactor using molasses as sole carbon source[J]. Appl Biochem Biotechnol, 2008, 151:700-710.
[8] Thakur S. Extracellular lipase producing bacterial strains[J]. Biochem J, 2014, 62:114-116.
[9] Arpigny J L, Jaeger K E. Bacterial lipolytic enzymes:classification and properties[J]. Biochem J, 1999, 343:177-183.
[10] Bayer S, Kunert A, Ballschmiter M, et al. Indication for a new lipolytic enzyme family:isolation and charac-terization of two esterases from a metagenomic library[J]. J Mol Microbiol Biotechnol, 2010, 18:181-187.
[11] Fu J, Leiros H K, de Pascale D, et al. Landfald B.Functional and structural studies of a novel cold adapted esterase from an arctic intertidal metagenomic library[J]. Appl Microbiol Biotechnol, 2012, 9:3965-3978.
[12] Lenfant N, Hotelier T, Velluet E, et al. Chatonnet A.ESTHER, the database of the a/b-hydrolase fold super-family of proteins:tools to explore diversity of functions[J]. Nucleic Acids Res, 2013, 41:423-429.
[13] Fischer M, Pleiss J. The lipase engineering database:a navigation and analysis tool for protein families[J]. Nu-cleic Acid Res, 2003, 31:319-321.
[14] Widmann M, Juhl P B, Pleiss J. Structural classifica-tion by the Lipase Engineering Database:a case study of Candida antarctica lipase A[J]. BMC Genomics,2010, 11:123-129.
[15] Beckerich J M, Boisrame-Baudevin A, Gaillardin C.Yarrowia lipolytica:a model organism for protein secre-tion studies[J]. International Microbiology, 1998, 1(2):123-130.
[16] Peters I I, Nelson F E. Factors influencing the produc-tion of lipase by Mycotorula lipolytica[J]. Journal of Bacteriology, 1948, 55(5):581-591.
[17] Mileto D, Brocca S, Lotti M, et al. Characterization of the Candida rugosa lipase system and overexpression of the Lip1 isoenzyme in a non-conventional yeast[J].Chemistry and Physics of Lipids, 1998, 93(1-2):47-55.
[18] Pignede G, Wang H, Fudalej F, et al. Characterization of an extracellular lipase encoded by LIP2 in Yarrowia lipolytica[J]. Journal of Bacteriology, 2000, 182(10):2802-2810.
[19] Kamoun J, SchuéM, Messaoud W, et al. Biochemical characterization of Yarrowia lipolytica LIP8, a secret-ed lipase with a cleavable C-terminal region[J]. Bio-chim Biophys Acta, 2015, 1851(2):129-140.