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| Identification of Glycerol Transporter in Pichia pastoris and Function Research |
| ZHAN Chun-jun1,2,3, LI Xiang1,2,3, LIU Guo-qiang1,2,3, LIU Xiu-xia1,2,3, YANG Yan-kun1,2,3, BAI Zhong-hu1,2,3 |
1. National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi 214122, China;
2. The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China;
3. The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China |
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Abstract Object Separation and identification glycerol transporter in Pichia pastoris (P. pastoris) and studing its function. Methods: the candidate glycerol transporter (gt1, GeneID:8197545) was found in NCBI database by bioinformatics, and transmembrance structure was predicted by DAS. Targeted gene fused with egfp was cloned into pPICZ B and pRS424 to study its location in cells; recombinant plasmid (gt1 + pRS424) was transformed into Schizosaccharomyces pombe (S. pombe) to study its function; In order to study the relationship between gt1 and aox1, gt1 was deleted by homologous recombination, and enzyme activity of AOX1 in wild and mutant were detected. Results: according to results of bioinformatics, it was found that GT1 share the same transmembrance regions with sugar transporter 1(stl1) which has been identified as a glycerol transporter in Saccharomuces cerevisiae (S. cerevisiae), results of subcellular localization showed that GT1 located on membrance, S. pombe transformed into gt1 could growth on glycerol medium, but wild type could not, which suggested that GT1 could absorbed glycerol from medium to support cells growth. Moreover, AOX1 achieved constitutive expression in Δgt1. Conclusion: glycerol transporter (gt1) in P. pastoris was separated and identified, and its function in glycerol repression was preliminarily clarified.
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Received: 23 February 2017
Published: 25 July 2017
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