title: Overexpression of Candida rugosa lipase Lip1 via combined strategies in Pichia pastoris.
abstract: In this study, combined strategies were employed to heterologously overexpress Candida rugosa lipase Lip1 (CRL1) in a Pichia pastoris system. The LIP1 gene was systematically codon-optimized and synthesized in vitro. The Lip1 activity of a recombinant strain harboring three copies of the codon-optimized LIP1 gene reached 1200 U/mL in a shake flask culture. Higher lipase activity, 1450 U/mL, was obtained using a five copy number construct. Co-expressing one copy of the ERO1p and BiP chaperones with Lip1p, the CRL1 lipase yield further reached 1758 U/mL, which was significantly higher than that achieved by expressing Lip1p alone or only co-expressing one molecular chaperone. When cultivated in a 3 L fermenter under optimal conditions, the recombinant strain GS115/87-ZA-ERO1p-BiP #7, expressing the molecular chaperones Ero1p and BiP, produced 13,490 U/mL of lipase activity at 130 h, which was greater than the 11,400 U/mL of activity for the recombinant strain GS115/pAO815-α-mCRL1 #87, which did not express a molecular chaperone. This study indicates that a strategy of combining codon optimization with co-expression of molecular chaperones has great potential for the industrial-scale production of pure CRL1.
authors.affiliation: Key Lab of Molecular Biophysics of Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, PR China. Electronic address: xuli@mail.hust.edu.cn.
authors.forename: Xu
authors.initials: X
authors.lastname: Li
authors.affiliation: Key Lab of Molecular Biophysics of Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, PR China.
authors.forename: Zimin
authors.initials: Z
authors.lastname: Liu
authors.affiliation: Key Lab of Molecular Biophysics of Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, PR China.
authors.forename: Guilong
authors.initials: G
authors.lastname: Wang
authors.affiliation: Key Lab of Molecular Biophysics of Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, PR China.
authors.forename: Dujie
authors.initials: D
authors.lastname: Pan
authors.affiliation: Key Lab of Molecular Biophysics of Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, PR China.
authors.forename: Liangcheng
authors.initials: L
authors.lastname: Jiao
authors.affiliation: Key Lab of Molecular Biophysics of Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, PR China. Electronic address: yanyunjun@mail.hust.edu.cn.
authors.forename: Yunjun
authors.initials: Y
authors.lastname: Yan
mesh_terms: D055162:Biocatalysis; D019149:Bioreactors; D002175:Candida / Q000201:enzymology*; D002265:Carboxylic Ester Hydrolases / Q000096:biosynthesis* / Q000302:isolation & purification; D003001:Cloning, Molecular / Q000379:methods*; D003062:Codon / Q000235:genetics; D005285:Fermentation; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D005822:Genetic Vectors / Q000235:genetics; D018832:Molecular Chaperones / Q000378:metabolism; D010843:Pichia / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification
chemical_list: D003062:Codon; D005656:Fungal Proteins; D018832:Molecular Chaperones; D011993:Recombinant Fusion Proteins; D002265:Carboxylic Ester Hydrolases
keywords: Candida rugosa lipase; Codon optimization; Fed-batch fermentation; Molecular chaperone; Pichia pastoris