title: [Expression of Vitreoscilla hemoglobin improves recombinant lipase production in Pichia pastoris].
abstract: Yarrowia lipolytica lipase Lip2 (YlLip2) is an important industrial enzyme with many potential applications. To alleviate the dissolved oxygen (DO) limitation and improve YlLip2 production during high-cell density fermentation, the YlLip2 gene lip2 and Vitreoscilla hemoglobin (VHb) gene vgb were co-expressed in Pichiapastoris under the control of AOX1 and PsADH2 promoter, respectively. The PsADH2 promoter from Pichia stipitis could be activated under oxygen limitation. The SDS-PAGE and CO-difference spectrum analysis indicated that VHb and YlLip2 had successfully co-expressed in recombinant strains. Compared with the control cells (VHb-, GS115/9Klip2), the expression levels of YlLip2 in VHb-expressing cells (VHb+, GS115/9Klip2-pZPVT) under oxygen limitation were improved 25% in shake-flask culture and 83% in a 10 L fermentor. Moreover, the VHb+ cells displayed higher biomass than VHb- cells at lower DO levels in a 10 L fermentor. In this study, we also achieved a VHb-expressing clone harboring multicopy lip2 gene (GS115/9Klip2-pZPVTlip2 49#), which showed the maximum lipolytic activity of 33 900 U/mL in a 10 L fermentor under lower DO conditions. Therefore, it can be seen that expression of VHb with PsADH2 promoter in P. pastoris combined with increasing copies of lip2 gene is an effective strategy to improve YlLip2 production.
authors.affiliation: Key Laboratory of Molecular Bio-physics, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China.
authors.forename: Xiaofeng
authors.initials: X
authors.lastname: Wang
authors.forename: Yongchuan
authors.initials: Y
authors.lastname: Sun
authors.forename: Xuguang
authors.initials: X
authors.lastname: Shen
authors.forename: Feng
authors.initials: F
authors.lastname: Ke
authors.forename: Li
authors.initials: L
authors.lastname: Xu
authors.forename: Yun
authors.initials: Y
authors.lastname: Liu
authors.forename: Yunjun
authors.initials: Y
authors.lastname: Yan
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D005285:Fermentation; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000235:genetics; D008049:Lipase / Q000096:biosynthesis* / Q000235:genetics; D010100:Oxygen / Q000032:analysis / Q000494:pharmacology; D010843:Pichia / Q000378:metabolism*; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D054793:Truncated Hemoglobins / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D005656:Fungal Proteins; D011994:Recombinant Proteins; D054793:Truncated Hemoglobins; C082613:hemoglobin protein, Vitreoscilla; C408971:LIP2 protein, Yarrowia lipolytica; D008049:Lipase; D010100:Oxygen