title: Codon optimization, promoter and expression system selection that achieved high-level production of Yarrowia lipolytica lipase in Pichia pastoris.
abstract: Lipase (EC 3.1.1.3) stands amongst the most important and promising biocatalysts for industrial applications. In this study, in order to realize a high-level expression of the Yarrowia lipolytica lipase gene in Pichia pastoris, we optimized the codon of LIP2 by de novo gene design and synthesis, which significantly improved the lipase expression when compared to the native lip2 gene. We also comparatively analyzed the effects of the promoter types (PAOX1 and PFLD1) and the Pichia expression systems, including the newly developed PichiaPink system, on lipase production and obtained the optimal recombinants. Bench-top scale fermentation studies indicated that the recombinant carrying the codon-optimized lipase gene syn-lip under the control of promoter PAOX1 has a significantly higher lipase production capacity in the fermenter than other types of recombinants. After undergoing methanol inducible expression for 96h, the wet cell weight of Pichia, the lipase activity and the protein content in the fermentation broth reached their highest values of 262g/L, 38,500U/mL and 2.82g/L, respectively. This study has not only greatly facilitated the bioapplication of lipase in industrial fields but the strategies utilized, such as de novo gene design and synthesis, the comparative analysis among promoters and different generations of Pichia expression systems will also be useful as references for future work in this field.
authors.affiliation: College of Biological and Pharmaceutical Engineering, Wuhan Polytechnic University, Wuhan 430023, China.
authors.forename: Wen-Jing
authors.initials: WJ
authors.lastname: Zhou
authors.affiliation: College of Biological and Pharmaceutical Engineering, Wuhan Polytechnic University, Wuhan 430023, China. Electronic address: jiangke.yang@gmail.com.
authors.forename: Jiang-Ke
authors.initials: JK
authors.lastname: Yang
authors.affiliation: College of Biological and Pharmaceutical Engineering, Wuhan Polytechnic University, Wuhan 430023, China.
authors.forename: Lin
authors.initials: L
authors.lastname: Mao
authors.affiliation: College of Biological and Pharmaceutical Engineering, Wuhan Polytechnic University, Wuhan 430023, China.
authors.forename: Li-Hong
authors.initials: LH
authors.lastname: Miao
mesh_terms: D003062:Codon / Q000235:genetics; D005285:Fermentation; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism*; D015870:Gene Expression; D005800:Genes, Fungal; D005818:Genetic Engineering; D007218:Industrial Microbiology; D008049:Lipase / Q000235:genetics* / Q000378:metabolism*; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D010843:Pichia / Q000201:enzymology* / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012331:RNA, Fungal / Q000737:chemistry / Q000235:genetics; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D025062:Yarrowia / Q000201:enzymology* / Q000235:genetics*
chemical_list: D003062:Codon; D005656:Fungal Proteins; D012331:RNA, Fungal; D012333:RNA, Messenger; D011994:Recombinant Proteins; C408971:LIP2 protein, Yarrowia lipolytica; D008049:Lipase
keywords: De novo design; Fermentation; Lipase; Pichiapink; Synthesis