Codon optimization, promoter and expression system selection that achieved high-level production of Yarrowia lipolytica lipase in Pichia pastoris

• Published in: Enzyme and microbial technology 2015-04, Vol.71, p.66-72
• Main Authors: Zhou, Wen-Jing, Yang, Jiang-Ke, Mao, Lin, Miao, Li-Hong
• Format: Article
• Language: English
• Subjects: Codon - genetics; De novo design; Fermentation; Fungal Proteins - genetics; Fungal Proteins - metabolism; Gene Expression; Genes, Fungal; Genetic Engineering; Industrial Microbiology; Lipase; Lipase - genetics; Lipase - metabolism; Models, Molecular; Nucleic Acid Conformation; Pichia - enzymology; Pichia - genetics; Pichiapink; Promoter Regions, Genetic; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; RNA, Fungal - chemistry; RNA, Fungal - genetics; RNA, Messenger - chemistry; RNA, Messenger - genetics; Synthesis; Yarrowia - enzymology; Yarrowia - genetics
• ISSN: 0141-0229; 1879-0909
• DOI: 10.1016/j.enzmictec.2014.10.007

•The codons of Y. lipolytica lipase gene were optimized by de novo gene design and synthesis.•Codon optimized lipase gene has significantly higher expression level than the native gene.•PAOX1 controlled lipase gene has higher expression level than PFLD1 controlled.•Lipase gene in Pichia GS115 has equal activity than newly developed PichiaPink system.•Lipase activity of codon optimized recombinants in fermenter reached 38,500U/mL. 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.