Development of a novel Pichia pastoris expression platform via genomic integration of lipase gene for sustained release of methanol from methyloleate

Abstract

A novel Lip⁺ Pichia pastoris expression platform was developed by integrating lipase Lip2 from Yarrowia lipolytica under constitutive Glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter. Effective expression of reporter protein amylase from Bacillus licheniformis was achieved utilizing methyloleate in Lip⁺Amy⁺host. Lipase hydrolyzed methyloleate into methanol that sustained P_AOX1 induction, and oleic acid, which was readily utilized as a carbon source. The protein expression achieved in presence of methyloleate was comparable to methanol-induced cells, along with an increase in productive biomass. In Lip⁺Amy⁺ host, total amylase production of 220.9 ± 13 U/mg biomass was achieved at 96 h using methyloleate supplemented every 24 h. While 206.0 ± 17 U/mg biomass was obtained at 108 h in an Amy⁺ host induced with methanol every 12 h. Further, lipase expression neither affected growth nor added additional burden on the cellular machinery and no oleic acid accumulation was observed at any time point due to its emulsification and efficient utilization by lipase positive host. Similar results obtained with the second reporter protein γ-cyclodextrin glycosyltransferase (CGTase) from Evansella caseinilytica validated the platform. An alternate lipase Lip11 from Y. lipolytica was also employed in developing a Lip⁺ host to validate disparity between lipase background and P_AOX1 induction in presence of methyloleate.

Keywords:

• AOX1 promoter
• Pichia pastoris
• lipase
• amylase
• Yarrowia lipolytica
• γ-CGTase

Acknowledgements

Amuliya Kashyap and Kuldeep Saini would like to thank the Council for Scientific & Industrial Research (CSIR), India and Meenu Saini would like to acknowledge Indian Council of Medical Research (ICMR), India for financial assistance. The authors would also like to thank Prof. T. Satyanarayana for his kindness in providing pPICKαA vector. Authors also duly acknowledge the Central Instrumentation Facility at UDSC for gene sequencing.

Disclosure statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Additional information

Funding

This work was financially supported by the Department of Biotechnology (DBT, Govt. of India. Grant No. BT/PR2020716/BBE/117/199/2016 dt. 15-06-2017).