Continuous cell recycling in methylotrophic yeast Pichia pastoris to enhance product yields: a case study with Yarrowia lipolytica lipase Lip2

• Published in: Biotechnology and bioprocess engineering Vol. 30; no. 1; pp. 116 - 131
• Main Authors: Mohanty, Shilpa, Babbal, Chauhan, Shivani, Talwar, Mohini, Khasa, Yogender Pal
• Format: Journal Article
• Language: English
• Published: Seoul The Korean Society for Biotechnology and Bioengineering 01.02.2025; Springer Nature B.V; 한국생물공학회
• Subjects: Biomass; Biotechnology; carboxylic ester hydrolases; case studies; Cell density; Chemistry; Chemistry and Materials Science; culture flasks; Cycle protein; Dry cells; E coli; Escherichia coli; fermenters; glycosylation; Inclusion bodies; Industrial and Production Engineering; Komagataella pastoris; LiP2 protein; Lipase; Pichia; Pichia pastoris; product quality; Proteins; Recycling; Research Paper; Retention; Saccharomyces cerevisiae; signal peptide; solubility; Specific yield; Yarrowia lipolytica; Yeast; yeasts; 생물공학; Cell retention; Glycosylation; Lip2 protein; High cell density fermentation
• ISSN: 1226-8372; 1976-3816
• DOI: 10.1007/s12257-024-00149-8

A robust cell recycling strategy based on the methylotrophic yeast, Pichia pastoris, was established to enhance product titers of the commercially important Yarrowia lipolytica lipase Lip2. The expression of Lip2 protein in the prokaryotic host Escherichia coli resulted in inclusion bodies, whereas utilization of SUMO fusion tag could not improve its solubility. Therefore, Lip2 extracellular expression was targeted via the Saccharomyces cerevisiae α -mating signal sequence in P. pastoris under methanol-inducible AOX1 promoter. Shake flask expression studies of hyper-producer Pichia clone under optimized conditions resulted in 438.83 and 420.09 mg/L of glycosylated Lip2 production after induction at an OD 600 of 10 and 20, respectively. A high Lip2 productivity was further targeted using a cell retention technique where the cell biomass was recycled to obtain higher product concentration with improved product quality. The biomass recycling at every 72 h followed a 3.8-fold enhanced Lip2 concentration with a cumulative volumetric product concentration of 1,794 mg/L. A high specific product yield (Y P/X ) in the range of 37.45–47.00 mg/g dry cell weight (DCW) was also maintained up to five retention cycles. Furthermore, higher cumulative protein yields were obtained from the 5-time recycled cells compared to five individual batch runs at shake flask up to 72 h. High cell density cultivation of recombinant P. pastoris in a 2.5-L fermenter yielded 5.25 g/L of Lip2 enzyme with a maximum specific yield of 51.97 mg/g DCW.