Demonstration-Scale High-Cell-Density Fermentation of Pichia pastoris

Abstract

Pichia pastoris has been one of the most successful heterologous overexpression systems in generating proteins for large-scale production through high-cell-density fermentation. However, optimizing conditions of the large-scale high-cell-density fermentation for biochemistry and industrialization is usually a laborious and time-consuming process. Furthermore, it is often difficult to produce authentic proteins in large quantities, which is a major obstacle for functional and structural features analysis and industrial application. For these reasons, we have developed a protocol for efficient demonstration-scale high-cell-density fermentation of P. pastoris, which employs a new methanol-feeding strategy-biomass-stat strategy and a strategy of increased air pressure instead of pure oxygen supplement. The protocol included three typical stages of glycerol batch fermentation (initial culture phase), glycerol fed-batch fermentation (biomass accumulation phase), and methanol fed-batch fermentation (induction phase), which allows direct online-monitoring of fermentation conditions, including broth pH, temperature, DO, anti-foam generation, and feeding of glycerol and methanol. Using this protocol, production of the recombinant β-xylosidase of Lentinula edodes origin in 1000-L scale fermentation can be up to ~900 mg/L or 9.4 mg/g cells (dry cell weight, intracellular expression), with the specific production rate and average specific production of 0.1 mg/g/h and 0.081 mg/g/h, respectively. The methodology described in this protocol can be easily transferred to other systems, and eligible to scale up for a large number of proteins used in either the scientific studies or commercial purposes.

Expression of multidrug transporter P‐glycoprotein in Pichia pastoris affects the host's methanol metabolism

“…Optical density at 600 nm (OD 600 ) of the appropriately diluted culture was determined using a spectrophotometer. Dry cell weight (DCW, mg ml −1 ) was measured by a moisture determination balance (MB 200; Ohaus Corporation, Florham Park, NJ, USA) after centrifugation and washing at 95°C (Liu and Zhu, ).…”

High cell density fermentation strategies for the production of bovine lactoferrin in Pichia pastoris

“…In the previous researches in fed-batch fermentation of P. pastoris strains [12,13,26] glycerol was supplied in two stages: Batch and fed-batch phases. At these stages, glycerol was utilized to boost cell biomass since it promotes cell development more effectively than methanol does [9,27]. The duration of glycerol feeding depends on the desired cell density before methanol induction.…”

“…High-cell density fermentation (HCDF) is a critical strategy in the large-scale engineering of host strains for industrial protein, peptide, and amino acid production [9]. Using the HCDF technique increases protein productivity, improves downstream yield, decreases culture volume, and reduces production costs.…”