Development of a New High-Cell Density Fermentation Strategy for Enhanced Production of a Fungus β-Glucosidase in Pichia pastoris

Abstract

Traditional diosgenin manufacturing process has led to serious environmental contamination and wastewater. Clean processes are needed that can alternate the diosgenin production. The β-glucosidase FBG1, cloned from Fusarium sp. CPCC 400709, can biotransform trillin and produce diosgenin. In this study, Pichia pastoris production of recombinant FBG1 was implemented to investigate various conventional methanol induction strategies, mainly including DO-stat (constant induction DO), μ-stat (constant exponential feeding rate) and m-stat (constant methanol concentration). The new co-stat strategy combining μ-stat and m-stat strategies was then developed for enhanced FBG1 production during fed-batch high-cell density fermentation on methanol. The fermentation process was characterized with respect to cell growth, methanol consumption, FBG1 production and methanol metabolism. It was found that large amounts of formaldehyde were released by the enhanced dissimilation pathway when the co-stat strategy was implemented, and therefore the energy generation was enhanced because of improved methanol metabolism. Using co-stat feeding, the highest volumetric activity reached ∼89 × 10⁴ U/L, with the maximum specific activity of ∼90 × 10² U/g. After 108 h induction, the highest volumetric production reached ∼403 mg/L, which was ∼91, 154, and 183 mg/L higher than the maximal production obtained at m-stat, μ-stat, and DO-stat strategies, respectively. FBG1 is the first P. pastoris produced recombinant enzyme for diosgenin production through the biotransformation of trillin. Moreover, this newly developed co-stat induction strategy represents the highest expression of FBG1 in P. pastoris, and the strategy can be used to produce FBG1 from similar Pichia strains harboring Fbg1 gene, which lays solid foundation for clean and sustainable production of diosgenin. The current work provides unique information on cell growth, substrate metabolism and protein biosynthesis for enhanced β-glucosidase production using a P. pastoris strain under controlled fermentation conditions. This information may be applicable for expression of similar proteins from P. pastoris strains.

Keywords: Pichia pastoris; biotransformation; diosgenin; energy generation; high-cell density fermentation; induction strategy; methanol metabolism.

FIGURE 1

Cell growth in response to different induction strategies. (A) Optical cell density (OD₆₀₀). (B) specific cell growth rate (μ, h^{– 1}). Time zero indicates the initiation of methanol addition. DO-stat strategy (filled green diamond), μ-stat strategy (open blue circle), m-stat strategy (open black triangle), co-stat (filled red square).

FIGURE 2

Methanol consumption at four induction strategies. (A) Methanol concentration (g/L). (B) specific methanol consumption rate (g/g/h). Time zero indicates the initiation of methanol addition. DO-stat strategy (filled green diamond), μ-stat strategy (open blue circle), m-stat strategy (open black triangle), co-stat (filled red square). Methanol concentrations at m-stat process (including the m-stat part at co-stat strategy) indicate the set-values.

FIGURE 3

Enzymatic activity at four induction strategies. (A) Volumetric activity (U/L). (B) specific activity (U/g). Time zero indicates the initiation of methanol addition. DO-stat strategy (filled green diamond), μ-stat strategy (open blue circle), m-stat strategy (open black triangle), co-stat (filled red square). Asterisk indicates the two-group have significant difference (^∗∗∗P < 0.001).

FIGURE 4

FBG1 production in response to different induction strategies. (A) Volumetric production (mg/L). (B) specific production (mg/g). (C) specific production rate (mg/g/h). Time zero indicates the initiation of methanol addition. DO-stat strategy (filled green diamond), μ-stat strategy (open blue circle), m-stat strategy (open black triangle), co-stat (filled red square). Asterisk indicates the two-group have significant difference (^∗∗∗P < 0.001).

FIGURE 5

Intracellular activities of AOX, FLD, FDH and formaldehyde concentration at four induction strategies. (A) Activity of alcohol oxidase (AOX, U/g-cell). (B) activity of formaldehyde dehydrogenase (FLD, U/g-cell). (C) activity of formate dehydrogenase (FDH, U/g-cell). (D) formaldehyde concentration (mM). Time zero indicates the initiation of methanol addition. DO-stat strategy (filled green diamond), μ-stat strategy (open blue circle), m-stat strategy (open black triangle), co-stat (filled red square). Asterisk indicates the two-group have significant difference (^∗∗∗P < 0.001).

FIGURE 6

Development of co-stat strategy and FBG1 expression at various induction strategies. (A) Fed-batch high-cell density fermentation process of co-stat strategy. (B) SDS-PAGE analysis. (C) FBG1 expression at DO-stat, μ-stat, m-stat and co-stat processes. Bands on SDS-PAGE represent the time points of FBG1 production maximums for each strategy. Twenty microliter samples were loaded into each lane. Arrow indicates the band of recombinant FBG1. Lane M, protein molecular marker. FBG1 expression at various strategies shows the enzyme activities and production at the peak time. Error bars show means ± S.D. of triplicate independent experiments. Asterisk indicates the two-group have significant difference (^∗∗∗P < 0.001).