Combined Strategies for Improving the Production of Recombinant Rhizopus oryzae Lipase in Pichia pastoris

Abstract

We have developed a yeast Pichia pastoris system for the high-level expression of recombinant Rhizopus oryzae lipase (ROL), which is a potentially effective catalyst in the solvent-free production of biodiesel fuel. In the glycerol fed-batch phase, the combination of the dissolvedoxygen-stat and gradient-control glycerol feeding strategies resulted in a higher cell biomass in the P. pastoris culture, with shorter feed times. In the methanol fed-batch phase, a constant methanol concentration of 0.3-0.5% (v/v) was found to be optimal for high ROL activity, maximum protein concentration, and a maximum biomass. Cells were grown at 30 °C and induced by methanol at 22 °C and a pH of 6.0 with the addition of 0.5% (w/v) casein, which reduced the proteolytic degradation of ROL. Using a combined glycerol step-increasing, on-line methanol feeding strategy and these reducing proteolytic degradation methods, a maximum biomass of 96 g DCW/l, a maximum concentration of 2.0 g/L, and an activity of 1302.2 U/mL ROL were attained. The simple culture process is the highest level of ROL expression reported in P. pastoris. Our results strongly demonstrate that the efficiency of the recombinant ROL expression is directly dependent on feed strategies, lower induction temperature, maintaining the proper pH, and the addition of casein.

Fed-Batch Production of Saccharomyces cerevisiae L-Asparaginase II by Recombinant Pichia pastoris MUTs Strain

“…Heterologous protein expression in shake flask cultures of P. pastoris is usually performed in complex BMGY medium, whose composition, however, is not optimized; therefore, modifications have been proposed (Stratton et al, 1998; Pichia Fermentation Process Guidelines, 2002; Ghosalkar et al, 2008; Li et al, 2013). Even though P. pastoris can grow in a large range of pH (from 3.0 to 7.0) (Ahmad et al, 2014), our previous results (not shown) demonstrated that ASNase II activity did not significantly vary in the pH range 5.0–6.0.…”

“…High cell density cultures of P. pastoris in bioreactor are usually carried out on chemically-defined high-salt concentration broth consisting of a modified basal salt medium (BSMm) enriched with a Pichia trace metal (PTM) solution (Ferrara et al, 2006; Julien, 2006; Ghosalkar et al, 2008; Li et al, 2013).…”

Rhizopus oryzae Lipase, a Promising Industrial Enzyme: Biochemical Characteristics, Production and Biocatalytic Applications

“…In order to improve rROL production and minimise the proteolysis in Mut S phenotype strain cultures, different operational strategies have been studied. Li et al [143] described that maintaining a constant methanol concentration during the fed-batch cultivation showed a better lipase production than adjusting methanol feeding rate to keep DO over 35% (DO-stat). However, the former strategy needs to be complemented to avoid proteases expression and lipase degradation.…”

Truncated Prosequence of Rhizopus oryzae Lipase: Key Factor for Production Improvement and Biocatalyst Stability

“…Using this yeast provides a number of advantages, including little endogenous protein secretion, the absence of endogenous lipases and esterases, the presence of a powerful, tightly regulated methanol-inducible alcohol oxidase 1 promoter (P AOX1 ), and the ability to grow at high cell densities on defined media [30]. The high suitability of P. pastoris for ROL production is confirmed by the vast amount of literature on its cloning and expression [31][32][33][34][35]. Previous studies have examined the effects of cloning and expressing the whole ROL prosequence (97 amino acids) with the mature sequence in P. pastoris [36,37].…”