Optimization of leghemoglobin expression conditions in Pichia pastoris

Abstract:

[Background] Leghemoglobin (LegH) can endow vegetarian meat products with beef-like reddish-brown color and meat texture, which has been approved by Food and Drug Administration as a colorant for vegetarian meat and has attracted extensive attention in recent years.[Objective] To optimize the expression conditions and increase the yield of LegH in P.pastoris. [Methods] Firstly, single-factor experiments were conducted to study the effects of peptones, peptone concentration, iron salts and hematin concentration on LegH production during the induction stage. Secondly, the three factors that have the greatest impact on LegH production were screened out through the Plackett-Burman design. Thirdly, the variation ranges and the optimum levels of the three factors were determined via the steepest ascent design and the response surface methodology (RSM), respectively. Finally, according to the RSM results, shake flask fermentation and high-density fermentation in a 5-L tank were carried out. [Results] The single-factor experiments demonstrated that with 4% soybean peptone as the main nitrogen source, the methanol concentration of 1.5%, and the hematin concentration of 5 μmol/L, the yield of LegH was higher. The RSM-optimized fermentation conditions were peptone concentration of 51.48 g/L, pH 5.66, and medium volume of 35.84 mL/250 mL. Under the optimized conditions, the yield of LegH in the shake flask was 0.191 mg/mL, close to the predicted value (0.183 mg/mL), and that in the 5-L tank reached 0.384 mg/mL. [Conclusion] Through optimization of the fermentation conditions for P.pastoris, higher yields of LegH were obtained,which had certain prospects for industrialization after further improvement in the tank fermentation.