Improved enzyme production on corncob hydrolysate by a xylose-evolved Pichia pastoris cell factory

Abstract

The global shift from the usage of crude oil in bio-production is receiving much attention owing to environmental concern associated with fossil fuel. Lignocellulosic biomass (LB) is a good carbon candidate for bio-production because it is environmental-friendly. Corncob being one of such LB is rich in glucose and xylose, which can be utilized for bio-production. We co-utilize these sugars for the production of enzymes from Pichia pastoris GS115 (Wild Type: WT). Glucose utilization was efficient from synthetic and real hydrolysate but xylose utilization was very low, hence, the need for optimization. Mutants were selected upon Adaptive Laboratory Evolution to efficiently utilize xylose. As expected, all the mutants examined showed improved xylose utilization but surprisingly, there was only 1.8 g/l residual xylose in the 50th generation (GS50). The 30th evolutionary generation (GS30) compared well with the WT by completely utilizing the glucose and also accumulated 48 OD₆₀₀ cell biomass, which is the highest among all the strains evaluated. More importantly, GS30 secreted 72.6 U/ml and 45.1 U/ml β-galactosidase and β-mannanase on hydrolysate respectively, which are higher than the titre for the WT. Conclusively, this study demonstrated the efficacy of corn corncob hydrolysate in biomanufacturing and gives insight for the optimization study.

Keywords: Adaptive Laboratory Evolution; Corncob; Pichia pastoris; Β-galactosidase; Β-mannanase.

Fig. 1

Effect of Adaptive evolution on xylose utilization in BMXY complex media. a Biomass accumulation in batch cultivation. b Residual xylose concentration. c Biomass accumulation in complex based medium in fed-batch phase cultivation. GS30: 30th generation of evolved strain, GS50: 50th generation of evolved strain, GS70: 70th generation of evolved strain, GS115: wild-type. Three parallel flasks are tested for each strain at 30 °C. Error bars represent deviations (n = 3). Statistically significant differences (P\0.05) were determined by student’s t test

Fig. 2

Batch cultivation on corncob hydrolysate for ALE-derived and wild-type strain a Biomass accumulation in batch cultivation. b Residual xylose concentration. c Residual glucose concentration. GS30: 30th generation of evolved strain, GS50: 50th generation of evolved strain, GS70: 70th generation of evolved strain, GS115: wild-type. Three parallel flasks are tested for each strain at 30 °C. Error bars represent deviations (n = 3). Statistically significant differences (P\0.05) were determined by student’s t test

Fig. 3

Fed-batch cultivation on corncob hydrolysate for ALE-derived and wild-type strain, GS30: 30th generation of evolved strain, GS50: 50th generation of evolved strain, GS70: 70th generation of evolved strain, GS115: wild-type. Three parallel flasks are tested for each strain at 30 °C. Error bars represent deviations (n = 3). Statistically significant differences (P\0.05) were determined by student’s t test

Fig. 4

Enzyme secretion of ALE-derived and wild-type strain. a β-galactosidase secretion on BMXY complex medium. b β-galactosidase secretion on CCH hydrolysate. c β–mannanase secretion on BMXY complex medium. d β–mannanase secretion on CCH hydrolysate. GS115-lac: Wild type strain integrated with β-galactosidase gene, GS30-lac: 30th generation of evolved strain integrated with β-galactosidase gene, GS115-man: Wild type strain integrated with β-mannanase gene, GS30-man: 30th generation of evolved strain integrated with β-mannanase gene. Three parallel flasks are tested for each strain at 30 °C. Error bars represent deviations (n = 3). Statistically significant differences (P\0.05) were determined by student’s t test

Fig. 5

a Cloning vector map for β-galactosidase and β-mannanase from Aspergillus oryzae and Bacillus subtilis. b Confirmation of correct clones for β-mannanase. c Confirmation of correct clones for β-galactosidase. d Protein expression of β-mannanase from GS30 on SDS-PAGE. e Protein expression of β-galactosidase from GS30 on SDS-PAGE