Constitutive expression of enzymes in Pichia pastoris for use in lignin valorisation

[摘要] ENGLISH SUMMARY: Lignin valorisation is a key breakthrough in developing efficient integrated biorefineries. Enzymatic modification of lignin wastes offers an environmentally benign, energy efficient avenue for lignin upgrading. However, for enzymatic lignin valorisation to be a viable option, large quantities of the selected enzymes are required at minimal cost. Heterologous enzyme production using recombinant P. pastoris is a well-studied method for the successful large-scale production of recombinant eukaryotic proteins. Considering the disadvantages associated with methanol-induction, the use of the constitutive PGAP promoter for heterologous protein production in P. pastoris is the preferred expression strategy.Three enzymes of interest for lignin degradation and modification were selected in this study for recombinant enzyme production: glucuronoyl esterase (GE), cellobiose dehydrogenase (CDH) and laccase (LCC). These enzymes were selected based on novelty and their broad range of functions in the lignocellulose biodegradation process. These enzymes are representative of carbohydrate degradation (GE, CDH: Fenton reaction) and lignin degradation and/or modification (LCC).The aim was to create, and provide fundamental data of, the capabilities of a constitutive P. pastoris expression system to produce three enzymes (CDH, LCC, and GE), that are associated with lignin valorisation. This study is rooted in aspects of molecular biology as well as bioprocess engineering.The DNA 2.0 (USA) pJexpress expression system, which is free of any proprietary restrictions, was used to create the expression constructs. The created recombinant P. pastoris strains were screened at shake-flask level, and based on these results, one strain for each enzyme was selected for further studies. The enzyme production process was scaled up to a 14 L bioreactor. A two-stage fermentation strategy, consisting of a batch phase, followed by a constant glycerol fed-batch stage was implemented. The fermentation culture was harvested and concentrated through a two-stage tangential filtration process, after which freezing was evaluated as a possible storage strategy. Glycerol was evaluated as a possible cryoprotectant.The constant glycerol feed strategy was shown to be effective, returning high biomass and protein yields. Results of the bioreactor fermentations showed similar biomass growth kinetics (μmax = 0.15 – 0.17 h-1) and biomass yields (119.54-136.47 gdcw/L) throughout the fermentation process for the three recombinant enzymes. High titres of recombinant protein were obtained, with the highest being glucuronoyl esterase, at 2778.01 mg/L, followed by cellobiose dehydrogenase, at 1489.3 mg/L, and lastly laccase, at 778.54 mg/L. The first incidence of constitutive expression of H. jecorina glucuronoyl esterase and N. crassa cellobiose dehydrogenase is reported here. In addition, the highest yield of constitutively expressed T. versicolor laccase lcc2 is also reported. Although the laccase fermentation returned the lowest productivity, preliminary experiments showed that lowering the fermentation temperature may improve this value by aiding in secretion and/or preventing temperature-related laccase degradation at 30 °C.No glycerol was accumulated during the glycerol fed-batch stage. A decline in growth rate was observed during the fed-batch stage due to constant feeding rate during biomass growth. However, since constitutive expression is largely growth associated, it may be beneficial to increase the glycerol feed rate in order to maintain the growth rate at values nearer to the maximum growth rate of the recombinant strains (0.15 – 0.17 h-1).Tangential flow filtration was successfully used to concentrate the cell-free enzyme extracts, where 5.20 – 6.01-fold increases in volumetric activity were obtained, with final volumetric activity yields of 49.57-60.04 %. It was found that glucuronoyl esterase was the most sensitive to volumetric activity loss after refrigeration and freezing, with a decrease of 10.71 % in volumetric activity after freezing at -20 °C. Cellobiose dehydrogenase showed no sensitivity to the freezing process, and laccase activity was successfully preserved with the use of 10 % glycerol as a cryoprotectant.This study has successfully reported the baseline capabilities of a constitutive P. pastoris expression system to produce three enzymes, namely cellobiose dehydrogenase, laccase, and glucuronoyl esterase, associated with lignin valorisation, using a bioreactor fermentation bioprocess. Even though an identical molecular and bioprocess strategy was used, there were significant differences in protein secretion of each enzyme, emphasising the effect of the selected gene on secretion levels in P. pastoris. The yields obtained in the current study may be improved at the microbiological level by improving secretion with the use of alternative secretion factors or gene coding sequences. The bioprocess may be improved by optimisation of the glycerol feed, to maintain the growth rate at a value nearer to the maximum growth rate of each strain. Further, preliminary data suggests that lowering the fermentation temperature may be beneficial for laccase production.

[发布机构] Stellenbosch University