Study On Efficient Heterologous Expression Of Pyrolysaccharide Monooxygenase Gene

An enzyme with synergistic effect with conventional cellulases was discovered in the study of lignocellulose degradation,which plays an important role in enhancing the degradation efficiency of cellulases as well as biomass enzyme conversion by catalyzing the oxidative cleavage of glycosidic bonds and destroying the recalcitrant crystal structure of cellulose.This enzyme is classified as lytic polysaccharide monooxygenase(LPMO)and has received much attention from researchers in recent years.In our laboratory,PdLPMO9 A,a lytic polysaccharide monooxygenase gene from Pleurotus djamor,was successfully expressed in pichia pastoris,but its expression was low,which posed great difficulties for the later isolation and purification.In this study,high yield recombinant strains were obtained through codon optimization,gene dosage increase and co-expression of molecular chaperone proteins,which laid the theoretical and methodological foundation for the large-scale production of recombinant lytic polysaccharide monooxygenase PdLPMO9 A.The main research contents and results are as follows:1.The PdLPMO9 A gene was added with a histidine tag(His-tag),and the pichia pastoris GS115-9A-H was constructed to investigate whether the histidine tag had an effect on the recombinant enzyme activity.The results showed that the shake flask culture and induction of expression of GS115-9A-H strain showed no significant effect of His-tag on the expression and activity of recombinase.In addition,the recombinant enzyme was found to degrade rice straw synergistically with cellulase,and the saccharification of rice straw at 72 h was elevated by89.8% compared with cellulase hydrolysis alone.2.The codon optimization of the PdLPMO9A-H gene containing His-tag and the construction of the pichia pastoris GS115-9A-M-H were carried out to investigate the effect of the codon optimization strategy on the expression of recombinant enzyme.The results showed that the codon-optimized strain GS115-9A-M-H was cultured in shake flasks to induce the expression of recombinase,and the protein content of the fermentation supernatant was 0.78 g/L and the recombinase activity was 13.51 U/L,which increased 11.42% and 27.21%,respectively,compared with the original strain GS115-9A.3.The multi copy expression vector of codon optimized gene was constructed and transformed into Pichia pastoris GS115.The strain GS115-9A-M-H-2 containing two copies of codon optimized gene was obtained,and the effect of this combination strategy on the expression of recombinase in Pichia pastoris was explored.The results showed that two copies of strain GS115-9A-M-H-2 were cultured in shake flask,and the recombinase was induced to express.The protein content of fermentation supernatant was 0.93 g/L,and the activity of recombinase was 14.94 U/L,which was 20.78% and 10.58% higher than that of strain GS115-9A-M-H,respectively.Compared to the original strain GS115-9A,it increased by36.76% and 40.41%.4.The Pichia pastoris helper protein expression vector was constructed,and the codon optimized gene of PdLPMO9 A and it were co expressed in Pichia pastoris to obtain the genetic engineering strain GS115-9A-M-H-P,and explore the impact of this combination strategy on the expression of recombinase in Pichia pastoris.The results showed that the codon optimized genetic engineering strain GS115-9A-M-H-P co expressing the auxiliary protein PDI was cultured in shake flask,and the recombinase was induced to express.The protein content of the fermentation supernatant was 1.19 g/L,and the recombinase activity was 17.76 U/L,which was52.56% and 31.46% higher than that of the strain GS115-9A-M-H,respectively.Compared to the original strain GS115-9A,it increased by 75% and 66.92%.