Study On Efficient Expression Of Heterologous Leghemoglobin In Microorganisms

As a plant hemoglobin,leghemoglobin(LegH)can greatly increase the authenticity of the taste and flavor of plant-based meat products.Since the process of extracting LegH from soybean nodules is complicated and has limited production capacity,which cannot achieve large-scale practical applications,it is necessary to develop efficient microbial fermentation process to produce LegH in our project.In this study,we firstly used the codon optimized gene of soybean LegH to construct an expression plasmid,which was then transformed into E.coli.The recombinant cells could produce 0.17 g/L leghemoglobin after primary condition optimization.Secondly,we also tried to prouduce the LegH in recombinant Pichia pastoris.After the construction of the expression plasmid,the effects of different promoters(AOX1,GAP,AOX1d1)were investigated with regard to the production of LegH.The results showed that the AOX1d1 promoter brought about the highest productivity.By futher screening of colonies with different copy number,the secretory expression of LegH was improved up to 0.12 g/L.Finally,the fermentation conditions were further optimized in Pichia pastoris at the shake flask level.Under the optimal conditions,the LegH secretory expression was further improved by 66.7%,and the final productivity attained 0.2 g/L in the broth.In conclusion,the recombinant expression of leghemoglobin was achieved in E.coli and Pichia pastoris,and the highest production of LegH in Pichia pastoris was 0.2 g/L.The present work paved one road to produce this kind magic protein in the future.