Combination Biosynthesis And Metabolic Regulation Of Genetic Recombination Phycocyanobilin

Phycocyanobilin(PCB)is a kind of unique pigments in some algae,which can be used as an excellent fluorescent probe for immune disease diagnosis and photodynamic therapy with its special optical properties.It also has significant potential application value in the field of food,cosmetics and health care products with antioxidant,free radical scavenging function.At present,phycocyanobilin extracted from natural algae occupies the main market,whose process is complicated and high cost.However,genetic engineering technology can make it simple and low-cost,having the potential value of large-scale preparationIn this paper,we first transform different expression vectors containing hox1 and pcyA genes into E.coli to build the biosynthetic pathway of phycocyanobilin.The results showed that the strain transformed with the double promoters controlling vector had the highest yield of exogenous enzyme expression and PCB compared with the strains transformed with the single promoter controlling vector.In order to further improve the production of PCB,we overpressed the heme upstream genes(hemB,hemG,hemH)and ALA upstream genes(hemA~s,hemL)and studied theirinfluences on the major metabolites,respectively.The results showed that the overexpression of hemB,hemG and hemH in the upstream genes of heme promoted the instantaneousaccumulation of ALA and heme,which were increased 1.49 and 3.77 times,respectively.However,the yield of ALA and heme could not be increased with time due to the feedback inhibition.The production of PCB were decreased due to reduced catalytic activity of Hox1and PcyA,which were affected by co-transgene expression.Overexpression of ALA upstream genes(hemA~s and hemL)could promote the accumulation of ALA and heme,which wereincreased by 2.23 and 2.18 times,but had little effect on the yield of PCB,which might be due to that the stable structure of HemA~s weakened the heme feedback.In addition,we also investigated the effect of increasing the copy number of hox1 and pcyA,which are the two key genes required for PCB biosynthesis in E.coli.However,the results showed that it could increase the yield of the two enzymes,not achieve an expected increase in PCB's yield.It might be due to some unknown feedback suppression of the PCB or intermediate metabolites,and indicated the complexity of the heme biosynthetic pathway.The study further clarifies the heme metabolic pathway in E.coli and provides an important theoretical and methodological basis for producing high value-added products.