Construction Of A High-Yield Melanin Engineering Strain Of Bacillus Thuringiensis

Bacillus thuringiensis（Bt）is a gram-positive bacteria that can produce insecticidal crystal protein.Bt-derived microbial insecticides have now been used worldwide to control crop pests,but due to ultraviolet radiation in the sunlight under field conditions,Bt insecticides will quickly lose their biological activity.Melanin can be used as a photoprotective agent to improve the ability of Bt to resist ultraviolet.Therefore,it is essential to obtain a Bt strain with high melanin production to protect the insecticidal crystal protein.Preliminary research in our laboratory found that there is an HGA melanin synthesis pathway in most Bt strains.In this pathway,L-tyrosine can be catalyzed to4-hydroxyphenylpyruvate under the action of aromatic amino acid transaminase（PhhC）.The product can generate homogentisic acid（HGA）under the action of hydroxyphenylpyruvate dioxygenase（HppD）.HGA is decomposed under the catalysis of homogentisate dioxygenase（HmgA）.When HmgA mutates and loses its activity,HGA will undergo oxidative polymerization to further produce pyomelanin.In the previous research work,our laboratory knocked out the hmgA gene and obtained a Bt strain BMB171（35）hmgA that can produce melanin,but the melanin production of the mutant strain is not high.In order to obtain a Bt strain with high melanin production,this study conducted a series of studies using BMB171（35）hmgA,which can produce melanin,as the starting strain.First,we explore the effect of the amount of L-tyrosine on the amount of melanin production.When the concentration of L-tyrosine in the medium was lower than 1.2 g/L,the OD₄₀₀ value increased with the gradual increase of L-tyrosine concentration.Compared with the control without L-tyrosine,the increase of the OD₄₀₀ value of fermentation supernatant with L-Tyrosine was smaller,and the maximum increase was 1.39 times,which indicated that the amount of L-tyrosine was not the main factor limiting melanin production.when L-tyrosine in the medium reached 1.4 g/L,melanin production decreased.Therefore,the production of melanin can be increased by adding 1.0-1.2 g/L L-tyrosine to the medium.Previous studies in our laboratory only confirmed that there is an HGA melanin synthesis pathway in Bt,but the role of the aromatic amino acid transaminase（PhhC）that works in this pathway has not been determined.PhhC is also an enzyme in the HGA pathway,which may have an impact on the production of melanin.We searched the BMB171 genome in NCBI and found that there are two genes that may code for PhhC（AQY41390,AQY40477）.Through the construction of overexpression and knockout strains of these two genes,it was confirmed that both two genes encode aromatic amino acid transaminases which operate in the HGA pathway.When phhc-2 overexpression strain（35）hmg A-P_rsi-phhC-2 was fermented in LB medium supplemented with L-Tyrosine（1.0 g/L）,the OD₄₀₀value increased to1.85 times compared with the control strain（35）hmg A-PCB in LB medium,indicating that increasing the amount of aromatic amino acid transaminase can also improve the production of melanin.In this study,a series of strong promoters were used to express HppD,and it was found that the strong promoter Prsi had the highest expression yield.The recombinant strain BMB171 Prsi-hpp D overexpressing HppD was constructed using this promoter.The melanin production of the recombinant strain in the fermentation medium containing 1.0 g/L L-tyrosine was approximately 3.77 times higher than that of the control strain（35）hmg A-pCB in the LB medium.On this basis,a strain BMB171 Pdc-2that overexpressed both phhC-2 and hppD was constructed in this study.It was found that the production of melanin was greatly increased.In the fermentation medium containing L-tyrosine,the OD₄₀₀ of BMB171 Pdc-2 is about 10.50,which is approximately 5.70 times higher than that of BMB171（35）hmg A-pCB cultured in LB medium,that is,the two key enzymes of phh C-2 and hppD in the HGA pathway are simultaneously overexpressed and also Without the limitation of L-tyrosine substrate,the production of melanin can be increased up to 5.70 times.In addition,we found that increasing the amount of dissolved oxygen in the culture medium can not increase the production of melanin,but it can accelerate the production of melanin and shorten the fermentation time..The Bt strain with high melanin production obtained in this study has important practical value for the industrial application of Bt pesticides.