The Studies On The Melanogenic Pathway In Bacillus Thuringiensis

Bacillus thuringiensis(Bt)–based insecticide is a widely used biological insecticide,but it has its certain disadvantages.One of them is that the insecticidal activity of a Bt-based formulation is unstable and rapidly loses under field conditions because of the UV radiation in sunlight.As a result,Bt-based formulations have to repeat spray,leading to they are more expensive than chemical pesticides.Melanin plays important roles in protecting living organisms against UV radiation,which is considered as an effective photoprotective agent for Bt-base bioinsecticides.However,B.thuringiensis strains that naturally produce melanin are very rare,and the melanogenic pathways of Bt are not clear,so it is very necessary to study the melanin synthesis pathway of Bt strains.Both wild strain Bt L-7601 and mutant strain Bt BMB181 were able to produce melanin in LB liquid medium.Bt BMB171 is the original strain of Bt BMB181,Bt HD73 could generate Cry1 AC.These two strains were chosed to study their melanin pathway.In this study,we demonstrated that Bt L-7601 and Bt BMB181 failed to produce melanin after treatment with mesotrione,an inhibitor of 4-hydroxyphenylpyruvate dioxygenase in the homogentisic acid pathway of melanin synthesis.Heterologous expression experiments suggested that homogentisate-1,2-dioxygenase(HmgA)in Bt BMB171 functions normally,yet HmgA in Bt L-7601 and Bt BMB181 had lost its activity,at least partly.Though whole-genome sequencing of Bt L-7601 and analysis of genomic data,the frameshift mutation of hmgA caused the gene to terminate prematurely in Bt L-7601.BLAST results indicated that the amino acid alteration(G272E)in HmgA of BMB181 was caused by a single point mutation(815G? A)compared with Bt Bt BMB171 The enzyme activity of purified HmgA171 was more than 10-fold higher than that of HmgA181.In addition,overexpression of its own hmgA gene in BMB181 also resulted in failure to produce the pigment.Using the CRISPR-Cas9 system,the hmgA gene in Bt BMB171 and Bt HD73 was knocked out and the mutant strain gained the ability to produce melanin.Furthermore,the complemented strain reverted to the wild-type phenotype.The mutant Bt HD73?hmgA had no significant difference in the formation efficiency of the spores,the morphology of the insecticidal crystal protein Cry1 AC and the amount of crystal protein compaired with original strain.This study confirmed that FahA of Bt BMB171 could still degrade maleoyl acetoacetic acid in the absence of MaiA,and the degradation of maleoyl acetoacetic acid was accelerated significantly when MaiA of Pseudomonas aeruginosa was added..It is indicated that the lack of MaiA in the Bt strain only reduces the degradation efficiency of homogentisic acid,rather than failure to degrade it.