Co-expression Of Validamycin And Chitinase And Its Evaluation Of Indoor Control Effects Against Rice Sheath Blight

Biotic pesticides are gradually edging out chemical pesticides in planting industry,animal husbandry and fishery industry due to their advantages of safety,no environmental pollution,ultra-low dosage,and high selectivity.Different biopesticides have their own advantages and disadvantages.The multifunctional biopesticides with stronger effects,lower toxicity,and slower resistance prepared by coupling or joint use of biopesticides with different mechanisms of action through chemical synthesis or genetic engineering methods,have become the main research direction of green pesticide development.Validamycin A and chitinase have strong antagonism effects on plant pathogenic fungi,but their antifungal mechanisms are different.This study aims to use genetic engineering methods to efficiently express validamycin A and chitinase simultaneously using Streptomyces as chassis cells,directly obtaining formulations containing these two biological pesticides and then testing their biological control effects.Firstly,based on the previously constructed heterologous expression strain of validamycin A,the eight key enzyme genes vlm ABC,vlm KLMN and vlm G in the validamycin A biosynthetic pathway were overexpressed by cloned into the downstream region of the constitutive strong promoters Perm E* and Pazi A4 in the Streptomyces self-replicative vectors p WHM4S(high copy)and p IJ903(low copy).The results showed that the overexpression strain Streptomyces lividans ZX1::9A9/p G02 constructed with the low copy vector yielded 18.33 mg/L of validamycin A,with an increase of 36.69%.The overexpression strain S.lividans ZX1::9A9/p LXY11 constructed with the high copy vector yielded 36.80 mg/L of validamycin A,with an increase of 174.26%,but the growth of the strain was somewhat inhibited.Meanwhile the optimized chassis cells of S.lividans ZAY4 were used to produce validamycin A.The vlm gene cluster was introduced into S.lividans ZAY4 for heterologous expression,and the yield of validamycin A went up by79.39%,indicating that the modification of chassis cells can further increase the production of validamycin.Secondly,the multi-dimensional structure,active sites and mode of action of Chi19 F and Chi18 b B with potential for antifungal activity in Streptomyces coelicolor were analyzed.The strong promoter Pazi A4,secretion signal sequence vsi,and chi18 b B were cloned into a high copy self-replicative vector p KC1139 by Gibson assembly and then the apramycin resistance gene on the vector was replaced with ampicillin resistance gene and thiostreptomycin resistance gene by λ-Red recombination system to obtain the expression plasmid p GEM-chi B.Subsequently,the gene chi19 F was inserted into the plasmid p GEM-chi B,giving rise to the plasmid p GEM-chi BF for coexpression of two chitinases.These two plasmids were introduced into the heterologous expression strain of validamycin A to obtain the genetic engineering strain S.lividans ZX1::9A9/p GEM-chi B and S lividans ZX1::9A9/p GEM-chi BF.Enzyme activity testing discovered that the secretion and expression of Chi18 b B significantly improved the ability to degrade chitin,while the co expression of Chi18 b B and Chi19 F could also increase enzyme activity,but lower than the only expression of Chi18 b B.In biological activity testing,the fermentation broth of S.lividans ZX1::9A9/p GEM-chi B completely inhibited the growth of the rice sheath blight pathogen,Rhizoctonia solani AG1-IA.The leaf and greenhouse pot experiments of rice and soybean also confirmed that application of the fermentation broth can significantly reduce the expansion of sheath blight,which indicated that the biological combination of validamycin A and chitinase in Streptomyces has the potential to develop a green,healthy and widely used agent against plant pathogenic fungi.