Whole Genome Analysis And Optimization Of Fermentation Technology For High-yield Chitinase Strain EBI-001

The cell walls of plant pathogens and fungi generally contain a large amount of chitin.Streptomyces is a high-yield strain of chitinase.In addition to producing a large amount of chitinase,its ability to produce antibiotics in plant diseases.The production practice of biological control has huge application potential.In this study,the conditions for producing chitinase of a strain of Streptomyces EBI-001 were optimized;at the genetic level,bioinformatics analysis of the high-yield chitinase of the strain was performed to aim at the molecular biological level of the chitinase gene.The above transformation,expression vector construction and engineering bacteria development provide more scientific basis and theoretical basis.The research group screened a biocontrol strain EBI-001 with high antibacterial activity and high chitinase production capacity in the early stage to conduct morphological observations,cultural characteristics observations and other physiological and biochemical tests of EBI-001.The results showed that the phylogenetic tree constructed with 16 S r DNA results and ANI analysis showed that EBI-001 belongs to a subspecies of Streptomyces albidoflavus.In order to further verify the biological function of EBI-001,the second generation sequencing of EBI-001 based on the Illumina Mi Seq sequencing platform,combined with the annotation results of the functional protein database,shows that the strong metabolic capacity of the strain makes it in engineering development Has a wider application prospect.The EBI-001 genome sequence was analyzed by anti SMASH to obtain a total of 24 secondary metabolic biosynthesis gene clusters with multiple antibiotic synthesis gene clusters.Therefore,it is predicted that the strain EBI-001 is a strain with biological control functions.In order to further investigate the activity of chitinase produced by EBI-001 and increase its enzyme production activity,this study identified the optimal fermentation medium and fermentation conditions for strain production by response surface analysis.First,the best carbon source is glucose through one factor,and the peptone is the nitrogen source.The key factors of the medium formulation are selected through the Plackett Burman experiment.Combined with the response surface experiment,the best medium composition formula was obtained.The fermentation conditions can be obtained by response surface analysis.The cultivation temperature is 30 ℃,the cultivation time is 3.9 d,the filling volume is 26.83% and the p H is 10.13,which is the optimal condition for the production of chitinase by EBI-001.The value is 6.02 U/m L.Finally,in order to further explore the structure and properties of the chitinase of strain EBI-001,this study analyzed the chitinase gene family of EBI-001 and the related metabolic pathways of chitin.Through NCBI blast comparison,strain EBI-001 has 8 potential chitinases(Chi A,Chi63,Chi C,Chia2 b,Chi B,Chi E and Chi6 and Exochi),and 24 of them have domains related to chitin degradation gene.Predict the physical and chemical properties,structure and function of chitinase.Among them,5 genes belong to the GH18 family,and 3 genes belong to the GH19 family.Among them,the chitinases encoded by Chi A,Chi63 and Chia2 b have relatively complete domains and stronger catalytic functions,so they are more suitable for genetic engineering.the study.By exploring the chitin metabolism pathway in the strain,the chitinase is crucial for the metabolism of chitin in EBI-001.It is inferred that the strain EBI-001 can obtain chitin by degrading chitin through two predicted pathways in vivo and in vitro.The degradation product Glc NAc is transported into the cell under the action of its specific transferase system,and then undergoes intracellular metabolism in EBI-001 bacteria in two ways.The first way is to decarboxylate and deaminate Glc NAc first,and then convert it into Fru-6-P to enter the glycolysis pathway for energy metabolism;the second way is to change into the activated state of monomeric sugar in the form of nucleoside sugar Can participate in the biosynthesis of antibiotics,and then polymerized into an important component of EBI-001-lipopolysaccharide.The improvement of the Glc NAc metabolic pathway in EBI-001 lays a good foundation for the use of genetic engineering such as gene activation or silencing to increase the production of chitinase,the reuse of chitin and the industrial production of its metabolite Glc NAc.