Fermentation Optimization And Biocontrol Functional Analysis Of Poly-γ-Glutamicacid Production Strain Bacillus Amyloliquefaciens JX-6

Polyglutamic acid(γ-PGA)is an anionic polymorphic amino acid.Previous studies have shown that γ-PGA has been used to increase crop yields,save fertiliser application and alleviate leaf chlorophyll reduction caused by aphid stress in small cabbage pots and field pepper experiments.A strain of Bacillus amyloliquefaciens JX-6,which has γ-PGA synthesis properties and potential biocontrol ability,has been obtained from agricultural soil samples in the laboratory.We hypothesise that this strain could be developed and optimised for use in agricultural production to reduce the application of chemical fertilisers and pesticides.In order to solve the problems of low productivity and unknown bacterial inhibition of this wild-type strain,and to enhance the application of JX-6,we have sequenced and analysed the genome ofγ-PGA-producing strain JX-6 and verified its ability to produce γ-PGA at the genetic level.In addition,we also investigated the fermentation optimization of the strain and the function of biocontrol.The main findings are as follows:(1)Whole genome analysis and function prediction of JX-6In this study,the complete genome sequence of B.amyloliquefaciens JX-6 strain was determined and annotated for gene function using the Biosign database.The results showed that the complete genome sequence of JX-6 was 4.06 Mb long,with an average GC content of46.31%,containing 4721 genes and 4156 protein-coding genes CDS.The functional annotation information showed that the genes were mainly involved in amino acid transport and metabolism,while the KEGG signalling pathway information suggested a pathway related to the synthesis of γ-PGA and the degradation of enzymatic genes.Antismash prediction analysis showed that JX-6 has the ability to synthesise surfactin,macrolactin,bacillaene,fengycin,daphycocyanin,bacteriocin and bacillin.difficidin,bacillibactin,bacilysin,mycosubtilin,lanthipeptide and other antibacterial compounds.In addition,JX-6 encodes plant growthpromoting related genes and has the potential to produce 12 classes of antibiotic substances with inhibitory activity against bacterial plant pathogens.This suggests that JX-6 is capable of synthesizing both γ-PGA and various antibacterial substances,and has good potential for application in agricultural cultivation.(2)Study on fermentation optimization,amplification and kinetics model for γ-PGA production by JX-6In order to enhance the fermentation yield of B.amyloliquefaciens JX-6,the study applied single-factor and orthogonal experimental methods to optimise the fermentation conditions of the strain.The optimum culture conditions for the shake flask fermentation of JX-6 were obtained as follows: inoculum volume 2 m L,temperature 35°C,speed 240 rpm,fermentation time 48 h,glucose 80 g/L,ammonium sulphate 8 g/L,citric acid 20 g/L,sodium glutamate 24g/L,dipotassium hydrogen phosphate 1 g/L,magnesium sulphate heptahydrate 0.5 g/L,calcium chloride anhydrous 0.2 g/L,ferric chloride hexahydrate The γ-PGA yield of JX-6 was increased from 17.45 g/L to 33.3 g/L.The strain was subjected to pilot experiments and kinetic model construction in a 100 L fermenter.The results showed that the error between the theoretical and experimental values of the derived kinetic model was within a reasonable range,which was consistent with the kinetic changes in the fermentation process and could be used to predict the biomass changes in the fermentation process.(3)Biocontrol activity and bacteriostatic mechanism of JX-6The in vitro antibacterial activity of JX-6 against 15 plant pathogens was determined.B.amyloliquefaciens JX-6 has a strong inhibitory effect on the growth of eight phytopathogenic fungi,including Thanatephorus cumulus,Cladosporium cumulus,Valsa mali,Alternaria solani,Colletotrichum lagenarium,Exserohilum turbine,Botrytis cinerea,and Fusarium solani.Scanning electron microscopy of the confrontation side pathogenic fungi showed irregular folded and constricted mycelium,twisted and folded,protoplasm leaking and swelling.A total of 9494 metabolites of fermentation broth species were detected by LC-MS,and 1513 metabolites were identified by analysis.The functional analysis of the compounds identified a total of 27 lipopeptides,pesticides and antibiotics as bacterial inhibitors.The results of the physical analysis indicated that these compounds have biological activities to inhibit fungi,bacteria,pests and diseases and induce resistance in plants.