| Rhizobacteria play an important role in promoting crop growth and disease resistant.Their agriculture function is degraded because of long-term synthetic pesticides and chemical fertilizers farming.In this study,we used non-specific UgRNA/Cas9 editing technology to disturb genome cis-acting elements CRES of a wild-type strain Bacillus pumilus,and restored its agricultural function,which was named LG3145.We found that LG3145 can secrete extracellular proteins and bacterial pigments,and exhibits good antibacterial properties.Using single nucleotide polymorphism(SNP)detection,transcriptome,metabolome and qPCR techniques,we further explored the relationship between plant-beneficial functions and CRES editing.SNP analysis showed that over 3300 genes in LG3145 genome were disturbed.DNA sequencing results of 35 limited genes involved in secondary metabolism,carbon metabolism,and amino acid metabolism revealed that the mutation sites mainly occurred at the [1,0],[2,0],and [0,2] CRES sites.The comparative analysis of the transcriptome of LG3145 and wild-type strains showed that the expression level of carbon metabolism genes was about 2.2 times higher than that of wild-type strain,such as glpK,ptsG,pgi,sucC and ykgB genes.The gene expression levels of carotenoid and riboflavin synthesis in the secondary metabolic pathway also increased2.6 and 4.1 times respectively,such as isp and rib operons.Therefore,when LG3145 uses glycerol as carbon source,it will produce orange-red secretions.Metabolomic analysis also confirmed that carbon sources are transferred to riboflavin metabolism(ko00740)and carotenoid biosynthesis(ko00906)through pentose phosphate and amino acid metabolism pathways.It is also transferred to the nonribosomal peptide biosynthesis pathway(ko01054)to generate surfactant,bacitracin and myxochelin B,which have good antibacterial properties.Among them,the content of surfactant can reach 1.75 g/L,and the expression of srfA gene cluster increases 6.6 flod.Moreover,LG3145 can colonize rhizosphere to assist wheat to against F.graminearum,which was likely due to the production of surfactin.In addition,LG3145 can promote wheat growth.So,CRES editing alters the carbon metabolic pathway,which is related with its agriculture function restoration. |
