UgRNA/Cas9 Assisted Multiple-Genes Editing To Construct Favorable Secretion Host Organism

Bacilli are genetically engineering strains used in modern industry,which produce abundant secondary metabolites and can be widely used in industry,agriculture and pharmacy.Bacillus,gram-positive soil bacterium(G~+),always utilize preferred sugars such as glucose over other carbon sources from ambient conditions,because the metabolite of glucose will repress the genes for other carbon metabolism,which will severely disrupt secondary metabolites production.This phenomenon is called carbon catabolite repression(CCR).CCR regulates target genes transcription with a cis-acting element named cre-box,which is a conserved sequence at the promoters in the carbon metabolism-related genes.Here,a novel CRISPR gene editing strategy was used to edit the multi-target cre-box in Bacillus pumilus SH-B9.Our aim is to relieve the multiple genes CCR simultaneously and construct a sugar tolerance,favorable secretion host organism.In this study,seven carbon metabolism genes of B.pumilus SH-B9 were targeted,which involved ack A,ycg E,pts H,bud A,acs A,xyl A,and suc C.According to the cre-box sequences and PAM position,gRNA of the seven genes were designed.And then,a series of unspecific gRNA(UgRNA)were designed according to 30～70%similarity with these gRNA,which were used to construct pCas9 recombinant vectors,pCas9-Cax,pCas9-Cac and pCas9-Cpt.B.pumilus were transformed by these plasmids with UgRNA:Cas9 systems,and then obtaining eight transformants,all of them were assayed by sanger sequencing and next-generation sequencing(NGS).The results indicated UgRNA:Cas9 system triggered a wobble editing at up/downstream of cre-box sites in the seven target genes,the mutant strain was named B.pumilus LG3145.After manifesting,the phenotypes of LG3145 obviously changed.Firstely,resistance to sugar,the cell density of LG3145 was two fold of SH-B9 as incubated in high sugar broth,at the sme time,an obvious polysaccharide capsule outside the cell observed by AFM,SEM and TEM while SH-B9 mostly lysed;Secondly,numerous secretions emerged,such as white flocculent protein appear on the upper layer of LB medium which is about 1.67g/L,and cytochrome overflowing,particularly glycerol as carbon source caused the orange-yellow broth with 309nm abs.On the other hand,these secretions of LG3145 could repress some pathomycetes with about 67%inhibition rate.All the results demonstrate CCR releasing of numerous genes may be the causes of the phenotypical changes.The mutations at multi-cre sites lead to target genes transcription changing and disrupt the carbon central pathway,which results in secondary metabolites increasing and extracellular pigment,protein and even forming polysaccharide capsule.