Establishment And Application Of CRISPR-Cas Based Gene Editing Tools In Clostridium Butyricum

Clostridium butyricum is a strict anaerobic Gram-positive bacteria with butyric acid biosynthesis ability.It has been widely used as probiotics to improve the intestinal health of human,livestock and poultry.Its excellent probiotic characteristics are mainly obtained by producing a variety of enzymes and short chain fatty acids during the fermentation.However,the lack of gene editing tools limits the research on improving the probiotic function of C.butyricum by metabolic engineering.Based on the three isolated C.butyricum strains,the exogenous CRISPR-Cas9 and endogenous CRISPR-Cas systems were successfully exploited for genome editing in C.butyricum.The main contents are as follows:The strains of C.butyricum MIYAIRI-588,CGMCC0313-1 and RH-2 were isolated from C.butyricum medicament samples.The fermentation experiments were carried out with glucose,Isomaltooligosaccharide,fructooligosaccharide,galactooligosaccharide,xylooligosaccharide and inulin as the sole carbon sources respectively,and the fermentation products were detected.Meanwhile,the conjugation transfer method of C.butyricum was explored.The fermentation results showed that C.butyricum could use a variety of carbon sources for growth,and the main fermentation products were butyric acid,acetic acid and ethanol,and the yield ratio was about 3:1:1.The results of the conjugation experiments showed that when the OD₆₀₀ of donor E.coli reached around 1.0 and the mating time was16 h,the transformation efficiency of the plasmid p MTL83151 containing the replicon of p CB102 was the highest,which was 2.51×10² CFU/m L.In this study,the exogenous CRISPR-Cas9 system was initially employed for gene editing in C.butyricum.Based on the high efficient conjugation method established above,the CRISPR-Cas9-based gene editing plasmids were successfully transformed into C.butyricum MIYAIRI-588,C.butyricum CGMCC0313-1 and C.butyricum RH-2.By inducing the expression of cas9 protein,the spo0A gene which involved in spore formation in C.butyricum MIYAIRI-588,C.butyricum CGMCC0313-1,C.butyricum RH-2 and the adh E gene which related to the ethanol production in C.butyricum RH-2 were successfully knocked out,with an editing efficiency of 100%and 12.5%,respectively.The results of sporulation test showed that the wild type strain could generate about 4.30×10⁵ CFU/m L of heat resistant colonies from spore germination,but the spo0A gene deleted mutant could not form spores.The fermentation results showed that the ethanol production of strain C.butyricum RH-2?adh Ewas decreased by 69.1%,compared to wild type strain.Whlie the butyric acid production was increased by61.9%.The endogenous CRISPR-Cas system of C.butyricum was analyzed and studied.Genome editing was successfully achieved in C.butyricum by using the endogenous CRISPR-Cas system.Firstly,the effective PAM sequences(5'-ACA-3'and 5'-TAA-3')which could be recognized by the endogenous CRISPR-Cas system of C.butyricum were identified by plasmid interference experiments.Secondly,the effect of spacer length on editing efficiency was investigated.The results show that high editing efficiency of endogenous CRISPR-Cas system was observed when spacers length ranged from 34 to 37nt.The spo0A gene of C.butyricum MIYAIRI-588,C.butyricum CGMCC0313-1 and C.butyricum RH-2 and aldh gene of C.butyricum RH-2 were successfully knocked out by using the endogenous CRISPR-Cas system.Fermentation results demonstrated that the aldh gene deleted mutant produced49.5%more butyric acid,compared to wild type strain.In conclusion,the conjugative plasmid transformation method was established for C.butyricum in this study.Based on the transformation method,the exogenous CRISPR-Cas9 system and endogenous CRISPR-Cas system were successfully developed and shown to exhibit high editing efficiency in all three tested C.butyricum strains.The two approaches developed herein remarkably expand the existing genetic toolbox available for investigation of C.butyricum,thus expediting the exploration of full potential of this versatile probiotic.