Establishment Of The Bacillus Subtilis Genome Editing System And The Genetic Regulation Of The Biosynthesis Of Three Anti-Fungal Compounds

So far, agrochemicals are the main approach to control soil borne diseases. However, intensive use of agrochemicals has led to the emergence of pathogen resistance and severe negative environmental impacts. There are also a number of soil-borne plant diseases for which chemical solutions are ineffective or non-existent. On the other hands, consumers need more and more pesticide-free foods. Thus, biological control based on the use of natural antagonistic microorganisms has emerged as a promising alternative to chemical pesticides for more rational and safe crop management.In this thesis, we described a genome editing system that allows multiple markerless modification of the Bacillus subtilis genome. We also reported the molecular basis for the antagonistic activity of Bacillus subtilis ATCC6633, Paenibacillus polymyxa SQR21and Streptomyces viridochomogenes ATCC29814.To equip the extensive genetic engineering of biocontrol bacteria toward the improved antifungal activities, we developed a simple and efficient Bacillus subtilis genome editing method in which targeted gene(s) could be inactivated by single-stranded PCR product(s) flanked by short homology regions and in-frame deletion could be achieved by incubating the transformants at42℃. In this process, homologous recombination (HR) was promoted by the lambda beta protein synthesized under the control of promoter PRM in the lambda cI857PRM-PR promoter system on a temperature sensitive plasmid pWY121. Promoter PR drove the expression of the recombinase gene cre at42℃for excising the floxed (lox sites flanked) disruption cassette that contained a bleomycin resistance marker and a heat inducible counter-selectable marker (hewl, encoding hen egg white lysozyme). Then, we amplified the single-stranded disruption cassette using the primers that carried70-nt homology extensions corresponding to the regions flanking the target gene. By transforming the respective PCR products into the B. subtilis that harbored pWY121and incubating the resultant mutants at42℃, we knocked out multiple genes in the same genetic background with no marker left. This process is simple and efficient and can be widely applied to large-scale genome analysis of recalcitrant Bacillus species.The genome editing system was applied to delete the mycosubtilin gene cluster myc and the gene abrB. Deletion of myc lead to the complete loss of antifungal activity while abrB deletion resulted in enhanced production of mycosubtilin. Pmyc-lacZ reporter system and ATCC6633genome library were constructed for the screening of the cosmid clone that was able to induce the expression of Pmyc-lacZ, which allowed us to conclude that the extracellular pheromone ComX could activate the expression of the myc gene cluster.Transposon mutagenesis of strain SQR21resulted in a mutant MUT-8that had completely lost the antifungal activity against Fusarium oxysporum. LC/MS analysis confirmed the incapability of fusaricidin production in the MUT-8strain. Comparison of the transposon disrupted sequence and the genome sequence of strain SQR21revealed a gene fusA, which was responsible for the biosynthesis of fusaricidin. The fusA gene encoded a polypeptide consisting of six modules in a single open-reading frame. Interestingly, the sixth module did not contain an epimerization domain, while all reported fusaricidins contained D-form alanines in their sixth amino acid residues, suggesting the sixth adenylation domain might directly recognize the D-form alanine. Bioinformatic analysis suggested that the genes fusGFEDCB located upstream of fusA were responsible for the biosynthesis of the fatty acid chain of fusaricidin. LacZ reporter system and gel retardation analysis demonstrated that the Pfus promoter was under negative control of AbrB, and inactivation of the gene abrB enhanced the production of fusaricidin.Genome sequencing technology also allowed us to identify the putative gene cluster responsible for the biosynthesis of laspartomycin in S. viridochomogenes ATCC29814. However, gene disruption was made difficult by the lack of genetic tools available for use in S. viridochomogenes ATCC29814, we therefore constructed a suicide vector pATKKA and established a transformation protocol, by which we successfully disrupted the lpmC gene and abolished the laspartomycin production in the corresponding mutant strain, demonstrating that the lpmC gene was involved in laspartomycin biosynthesis. Sequence alignment between lpmC gene sequence and the genome sequence of ATCC29814showed that the biosynthetic gene cluster for laspartomycins spanned approximately60kb, and consisted of21open reading frames. Interestingly, the dab A, dabB and dabC genes predicted to code for the biosynthesis of the unusual amino acid diaminobutyric acid (Dab) were organized into the lpm cluster even though the Dab residue was not found in the laspartomycins.Mycosubtilins, fusaricidins and laspartomycins were non-ribosomally synthesized, and each was constituted with several variants. These findings highlighted that B. subtilis ATCC6633, P. polymyxa SQR21and S. viridochomogenes ATCC29814could be as good candidates for the development of biocontrol agents, and allowed molecular engineering and combinatorial biosynthesis approaches to expand the structural diversity of these antibiotics.In conclusion, we developed the Bacillus genome editing system, and disrupted multiple genes with this system. By using the genome editing system together with several other techniques, we observed that the production of mycosubtilin was subject to negative control by AbrB, and positive regulation by ComX; we cloned and characterized two gene clusters responsible for the biosynthesis of the antifungal metabolites fusaricidin and laspartomycins, and analyzed the gene functions using bioinformatics prediction; we also created the vector pATKK for gene modification in S. viridochomogenes ATCC29814together with the DNA transformation protocol, and disrupted the lpmC gene of strain ATCC29814using this system. With the fast development of genome sequencing technology, the genome editing system will be widely used for genome analysis and engineering in the post-genome era.