Mahipant Gumpanat, Kato Junichi, Kataoka Naoya, Vangnai Alisa S
Biological Sciences Program, Faculty of Science, Chulalongkorn University.
Biocatalyst and Environmental Biotechnology Research Unit, Department of Biochemistry, Faculty of Science, Chulalongkorn University.
J Gen Appl Microbiol. 2019 May 21;65(2):96-105. doi: 10.2323/jgam.2018.06.001. Epub 2018 Nov 27.
Given their applicability in genetic engineering, undomesticated Bacillus strains are extensively used as non-natural hosts for chemical production due to their high tolerance of toxic substrates or products. However, they are difficult to genomically modify due to their low transformation efficiencies. In this study, the Bacillus-E. coli shuttle vector pHY300PLK, which is widely used in gram-positive bacteria, was adopted for genome integration in organic solvent-tolerant Bacillus isolates. The Bacillus-replicative vector was used to deliver homologous recombinant DNA and propagate itself inside the host cell, increasing the likelihood of genome integration of the recombinant DNA. Then, the unintegrated vectors were cured by cell cultivation in antibiotic-free medium with facilitation of nickel ions. The developed protocol was successfully demonstrated and validated by the disruption of amyE gene in B. subtilis 168. With an improved clonal selection protocol, the probability of clonal selection of the amyE::cat genome-integrated mutants was increased up to 42.0 ± 10.2%. Genome integration in undomesticated, organic solvent tolerant Bacillus strains was also successfully demonstrated with amyE as well as proB gene creating the gene-disrupted mutants with the corresponding phenotype and genotype. Not only was this technique effectively applied to several strains of undomesticated B. subtilis, but it was also successfully applied to B. cereus. This study validates the possibility of the application of Bacillus-replicative vector as well as the developed protocol in a variety of genome modification of undomesticated Bacillus species.
鉴于其在基因工程中的适用性，未驯化的芽孢杆菌菌株因其对有毒底物或产物具有高耐受性，被广泛用作化学品生产的非天然宿主。然而，由于其转化效率低，难以对其进行基因组修饰。在本研究中，将广泛应用于革兰氏阳性菌的芽孢杆菌-大肠杆菌穿梭载体pHY300PLK用于耐有机溶剂芽孢杆菌分离株的基因组整合。芽孢杆菌复制型载体用于递送同源重组DNA并在宿主细胞内自我复制，增加了重组DNA基因组整合的可能性。然后，通过在含有镍离子的无抗生素培养基中进行细胞培养，消除未整合的载体。通过枯草芽孢杆菌168中amyE基因的破坏，成功证明并验证了所开发的方案。通过改进的克隆筛选方案，amyE::cat基因组整合突变体的克隆筛选概率提高到了42.0±10.2%。利用amyE以及proB基因成功证明了在未驯化的、耐有机溶剂的芽孢杆菌菌株中进行基因组整合，产生了具有相应表型和基因型的基因破坏突变体。该技术不仅有效地应用于几种未驯化的枯草芽孢杆菌菌株，还成功地应用于蜡样芽孢杆菌。本研究验证了芽孢杆菌复制型载体以及所开发的方案在未驯化芽孢杆菌物种的各种基因组修饰中的应用可能性。
