Establishment and application of CRISPR interference to affect sporulation, hydrogen peroxide detoxification, and mannitol catabolism in the methylotrophic thermophile Bacillus methanolicus.

Establishment and application of CRISPR interference to affect sporulation, hydrogen peroxide detoxification, and mannitol catabolism in the methylotrophic thermophile Bacillus methanolicus. Appl Microbiol Biotechnol. 2019 May 28;: Authors: Schultenkämper K, Brito LF, López MG, Brautaset T, Wendisch VF Abstract Bacillus methanolicus is a thermophilic, Gram-positive, rod-shaped bacterium. It is a facultative methylotroph which can use carbon and energy sources including mannitol and the one-carbon (C1) and non-food substrate methanol for growth and overproduction of amino acids, which makes it a promising candidate for biotechnological applications. Despite a growing tool box for gene cloning and expression, tools for targeted chromosomal gene knockouts and gene repression are still missing for this organism. Here, the CRISPRi-dCas9 technique for gene repression was established in B. methanolicus MGA3. Significantly reduced spore formation on the one hand and increased biofilm formation on the other hand could be demonstrated when the stage zero sporulation protein A gene spo0A was targeted. Furthermore, when the mannitol-1-phosphate 5-dehydrogenase gene mtlD was targeted by CRISPRi, mtlD RNA levels, and MtlD specific activities in crude extracts were decreased to about 50 % which resulted in reduced biomass formation from mannitol. As a third target, the catalase gene katA was chosen. Upon targeting katA by CRISPRi, catalase activ...
Source: Applied Microbiology and Biotechnology - Category: Microbiology Authors: Tags: Appl Microbiol Biotechnol Source Type: research