Engineered CRISPR/Cas9 System for Transcriptional Gene Silencing in Arthrobacter Species Indicates Bacterioruberin is Indispensable for Growth at Low Temperatures

Abstract

Pink-pigmented Arthrobacter species produce the rare C50 carotenoid bacterioruberin, which is suspected to be part of the cold adaptation mechanism. In silico analysis of the repertoire of genes encoded by the Arthrobacter agilis and Arthrobacter bussei genome revealed the biosynthetic pathway of bacterioruberin. Although genetic analysis is an essential tool for studying the physiology of Arthrobacter species, genetic manipulation of Arthrobacter is always time and labor intensive due to the lack of genetic engineering tools. Here we report the construction and application of a CRISPR/deadCas9 system (pCasiART) for gene silencing in Arthrobacter species. The engineered system pCasiART is suitable for the Golden Gate assembly of spacers, enabling rapid and accurate construction of adapted systems. In addition, pCasiART has been developed to provide an efficient transcription inhibition system for genome-wide gene silencing. The gene silencing of the phytoene synthase (CrtB), the first enzyme in bacterioruberin biosynthesis, suppressed bacterioruberin biosynthesis in Arthrobacter agilis and Arthrobacter bussei, resulting in a lack of pink pigmentation, reduction of biomass production, and growth rates at low temperatures.