Degradação de quitina em Chromobacterium: proposição da via catabólica e expressão heteróloga de uma monooxigenase lítica de polissacarídeo de C. violaceum ATCC 12472

Abstract

The genus Chromobacterium is formed by Betaproteobacteria, Gram-negative, saprophytes, free living forms and a few of pathogenic bacteria. This group has shown significant growth between 2007 and 2017 with the discovery of ten new species. Chromobacterium violaceum was the first to be described and it is the most studied of the group. The species is commonly found in waters and soils of tropical and subtropical regions such as saprophytes, being able to use only chitin as an energy source. Chitin is the second most abundant biopolymer in nature and can be used as a source of carbon and nitrogen by multiple organisms, such as bacteria. Its degradation begins by lytic polysaccharide monooxygenases (LPMOs) active in crystalline substrate. This work focuses on the construction, by comparative genomic analysis, of chitin catabolism of Cromobacterium and the expression of one LPMO of C. violaceum ATCC 12472. One likely catabolic pathway of C. violaceum ATCC 12472 was obtained from the one described for the family Vibrionaceae, using information gathered about the regulon NagQ from the species and from in silico analysis of subcelular locations of each selected protein. The pathway was extended to genus level by the localization of ortologs on the 41 genomes of Cromobacterium available on NCBI database. Five sequences of LPMOs were found in Chromobacterium ATCC 12472: CV0553, CV0554, CV2592, CV3323 e CV3489. Among those, CV0553 e CV2592 had improved codons on its synthetic coding sequences being afterwards inserted into pET-Sumo modified plasmids. The vectors were introduced into strains of Escherichia coli BL21(DE3), SHuffle® T7 Express Competent e ArcticExpress (DE3). Of the two proteins produced, rCv2592-Sumo was the only one to be found in the soluble way on the intracellular fraction of the three analyzed hosts. The recombinant protein was purified in chitin substrate and was able to degrade α-chitin. We conclude that chitin catabolism is conserved in Chromobacterium. This is an indicative of its importance on the use of chitin as an alternative source of energy. The gene for NagQ regulator its well conserved on the genus level, with its most plesiomorphic form being found on C. haemolyticum and the most recent on C. violaceum. Finally, the production of rCv2592 can be prove to be an efficient alternative for the synthesis of chitin derivatives, thus further information need to be gathered about it.