Molecular identification of a cyclodextrin glycosyltransferase-producing microorganism and phylogenetic assessment of enzymatic activities.

Soledad Caminata Landriel,Hernán Costa,Alexandra M Gottlieb,Oscar A Taboga,Susana A Ferrarotti,Julieta D.L.M Castillo

doi:10.1590/0001-3765201920180568

Abstract

Cyclodextrin glycosyltransferases (CGTases) are important enzymes in the biotechnology field because they catalyze starch conversion into cyclodextrins and linear oligosaccharides, which are used in food, pharmaceutical and cosmetic industries. The CGTases are classified according to their product specificity in α-, β-, α/β- and γ-CGTases. As molecular markers are the preferred tool for bacterial identification, we employed six molecular markers (16S rRNA, dnaK, gyrB, recA, rpoB and tufA) to test the identification of a CGTase-producing bacterial strain (DF 9R) in a phylogenetic context. In addition, we assessed the phylogenetic relationship of CGTases along bacterial evolution. The results obtained here allowed us to identify the strain DF 9R as Paenibacillus barengoltzii, and to unveil a complex origin for CGTase types during archaeal and bacterial evolution. We postulate that the α-CGTase activity represents the ancestral type, and that the γ-activity may have derived from β-CGTases.

Highlights

During a prospection of cyclodextrin glycosyltransferases (CGTase)-producing soil microorganisms, several bacteria were identified through biochemical tests (Ferrarotti S.A., unpublished data)
Two variants of the 16S rRNA gene were detected in the bacterial genome of strain DF 9R, differing in six positions along the gene
Phylogenetic analyses performed on singlemarker matrices generated topologies which were congruent in the unequivocal location of strain DF 9R within Paenibacillus clades (97.6-100% of bootstrap support, BS) (Supplementary Material, Figures S1-S6)

Summary

Introduction

During a prospection of cyclodextrin glycosyltransferases (CGTase)-producing soil microorganisms, several bacteria were identified through biochemical tests (Ferrarotti S.A., unpublished data). A peculiar strain -DF 9R- attracted our attention because of its high cyclodextrin (CD) producing activity and its concomitant potential for industrial exploitation. This strain produces a unique CGTase whose structure allowed clarifying the induced fit mechanism of these enzymes (Costa et al 2012). The classification of CGTase-producing bacteria has been under review (Kaulpiboon et al 2010) All these revisions were stimulated by the availability of molecular tools (Weisburg et al 1991, Rajendhran and Gunasekaran 2011). Nowadays, housekeeping genes have become the preferred complementary tools for modern bacterial taxonomy (Tanabe et al 2007, Porwal et al 2009, Hwang et al 2011, Vos et al 2012, Gomes et al 2018); they are single copy markers that evolve faster than the 16S rRNA and show scarce indel events (Santos and Ochman 2004)

Methods

Results

Conclusion