Tolerance engineering in Deinococcus geothermalis by heterologous efflux pumps

Erika Boulant,Olivier Gorgé,Julia Vergalli,Flora Nolent,Rémi Bernard,Jean-Paul Leonetti,Emmanuelle Cambon,Fabienne Neulat-Ripoll,Anne-Laure Favier,Jean Michel Bolla,Maria Girleanu

doi:10.1038/s41598-021-83339-1

Abstract

Producing industrially significant compounds with more environmentally friendly represents a challenging task. The large-scale production of an exogenous molecule in a host microfactory can quickly cause toxic effects, forcing the cell to inhibit production to survive. The key point to counter these toxic effects is to promote a gain of tolerance in the host, for instance, by inducing a constant flux of the neo-synthetized compound out of the producing cells. Efflux pumps are membrane proteins that constitute the most powerful mechanism to release molecules out of cells. We propose here a new biological model, Deinococcus geothermalis, organism known for its ability to survive hostile environment; with the aim of coupling the promising industrial potential of this species with that of heterologous efflux pumps to promote engineering tolerance. In this study, clones of D. geothermalis containing various genes encoding chromosomal heterologous efflux pumps were generated. Resistant recombinants were selected using antibiotic susceptibility tests to screen promising candidates. We then developed a method to determine the efflux efficiency of the best candidate, which contains the gene encoding the MdfA of Salmonella enterica serovar Choleraesuis. We observe 1.6 times more compound in the external medium of the hit recombinant than that of the WT at early incubation time. The data presented here will contribute to better understanding of the parameters required for efficient production in D. geothermalis.

Highlights

Deinococcus geothermalis, initially isolated from hot s prings[1], is an aerobic, non-pathogenic, non-sporulating, non-flagellar, pinkish-red bacterium that typically exists as dyads and tetrads
Bacteria from Deinococcus spp. have been the subject of other industrial applications, where specific enzymes of D. geothermalis or D. radiodurans are used for the production of molecules, such as α-arbutin (KR20110028169, Kyung Hee University—Industry Cooperation Group), nucleoside triphosphates and ribonucleic acid (WO2017176963 and WO2019075167, Greenlight Biosciences Inc.)
Twenty-seven efflux pump genes were cloned in D. geothermalis by homologous recombination at the same locus on the chromosome under the control of a constitutive promoter optimized for expression

Summary

Introduction

Deinococcus geothermalis, initially isolated from hot s prings[1], is an aerobic, non-pathogenic, non-sporulating, non-flagellar, pinkish-red bacterium that typically exists as dyads and tetrads. D. geothermalis is the best candidate for the bioremediation of soils contaminated with radionuclide w astes[1,17,19,20] or x enobiotics[21] This bacterium was recently chosen as a new model organism to determine the impact of exposure in space or Martian conditions on a bacterial biofilm[22]. D. geothermalis is a thermophilic organism with promising applications in human therapy due to its ability to produce UV-resistant extremolytes as well as carotenoids with significant antioxidant p otential[23,24]. For all these reasons, D. geothermalis has been selected and used as a cellular microfactory by the biotechnology company Deinove specialized in the synthesis of innovative compounds from extremophilic organisms. Bacteria from Deinococcus spp. have been the subject of other industrial applications, where specific enzymes of D. geothermalis or D. radiodurans are used for the production of molecules, such as α-arbutin (KR20110028169, Kyung Hee University—Industry Cooperation Group), nucleoside triphosphates and ribonucleic acid (WO2017176963 and WO2019075167, Greenlight Biosciences Inc.)

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Conclusion