Production of New Isoflavone Diglucosides from Glycosylation of 8-Hydroxydaidzein by Deinococcus geothermalis Amylosucrase

8-Hydroxydaidzein (8-OHDe) is a non-natural isoflavone polyphenol isolated from fermented soybean foods. 8-OHDe exhibits a wide range of pharmaceutical activities. However, both the poor solubility and instability of 8-OHDe limit its applications. To resolve the limitations of 8-OHDe, Deinococcus geothermalis amylosucrase (DgAS) has previously been used to glycosylate 8-OHDe to produce soluble and stable 8-OHDe-7-O-α-glucopyranoside (8-OHDe-7-G) in a 0.5 h reaction time. In this study, we aimed to use DgAS and an extended reaction time to produce 8-OHDe diglucosides. At least three 8-OHDe derivatives were produced after a 24 h reaction time, and two major products were successfully purified and identified as new compounds: 8-OHDe-7-O-[α-glucopyranosyl-(1®6)-α-glucopyranoside] (8-OHDe-7-G2) and 8-OHDe-7,4′-O-α-diglucopyranoside (8-OHDe-7-G-4′-G). 8-OHDe-7-G-4′-G showed a 4619-fold greater aqueous solubility than 8-OHDe. In addition, over 92% of the 8-OHDe diglucosides were stable after 96 h, while only 10% of the 8-OHDe could be detected after being subjected to the same conditions. The two stable 8-OHDe diglucoside derivatives have the potential for pharmacological usage in the future.

An Antioxidant Defense System in Radiation-Resistant Bacterium Deinococcus geothermalis against Oxidative Stress

A radiation-resistant bacterium, Deinococcus geothermalis has various stress response mechanisms, including antioxidation. Features that maintain vitality at high radiation doses include the following: enzymatic scavengers of ROS such as catalase, SOD, and peroxidase; strain-specific DNA repair systems such as Deinococcal unique proteins; non-enzymatic responses such as manganese complexes, carotenoids, and DNA-binding proteins. This chapter summarizes the primary response mechanism by redox balance centered on the cystine transporter. It also reviews action characteristics of DNA-binding protein Dps and a putative LysR family protein, and effects on loss of function of the carotenoid biosynthesis genes by transposition of insertion sequences. Environmental adaptation and molecular evolution of radiation-resistant bacterium are also considered to explain the potentials of molecular behavior induced by oxidative stress.

Novel DNA-protection Protein (Dps) Originated From Deinococcus Geothermalis

We characterized two interrelated proteins from the Deinococcus geothermalis strain with regard to the role of DNA-binding and protection, such as a novel Dps, Dgeo_0257, and a Dps orthologous protein, Dgeo_0281. Despite the lack of conserved amino acid sequence for ferroxidase activity, Dgeo_0257 exhibited high DNA-binding affinity and formed a dodecameric conformation. In contrast, Dgeo_0281 protein showed less effective DNA-binding and was mainly observed monomeric or dimeric forms. Electrophoretic mobility shift assay showed that both purified proteins are non-specifically bound to DNA to protect against DNA degradation and the properties of Dps responding to specific metal ions. In the presence of ferrous and ferric ions, Dgeo_0257 or Dgeo_0281 protein does not readily bind to DNA. However, in Zn and Mn ions are present, both proteins had more stable DNA-binding. From the Dps gene disrupted mutant test, each showed a higher sensitivity to oxidative stress than the wild-type strain. In addition, the expression level of each gene was correlated with the presence of each other. In this study, we characterized and confirmed the functional roles of Dgeo_0257 for sensing metal ions and binding to DNAs along with the Dps orthologous Dgeo_0281. Based on various observational evidence, we propose that Dgeo_0257 is a novel Dps protein with no ferroxidase activity.

Tolerance engineering in Deinococcus geothermalis by heterologous efflux pumps

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.

Genome Plasticity by Insertion Sequences Learned From a Case of Radiation-Resistant Bacterium Deinococcus geothermalis

The genome of the radiation-resistant bacterium Deinococcus geothermalis contains 19 types of insertion sequences (ISs), including 93 total transposases (Tpases) in 73 full-length ISs from the main chromosome and 2 mega plasmids. In this study, 68 ISs from the D. geothermalis genome were extracted to implicate the earlier genome before its mutation by transposition of ISs. The total size of eliminated ISs from genome was 78.85 kb. From these in silico corrections of mutation by the ISs, we have become aware of some bioinformatics factualness as follows: (1) can reassemble the disrupted genes if the exact IS region was eliminated, (2) can configure the schematic clustering of major DDE type Tpases, (3) can determine IS integration order across multiple hot spots, and (4) can compare genetic relativeness by the partial synteny analysis between D. geothermalis and Deinococcus strain S9. Recently, we found that several IS elements actively transferred to other genomic sites under hydrogen peroxide-induced oxidative stress conditions, resulting in the inactivation of functional genes. Therefore, the single species genome’s mobilome study provides significant support to define bacterial genome plasticity and molecular evolution from past and present progressive transposition events.