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Molecular and Biochemical Characterization of Salt-Tolerant Trehalose-6-Phosphate Hydrolases Identified by Screening and Sequencing Salt-Tolerant Clones From the Metagenomic Library of the Gastrointestinal Tract.

ABSTRACT: The exploration and utilization of microbial salt-tolerant enzymatic and genetic resources are of great significance in the field of biotechnology and for the research of the adaptation of microorganisms to extreme environments. The presence of new salt-tolerant genes and enzymes in the microbial metagenomic library of the gastrointestinal tract has been confirmed through metagenomic technology. This paper aimed to identify and characterize enzymes that confer salt tolerance in the gastrointestinal tract microbe. By screening the fecal metagenomic library, 48 salt-tolerant clones were detected, of which 10 salt-tolerant clones exhibited stronger tolerance to 7% (wt/vol) NaCl and stability in different concentrations of NaCl [5%-9% (wt/vol)]. High-throughput sequencing and biological information analysis showed that 91 potential genes encoded proteins and enzymes that were widely involved in salt tolerance. Furthermore, two trehalose-6-phosphate hydrolase genes, namely, tre_P2 and tre_P3, were successfully cloned and expressed in Escherichia coli BL21 (DE3). By virtue of the substrate of p-nitrophenyl-?-D-glucopyranoside (pNPG) which can be specifically hydrolyzed by trehalose-6-phosphate hydrolase to produce glucose and p-nitrophenol, the two enzymes can act optimally at pH 7.5 and 30°C. Steady-state kinetics with pNPG showed that the K M and K cat values were 15.63 mM and 10.04 s-1 for rTRE_P2 and 12.51 mM and 10.71 s-1 for rTRE_P3, respectively. Characterization of enzymatic properties demonstrated that rTRE_P2 and rTRE_P3 were salt-tolerant. The enzymatic activity increased with increasing NaCl concentration, and the maximum activities of rTRE_P2 and rTRE_P3 were obtained at 4 and 3 M NaCl, respectively. The activities of rTRE_P2 increased by approximately 43-fold even after 24 h of incubation with 5 M NaCl. This study is the first to report the identification as well as molecular and biochemical characterization of salt-tolerant trehalose-6-phosphate hydrolase from the metagenomic library of the gastrointestinal tract. Results indicate the existence of numerous salt-tolerant genes and enzymes in gastrointestinal microbes and provide new insights into the salt-tolerant mechanisms in the gastrointestinal environment.

SUBMITTER: Yang Y

PROVIDER: S-EPMC7358406 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Molecular and Biochemical Characterization of Salt-Tolerant Trehalose-6-Phosphate Hydrolases Identified by Screening and Sequencing Salt-Tolerant Clones From the Metagenomic Library of the Gastrointestinal Tract.

Yang Yanxia Y Yang Yunjuan Y Fan Qin Q Huang Zunxi Z Li Junjun J Wu Qian Q Tang Xianghua X Ding Junmei J Han Nanyu N Xu Bo B

Frontiers in microbiology 20200707

The exploration and utilization of microbial salt-tolerant enzymatic and genetic resources are of great significance in the field of biotechnology and for the research of the adaptation of microorganisms to extreme environments. The presence of new salt-tolerant genes and enzymes in the microbial metagenomic library of the gastrointestinal tract has been confirmed through metagenomic technology. This paper aimed to identify and characterize enzymes that confer salt tolerance in the gastrointesti ...[more]

PMID: 32733411

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Project description:Here, we report on the construction of a metagenomic library from a chitin-amended disease-suppressive agricultural soil and its screening for genes that encode novel chitinolytic enzymes. The library, constructed in fosmids in an Escherichia coli host, comprised 145,000 clones containing inserts of sizes of 21 to 40 kb, yielding a total of approximately 5.8 GB of cloned soil DNA. Using genetic screenings by repeated PCR cycles aimed to detect gene sequences of the bacterial chitinase A-class (hereby named chi A genes), we identified and characterized five fosmids carrying candidate genes for chitinolytic enzymes. The analysis thus allowed access to the genomic (fosmid-borne) context of these genes. Using the chiA-targeted PCR, which is based on degenerate primers, the five fosmids all produced amplicons, of which the sequences were related to predicted chitinolytic enzyme-encoding genes of four different host organisms, including Stenotrophomonas maltophilia. Sequencing and de novo annotation of the fosmid inserts confirmed that each one of these carried one or more open reading frames that were predicted to encode enzymes active on chitin, including one for a chitin deacetylase. Moreover, the genetic contexts in which the putative chitinolytic enzyme-encoding genes were located were unique per fosmid. Specifically, inserts from organisms related to Burkholderia sp., Acidobacterium sp., Aeromonas veronii, and the chloroflexi Nitrolancetus hollandicus and/or Ktedonobacter racemifer were obtained. Remarkably, the S. maltophilia chiA-like gene was found to occur in two different genetic contexts (related to N. hollandicus/K. racemifer), indicating the historical occurrence of genetic reshufflings in this part of the soil microbiota. One fosmid containing the insert composed of DNA from the N. hollandicus-like organism (denoted 53D1) was selected for further work. Using subcloning procedures, its putative gene for a chitinolytic enzyme was successfully brought to expression in an E. coli host. On the basis of purified protein preparations, the produced protein was characterized as a chitobiosidase of 43.6 kDa, with a pI of 4.83. Given its activity spectrum, it can be typified as a halotolerant chitobiosidase.

Project description:The crenarchaeon Sulfolobus acidocaldarius has been described to synthesize trehalose via the maltooligosyltrehalose synthase (TreY) and maltooligosyltrehalose trehalohydrolase (TreZ) pathway, and the trehalose glycosyltransferring synthase (TreT) pathway has been predicted. Deletion mutant analysis of strains with single and double deletions of ΔtreY and ΔtreT in S. acidocaldarius revealed that in addition to these two pathways, a third, novel trehalose biosynthesis pathway is operative in vivo: the trehalose-6-phosphate (T6P) synthase/T6P phosphatase (TPS/TPP) pathway. In contrast to known TPS proteins, which belong to the GT20 family, the S. acidocaldarius TPS belongs to the GT4 family, establishing a new function within this group of enzymes. This novel GT4-like TPS was found to be present mainly in the Sulfolobales The ΔtreY ΔtreT Δtps triple mutant of S. acidocaldarius, which lacks the ability to synthesize trehalose, showed no altered phenotype under standard conditions or heat stress but was unable to grow under salt stress. Accordingly, in the wild-type strain, a significant increase of intracellular trehalose formation was observed under salt stress. Quantitative real-time PCR showed a salt stress-mediated induction of all three trehalose-synthesizing pathways. This demonstrates that in Archaea, trehalose plays an essential role for growth under high-salt conditions.IMPORTANCE The metabolism and function of trehalose as a compatible solute in Archaea was not well understood. This combined genetic and enzymatic approach at the interface of microbiology, physiology, and microbial ecology gives important insights into survival under stress, adaptation to extreme environments, and the role of compatible solutes in Archaea Here, we unraveled the complexity of trehalose metabolism, and we present a comprehensive study on trehalose function in stress response in S. acidocaldarius This sheds light on the general microbiology and the fascinating metabolic repertoire of Archaea, involving many novel biocatalysts, such as glycosyltransferases, with great potential in biotechnology.

Dataset Information

Molecular and Biochemical Characterization of Salt-Tolerant Trehalose-6-Phosphate Hydrolases Identified by Screening and Sequencing Salt-Tolerant Clones From the Metagenomic Library of the Gastrointestinal Tract.

Publications

Molecular and Biochemical Characterization of Salt-Tolerant Trehalose-6-Phosphate Hydrolases Identified by Screening and Sequencing Salt-Tolerant Clones From the Metagenomic Library of the Gastrointestinal Tract.

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