Dataset Information

Expression and characterization of a 9-cis-epoxycarotenoid dioxygenase from Serratia sp. ATCC 39006 capable of biotransforming isoeugenol and 4-vinylguaiacol to vanillin.

ABSTRACT: A 9-cis-epoxycarotenoid dioxygenase gene from Serratia sp. ATCC 39,006 (SeNCED) was overexpressed in soluble form in E.coli. SeNCED showed the maximum activity at 30 °C and pH 8.0, and it was stable relatively at range of pH 5-10 and temperature of 20 °C to 30 °C. SeNCED effectively catalyzes the side chain double bond cleavage of isoeugenol and 4-vinylguaiacol to vanillin. The kinetic constant K_m values toward isoeugenol and 4-vinylguaiacol were 18.92 mM and 6.31 mM and V_max values were 50.73 IU/g and 4.77 IU/g, respectively. Moreover, the SeNCED exhibited an excellent organic solvent tolerance and the enzyme activity was substantially improved at presence of 10% of trichloromethane. The produced vanillin was achieved at an around 0.53 g/L (3.47 mM) and 0.33 g/L (2.17 mM) after 8 h reaction at 4 mM of isoeugenol and 4-vinylguaiacol, respectively, using transformed Escherichia coli cells harboring SeNCED in the presence of trichloromethane.

SUBMITTER: Tang J

PROVIDER: S-EPMC5989660 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Expression and characterization of a 9-cis-epoxycarotenoid dioxygenase from Serratia sp. ATCC 39006 capable of biotransforming isoeugenol and 4-vinylguaiacol to vanillin.

Tang Jiao J Shi Lei L Li Lulu L Long Liangkun L Ding Shaojun S

Biotechnology reports (Amsterdam, Netherlands) 20180418

A 9-cis-epoxycarotenoid dioxygenase gene from Serratia sp. ATCC 39,006 (SeNCED) was overexpressed in soluble form in E.coli. SeNCED showed the maximum activity at 30 °C and pH 8.0, and it was stable relatively at range of pH 5-10 and temperature of 20 °C to 30 °C. SeNCED effectively catalyzes the side chain double bond cleavage of isoeugenol and 4-vinylguaiacol to vanillin. The kinetic constant Km values toward isoeugenol and 4-vinylguaiacol were 18.92 mM a ...[more]

PMID: 29881703

Similar Datasets

Project description:The biosynthesis of prodigiosin in the model prodigiosin-producing strain, Serratia sp. ATCC 39006, is significantly influenced by environmental and cellular signals. However, a comprehensive regulatory mechanism for this process has not been well established. In the present study, we demonstrate that organic hydroperoxide activates prodigiosin biosynthesis in an OhrR-dependent manner. Specifically, the MarR-family transcriptional repressor OhrR (Ser39006_RS05455) binds to its operator located far upstream of the promoter region of the prodigiosin biosynthesis operon (319 to 286 nucleotides [nt] upstream of the transcription start site) and negatively regulates the expression of prodigiosin biosynthesis genes. Organic hydroperoxide disassociates the binding between OhrR and its operator, thereby promoting the prodigiosin production. Moreover, OhrR modulates the resistance of Serratia sp. ATCC 39006 to organic hydroperoxide by regulating the transcription of its own gene and the downstream cotranscribed ohr gene. These results demonstrate that OhrR is a pleiotropic repressor that modulates the prodigiosin production and the resistance of Serratia sp. ATCC 39006 to organic hydroperoxide stress. IMPORTANCE Bacteria naturally encounter various environmental and cellular stresses. Organic hydroperoxides generated from the oxidation of polyunsaturated fatty acids are widely distributed and usually cause lethal oxidative stress by damaging cellular components. OhrR is known as a regulator that modulates the resistance of bacteria to organic hydroperoxide stress. In the current study, organic hydroperoxide disassociates OhrR from the promoter of prodigiosin biosynthesis gene cluster, thus promoting transcription of pigA to -O genes. In this model, organic hydroperoxide acts as an inducer of prodigiosin synthesis in Serratia sp. ATCC 39006. These results improve our understanding of the regulatory network of prodigiosin synthesis and serve as an example for identifying the cross talk between the stress responses and the regulation of secondary metabolism.

Project description:Serratia sp. ATCC 39006 is an important model strain for the study of prodigiosin production, whose prodigiosin biosynthesis genes (pigA-O) are arranged in an operon. Several transcription factors have been shown to control the transcription of the pig operon. However, since the regulation of prodigiosin biosynthesis is complex, the regulatory mechanism for this process has not been well established. In most γ-proteobacteria, the ROK family regulator NagC acts as a global transcription factor in response to N-acetylglucosamine (GlcNAc). In Serratia sp. ATCC 39006, NagC represses the transcription of two divergent operons, nagE and nagBAC, which encode proteins involved in the transport and metabolism of GlcNAc. Moreover, NagC directly binds to a 21-nt region that partially overlaps the -10 and -35 regions of the pig promoter and promotes the transcription of prodigiosin biosynthesis genes, thereby increasing prodigiosin production. Although NagC still acts as both repressor and activator in Serratia sp. ATCC 39006, its transcriptional regulatory activity is independent of GlcNAc. NagC was first found to regulate antibiotic biosynthesis in Gram-negative bacteria, and NagC-mediated regulation is not responsive to GlcNAc, which contributes to future studies on the regulation of secondary metabolism by NagC in other bacteria.ImportanceThe ROK family transcription factor NagC is an important global regulator in the γ-proteobacteria. A large number of genes involved in the transport and metabolism of sugars, as well as those associated with biofilm formation and pathogenicity, are regulated by NagC. In all of these regulations, the transcriptional regulatory activity of NagC responds to the supply of GlcNAc in the environment. Here, we found for the first time that NagC can regulate antibiotic biosynthesis, whose transcriptional regulatory activity is independent of GlcNAc. This suggests that NagC may respond to more signals and regulate more physiological processes in Gram-negative bacteria.

Project description:The opportunistic pathogen Serratia sp. ATCC 39006 (S39006) is a rod-shaped, motile, Gram-negative bacterium that produces a 𝛽-lactam antibiotic (a carbapenem) and a bioactive red-pigmented tripyrrole antibiotic, prodigiosin. It is also the only known enterobacterium that naturally produces intracellular gas vesicles (GVs), enabling cells to float in static water columns. Regulation of GVs and secondary metabolites in S39006 can be coordinated but such pleiotropy is still poorly understood. To uncover novel inputs to this complex regulatory network, we used transposon mutagenesis to identify a mutant with an insertion in an IclR-type transcriptional regulator gene. The iclR mutant showed diminished production of carbapenem, prodigiosin, GVs and cellulase. Furthermore, the mutant also showed increased swimming and swarming motilities but exhibited attenuated virulence in planta and ability to kill the nematode C. elegans. Using differential expression analysis of the intracellular proteomes of the wild type and iclR mutant, we confirmed that the mutation negatively impacted expression of the corresponding GV, carbapenem and prodigiosin gene clusters. In contrast, flagellar and chemotaxis proteins were overexpressed, consistent with the increased motility of the mutant. We also found that the proteins encoded by a putative yagEF-yjhF operon, involved in xylonate catabolism and transport, showed a 5- to 7-fold increase in expression. Finally, we show that IclR is a repressor of xylonate catabolism in S39006 and suggest that xylonate is potentially involved in controlling carbapenem and prodigiosin biosynthesis. Our results indicate that IclR is a global regulator that controls antibiotic biosynthesis, flotation through modulating GV assembly, and has pleiotropic impacts on the physiology and virulence of S39006. Based on these findings, we propose the designation of this IclR-family transcriptional regulator as XyrR (Xylonate response Regulator).

Project description:BACKGROUND:Serratia sp. ATCC 39006 (S39006) is a Gram-negative enterobacterium that is virulent in plant and animal models. It produces a red-pigmented trypyrrole secondary metabolite, prodigiosin (Pig), and a carbapenem antibiotic (Car), as well as the exoenzymes, pectate lyase and cellulase. Secondary metabolite production in this strain is controlled by a complex regulatory network involving quorum sensing (QS). Hfq and RsmA (two RNA binding proteins and major post-transcriptional regulators of gene expression) play opposing roles in the regulation of several key phenotypes within S39006. Prodigiosin and carbapenem production was abolished, and virulence attenuated, in an S39006 ?hfq mutant, while the converse was observed in an S39006 rsmA transposon insertion mutant. RESULTS:In order to define the complete regulon of Hfq and RsmA, deep sequencing of cDNA libraries (RNA-seq) was used to analyse the whole transcriptome of S39006 ?hfq and rsmA::Tn mutants. Moreover, we investigated global changes in the proteome using an LC-MS/MS approach. Analysis of differential gene expression showed that Hfq and RsmA directly or indirectly regulate (at the level of RNA) 4% and 19% of the genome, respectively, with some correlation between RNA and protein expression. Pathways affected include those involved in antibiotic regulation, virulence, flagella synthesis, and surfactant production. Although Hfq and RsmA are reported to activate flagellum production in E. coli and an adherent-invasive E. coli hfq mutant was shown to have no flagella by electron microscopy, we found that flagellar production was increased in the S39006 rsmA and hfq mutants. Additionally, deletion of rsmA resulted in greater genomic flux with increased activity of two mobile genetic elements. This was confirmed by qPCR and analysis of rsmA culture supernatant revealed the presence of prophage DNA and phage particles. Finally, expression of a hypothetical protein containing DUF364 increased prodigiosin production and was controlled by a putative 5' cis-acting regulatory RNA element. CONCLUSION:Using a combination of transcriptomics and proteomics this study provides a systems-level understanding of Hfq and RsmA regulation and identifies similarities and differences in the regulons of two major regulators. Additionally our study indicates that RsmA regulates both core and variable genome regions and contributes to genome stability.

Dataset Information

Expression and characterization of a 9-cis-epoxycarotenoid dioxygenase from Serratia sp. ATCC 39006 capable of biotransforming isoeugenol and 4-vinylguaiacol to vanillin.

Publications

Expression and characterization of a 9-cis-epoxycarotenoid dioxygenase from <i>Serratia</i> sp. ATCC 39006 capable of biotransforming isoeugenol and 4-vinylguaiacol to vanillin.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets