Towards a Biocatalyst for (S)-Styrene Oxide Production: Characterization of the Styrene Degradation Pathway of Pseudomonas sp. Strain VLB120
Ontology highlight
ABSTRACT:
SUBMITTER:
PROVIDER: S-EPMC106767 | biostudies-literature | 1998 Sep
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:In order to design a biocatalyst for the production of optically pure styrene oxide, an important building block in organic synthesis, the metabolic pathway and molecular biology of styrene degradation in Pseudomonas sp. strain VLB120 was investigated. A 5.7-kb XhoI fragment, which contained on the same strand of DNA six genes involved in styrene degradation, was isolated from a gene library of this organism in Escherichia coli by screening for indigo formation. T7 RNA polymerase expression experiments indicated that this fragment coded for at least five complete polypeptides, StyRABCD, corresponding to five of the six genes. The first two genes encoded the potential carboxy-terminal part of a sensor, named StySc, and the complete response regulator StyR. Fusion of the putative styAp promoter to a lacZ reporter indicated that StySc and StyR together regulate expression of the structural genes at the transcriptional level. Expression of styScR also alleviated a block that prevented translation of styA mRNA when a heterologous promoter was used. The structural genes styA and styB produced a styrene monooxygenase that converted styrene to styrene oxide, which was then converted to phenylacetaldehyde by StyC. Sequence homology analysis of StyD indicated a probable function as a phenylacetaldehyde dehydrogenase. To assess the usefulness of the enzymes for the production of enantiomerically pure styrene oxide, we investigated the enantiospecificities of the reactions involved. Kinetic resolution of racemic styrene oxide by styrene oxide isomerase was studied with E. coli recombinants carrying styC, which converted styrene oxide at a very high rate but with only a slight preference for the S enantiomer. However, recombinants producing styrene monooxygenase catalyzed the formation of (S)-styrene oxide from inexpensive styrene with an excellent enantiomeric excess of more than 99% at rates up to 180 U g (dry weight) of cells-1.
| S-EPMC106275 | biostudies-literature
Project description:BackgroundOver the recent years the production of Ehrlich pathway derived chemicals was shown in a variety of hosts such as Escherichia coli, Corynebacterium glutamicum, and yeast. Exemplarily the production of isobutyric acid was demonstrated in Escherichia coli with remarkable titers and yields. However, these examples suffer from byproduct formation due to the fermentative growth mode of the respective organism. We aim at establishing a new aerobic, chassis for the synthesis of isobutyric acid and other interesting metabolites using Pseudomonas sp. strain VLB120, an obligate aerobe organism, as host strain.ResultsThe overexpression of kivd, coding for a 2-ketoacid decarboxylase from Lactococcus lactis in Ps. sp. strain VLB120 enabled for the production of isobutyric acid and isobutanol via the valine synthesis route (Ehrlich pathway). This indicates the existence of chromosomally encoded alcohol and aldehyde dehydrogenases catalyzing the reduction and oxidation of isobutyraldehyde. In addition we showed that the strain possesses a complete pathway for isobutyric acid metabolization, channeling the compound via isobutyryl-CoA into valine degradation. Three key issues were addressed to allow and optimize isobutyric acid synthesis: i) minimizing isobutyric acid degradation by host intrinsic enzymes, ii) construction of suitable expression systems and iii) streamlining of central carbon metabolism finally leading to production of up to 26.8 ± 1.5 mM isobutyric acid with a carbon yield of 0.12 ± 0.01 g g(glc)⁻¹.ConclusionThe combination of an increased flux towards isobutyric acid using a tailor-made expression system and the prevention of precursor and product degradation allowed efficient production of isobutyric acid in Ps. sp. strain VLB120. This will be the basis for the development of a continuous reaction process for this bulk chemicals.
| S-EPMC3897908 | biostudies-literature
Project description:Biofilms are ubiquitous surface-associated microbial communities embedded in an extracellular polymeric (EPS) matrix, which gives the biofilm structural integrity and strength. It is often reported that biofilm-grown cells exhibit enhanced tolerance toward adverse environmental stress conditions, and thus there has been a growing interest in recent years to use biofilms for biotechnological applications. We present a time- and locus-resolved, noninvasive, quantitative approach to study biofilm development and its response to the toxic solvent styrene. Pseudomonas sp. strain VLB120ΔC-BT-gfp1 was grown in modified flow-cell reactors and exposed to the solvent styrene. Biofilm-grown cells displayed stable catalytic activity, producing (S)-styrene oxide continuously during the experimental period. The pillar-like structure and growth rate of the biofilm was not influenced by the presence of the solvent. However, the cells experience severe membrane damage during styrene treatment, although they obviously are able to adapt to the solvent, as the amount of permeabilized cells decreased from 75 to 80% down to 40% in 48 h. Concomitantly, the fraction of concanavalin A (ConA)-stainable EPS increased, substantiating the assumption that those polysaccharides play a major role in structural integrity and enhanced biofilm tolerance toward toxic environments. Compared to control experiments with planktonic grown cells, the Pseudomonas biofilm adapted much better to toxic concentrations of styrene, as nearly 65% of biofilm cells were not permeabilized (viable), compared to only 7% in analogous planktonic cultures. These findings underline the robustness of biofilms under stress conditions and its potential for fine chemical syntheses.
| S-EPMC3067263 | biostudies-literature
Project description:Pseudomonas sp. VLB120 uses styrene as a sole source of carbon and energy. The first step in this metabolic pathway is catalyzed by an oxygenase (StyA) and a NADH-flavin oxidoreductase (StyB). Both components have been isolated from wild-type Pseudomonas strain VLB120 as well as from recombinant Escherichia coli. StyA from both sources is a dimer, with a subunit size of 47 kDa, and catalyzes the enantioselective epoxidation of CC double bonds. Styrene is exclusively converted to S-styrene oxide with a specific activity of 2.1 U mg(-1) (k(cat) = 1.6 s(-1)) and K(m) values for styrene of 0.45 +/- 0.05 mM (wild type) and 0.38 +/- 0.09 mM (recombinant). The epoxidation reaction depends on the presence of a NADH-flavin adenine dinucleotide (NADH-FAD) oxidoreductase for the supply of reduced FAD. StyB is a dimer with a molecular mass of 18 kDa and a NADH oxidation activity of 200 U mg(-1) (k(cat) [NADH] = 60 s(-1)). Steady-state kinetics determined for StyB indicate a mechanism of sequential binding of NADH and flavin to StyB. This enzyme reduces FAD as well as flavin mononucleotide and riboflavin. The NADH oxidation activity does not depend on the presence of StyA. During the epoxidation reaction, no formation of a complex of StyA and StyB has been observed, suggesting that electron transport between reductase and oxygenase occurs via a diffusing flavin.
| S-EPMC490909 | biostudies-literature
Project description:The application of whole cells as biocatalysts is often limited by the toxicity of organic solvents, which constitute interesting substrates/products or can be used as a second phase for in situ product removal and as tools to control multistep biocatalysis. Solvent-tolerant bacteria, especially Pseudomonas strains, are proposed as promising hosts to overcome such limitations due to their inherent solvent tolerance mechanisms. However, potential industrial applications suffer from tedious, unproductive adaptation processes, phenotypic variability, and instable solvent-tolerant phenotypes. In this study, genes described to be involved in solvent tolerance were identified in Pseudomonas taiwanensis VLB120, and adaptive solvent tolerance was proven by cultivation in the presence of 1% (vol/vol) toluene. Deletion of ttgV, coding for the specific transcriptional repressor of solvent efflux pump TtgGHI gene expression, led to constitutively solvent-tolerant mutants of P. taiwanensis VLB120 and VLB120ΔC. Interestingly, the increased amount of solvent efflux pumps enhanced not only growth in the presence of toluene and styrene but also the biocatalytic performance in terms of stereospecific styrene epoxidation, although proton-driven solvent efflux is expected to compete with the styrene monooxygenase for metabolic energy. Compared to that of the P. taiwanensis VLB120ΔC parent strain, the maximum specific epoxidation activity of P. taiwanensis VLB120ΔCΔttgV doubled to 67 U/g of cells (dry weight). This study shows that solvent tolerance mechanisms, e.g., the solvent efflux pump TtgGHI, not only allow for growth in the presence of organic compounds but can also be used as tools to improve redox biocatalysis involving organic solvents.
| S-EPMC4178659 | biostudies-literature
Project description:The chromosomal region of Pseudomonas sp. strain Y2 involved in the conversion of styrene to phenylacetate (upper catabolic pathway) has been cloned and sequenced. Four catabolic genes, styABCD, and two regulatory genes, stySR, were identified. This gene cluster when transferred to Escherichia coli W confers to this phenylacetate-degrading host the ability to grow on styrene as the sole carbon and energy source. Genes styABCD are homologous to those encoding the styrene upper catabolic pathway in Pseudomonas fluorescens ST. Northern blot analyses have confirmed that genes styABCD constitute a transcription unit. The transcription start site of the sty operon was mapped 33 nucleotides upstream of the styA translational start codon. The styS and styR genes, which form an independent transcriptional unit, are located upstream of the styABCD operon, and their gene products show high similarity to members of the superfamily of two-component signal transduction systems. The styS gene product is homologous to histidine kinase proteins, whereas the styR gene product exhibits similarity at its N-terminal domain with cluster 1 of receiver modules and at its C terminus with the LuxR/FixJ family 3 of DNA-binding domains. Expression of the catabolic operon decreased significantly in the absence of the stySR genes and was restored when the stySR genes were provided in trans in the presence of styrene, suggesting that the stySR system behaves as a styrene-inducible positive regulator of the styABCD operon. Finally, a gene encoding a phenylacetyl-coenzyme A ligase that catalyzes the first step in the phenylacetate catabolism (styrene lower catabolic pathway) has been identified upstream of the styS gene. This activity was found to be induced in Pseudomonas sp. strain Y2 cells grown on styrene but not present in cells grown on glycerol. These results strongly suggest that the genes responsible for the complete mineralization of styrene are clustered in the chromosome of Pseudomonas sp. strain Y2.
| S-EPMC106992 | biostudies-literature
Project description:Pseudomonas sp. VLB120 Genome sequencing
| PRJNA178195 | ENA
Project description:Pseudomonas sp. strain ADP is the model strain for studying bacterial degradation of the s-triazine herbicide atrazine. In this work, we focused on the expression of the atzDEF operon, involved in mineralization of the central intermediate of the pathway, cyanuric acid. Expression analysis of atzD-lacZ fusions in Pseudomonas sp. strain ADP and Pseudomonas putida showed that atzDEF is subjected to dual regulation in response to nitrogen limitation and cyanuric acid. The gene adjacent to atzD, orf99 (renamed here atzR), encoding a LysR-like regulator, was found to be required for both responses. Expression of atzR-lacZ was induced by nitrogen limitation and repressed by AtzR. Nitrogen regulation of atzD-lacZ and atzR-lacZ expression was dependent on the alternative sigma factor sigmaN and NtrC, suggesting that the cyanuric acid degradation operon may be subject to general nitrogen control. However, while atzR is transcribed from a sigmaN-dependent promoter, atzDEF transcription appears to be driven from a sigma70-type promoter. Expression of atzR from a heterologous promoter revealed that although NtrC regulation of atzD-lacZ requires the AtzR protein, it is not the indirect result of NtrC-activated AtzR synthesis. We propose that expression of the cyanuric acid degradation operon atzDEF is controlled by means of a complex regulatory circuit in which AtzR is the main activator. AtzR activity is in turn modulated by the presence of cyanuric acid and by a nitrogen limitation signal transduced by the Ntr system.
| S-EPMC538813 | biostudies-literature
Project description:Pseudomonas sp. strain M1 is a soil isolate with remarkable biotechnological potential. The genome of Pseudomonas sp. M1 was sequenced using both 454 and Illumina technologies. A customized genome assembly pipeline was used to reconstruct its genome sequence to a single scaffold.
| S-EPMC3569285 | biostudies-literature
Project description:BackgroundBio-conversion of lignocellulosic biomass to high-value products offers numerous benefits; however, its development is hampered by chemical inhibitors generated during the pretreatment process. A better understanding of how microbes naturally respond to those inhibitors is valuable in the process of designing microorganisms with improved tolerance. Pseudomonas taiwanensis VLB120 is a natively tolerant strain that utilizes a wide range of carbon sources including pentose and hexose sugars. To this end, we investigated the tolerance and metabolic response of P. taiwanensis VLB120 towards biomass hydrolysate-derived inhibitors including organic acids (acetic acid, formic acid, and levulinic acid), furans (furfural, 5-hydroxymethylfurfural), and phenols (vanillin).ResultsThe inhibitory effect of the tested compounds varied with respect to lag phase, specific growth rate, and biomass yield compared to the control cultures grown under the same conditions without addition of inhibitors. However, P. taiwanensis was able to oxidize vanillin and furfural to vanillic acid and 2-furoic acid, respectively. Vanillic acid was further metabolized, whereas 2-furoic acid was secreted outside the cells and remained in the fermentation broth without further conversion. Acetic acid and formic acid were completely consumed from the fermentation broth, while concentration of levulinic acid remained constant throughout the fermentation process. Analysis of free intracellular metabolites revealed varying levels when P. taiwanensis VLB120 was exposed to inhibitory compounds. This resulted in increased levels of ATP to export inhibitors from the cell and NADPH/NADP ratio that provides reducing power to deal with the oxidative stress caused by the inhibitors. Thus, adequate supply of these metabolites is essential for the survival and reproduction of P. taiwanensis in the presence of biomass-derived inhibitors.ConclusionsIn this study, the tolerance and metabolic response of P. taiwanensis VLB120 to biomass hydrolysate-derived inhibitors was investigated. P. taiwanensis VLB120 showed high tolerance towards biomass hydrolysate-derived inhibitors compared to most wild-type microbes reported in the literature. It adopts different resistance mechanisms, including detoxification, efflux, and repair, which require additional energy and resources. Thus, targeting redox and energy metabolism in strain engineering may be a successful strategy to overcome inhibition during biomass hydrolysate conversion and lead to development of more robust strains.
| S-EPMC6052574 | biostudies-literature