Dehydrogenation Mechanism of Three Stereoisomers of Butane-2,3-Diol in Pseudomonas putida KT2440
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SUBMITTER: Liu Y
PROVIDER: S-EPMC8427195 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:D-Amino acids have been shown to play an increasingly diverse role in bacterial physiology, yet much remains to be learned about their synthesis and catabolism. Here we used the model soil- and rhizosphere-dwelling organism Pseudomonas putida KT2440 to elaborate on the genomics and enzymology of d-amino acid metabolism. P. putida KT2440 catabolized the d-stereoisomers of lysine, phenylalanine, arginine, alanine, and hydroxyproline as the sole carbon and nitrogen sources. With the exception of phenylalanine, each of these amino acids was racemized by P. putida KT2440 enzymes. Three amino acid racemases were identified from a genomic screen, and the enzymes were further characterized in vitro. The putative biosynthetic alanine racemase Alr showed broad substrate specificity, exhibiting measurable racemase activity with 9 of the 19 chiral amino acids. Among these amino acids, activity was the highest with lysine, and the k(cat)/K(m) values with l- and d-lysine were 3 orders of magnitude greater than the k(cat)/K(m) values with l- and d-alanine. Conversely, the putative catabolic alanine racemase DadX showed narrow substrate specificity, clearly preferring only the alanine stereoisomers as the substrates. However, DadX did show 6- and 9-fold higher k(cat)/K(m) values than Alr with l- and d-alanine, respectively. The annotated proline racemase ProR of P. putida KT2440 showed negligible activity with either stereoisomer of the 19 chiral amino acids but exhibited strong epimerization activity with hydroxyproline as the substrate. Comparative genomic analysis revealed differences among pseudomonads with respect to alanine racemase genes that may point to different roles for these genes among closely related species.
| S-EPMC3811597 | biostudies-literature
Project description:Plastics, in all forms, are a ubiquitous cornerstone of modern civilization. Although humanity undoubtedly benefits from the versatility and durability of plastics, they also cause a tremendous burden for the environment. Bio-upcycling is a promising approach to reduce this burden, especially for polymers that are currently not amenable to mechanical recycling. Wildtype P. putida KT2440 is able to grow on 1,4-butanediol as sole carbon source, but only very slowly. Adaptive laboratory evolution (ALE) led to the isolation of several strains with significantly enhanced growth rate and yield. Genome re-sequencing and proteomic analysis were applied to characterize the genomic and metabolic basis of efficient 1,4-butanediol metabolism. Initially, 1,4-butanediol is oxidized to 4-hydroxybutyrate, in which the highly expressed dehydrogenase enzymes encoded within the PP_2674-2680 ped gene cluster play an essential role. The resulting 4-hydroxybutyrate can be metabolized through three possible pathways: (i) oxidation to succinate, (ii) CoA activation and subsequent oxidation to succinyl-CoA, and (iii) beta oxidation to glycolyl-CoA and acetyl-CoA. The evolved strains were both mutated in a transcriptional regulator (PP_2046) of an operon encoding both beta-oxidation related genes and an alcohol dehydrogenase. When either the regulator or the alcohol dehydrogenase is deleted, no 1,4-butanediol uptake or growth could be detected. Using a reverse engineering approach, PP_2046 was replaced by a synthetic promotor (14g) to overexpress the downstream operon (PP_2047-2051), thereby enhancing growth on 1,4-butanediol. This work provides a deeper understanding of microbial 1,4-butanediol metabolism in P. putida, which is also expandable to other aliphatic alpha-omega diols. It enables the more efficient metabolism of these diols, thereby enabling biotechnological valorization of plastic monomers in a bio-upcycling approach.
| S-EPMC7090098 | biostudies-literature
Project description:Pseudomonas putida is rapidly becoming a workhorse for industrial production due to its metabolic versatility, genetic accessibility and stress-resistance properties. The P. putida strain KT2440 is often described as Generally Regarded as Safe, or GRAS, indicating the strain is safe to use as food additive. This description is incorrect. P. putida KT2440 is classified by the FDA as HV1 certified, indicating it is safe to use in a P1 or ML1 environment.
| S-EPMC6680625 | biostudies-literature
Project description:Pseudomonas putida KT2440 is a well-established chassis in industrial biotechnology. To increase the substrate spectrum, we implemented three alternative xylose utilization pathways, namely the Isomerase, Weimberg, and Dahms pathways. The synthetic operons contain genes from Escherichia coli and Pseudomonas taiwanensis. For isolating the Dahms pathway in P. putida KT2440 two genes (PP_2836 and PP_4283), encoding an endogenous enzyme of the Weimberg pathway and a regulator for glycolaldehyde degradation, were deleted. Before and after adaptive laboratory evolution, these strains were characterized in terms of growth and synthesis of mono-rhamnolipids and pyocyanin. The engineered strain using the Weimberg pathway reached the highest maximal growth rate of 0.30 h?1. After adaptive laboratory evolution the lag phase was reduced significantly. The highest titers of 720 mg L?1 mono-rhamnolipids and 30 mg L?1 pyocyanin were reached by the evolved strain using the Weimberg or an engineered strain using the Isomerase pathway, respectively. The different stoichiometries of the three xylose utilization pathways may allow engineering of tailored chassis for valuable bioproduct synthesis.
| S-EPMC6978631 | biostudies-literature
Project description:Targeted gene regulation is indispensable for reprogramming a cellular network to modulate a microbial phenotype. Here, we adopted the type II CRISPR interference (CRISPRi) system for simple and efficient regulation of target genes in Pseudomonas putida KT2440. A single CRISPRi plasmid was generated to express a nuclease-deficient Cas9 gene and a designed single guide RNA, under control of l-rhamnose-inducible Prha BAD and the constitutive Biobrick J23119 promoter respectively. Two target genes were selected to probe the CRISPRi-mediated gene regulation: exogenous green fluorescent protein on the multicopy plasmid and endogenous glpR on the P. putida KT2440 chromosome, encoding GlpR, a transcriptional regulator that represses expression of the glpFKRD gene cluster for glycerol utilization. The CRISPRi system successfully repressed the two target genes, as evidenced by a reduction in the fluorescence intensity and the lag phase of P. putida KT2440 cell growth on glycerol. Furthermore, CRISPRi-mediated repression of glpR improved both the cell growth and glycerol utilization, resulting in the enhanced production of mevalonate in an engineered P. putida KT2440 harbouring heterologous genes for the mevalonate pathway. CRISPRi is expected to become a robust tool to reprogram P. putida KT2440 for the development of microbial cell factories producing industrially valuable products.
| S-EPMC6922533 | biostudies-literature
Project description:Pyrolysis offers a straightforward approach for the deconstruction of plant cell wall polymers into bio-oil. Recently, there has been substantial interest in bio-oil fractionation and subsequent use of biological approaches to selectively upgrade some of the resulting fractions. A fraction of particular interest for biological upgrading consists of polysaccharide-derived substrates including sugars and sugar dehydration products such as levoglucosan and cellobiosan, which are two of the most abundant pyrolysis products of cellulose. Levoglucosan can be converted to glucose-6-phosphate through the use of a levoglucosan kinase (LGK), but to date, the mechanism for cellobiosan utilization has not been demonstrated. Here, we engineer the microbe Pseudomonas putida KT2440 to use levoglucosan as a sole carbon and energy source through LGK integration. Moreover, we demonstrate that cellobiosan can be enzymatically converted to levoglucosan and glucose with β-glucosidase enzymes from both Glycoside Hydrolase Family 1 and Family 3. β-glucosidases are commonly used in both natural and industrial cellulase cocktails to convert cellobiose to glucose to relieve cellulase product inhibition and to facilitate microbial uptake of glucose. Using an exogenous β-glucosidase, we demonstrate that the engineered strain of P. putida can grow on levoglucosan up to 60 g/L and can also utilize cellobiosan. Overall, this study elucidates the biological pathway to co-utilize levoglucosan and cellobiosan, which will be a key transformation for the biological upgrading of pyrolysis-derived substrates.
| S-EPMC5779712 | biostudies-literature
Project description:Pseudomonas putida KT2440 is a chloramphenicol-resistant bacterium that is able to grow in the presence of this antibiotic at a concentration of up to 25 μg/ml. Transcriptomic analyses revealed that the expression profile of 102 genes changed in response to this concentration of chloramphenicol in the culture medium. The genes that showed altered expression include those involved in general metabolism, cellular stress response, gene regulation, efflux pump transporters, and protein biosynthesis. Analysis of a genome-wide collection of mutants showed that survival of a knockout mutant in the TtgABC resistance-nodulation-division (RND) efflux pump and mutants in the biosynthesis of pyrroloquinoline (PQQ) were compromised in the presence of chloramphenicol. The analysis also revealed that an ABC extrusion system (PP2669/PP2668/PP2667) and the AgmR regulator (PP2665) were needed for full resistance toward chloramphenicol. Transcriptional arrays revealed that AgmR controls the expression of the pqq genes and the operon encoding the ABC extrusion pump from the promoter upstream of open reading frame (ORF) PP2669.
| S-EPMC3264264 | biostudies-literature
Project description:Water deprivation can be a major stressor to microbial life in surface and subsurface soil. In unsaturated soils, the matric potential (Ψ(m)) is often the main component of the water potential, which measures the thermodynamic availability of water. A low matric potential usually translates into water forming thin liquid films in the soil pores. Little is known of how bacteria respond to such conditions, where, in addition to facing water deprivation that might impair their metabolism, they have to adapt their dispersal strategy as swimming motility may be compromised. Using the pressurized porous surface model (PPSM), which allows creation of thin liquid films by controlling Ψ(m), we examined the transcriptome dynamics of Pseudomonas putida KT2440. We identified the differentially expressed genes in cells exposed to a mild matric stress (-0.4 MPa) for 4, 24, or 72 h. The major response was detected at 4 h before gradually disappearing. Upregulation of alginate genes was notable in this early response. Flagellar genes were not downregulated, and the microarray data even suggested increasing expression as the stress prolonged. Moreover, we tested the effect of polyethylene glycol 8000 (PEG 8000), a nonpermeating solute often used to simulate Ψ(m), on the gene expression profile and detected a different profile than that observed by directly imposing Ψ(m). This study is the first transcriptome profiling of KT2440 under directly controlled Ψ(m) and also the first to show the difference in gene expression profiles between a PEG 8000-simulated and a directly controlled Ψ(m).
| S-EPMC3264132 | biostudies-literature
Project description:The metabolically versatile soil bacterium Pseudomonas putida has to cope with numerous abiotic stresses in its habitats. The stress responses of P. putida KT2440 to 4 degrees C, pH 4.5, 0.8 M urea, and 45 mM sodium benzoate were analyzed by determining the global mRNA expression profiles and screening for stress-intolerant nonauxotrophic Tn5 transposon mutants. In 392 regulated genes or operons, 36 gene regions were differentially expressed by more than 2.5-fold, and 32 genes in 23 operons were found to be indispensable for growth during exposure to one of the abiotic stresses. The transcriptomes of the responses to urea, benzoate, and 4 degrees C correlated positively with each other but negatively with the transcriptome of the mineral acid response. The CbrAB sensor kinase, the cysteine synthase CysM, PcnB and VacB, which control mRNA stability, and BipA, which exerts transcript-specific translational control, were essential to cope with cold stress. The cyo operon was required to cope with acid stress. A functional PhoP, PtsP, RelA/SpoT modulon, and adhesion protein LapA were necessary for growth in the presence of urea, and the outer membrane proteins OmlA and FepA and the phosphate transporter PstBACS were indispensable for growth in the presence of benzoate. A lipid A acyltransferase (PP0063) was a mandatory component of the stress responses to cold, mineral acid, and benzoate. Adaptation of the membrane barrier, uptake of phosphate, maintenance of the intracellular pH and redox status, and translational control of metabolism are key mechanisms of the response of P. putida to abiotic stresses.
| S-EPMC1482902 | biostudies-literature
Project description:Pseudomonas putida KT2440 is an emerging microbial chassis for biobased chemical production from renewable feedstocks and environmental bioremediation. However, tools for studying, engineering, and modulating protein complexes and biosynthetic enzymes in this organism are largely underdeveloped. Genetic code expansion for the incorporation of unnatural amino acids (unAAs) into proteins can advance such efforts and, furthermore, enable additional controls of biological processes of the strain. In this work, we established the orthogonality of two widely used archaeal tRNA synthetase and tRNA pairs in KT2440. Following the optimization of decoding systems, four unAAs were incorporated into proteins in response to a UAG stop codon at 34.6-78% efficiency. In addition, we demonstrated the utility of genetic code expansion through the incorporation of a photocross-linking amino acid, p-benzoyl-l-phenylalanine (pBpa), into glutathione S-transferase (GstA) and a chemosensory response regulator (CheY) for protein-protein interaction studies in KT2440. This work reported the successful genetic code expansion in KT2440 for the first time. Given the diverse structure and functions of unAAs that have been added to protein syntheses using the archaeal systems, our research lays down a solid foundation for future work to study and enhance the biological functions of KT2440.
| S-EPMC10656745 | biostudies-literature