Unravelling the thioesterases responsible for propionate formation in engineered Pseudomonas putida KT2440.
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ABSTRACT:
SUBMITTER: Ma C
PROVIDER: S-EPMC8085926 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundEfficient conversion of plant biomass to commodity chemicals is an important challenge that needs to be solved to enable a sustainable bioeconomy. Deconstruction of biomass to sugars and lignin yields a wide variety of low molecular weight carbon substrates that need to be funneled to product. Pseudomonas putida KT2440 has emerged as a potential platform for bioconversion of lignin and the other components of plant biomass. However, P. putida is unable to natively utilize several of the common sugars in hydrolysate streams, including galactose.ResultsIn this work, we integrated a De Ley-Doudoroff catabolic pathway for galactose catabolism into the chromosome of P. putida KT2440, using genes from several different organisms. We found that the galactonate catabolic pathway alone (DgoKAD) supported slow growth of P. putida on galactose. Further integration of genes to convert galactose to galactonate and to optimize the transporter expression level resulted in a growth rate of 0.371 h-1. Additionally, the best-performing strain was demonstrated to co-utilize galactose with glucose.ConclusionsWe have engineered P. putida to catabolize galactose, which will allow future engineered strains to convert more plant biomass carbon to products of interest. Further, by demonstrating co-utilization of glucose and galactose, continuous bioconversion processes for mixed sugar streams are now possible.
| S-EPMC6927180 | biostudies-literature
Project description:Pseudomonas species have become promising cell factories for the production of natural products due to their inherent robustness. Although these bacteria have naturally evolved strategies to cope with different kinds of stress, many biotechnological applications benefit from engineering of optimised chassis strains with specially adapted tolerance traits. Here, we explored the formation of outer membrane vesicles (OMV) of Pseudomonas putida KT2440. We found OMV production to correlate with the recombinant production of a natural compound with versatile beneficial properties, the tripyrrole prodigiosin. Further, several P. putida genes were identified, whose up- or down-regulated expression allowed controlling OMV formation. Finally, genetically triggering vesiculation in production strains of the different alkaloids prodigiosin, violacein, and phenazine-1-carboxylic acid, as well as the carotenoid zeaxanthin, resulted in up to three-fold increased product yields. Consequently, our findings suggest that the construction of robust strains by genetic manipulation of OMV formation might be developed into a useful tool which may contribute to improving limited biotechnological applications.
| S-EPMC10832525 | biostudies-literature
Project description:Propionic acid (PA) has been widely used as a food preservative and chemical intermediate in the agricultural and pharmaceutical industries. Environmental and friendly biotechnological production of PA from biomass has been considered as an alternative to the traditional petrochemical route. However, because PA is a strong inhibitor of cell growth, the biotechnological host should be not only able to produce the compound but the host should be robust. In this study, we identified key PA tolerance factors in Pseudomonas putida KT2440 strain by comparative transcriptional analysis in the presence or absence of PA stress. The identified major facilitator superfamily (MFS) transporter gene cluster of PP_1271, PP_1272 and PP_1273 was experimentally verified to be involved in PA tolerance in P. putida strains. Overexpression of this cluster improved tolerance to PA in a PA producing strain, what is useful to further engineer this robust platform not only for PA synthesis but for the production of other weak acids.
| S-EPMC7936288 | biostudies-literature
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:Bacterial toxin/antitoxin (TA) systems have received increasing attention due to their prevalence, diverse structures, and important physiological functions. In this study, we identified and characterized a type II TA system in a soil bacterium Pseudomonas putida KT2440. This TA system belongs to the MqsR/MqsA family. We found that PP_4205 (MqsR) greatly inhibits cell growth in P. putida KT2440 and Escherichia coli, the antitoxin PP_4204 (MqsA) neutralizes the toxicity of the toxin MqsR, and the two genes encoding them are co-transcribed. MqsR and MqsA interact with each other directly in vivo and MqsA is a negative regulator of the TA operon through binding to the promoter. Consistent with the MqsR/MqsA pair in E. coli, the binding of the toxin MqsR to MqsA inhibits the DNA binding ability of MqsA in P. putida KT2440. Disruption of the mqsA gene which induces mqsR expression increases persister cell formation 53-fold, while overexpressing mqsA which represses mqsR expression reduces persister cell formation 220-fold, suggesting an important role of MqsR in persistence in P. putida KT2440. Furthermore, both MqsR and MqsA promote biofilm formation. As a DNA binding protein, MqsA can also negatively regulate an ECF sigma factor AlgU and a universal stress protein PP_3288. Thus, we revealed an important regulatory role of MqsR/MqsA in persistence and biofilm formation in P. putida KT2440.
| S-EPMC5422877 | 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: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