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Metabolic Engineering Mar 2023Deciphering the mechanisms of bacterial fatty acid biosynthesis is crucial for both the engineering of bacterial hosts to produce fatty acid-derived molecules and the...
Deciphering the mechanisms of bacterial fatty acid biosynthesis is crucial for both the engineering of bacterial hosts to produce fatty acid-derived molecules and the development of new antibiotics. However, gaps in our understanding of the initiation of fatty acid biosynthesis remain. Here, we demonstrate that the industrially relevant microbe Pseudomonas putida KT2440 contains three distinct pathways to initiate fatty acid biosynthesis. The first two routes employ conventional β-ketoacyl-ACP synthase III enzymes, FabH1 and FabH2, that accept short- and medium-chain-length acyl-CoAs, respectively. The third route utilizes a malonyl-ACP decarboxylase enzyme, MadB. A combination of exhaustive in vivo alanine-scanning mutagenesis, in vitro biochemical characterization, X-ray crystallography, and computational modeling elucidate the presumptive mechanism of malonyl-ACP decarboxylation via MadB. Given that functional homologs of MadB are widespread throughout domain Bacteria, this ubiquitous alternative fatty acid initiation pathway provides new opportunities to target a range of biotechnology and biomedical applications.
Topics: Pseudomonas putida; 3-Oxoacyl-(Acyl-Carrier-Protein) Synthase; Mutagenesis; Fatty Acids
PubMed: 36796578
DOI: 10.1016/j.ymben.2023.02.006 -
Microbial Biotechnology Jan 2022To meet the needs of synthetic biologists, DNA assembly methods have transformed from simple 'cut-and-paste' procedures to highly advanced, standardised assembly...
To meet the needs of synthetic biologists, DNA assembly methods have transformed from simple 'cut-and-paste' procedures to highly advanced, standardised assembly techniques. Implementing these standardised DNA assembly methods in biotechnological research conducted in non-model hosts, including Pseudomonas putida and Pseudomonas aeruginosa, could greatly benefit reproducibility and predictability of experimental results. SEVAtile is a Type IIs-based assembly approach, which enables the rapid and standardised assembly of genetic parts - or tiles - to create genetic circuits in the established SEVA-vector backbone. Contrary to existing DNA assembly methods, SEVAtile is an easy and straightforward method, which is compatible with any vector, both SEVA- and non-SEVA. To prove the efficiency of the SEVAtile method, a three-vector system was successfully generated to independently co-express three different proteins in P. putida and P. aeruginosa. More specifically, one of the vectors, pBGDes, enables genomic integration of assembled circuits in the Tn7 landing site, while self-replicatory vectors pSTDesX and pSTDesR enable inducible expression from the XylS/Pm and RhaRS/PrhaB expression systems, respectively. Together, we hope these vector systems will support research in both the microbial SynBio and Pseudomonas field.
Topics: DNA; Genetic Vectors; Plasmids; Pseudomonas; Pseudomonas putida; Reproducibility of Results
PubMed: 34651450
DOI: 10.1111/1751-7915.13922 -
Toxics Apr 2023Surfactants can be found in an ever-widening variety of products and applications, in which the combination of several types of surfactants is used to reinforce their...
Surfactants can be found in an ever-widening variety of products and applications, in which the combination of several types of surfactants is used to reinforce their properties, looking for synergistic effects between them. After use, they tend to be discarded into wastewater, ending up in aquatic bodies with concerning harmful and toxic effects. The aim of this study is the toxicological assessment of three anionic surfactants (ether carboxylic derivative, EC) and three amphoteric surfactants (amine-oxide-based, AO), individually and in binary mixtures of them (1:1 /), to bacteria and marine microalgae . Critical Micelle Concentration (CMC) was determined to demonstrate the capacity to reduce surface tension and the toxicity of the surfactants and mixtures. Zeta potential (ζ-potential) and micelle diameter (MD) were also determined to confirm the formation of mixed surfactant micelles. The Model of Toxic Units (MTUs) was used to quantify the interactions of surfactants in binary mixtures and to predict if the concentration addition or response addition principle can be assumed for each mixture. The results showed a higher sensitivity of microalgae to the surfactants tested and their mixtures than bacteria . Antagonism toxic effects have been detected in the mixture of EC + AO and in one binary mixture of different AOs; this is to say, the mixtures showed lower toxicity than expected.
PubMed: 37112571
DOI: 10.3390/toxics11040344 -
Microbiology Spectrum Jun 2023Biotransformation of plastics or their depolymerization monomers as raw materials would offer a better end-of-life solutions to the plastic waste dilemma. 1,4-butanediol...
Biotransformation of plastics or their depolymerization monomers as raw materials would offer a better end-of-life solutions to the plastic waste dilemma. 1,4-butanediol (BDO) is one of the major depolymerization monomers of many plastics polymers. BDO valorization presents great significance for waste plastic up-recycling and fermenting feedstock exploitation. In the present study, atmospheric pressure room temperature plasma (ARTP)-induced mutation combined with adaptive laboratory evolution (ALE) was used to improve the BDO utilization capability of Pseudomonas putida KT2440. The excellent mutant P. putida NB10 was isolated and stored in the China Typical Culture Preservation Center (CCTCC) with the deposit number M 2021482. Whole-genome resequencing and transcriptome analysis revealed that the BDO degradation process consists of β-oxidation, glyoxylate carboligase (GCL) pathway, glyoxylate cycle and gluconeogenesis pathway. The imbalance between the two key intermediates (acetyl-CoA and glycolyl-CoA) and the accumulation of cytotoxic aldehydes resulted in the weak metabolism performance of KT2440 in the utilization of BDO. The balance of the carbon flux and enhanced tolerance to cytotoxic intermediates endow NB10 with great BDO degradation capability. This study deeply revealed the metabolic mechanism behind BDO degradation and provided an excellent chassis cell for BDO further up-cycling to high-value chemicals. Plastic waste represents not only a global pollution problem but also a carbon-rich, low-cost, globally renewable feedstock for industrial biotechnology. BDO is the basic material for polybutylene terephthalate (PBT), poly butylene adipate-co-terephthalate (PBAT), poly (butylene succinate) (PBS), etc. Herein, the construction of BDO valorization cell factory presents great significance for waste plastic up-recycling and novel fermentation feedstock exploitation. However, BDO is hard to be metabolized and its metabolic pathway is unclear. This study presents a P. putida mutant NB10, obtained through the integration of ARTP and ALE, displaying significant growth improvement with BDO as the sole carbon source. Further genome resequencing, transcriptome analysis and genetic engineering deeply revealed the metabolic mechanism behind BDO degradation in P. putida, this study offers an excellent microbial chassis and modification strategy for plastic waste up-cycling.
Topics: Pseudomonas putida; Mutation; Carbon; Plastics
PubMed: 37067433
DOI: 10.1128/spectrum.04988-22 -
Metabolic Engineering Communications Dec 2020KT2440 is a promising bacterial chassis for the conversion of lignin-derived aromatic compound mixtures to biofuels and bioproducts. Despite the inherent robustness of...
KT2440 is a promising bacterial chassis for the conversion of lignin-derived aromatic compound mixtures to biofuels and bioproducts. Despite the inherent robustness of this strain, further improvements to aromatic catabolism and toxicity tolerance of will be required to achieve industrial relevance. Here, tolerance adaptive laboratory evolution (TALE) was employed with increasing concentrations of the hydroxycinnamic acids -coumaric acid (CA) and ferulic acid (FA) individually and in combination (CA + FA). The TALE experiments led to evolved strains with increased tolerance to the targeted acids as compared to wild type. Specifically, a 37 h decrease in lag phase in 20 g/L CA and a 2.4-fold increase in growth rate in 30 g/L FA was observed. Whole genome sequencing of intermediate and endpoint evolved populations revealed several expected and non-intuitive genetic targets underlying these aromatic catabolic and toxicity tolerance enhancements. and were among the most frequently mutated genes, and the beneficial contributions of these mutations were verified via gene knockouts. Deletion of encoding a hypothetical protein, recapitulated improved toxicity tolerance to high concentrations of CA, but not an improved growth rate in high concentrations of FA. Deletion of part of the TtgABC efflux pump, severely inhibited growth in CA + FA TALE-derived strains but did not affect growth in CA + FA in a wild type background, suggesting epistatic interactions. Genes involved in flagellar movement and transcriptional regulation were often mutated in the TALE experiments on multiple substrates, reinforcing ideas of a minimal and deregulated cell as optimal for domesticated growth. Overall, this work demonstrates increased tolerance towards and growth rate at the expense of hydroxycinnamic acids and presents new targets for improving for microbial lignin valorization.
PubMed: 32963959
DOI: 10.1016/j.mec.2020.e00143 -
BMC Genomics Jan 2024Pseudomonas putida S12 is a gram-negative bacterium renowned for its high tolerance to organic solvents and metabolic versatility, making it attractive for various...
BACKGROUND
Pseudomonas putida S12 is a gram-negative bacterium renowned for its high tolerance to organic solvents and metabolic versatility, making it attractive for various applications, including bioremediation and the production of aromatic compounds, bioplastics, biofuels, and value-added compounds. However, a metabolic model of S12 has yet to be developed.
RESULTS
In this study, we present a comprehensive and highly curated genome-scale metabolic network model of S12 (iSH1474), containing 1,474 genes, 1,436 unique metabolites, and 2,938 metabolic reactions. The model was constructed by leveraging existing metabolic models and conducting comparative analyses of genomes and phenomes. Approximately 2,000 different phenotypes were measured for S12 and its closely related KT2440 strain under various nutritional and environmental conditions. These phenotypic data, combined with the reported experimental data, were used to refine and validate the reconstruction. Model predictions quantitatively agreed well with in vivo flux measurements and the batch cultivation of S12, which demonstrated that iSH1474 accurately represents the metabolic capabilities of S12. Furthermore, the model was simulated to investigate the maximum theoretical metabolic capacity of S12 growing on toxic organic solvents.
CONCLUSIONS
iSH1474 represents a significant advancement in our understanding of the cellular metabolism of P. putida S12. The combined results of metabolic simulation and comparative genome and phenome analyses identified the genetic and metabolic determinants of the characteristic phenotypes of S12. This study could accelerate the development of this versatile organism as an efficient cell factory for various biotechnological applications.
Topics: Solvents; Pseudomonas putida; Genome, Bacterial; Genomics; Metabolic Networks and Pathways
PubMed: 38229031
DOI: 10.1186/s12864-023-09940-y -
Microbiology (Reading, England) Nov 2020Microbial bioproduction of the aromatic acid anthranilate (-aminobenzoate) has the potential to replace its current, environmentally demanding production process. The...
Microbial bioproduction of the aromatic acid anthranilate (-aminobenzoate) has the potential to replace its current, environmentally demanding production process. The host organism employed for such a process needs to fulfil certain demands to achieve industrially relevant product levels. As anthranilate is toxic for microorganisms, the use of particularly robust production hosts can overcome issues from product inhibition. The microorganisms and are known for high tolerance towards a variety of chemicals and could serve as promising platform strains. In this study, the resistance of both wild-type strains towards anthranilate was assessed. To further enhance their native tolerance, adaptive laboratory evolution (ALE) was applied. Sequential batch fermentation processes were developed, adapted to the cultivation demands for and to enable long-term cultivation in the presence of anthranilate. Isolation and analysis of single mutants revealed phenotypes with improved growth behaviour in the presence of anthranilate for both strains. The characterization and improvement of both potential hosts provide an important basis for further process optimization and will aid the establishment of an industrially competitive method for microbial synthesis of anthranilate.
Topics: Adaptation, Physiological; Bioreactors; Corynebacterium glutamicum; Directed Molecular Evolution; Industrial Microbiology; Mutation; Pseudomonas putida; ortho-Aminobenzoates
PubMed: 33095135
DOI: 10.1099/mic.0.000982 -
PloS One 2022Manual proteomic sample preparation methods limit sample throughput and often lead to poor data quality when thousands of samples must be analyzed. Automated liquid...
Manual proteomic sample preparation methods limit sample throughput and often lead to poor data quality when thousands of samples must be analyzed. Automated liquid handler systems are increasingly used to overcome these issues for many of the sample preparation steps. Here, we detail a step-by-step protocol to prepare samples for bottom-up proteomic analysis for Gram-negative bacterial and fungal cells. The full modular protocol consists of three optimized protocols to: (A) lyse Gram-negative bacteria and fungal cells; (B) quantify the amount of protein extracted; and (C) normalize the amount of protein and set up tryptic digestion. These protocols have been developed to facilitate rapid, low variance sample preparation of hundreds of samples, be easily implemented on widely-available Beckman-Coulter Biomek automated liquid handlers, and allow flexibility for future protocol development. By using this workflow 50 micrograms of protein from 96 samples can be prepared for tryptic digestion in under an hour. We validate these protocols by analyzing 47 Pseudomonas putida and Rhodosporidium toruloides samples and show that this modular workflow provides robust, reproducible proteomic samples for high-throughput applications. The expected results from these protocols are 94 peptide samples from Gram-negative bacterial and fungal cells prepared for bottom-up quantitative proteomic analysis without the need for desalting column cleanup and with protein relative quantity variance (CV%) below 15%.
Topics: Automation; Chromatography, High Pressure Liquid; Mass Spectrometry; Proteome; Proteomics; Pseudomonas putida; Reproducibility of Results; Rhodotorula; Specimen Handling
PubMed: 35213656
DOI: 10.1371/journal.pone.0264467 -
Journal of Microbiology and... Feb 2021Many bacteria metabolize aromatic compounds via catechol as a catabolic intermediate, and possess multiple genes or clusters encoding catechol-cleavage enzymes. The...
Many bacteria metabolize aromatic compounds via catechol as a catabolic intermediate, and possess multiple genes or clusters encoding catechol-cleavage enzymes. The presence of multiple isozyme-encoding genes is a widespread phenomenon that seems to give the carrying strains a selective advantage in the natural environment over those with only a single copy. In the naphthalene-degrading strain ND6, catechol can be converted into intermediates of the tricarboxylic acid cycle via either the - or -cleavage pathways. In this study, we demonstrated that the catechol ortho-cleavage pathway genes ( and ) on the chromosome play an important role. The and operons are co-transcribed, whereas and are under independent transcriptional regulation. We examined the binding of regulatory proteins to promoters. In the presence of -muconate, a well-studied inducer of the cat gene cluster, CatR and CatR occupy an additional downstream site, designated as the activation binding site. Notably, CatR binds to both the and promoters with high affinity, while CatR binds weakly. This is likely caused by a T to G mutation in the G/T-N-A motif. Specifically, we found that CatR and CatR regulate and in a cooperative manner, which provides new insights into naphthalene degradation.
Topics: Bacterial Proteins; Catechols; Gene Expression Regulation, Bacterial; Multigene Family; Operon; Promoter Regions, Genetic; Pseudomonas putida
PubMed: 33323670
DOI: 10.4014/jmb.2009.09026 -
International Journal of Molecular... May 2022The biofilm of is complexly regulated by several intercellular and extracellular factors. The cell surface adhesin LapA of this bacterium is a central factor for the...
The biofilm of is complexly regulated by several intercellular and extracellular factors. The cell surface adhesin LapA of this bacterium is a central factor for the biofilm and, consequently, the regulation of expression, for example, by Fis. It has been recently shown that peptides in growth media enhance the formation of biofilm, but not as a source of carbon and nitrogen. Moreover, the peptide-dependent biofilm appeared especially clearly in the -overexpression strain, which also has increased LapA. Therefore, we investigate here whether there is a relationship between LapA and peptide-dependent biofilm. The strains with inducible expression and LapA without the vWFa domain, which is described as a domain similar to von Willebrand factor domain A, were constructed. Thereafter, the biofilm of these strains was assessed in growth media containing extracellular peptides in the shape of tryptone and without it. We show that the vWFa domain in LapA is necessary for biofilm enhancement by the extracellular peptides in the growth medium. The importance of vWFa in LapA was particularly evident for the -overexpression strain F15. The absence of the vWFa domain diminished the positive effect of Fis on the F15 biofilm.
Topics: Bacterial Proteins; Biofilms; Gene Expression Regulation, Bacterial; Peptides; Pseudomonas putida
PubMed: 35682576
DOI: 10.3390/ijms23115898