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Communications Biology Dec 2022Despite advances in understanding the metabolism of Pseudomonas putida KT2440, a promising bacterial host for producing valuable chemicals from plant-derived feedstocks,...
Despite advances in understanding the metabolism of Pseudomonas putida KT2440, a promising bacterial host for producing valuable chemicals from plant-derived feedstocks, a strain capable of producing free fatty acid-derived chemicals has not been developed. Guided by functional genomics, we engineered P. putida to produce medium- and long-chain free fatty acids (FFAs) to titers of up to 670 mg/L. Additionally, by taking advantage of the varying substrate preferences of paralogous native fatty acyl-CoA ligases, we employed a strategy to control FFA chain length that resulted in a P. putida strain specialized in producing medium-chain FFAs. Finally, we demonstrate the production of oleochemicals in these strains by synthesizing medium-chain fatty acid methyl esters, compounds useful as biodiesel blending agents, in various media including sorghum hydrolysate at titers greater than 300 mg/L. This work paves the road to produce high-value oleochemicals and biofuels from cheap feedstocks, such as plant biomass, using this host.
Topics: Pseudomonas putida; Fatty Acids, Nonesterified; Biofuels; Biomass; Fatty Acids
PubMed: 36509863
DOI: 10.1038/s42003-022-04336-2 -
Microbial Cell Factories Jun 2022Biocatalysis offers a promising path for plastic waste management and valorization, especially for hydrolysable plastics such as polyethylene terephthalate (PET)....
BACKGROUND
Biocatalysis offers a promising path for plastic waste management and valorization, especially for hydrolysable plastics such as polyethylene terephthalate (PET). Microbial whole-cell biocatalysts for simultaneous PET degradation and growth on PET monomers would offer a one-step solution toward PET recycling or upcycling. We set out to engineer the industry-proven bacterium Pseudomonas putida for (i) metabolism of PET monomers as sole carbon sources, and (ii) efficient extracellular expression of PET hydrolases. We pursued this approach for both PET and the related polyester polybutylene adipate co-terephthalate (PBAT), aiming to learn about the determinants and potential applications of bacterial polyester-degrading biocatalysts.
RESULTS
P. putida was engineered to metabolize the PET and PBAT monomer terephthalic acid (TA) through genomic integration of four tphII operon genes from Comamonas sp. E6. Efficient cellular TA uptake was enabled by a point mutation in the native P. putida membrane transporter MhpT. Metabolism of the PET and PBAT monomers ethylene glycol and 1,4-butanediol was achieved through adaptive laboratory evolution. We then used fast design-build-test-learn cycles to engineer extracellular PET hydrolase expression, including tests of (i) the three PET hydrolases LCC, HiC, and IsPETase; (ii) genomic versus plasmid-based expression, using expression plasmids with high, medium, and low cellular copy number; (iii) three different promoter systems; (iv) three membrane anchor proteins for PET hydrolase cell surface display; and (v) a 30-mer signal peptide library for PET hydrolase secretion. PET hydrolase surface display and secretion was successfully engineered but often resulted in host cell fitness costs, which could be mitigated by promoter choice and altering construct copy number. Plastic biodegradation assays with the best PET hydrolase expression constructs genomically integrated into our monomer-metabolizing P. putida strains resulted in various degrees of plastic depolymerization, although self-sustaining bacterial growth remained elusive.
CONCLUSION
Our results show that balancing extracellular PET hydrolase expression with cellular fitness under nutrient-limiting conditions is a challenge. The precise knowledge of such bottlenecks, together with the vast array of PET hydrolase expression tools generated and tested here, may serve as a baseline for future efforts to engineer P. putida or other bacterial hosts towards becoming efficient whole-cell polyester-degrading biocatalysts.
Topics: Biocatalysis; Hydrolases; Plastics; Polyethylene Terephthalates; Pseudomonas putida
PubMed: 35717313
DOI: 10.1186/s12934-022-01849-7 -
ACS Synthetic Biology Nov 2022KT2440 is an emerging microbial chassis for biobased chemical production from renewable feedstocks and environmental bioremediation. However, tools for studying,...
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, -benzoyl-l-phenylalanine (pBpa), into glutathione -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.
Topics: Pseudomonas putida; Genetic Code; Amino Acyl-tRNA Synthetases; RNA, Transfer; Amino Acids
PubMed: 36287825
DOI: 10.1021/acssynbio.2c00325 -
FEMS Microbiology Reviews Mar 2011Pseudomonas putida is a gram-negative rod-shaped gammaproteobacterium that is found throughout various environments. Members of the species P. putida show a diverse... (Comparative Study)
Comparative Study Review
Pseudomonas putida is a gram-negative rod-shaped gammaproteobacterium that is found throughout various environments. Members of the species P. putida show a diverse spectrum of metabolic activities, which is indicative of their adaptation to various niches, which includes the ability to live in soils and sediments contaminated with high concentrations of heavy metals and organic contaminants. Pseudomonas putida strains are also found as plant growth-promoting rhizospheric and endophytic bacteria. The genome sequences of several P. putida species have become available and provide a unique tool to study the specific niche adaptation of the various P. putida strains. In this review, we compare the genomes of four P. putida strains: the rhizospheric strain KT2440, the endophytic strain W619, the aromatic hydrocarbon-degrading strain F1 and the manganese-oxidizing strain GB-1. Comparative genomics provided a powerful tool to gain new insights into the adaptation of P. putida to specific lifestyles and environmental niches, and clearly demonstrated that horizontal gene transfer played a key role in this adaptation process, as many of the niche-specific functions were found to be encoded on clearly defined genomic islands.
Topics: Adaptation, Physiological; Bacterial Proteins; Gene Expression Regulation, Bacterial; Genome, Bacterial; Genomics; Pseudomonas putida
PubMed: 20796030
DOI: 10.1111/j.1574-6976.2010.00249.x -
Metabolic Engineering Nov 2018The itinerary followed by Pseudomonas putida from being a soil-dweller and plant colonizer bacterium to become a flexible and engineer-able platform for metabolic... (Review)
Review
The itinerary followed by Pseudomonas putida from being a soil-dweller and plant colonizer bacterium to become a flexible and engineer-able platform for metabolic engineering stems from its natural lifestyle, which is adapted to harsh environmental conditions and all sorts of physicochemical stresses. Over the years, these properties have been capitalized biotechnologically owing to the expanding wealth of genetic tools designed for deep-editing the P. putida genome. A suite of dedicated vectors inspired in the core tenets of synthetic biology have enabled to suppress many of the naturally-occurring undesirable traits native to this species while enhancing its many appealing properties, and also to import catalytic activities and attributes from other biological systems. Much of the biotechnological interest on P. putida stems from the distinct architecture of its central carbon metabolism. The native biochemistry is naturally geared to generate reductive currency [i.e., NAD(P)H] that makes this bacterium a phenomenal host for redox-intensive reactions. In some cases, genetic editing of the indigenous biochemical network of P. putida (cis-metabolism) has sufficed to obtain target compounds of industrial interest. Yet, the main value and promise of this species (in particular, strain KT2440) resides not only in its capacity to host heterologous pathways from other microorganisms, but also altogether artificial routes (trans-metabolism) for making complex, new-to-Nature molecules. A number of examples are presented for substantiating the worth of P. putida as one of the favorite workhorses for sustainable manufacturing of fine and bulk chemicals in the current times of the 4th Industrial Revolution. The potential of P. putida to extend its rich native biochemistry beyond existing boundaries is discussed and research bottlenecks to this end are also identified. These aspects include not just the innovative genetic design of new strains but also the incorporation of novel chemical elements into the extant biochemistry, as well as genomic stability and scaling-up issues.
Topics: Biocatalysis; Metabolic Engineering; Metabolic Networks and Pathways; Oxidation-Reduction; Pseudomonas putida
PubMed: 29758287
DOI: 10.1016/j.ymben.2018.05.005 -
MBio Dec 2021Perfluorinated carbon atoms in a diether linkage are common in commercial anesthetics, drugs, fungicides, and insecticides. An important chemical group comprising...
Perfluorinated carbon atoms in a diether linkage are common in commercial anesthetics, drugs, fungicides, and insecticides. An important chemical group comprising perfluorodiethers is the 2,2-fluoro-1,3-benzodioxole (DFBD) moiety. The fluorine atoms stabilize the molecule by mitigating against metabolism by humans and microbes, as used in drugs and pesticides, respectively. Pseudomonas putida F1 catalyzed defluorination of DFBD at an initial rate of 2,100 nmol/h per mg cellular protein. This is orders of magnitude higher than previously reported microbial defluorination rates with multiply fluorinated carbon atoms. Defluorination rates declined after several hours, and the medium darkened. Significant defluorination activity was observed with cells grown on toluene but not l-arginine. Defluorination required only toluene dioxygenase. Pseudomonas and recombinant Escherichia coli cells expressing toluene dioxygenase oxidized DFBD to DFBD-4,5-dihydrodiol. The dihydrodiol could be oxidized to 4,5-dihydroxy-DFBD via the dihydrodiol dehydrogenase from P. putida F1. The dihydrodiol dehydrated with acid to yield a mixture of 4-hydroxy-DFBD and 5-hydroxy-DFBD. All those metabolites retained the difluoromethylene group; no fluoride or dark color was observed. The major route of DFBD-4,5-dihydrodiol decomposition produced fluoride and 1,2,3-trihydroxybenzene, or pyrogallol, and that was shown to be the source of the dark colors in the medium. A mechanism for DFBD-4,5-dihydrodiol transformation to two fluoride ions and pyrogallol is proposed. The Pseudomonas genome database and other databases revealed hundreds of bacteria with enzymes sharing high amino acid sequence identity to toluene dioxygenase from P. putida F1, suggesting the mechanism revealed here may apply to the defluorination of DFBD-containing compounds in the environment. There are more than 9,000 polyfluorinated compounds developed for commercial use, some negatively impacting human health, and they are generally considered to be resistant to biodegradation. Only a limited number of studies have identified microbes with enzymes sufficiently reactive to defluorinate difluoromethylene carbon groups. The present study examined one important group of commercial fluorinated chemicals and showed its rapid defluorination by a bacterium and its key enzyme, a Rieske dioxygenase. Rieske dioxygenases are common in environmental bacteria, and those closely resembling toluene dioxygenase from Pseudomonas putida F1 are candidates for biodegradative defluorination of the common 2,2-fluoro-1,3-benzodioxole (DFBD) moiety.
Topics: Bacterial Proteins; Biodegradation, Environmental; Dioxoles; Halogenation; Oxygenases; Pseudomonas putida
PubMed: 34781746
DOI: 10.1128/mBio.03001-21 -
Biotechnology and Bioengineering Sep 2022Lignin is a largely untapped source for the bioproduction of value-added chemicals. Pseudomonas putida KT2440 has emerged as a strong candidate for bioprocessing of...
Lignin is a largely untapped source for the bioproduction of value-added chemicals. Pseudomonas putida KT2440 has emerged as a strong candidate for bioprocessing of lignin feedstocks due to its resistance to several industrial solvents, broad metabolic capabilities, and genetic amenability. Here we demonstrate the engineering of P. putida for the ability to metabolize syringic acid, one of the major products that comes from the breakdown of the syringyl component of lignin. The rational design was first applied for the construction of strain Sy-1 by overexpressing a native vanillate demethylase. Subsequent adaptive laboratory evolution (ALE) led to the generation of mutations that achieved robust growth on syringic acid as a sole carbon source. The best mutant showed a 30% increase in the growth rate over the original engineered strain. Genomic sequencing revealed multiple mutations repeated in separate evolved replicates. Reverse engineering of mutations identified in agmR, gbdR, fleQ, and the intergenic region of gstB and yadG into the parental strain recaptured the improved growth of the evolved strains to varied extent. These findings thus reveal the ability of P. putida to utilize lignin more fully as a feedstock and make it a more economically viable chassis for chemical production.
Topics: Base Sequence; Carbon; Lignin; Metabolic Engineering; Pseudomonas putida
PubMed: 35524438
DOI: 10.1002/bit.28131 -
Microbial Cell Factories Jan 2023Pseudomonas putida has received increasing interest as a cell factory due to its remarkable features such as fast growth, a versatile and robust metabolism, an extensive...
BACKGROUND
Pseudomonas putida has received increasing interest as a cell factory due to its remarkable features such as fast growth, a versatile and robust metabolism, an extensive genetic toolbox and its high tolerance to oxidative stress and toxic compounds. This interest is driven by the need to improve microbial performance to a level that enables biologically possible processes to become economically feasible, thereby fostering the transition from an oil-based economy to a more sustainable bio-based one. To this end, one of the current strategies is to maximize the product-substrate yield of an aerobic biocatalyst such as P. putida during growth on glycolytic carbon sources, such as glycerol and xylose. We demonstrate that this can be achieved by implementing the phosphoketolase shunt, through which pyruvate decarboxylation is prevented, and thus carbon loss is minimized.
RESULTS
In this study, we introduced the phosphoketolase shunt in the metabolism of P. putida KT2440. To maximize the effect of this pathway, we first tested and selected a phosphoketolase (Xfpk) enzyme with high activity in P. putida. Results of the enzymatic assays revealed that the most efficient Xfpk was the one isolated from Bifidobacterium breve. Using this enzyme, we improved the P. putida growth rate on glycerol and xylose by 44 and 167%, respectively, as well as the biomass yield quantified by OD by 50 and 30%, respectively. Finally, we demonstrated the impact on product formation and achieved a 38.5% increase in mevalonate and a 25.9% increase in flaviolin yield from glycerol. A similar effect was observed on the mevalonate-xylose and flaviolin-xylose yields, which increased by 48.7 and 49.4%, respectively.
CONCLUSIONS
Pseudomonas putida with the implemented Xfpk shunt grew faster, reached a higher final OD and provided better product-substrate yields than the wild type. By reducing the pyruvate decarboxylation flux, we significantly improved the performance of this important workhorse for industrial applications. This work encompasses the first steps towards full implementation of the non-oxidative glycolysis (NOG) or the glycolysis alternative high carbon yield cycle (GATCHYC), in which a substrate is converted into products without CO loss These enhanced properties of P. putida will be crucial for its subsequent use in a range of industrial processes.
Topics: Pseudomonas putida; Xylose; Glycerol; Mevalonic Acid; Pyruvates; Carbon
PubMed: 36658566
DOI: 10.1186/s12934-022-02015-9 -
Metabolic Engineering Jan 2023Medium-chain-length fatty alcohols have broad applications in the surfactant, lubricant, and cosmetic industries. Their acetate esters are widely used as flavoring and...
Medium-chain-length fatty alcohols have broad applications in the surfactant, lubricant, and cosmetic industries. Their acetate esters are widely used as flavoring and fragrance substances. Pseudomonas putida KT2440 is a promising chassis for fatty alcohol and ester production at the industrial scale due to its robustness, versatility, and high oxidative capacity. However, P. putida has also numerous native alcohol dehydrogenases, which lead to the degradation of these alcohols and thereby hinder its use as an effective biocatalyst. Therefore, to harness its capacity as a producer, we constructed two engineered strains (WTΔpedFΔadhP, GN346ΔadhP) incapable of growing on mcl-fatty alcohols by deleting either a cytochrome c oxidase PedF and a short-chain alcohol dehydrogenase AdhP in P. putida or AdhP in P. putida GN346. Carboxylic acid reductase, phosphopantetheinyl transferase, and alcohol acetyltransferase were expressed in the engineered P. putida strains to produce hexyl acetate. Overexpression of transporters further increased 1-hexanol and hexyl acetate production. The optimal strain G23E-MPAscTP produced 93.8 mg/L 1-hexanol and 160.5 mg/L hexyl acetate, with a yield of 63.1%. The engineered strain is applicable for C-C fatty alcohols and their acetate ester production. This study lays a foundation for P. putida being used as a microbial cell factory for sustainable synthesis of a broad range of products based on medium-chain-length fatty alcohols.
Topics: Pseudomonas putida; Fatty Acids; Metabolic Engineering; Esters; Fatty Alcohols; Acetates
PubMed: 36494025
DOI: 10.1016/j.ymben.2022.11.006 -
Applied and Environmental Microbiology Oct 2023have emerged as promising biocatalysts for the conversion of sugars and aromatic compounds obtained from lignocellulosic biomass. Understanding the role of carbon...
have emerged as promising biocatalysts for the conversion of sugars and aromatic compounds obtained from lignocellulosic biomass. Understanding the role of carbon catabolite repression (CCR) in these strains is critical to optimize biomass conversion to fuels and chemicals. The CCR functioning in M2, a strain capable of consuming both hexose and pentose sugars as well as aromatic compounds, was investigated by cultivation experiments, proteomics, and CRISPRi-based gene repression. Strain M2 co-utilized sugars and aromatic compounds simultaneously; however, during cultivation with glucose and aromatic compounds (coumarate and ferulate) mixture, intermediates (4-hydroxybenzoate and vanillate) accumulated, and substrate consumption was incomplete. In contrast, xylose-aromatic consumption resulted in transient intermediate accumulation and complete aromatic consumption, while xylose was incompletely consumed. Proteomics analysis revealed that glucose exerted stronger repression than xylose on the aromatic catabolic proteins. Key glucose (Eda) and xylose (XylX) catabolic proteins were also identified at lower abundance during cultivation with aromatic compounds implying simultaneous catabolite repression by sugars and aromatic compounds. Reduction of expression via CRISPRi led to faster growth and glucose and -coumarate uptake in the CRISPRi strains compared to the control, while no difference was observed on xylose+-coumarate. The increased abundances of Eda and amino acid biosynthesis proteins in the CRISPRi strain further supported these observations. Lastly, small RNAs (sRNAs) sequencing results showed that CrcY and CrcZ homologues levels in M2, previously identified in strains, were lower under strong CCR (glucose+-coumarate) condition compared to when repression was absent (-coumarate or glucose only).IMPORTANCEA newly isolated strain, M2, can utilize both hexose and pentose sugars as well as aromatic compounds making it a promising host for the valorization of lignocellulosic biomass. Pseudomonads have developed a regulatory strategy, carbon catabolite repression, to control the assimilation of carbon sources in the environment. Carbon catabolite repression may impede the simultaneous and complete metabolism of sugars and aromatic compounds present in lignocellulosic biomass and hinder the development of an efficient industrial biocatalyst. This study provides insight into the cellular physiology and proteome during mixed-substrate utilization in M2. The phenotypic and proteomics results demonstrated simultaneous catabolite repression in the sugar-aromatic mixtures, while the CRISPRi and sRNA sequencing demonstrated the potential role of the gene and small RNAs in carbon catabolite repression.
Topics: Sugars; Catabolite Repression; Xylose; Pseudomonas putida; Glucose; Hexoses; Pentoses; Carbon
PubMed: 37724856
DOI: 10.1128/aem.00852-23