Metabolic engineering of Pseudomonas putida for production of vanillylamine from lignin-derived substrates.
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ABSTRACT:
SUBMITTER: Manfrao-Netto JHC
PROVIDER: S-EPMC8601178 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Microbial conversion offers a promising strategy for overcoming the intrinsic heterogeneity of the plant biopolymer, lignin. Soil microbes that natively harbour aromatic-catabolic pathways are natural choices for chassis strains, and Pseudomonas putida KT2440 has emerged as a viable whole-cell biocatalyst for funnelling lignin-derived compounds to value-added products, including its native carbon storage product, medium-chain-length polyhydroxyalkanoates (mcl-PHA). In this work, a series of metabolic engineering targets to improve mcl-PHA production are combined in the P. putida chromosome and evaluated in strains growing in a model aromatic compound, p-coumaric acid, and in lignin streams. Specifically, the PHA depolymerase gene phaZ was knocked out, and the genes involved in ?-oxidation (fadBA1 and fadBA2) were deleted. Additionally, to increase carbon flux into mcl-PHA biosynthesis, phaG, alkK, phaC1 and phaC2 were overexpressed. The best performing strain - which contains all the genetic modifications detailed above - demonstrated a 53% and 200% increase in mcl-PHA titre (g l-1 ) and a 20% and 100% increase in yield (g mcl-PHA per g cell dry weight) from p-coumaric acid and lignin, respectively, compared with the wild type strain. Overall, these results present a promising strain to be employed in further process development for enhancing mcl-PHA production from aromatic compounds and lignin.
| S-EPMC6922519 | biostudies-literature
Project description:Bioconversion of a heterogeneous mixture of lignin-related aromatic compounds (LRCs) to a single product via microbial biocatalysts is a promising approach to valorize lignin. Here, Pseudomonas putida KT2440 was engineered to convert mixed p-coumaroyl- and coniferyl-type LRCs to β-ketoadipic acid, a precursor for performance-advantaged polymers. Expression of enzymes mediating aromatic O-demethylation, hydroxylation, and ring-opening steps was tuned, and a global regulator was deleted. β-ketoadipate titers of 44.5 and 25 grams per liter and productivities of 1.15 and 0.66 grams per liter per hour were achieved from model LRCs and corn stover-derived LRCs, respectively, the latter representing an overall yield of 0.10 grams per gram corn stover-derived lignin. Technoeconomic analysis of the bioprocess and downstream processing predicted a β-ketoadipate minimum selling price of $2.01 per kilogram, which is cost competitive with fossil carbon-derived adipic acid ($1.10 to 1.80 per kilogram). Overall, this work achieved bioproduction metrics with economic relevance for conversion of lignin-derived streams into a performance-advantaged bioproduct.
| S-EPMC10482344 | biostudies-literature
Project description:Muconic acid is a bioprivileged molecule that can be converted into direct replacement chemicals for incumbent petrochemicals and performance-advantaged bioproducts. In this study, Pseudomonas putida KT2440 is engineered to convert glucose and xylose, the primary carbohydrates in lignocellulosic hydrolysates, to muconic acid using a model-guided strategy to maximize the theoretical yield. Using adaptive laboratory evolution (ALE) and metabolic engineering in a strain engineered to express the D-xylose isomerase pathway, we demonstrate that mutations in the heterologous D-xylose:H+ symporter (XylE), increased expression of a major facilitator superfamily transporter (PP_2569), and overexpression of aroB encoding the native 3-dehydroquinate synthase, enable efficient muconic acid production from glucose and xylose simultaneously. Using the rationally engineered strain, we produce 33.7 g L-1 muconate at 0.18 g L-1 h-1 and a 46% molar yield (92% of the maximum theoretical yield). This engineering strategy is promising for the production of other shikimate pathway-derived compounds from lignocellulosic sugars.
| S-EPMC9395534 | biostudies-literature
Project description:Lignin is an abundant and recalcitrant component of plant cell walls. While lignin degradation in nature is typically attributed to fungi, growing evidence suggests that bacteria also catabolize this complex biopolymer. However, the spatiotemporal mechanisms for lignin catabolism remain unclear. Improved understanding of this biological process would aid in our collective knowledge of both carbon cycling and microbial strategies to valorize lignin to value-added compounds. Here, we examine lignin modifications and the exoproteome of three aromatic-catabolic bacteria: Pseudomonas putida KT2440, Rhodoccocus jostii RHA1, and Amycolatopsis sp. ATCC 39116. P. putida cultivation in lignin-rich media is characterized by an abundant exoproteome that is dynamically and selectively packaged into outer membrane vesicles (OMVs). Interestingly, many enzymes known to exhibit activity toward lignin-derived aromatic compounds are enriched in OMVs from early to late stationary phase, corresponding to the shift from bioavailable carbon to oligomeric lignin as a carbon source. In vivo and in vitro experiments demonstrate that enzymes contained in the OMVs are active and catabolize aromatic compounds. Taken together, this work supports OMV-mediated catabolism of lignin-derived aromatic compounds as an extracellular strategy for nutrient acquisition by soil bacteria and suggests that OMVs could potentially be useful tools for synthetic biology and biotechnological applications.
| S-EPMC7196908 | biostudies-literature
Project description:para-Hydroxy benzoic acid (PHBA) is the key component for preparing parabens, a common preservatives in food, drugs, and personal care products, as well as high-performance bioplastics such as liquid crystal polymers. Pseudomonas putida KT2440 was engineered to produce PHBA from glucose via the shikimate pathway intermediate chorismate. To obtain the PHBA production strain, chorismate lyase UbiC from Escherichia coli and a feedback resistant 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase encoded by gene aroGD146N were overexpressed individually and simultaneously. In addition, genes related to product degradation (pobA) or competing for the precursor chorismate (pheA and trpE) were deleted from the genome. To further improve PHBA production, the glucose metabolism repressor hexR was knocked out in order to increase erythrose 4-phosphate and NADPH supply. The best strain achieved a maximum titer of 1.73?g L-1 and a carbon yield of 18.1% (C-mol C-mol-1) in a non-optimized fed-batch fermentation. This is to date the highest PHBA concentration produced by P.?putida using a chorismate lyase.
| S-EPMC5124731 | biostudies-literature
Project description:Pseudomonas putida KT2440 has received increasing attention as an important biocatalyst for the conversion of diverse carbon sources to multiple products, including the olefinic diacid, cis,cis-muconic acid (muconate). P. putida has been previously engineered to produce muconate from glucose; however, periplasmic oxidation of glucose causes substantial 2-ketogluconate accumulation, reducing product yield and selectivity. Deletion of the glucose dehydrogenase gene (gcd) prevents 2-ketogluconate accumulation, but dramatically slows growth and muconate production. In this work, we employed adaptive laboratory evolution to improve muconate production in strains incapable of producing 2-ketogluconate. Growth-based selection improved growth, but reduced muconate titer. A new muconate-responsive biosensor was therefore developed to enable muconate-based screening using fluorescence activated cell sorting. Sorted clones demonstrated both improved growth and muconate production. Mutations identified by whole genome resequencing of these isolates indicated that glucose metabolism may be dysregulated in strains lacking gcd. Using this information, we used targeted engineering to recapitulate improvements achieved by evolution. Deletion of the transcriptional repressor gene hexR improved strain growth and increased the muconate production rate, and the impact of this deletion was investigated using transcriptomics. The genes gntZ and gacS were also disrupted in several evolved clones, and deletion of these genes further improved strain growth and muconate production. Together, these targets provide a suite of modifications that improve glucose conversion to muconate by P. putida in the context of gcd deletion. Prior to this work, our engineered strain lacking gcd generated 7.0 g/L muconate at a productivity of 0.07 g/L/h and a 38% yield (mol/mol) in a fed-batch bioreactor. Here, the resulting strain with the deletion of hexR, gntZ, and gacS achieved 22.0 g/L at 0.21 g/L/h and a 35.6% yield (mol/mol) from glucose in similar conditions. These strategies enabled enhanced muconic acid production and may also improve production of other target molecules from glucose in P. putida.
2022-03-26 | GSE198795 | GEO
Project description:Sucrose is an important disaccharide used as a substrate in many industrial applications. It is a major component of molasses, a cheap by-product of the sugar industry. Unfortunately, not all industrially relevant organisms, among them Pseudomonas putida, are capable of metabolizing sucrose. We chose a metabolic engineering approach to circumvent this blockage and equip P. putida with the activities necessary to consume sucrose. Therefore, we constructed a pair of broad-host range mini-transposons (pSST - sucrose splitting transposon), carrying either cscA, encoding an invertase able to split sucrose into glucose and fructose, or additionally cscB, encoding a sucrose permease. Introduction of cscA was sufficient to convey sucrose consumption and the additional presence of cscB had no further effect, though the sucrose permease was built and localized to the membrane. Sucrose was split extracellularly by the activity of the invertase CscA leaking out of the cell. The transposons were also used to confer sucrose consumption to Cupriavidus necator. Interestingly, in this strain, CscB acted as a glucose transporter, such that C. necator also gained the ability to grow on glucose. Thus, the pSST transposons are functional tools to extend the substrate spectrum of Gram-negative bacterial strains toward sucrose.
| S-EPMC5552902 | biostudies-literature
Project description:BackgroundEfficient utilization of all available carbons from lignocellulosic biomass is critical for economic efficiency of a bioconversion process to produce renewable bioproducts. However, the metabolic responses that enable Pseudomonas putida to utilize mixed carbon sources to generate reducing power and polyhydroxyalkanoate (PHA) remain unclear. Previous research has mainly focused on different fermentation strategies, including the sequential feeding of xylose as the growth stage substrate and octanoic acid as the PHA-producing substrate, feeding glycerol as the sole carbon substrate, and co-feeding of lignin and glucose. This study developed a new strategy-co-feeding glycerol and lignin derivatives such as benzoate, vanillin, and vanillic acid in Pseudomonas putida KT2440-for the first time, which simultaneously improved both cell biomass and PHA production.ResultsCo-feeding lignin derivatives (i.e. benzoate, vanillin, and vanillic acid) and glycerol to P. putida KT2440 was shown for the first time to simultaneously increase cell dry weight (CDW) by 9.4-16.1% and PHA content by 29.0-63.2%, respectively, compared with feeding glycerol alone. GC-MS results revealed that the addition of lignin derivatives to glycerol decreased the distribution of long-chain monomers (C10 and C12) by 0.4-4.4% and increased the distribution of short-chain monomers (C6 and C8) by 0.8-3.5%. The 1H-13C HMBC, 1H-13C HSQC, and 1H-1H COSY NMR analysis confirmed that the PHA monomers (C6-C14) were produced when glycerol was fed to the bacteria alone or together with lignin derivatives. Moreover, investigation of the glycerol/benzoate/nitrogen ratios showed that benzoate acted as an independent factor in PHA synthesis. Furthermore, 1H, 13C and 31P NMR metabolite analysis and mass spectrometry-based quantitative proteomics measurements suggested that the addition of benzoate stimulated oxidative-stress responses, enhanced glycerol consumption, and altered the intracellular NAD+/NADH and NADPH/NADP+ ratios by up-regulating the proteins involved in energy generation and storage processes, including the Entner-Doudoroff (ED) pathway, the reductive TCA route, trehalose degradation, fatty acid β-oxidation, and PHA biosynthesis.ConclusionsThis work demonstrated an effective co-carbon feeding strategy to improve PHA content/yield and convert lignin derivatives into value-added products in P. putida KT2440. Co-feeding lignin break-down products with other carbon sources, such as glycerol, has been demonstrated as an efficient way to utilize biomass to increase PHA production in P. putida KT2440. Moreover, the involvement of aromatic degradation favours further lignin utilization, and the combination of proteomics and metabolomics with NMR sheds light on the metabolic and regulatory mechanisms for cellular redox balance and potential genetic targets for a higher biomass carbon conversion efficiency.
| S-EPMC7792162 | biostudies-literature
Project description:An industrial waste, 1,2,3-trichloropropane (TCP), is toxic and extremely recalcitrant to biodegradation. To date, no natural TCP degraders able to mineralize TCP aerobically have been isolated. In this work, we engineered a biosafety Pseudomonas putida strain KT2440 for aerobic mineralization of TCP by implantation of a synthetic biodegradation pathway into the chromosome and further improved TCP mineralization using combinatorial engineering strategies. Initially, a synthetic pathway composed of haloalkane dehalogenase, haloalcohol dehalogenase and epoxide hydrolase was functionally assembled for the conversion of TCP into glycerol in P. putida KT2440. Then, the growth lag-phase of using glycerol as a growth precursor was eliminated by deleting the glpR gene, significantly enhancing the flux of carbon through the pathway. Subsequently, we improved the oxygen sequestering capacity of this strain through the heterologous expression of Vitreoscilla hemoglobin, which makes this strain able to mineralize TCP under oxygen-limited conditions. Lastly, we further improved intracellular energy charge (ATP/ADP ratio) and reducing power (NADPH/NADP+ ratio) by deleting flagella-related genes in the genome of P. putida KT2440. The resulting strain (named KTU-TGVF) could efficiently utilize TCP as the sole source of carbon for growth. Degradation studies in a bioreactor highlight the value of this engineered strain for TCP bioremediation.
| S-EPMC5539299 | biostudies-literature
Project description:Background:Pseudomonas putida is a promising candidate for the industrial production of biofuels and biochemicals because of its high tolerance to toxic compounds and its ability to grow on a wide variety of substrates. Engineering this organism for improved performances and predicting metabolic responses upon genetic perturbations requires reliable descriptions of its metabolism in the form of stoichiometric and kinetic models. Results:In this work, we developed kinetic models of P. putida to predict the metabolic phenotypes and design metabolic engineering interventions for the production of biochemicals. The developed kinetic models contain 775 reactions and 245 metabolites. Furthermore, we introduce here a novel set of constraints within thermodynamics-based flux analysis that allow for considering concentrations of metabolites that exist in several compartments as separate entities. We started by a gap-filling and thermodynamic curation of iJN1411, the genome-scale model of P. putida KT2440. We then systematically reduced the curated iJN1411 model, and we created three core stoichiometric models of different complexity that describe the central carbon metabolism of P. putida. Using the medium complexity core model as a scaffold, we generated populations of large-scale kinetic models for two studies. In the first study, the developed kinetic models successfully captured the experimentally observed metabolic responses to several single-gene knockouts of a wild-type strain of P. putida KT2440 growing on glucose. In the second study, we used the developed models to propose metabolic engineering interventions for improved robustness of this organism to the stress condition of increased ATP demand. Conclusions:The study demonstrates the potential and predictive capabilities of the kinetic models that allow for rational design and optimization of recombinant P. putida strains for improved production of biofuels and biochemicals. The curated genome-scale model of P. putida together with the developed large-scale stoichiometric and kinetic models represents a significant resource for researchers in industry and academia.
| S-EPMC7048048 | biostudies-literature