Engineered Pseudomonas Putida Research Articles

Production of bio-indigo from engineered Pseudomonas putida KT2440 harboring tryptophanase and flavin-containing monooxygenase

Production of bio-indigo from engineered Pseudomonas putida KT2440 harboring tryptophanase and flavin-containing monooxygenase

Microbial production of levulinic acid from glucose by engineered Pseudomonas putida KT2440

Levulinic acid(LA) is produced through acid-catalyzed hydrolysis and dehydration of lignocellulosic biomass. It is a key platform chemical used as an intermediate in various industries including biofuels, cosmetics, pharmaceuticals, and polymers. Traditional LA production uses chemical conversion, which requires high temperatures and pressures, strong acids, and produces undesirable side reactions, repolymerization products, and waste problems Therefore, we designed an integrated process to produce LA from glucose through metabolic engineering of Pseudomonas putida KT2440. As a metabolic engineering strategy, codon optimized phospho-2-dehydro-3-deoxyheptonate aldolase (AroG), 3-dehydroshikimate dehydratase (AsbF), and acetoacetate decarboxylase (Adc) were introduced to express genes of the shikimate and β-ketoadipic acid pathways, and the 3-oxoadipate CoA-transferase (pcaIJ) gene was deleted to prevent loss of biosynthetic intermediates. To increase the accumulation of the produced LA, the lva operon encoding levulinyl-CoA synthetase (LvaE) was deleted resulting in the high LA-producing strain P. putida HP203. Culture conditions such as medium, temperature, glucose concentration, and nitrogen source were optimized, and under optimal conditions, P. putida HP203 strain biosynthesized 36.3 mM (4.2 g/L) LA from glucose in a fed-batch fermentation system. When lignocellulosic biomass hydrolysate was used as the substrate, this strain produced 7.31 mM of LA. This is the first report of microbial production of LA from glucose by P. putida. This study suggests the possibility of manipulating biosynthetic pathway to produce biological products from glucose for various applications.

Production of bio-based adipic acid using a combination of engineered Pseudomonas putida strains

Adipic acid is an industrially important dicarboxylic acid and an essential precursor in the synthesis of Nylon-6,6. Due to the environmental impact associated with the current synthetic methods for Nylon-6,6 production, greener alternatives based on renewable and bio-based feedstocks are needed. In the present study, the potential of two Pseudomonas putida strains engineered for muconic acid accumulation was used to produce adipic acid from the lignin-derived aromatic compound guaiacol. This was achieved by expressing genes enabling the conversion of guaiacol to catechol in the first strain, and the conversion of muconic acid to adipic acid via expression of the Bacillus coagulans enoate reductase gene in the second strain. Through process engineering, a 2-step production was designed in which 2.93 mM bio-adipic acid was produced, with a yield of 0.57 mol/mol. This is the first demonstration that process and strain engineering can be combined to obtain a complete bio-based route for adipic acid production from depolymerized lignin compounds using the robust P. putida chassis.

Funneling lignin hydrolysates into β-ketoadipic acid by engineered Pseudomonas putida KT2440

Bioconversion of lignin into value-added chemicals could promote lignin valorization and the lignin-based bioeconomy. The heterogeneity nature of lignin and the lack of efficient conversion pathways pose limitations on the efficiency of lignin conversion. In this study, Pseudomonas putida KT2440 was successfully engineered for converting lignin to β-ketoadipic acid, a C6 keto-diacid that functions as building block for high performance polymers. Blocking the degradation pathway of β-ketoadipic acid enabled its biosynthesis from both H- and G-type lignin-derived monomers. The aromatic hydroxylation and O-demethylation was identified as the rate-limiting step for producing β-ketoadipic acid from H- and G- type lignin-derived monomers, respectively. Overexpressing pobA and vanAB successfully vanished the accumulation of 4-hydroxybenzoic acid and vanillic acid within 36 h and 30 h, respectively. The beneficial gene operations were integrated into the chromosome to create the strain HE65. Employing this strain, fed-batch strategies were used to achieve a record β-ketoadipic acid production of 14.80 g/L from mixed lignin-derived aromatics in shake-flask experiments. Remarkably, the strain could also thrive in actual lignin hydrolysates from corn stover, yielding a record β-ketoadipic acid output of 2.48 g/L without the need for separation or detoxification processes. Overall, these results underscore the significant potential of engineering P. putida for converting lignin into β-ketoadipic acid, offering a promising pathway for feasible lignin valorization.

Bioaugmentation of Bio-Slurry Reactor Containing Pyrene Contaminated Soil by Engineered Pseudomonas putida KT2440

Bioaugmentation of Bio-Slurry Reactor Containing Pyrene Contaminated Soil by Engineered Pseudomonas putida KT2440

Biological Valorization of Lignin-Derived Aromatics in Hydrolysate to Protocatechuic Acid by Engineered Pseudomonas putida KT2440.

Alongside fermentable sugars, weak acids, and furan derivatives, lignocellulosic hydrolysates contain non-negligible amounts of lignin-derived aromatic compounds. The biological funnel of lignin offers a new strategy for the "natural" production of protocatechuic acid (PCA). Herein, Pseudomonas putida KT2440 was engineered to produce PCA from lignin-derived monomers in hydrolysates by knocking out protocatechuate 3,4-dioxygenase and overexpressing vanillate-O-demethylase endogenously, while acetic acid was used for cell growth. The sugar catabolism was further blocked to prevent the loss of fermentable sugar. Using the engineered strain, a total of 253.88 mg/L of PCA was obtained with a yield of 70.85% from corncob hydrolysate 1. The highest titer of 433.72 mg/L of PCA was achieved using corncob hydrolysate 2 without any additional nutrients. This study highlights the potential ability of engineered strains to address the challenges of PCA production from lignocellulosic hydrolysate, providing novel insights into the utilization of hydrolysates.

Bio-upcycling of even and uneven medium-chain-length diols and dicarboxylates to polyhydroxyalkanoates using engineered Pseudomonas putida

Bio-upcycling of plastics is an emerging alternative process that focuses on extracting value from a wide range of plastic waste streams. Such streams are typically too contaminated to be effectively processed using traditional recycling technologies. Medium-chain-length (mcl) diols and dicarboxylates (DCA) are major products of chemically or enzymatically depolymerized plastics, such as polyesters or polyethers. In this study, we enabled the efficient metabolism of mcl-diols and -DCA in engineered Pseudomonas putida as a prerequisite for subsequent bio-upcycling. We identified the transcriptional regulator GcdR as target for enabling metabolism of uneven mcl-DCA such as pimelate, and uncovered amino acid substitutions that lead to an increased coupling between the heterologous β-oxidation of mcl-DCA and the native degradation of short-chain-length DCA. Adaptive laboratory evolution and subsequent reverse engineering unravelled two distinct pathways for mcl-diol metabolism in P. putida, namely via the hydroxy acid and subsequent native β-oxidation or via full oxidation to the dicarboxylic acid that is further metabolized by heterologous β-oxidation. Furthermore, we demonstrated the production of polyhydroxyalkanoates from mcl-diols and -DCA by a single strain combining all required metabolic features. Overall, this study provides a powerful platform strain for the bio-upcycling of complex plastic hydrolysates to polyhydroxyalkanoates and leads the path for future yield optimizations.Graphical

Simultaneous utilization of glucose and xylose by metabolically engineered Pseudomonas putida for the production of 3-hydroxypropionic acid

Pseudomonas putida,a robust candidate for lignocellulosicbiomass-based biorefineries, encounters challenges in metabolizing xylose. In this study, Weimberg pathway was introduced intoP. putidaEM42 under a xylose-inducible promoter, resulting in slow cell growth (0.05 h−1) on xylose.Through adaptive laboratory evolution, an evolved strain exhibited highly enhanced growth on xylose (0.36 h−1), comparable to that on glucose (0.39 h−1). Whole genome sequencing identified four mutations, with two key mutations located inPP3380andPP2219. Reverse-engineered strain 8EM42_Xyl, harboring these two mutations, showed enhanced growth on xylose but co-utilizing glucose and xylose at a rate of 0.3 g/L/h. Furthermore, 8EM42_Xyl was employed for 3-hydroxypropionic acid (3HP) production from glucose and xylose by expressing malonyl-CoA reductase and acetyl-CoA carboxylase, yielding 29 g/L in fed-batch fermentation. Moreover, the engineered strain exhibited promising performance in 3HP production from empty palm fruit bunch hydrolysate, demonstrating its potential as a promising cell factory forbiorefineries.

A novel strategy for extraction of intracellular poly(3-hydroxybutyrate) from engineered Pseudomonas putida using deep eutectic solvents: Comparison with traditional biobased organic solvents

Polyhydroxyalkanoates (PHAs) represent a category of microbial polyesters that offer both biodegradability and biocompatibility, if produced in sufficient quantities, they could serve as an alternative to many conventional plastics in use today. However, these microbial polymers are intracellularly stored, necessitating a more complex downstream extraction and purification process. Downstream processes often constitute the most financially burdensome stage in biomolecule production. One significant drawback of many existing extraction processes is their reliance on harsh organic solvents, such as chloroform, and high temperatures. This study presents and compares two novel downstream processes for the extraction and purification of poly(3-hydroxybutyrate) (PHB), a type of short-chain-length PHA, utilizing bio-based green solvents and natural deep eutectic solvents (NADES), respectively. The soil bacterium Pseudomonas putida, engineered to produce PHB from sugars, was adopted as a model for testing these extraction procedures. Initially, biomass was disrupted using a hypotonic buffer containing lysozyme to enhance the extraction efficiency in the downstream process. After extensive screening, the bio-based solvent ethyl acetate was selected for PHB extraction from P. putida biomass, yielding ∼ 95 wt% of the homo-polymer with a purity of ∼ 97 wt%, results comparable to those achieved with the traditional benchmark solvent, chloroform. Furthermore, a hydrophobic natural deep eutectic solvent (hydrophobic NADES) was synthesized, comprising L-menthol and acetic acid in a 1:3 M ratio, and employed as the extraction solvent in combination with methanol as the anti-solvent. The optimized extraction process resulted in a homo-polymer yield of ∼ 66 wt% with a high purity of ∼ 85 wt%. These results are promising considering the benefits associated with the use of NADES, they are less toxic and much easier to handle than ethyl acetate and have the potential to be recycled. Therefore, it represents a promising avenue for a more sustainable PHB extraction process, devoid of harmful organic solvents.

Rapid Degradation of Hazardous Amides by Immobilized Engineered Pseudomonas putida KT2440 Based on a Novel Gene Expression Vector.

The amides 4-trifluoromethylnicotinamide, acrylamide, and benzamide are widely used in agriculture and industry, posing hazards to the environment and animals. Immobilized bacteria are preferred in wastewater treatment, but degradation of these amides by immobilized engineered bacteria has not been explored. Here, engineered Pseudomonas putida KT2440 pLSJ15-amiA was constructed by introducing a new amidase gene expression vector into environmentally safe P. putida KT2440. P. putida KT2440 pLSJ15-amiA had high amidase activity, even at 80 °C. P. putida KT2440 pLSJ15-amiA immobilized with calcium alginate exhibited a greater environmental tolerance than free cells. The amides were rapidly degraded by the immobilized cells, but the activity was inhibited by high concentrations of substrates. The substrate inhibition model revealed that the optimum initial concentrations of 4-trifluoromethylnicotinamide, acrylamide, and benzamide for degradation by immobilized cells were 197.65, 350.76, and 249.40 μmol/L, respectively. This study develops a novel and excellent immobilized biocatalyst for remediation of wastewater containing hazardous amides.

An efficient chemoenzymatic approach to produce galactose from red macroalgae biomass and its valorization

Marine biomass is receiving much attention as the third-generation feedstock owing to its advantages over terrestrial biomass. Red macroalgae have an impressive high carbohydrate content, with galactose as the most abundant monosaccharide. However, the technology for producing galactose with a high yield and its valorization is not yet available. This study described an efficient chemoenzymatic approach toward upgrading of red macroalgae (Gelidium amansii) into value-added products (taking galactonic acid as an example). With the aid of microwave-assisted maleic acid pretreatment, galactose was obtained with an excellent yield of 92.7%. Various inhibitors were simultaneously generated and their inhibition effects on biocatalysis were overcame by genetic and bioprocess engineering. Finally, the galactose in the hydrolysate was converted into galactonic acid with a yield of 100% by engineered Pseudomonas putida. This approach can be easily expanded to other galactose-derived products and promotes green development of marine biomass biorefinery.

γ-Glutamylation of Isopropylamine by Fermentation.

Glutamylation yields N-functionalized amino acids in several natural pathways. γ-Glutamylated amino acids may exhibit improved properties for their industrial application, e. g., as taste enhancers or in peptide drugs. γ-Glutamyl-isopropylamide (GIPA) can be synthesized from isopropylamine (IPA) and l-glutamate. In Pseudomonas sp. strain KIE171, GIPA is an intermediate in the biosynthesis of l-alaninol (2-amino-1-propanol), a precursor of the fluorochinolone antibiotic levofloxacin and of the chloroacetanilide herbicide metolachlor. In this study, fermentative production of GIPA with metabolically engineered Pseudomonas putida KT2440 using γ-glutamylmethylamide synthetase (GMAS) from Methylorubrum extorquens was established. Upon addition of IPA during growth with glycerol as carbon source in shake flasks, the recombinant strain produced up to 21.8 mM GIPA. In fed-batch bioreactor cultivations, GIPA accumulated to a titer of 11 g L-1 with a product yield of 0.11 g g-1 glycerol and a volumetric productivity of 0.24 g L-1 h-1 . To the best of our knowledge, this is the first fermentative production of GIPA.

Production of tailored hydroxylated prodiginine showing combinatorial activity with rhamnolipids against plant-parasitic nematodes.

Bacterial secondary metabolites exhibit diverse remarkable bioactivities and are thus the subject of study for different applications. Recently, the individual effectiveness of tripyrrolic prodiginines and rhamnolipids against the plant-parasitic nematode Heterodera schachtii, which causes tremendous losses in crop plants, was described. Notably, rhamnolipid production in engineered Pseudomonas putida strains has already reached industrial implementation. However, the non-natural hydroxyl-decorated prodiginines, which are of particular interest in this study due to a previously described particularly good plant compatibility and low toxicity, are not as readily accessible. In the present study, a new effective hybrid synthetic route was established. This included the engineering of a novel P. putida strain to provide enhanced levels of a bipyrrole precursor and an optimization of mutasynthesis, i.e., the conversion of chemically synthesized and supplemented monopyrroles to tripyrrolic compounds. Subsequent semisynthesis provided the hydroxylated prodiginine. The prodiginines caused reduced infectiousness of H. schachtii for Arabidopsis thaliana plants resulting from impaired motility and stylet thrusting, providing the first insights on the mode of action in this context. Furthermore, the combined application with rhamnolipids was assessed for the first time and found to be more effective against nematode parasitism than the individual compounds. To obtain, for instance, 50% nematode control, it was sufficient to apply 7.8 μM hydroxylated prodiginine together with 0.7 μg/ml (~ 1.1 μM) di-rhamnolipids, which corresponded to ca. ¼ of the individual EC50 values. In summary, a hybrid synthetic route toward a hydroxylated prodiginine was established and its effects and combinatorial activity with rhamnolipids on plant-parasitic nematode H. schachtii are presented, demonstrating potential application as antinematodal agents. Graphical Abstract.

Evaluation of water management on arsenic methylation and volatilization in arsenic-contaminated soils strengthened by bioaugmentation and biostimulation

Arsenic (As) fate in paddy fields has been one of the most significant current issues due to the strong As accumulation potential of rice plants under flooded conditions. However, no attempt was done to explore As methylation and volatilization under non-flooded conditions. Herein, we investigated the effects of water management on As methylation and volatilization in three arsenic-contaminated soils enhanced by biostimulation with straw-derived organic matter and bioaugmentation with genetic engineered Pseudomonas putida KT2440 (GE P. putida). Under flooded conditions, the application of biochar (BC), rice straw (RS) and their combination (BC+RS) increased total As in porewater. However, these effects were greatly attenuated under non-flooded conditions. Compared with RS amendment alone, the combination of GE P. putida and RS further promoted the As methylation and volatilization, and the promotion percentage under non-flooded conditions were significantly higher than that under flooded conditions. The combined GE P. putida and RS showed the highest efficiency in As methylation (88 µg/L) and volatilization (415.4 µg/(kg·year)) in the non-flooded soil with moderate As contamination. Finally, stepwise multiple linear regression analysis presented that methylated As, DOC and pH in porewater were the most important factors contributing to As volatilization. Overall, our findings suggest that combination of bioaugmentation with GE P. putida and biostimulation with RS/BC+RS is a potential strategy for bioremediation of arsenic-contaminated soils by enhancing As methylation and volatilization under non-flooded conditions.

Improved terephthalic acid production from p-xylene using metabolically engineered Pseudomonas putida

Terephthalic acid (TPA) is an important commodity chemical used as a monomer of polyethylene terephthalate (PET). Since a large quantity of PET is routinely manufactured and consumed worldwide, the development of sustainable biomanufacturing processes for its monomers (i.e. TPA and ethylene glycol) has recently gained much attention. In a previous study, we reported the development of a metabolically engineered Escherichia coli strain producing 6.7 g/L of TPA from p-xylene (pX) with a productivity and molar conversion yield of 0.278 g/L/h and 96.7 mol%, respectively. Here, we report metabolic engineering of Pseudomonas putida KT2440, a microbial chassis particularly suitable for the synthesis of aromatic compounds, for improved biocatalytic conversion of pX to TPA. To develop a plasmid-free, antibiotic-free, and inducer-free biocatalytic process for cost-competitive TPA production, all heterologous genes required for the synthetic pX-to-TPA bioconversion pathway were integrated into the chromosome of P. putida KT2440 by RecET-based markerless recombineering and overexpressed under the control of constitutive promoters. Next, TPA production was enhanced by integrating multiple copies of the heterologous genes to the ribosomal RNA genes through iteration of recombineering-based random integration and subsequent screening of high-performance strains. Finally, fed-batch fermentation process was optimized to further improve the performance of the engineered P. putida strain. As a result, 38.25 ± 0.11 g/L of TPA was produced from pX with a molar conversion yield of 99.6 ± 0.6%, which is equivalent to conversion of 99.3 ± 0.8 g pX to 154.6 ± 0.5 g TPA. This superior pX-to-TPA biotransformation process based on the engineered P. putida strain will pave the way to the commercial biomanufacturing of TPA in an industrial scale.

Aerobic Degradation of the Antidiabetic Drug Metformin by Aminobacter sp. Strain NyZ550.

Metformin is becoming one of the most common emerging contaminants in surface and wastewater. Its biodegradation generally leads to the accumulation of guanylurea in the environment, but the microorganisms and mechanisms involved in this process remain elusive. Here, Aminobacter sp. strain NyZ550 was isolated and characterized for its ability to grow on metformin as a sole source of carbon, nitrogen, and energy under oxic conditions. This isolate also assimilated a variety of nitrogenous compounds, including dimethylamine. Hydrolysis of metformin by strain NyZ550 was accompanied by a stoichiometric accumulation of guanylurea as a dead-end product. Based on ion chromatography, gas chromatography-mass spectrometry, and comparative transcriptomic analyses, dimethylamine was identified as an additional hydrolytic product supporting the growth of the strain. Notably, a microbial mixture consisting of strain NyZ550 and an engineered Pseudomonas putida PaW340 expressing a guanylurea hydrolase was constructed for complete elimination of metformin and its persistent product guanylurea. Overall, our results not only provide new insights into the metformin biodegradation pathway, leading to the commonly observed accumulation of guanylurea in the environment, but also open doors for the complete degradation of the new pollutant metformin.

Biological conversion of cyclic ketones from catalytic fast pyrolysis with Pseudomonas putida KT2440

A chemical fraction enriched in cyclic ketones, was isolated from ex situ catalytic fast pyrolysis (CFP) bio-oil and valorized to hydroxy and dicarboxylic acids by an engineered Pseudomonas putida strain.

Unraveling the mechanism of furfural tolerance in engineered Pseudomonas putida by genomics.

As a dehydration product of pentoses in hemicellulose sugar streams derived from lignocellulosic biomass, furfural is a prevalent inhibitor in the efficient microbial conversion process. To solve this obstacle, exploiting a biorefinery strain with remarkable furfural tolerance capability is essential. Pseudomonas putida KT2440 (P. putida) has served as a valuable bacterial chassis for biomass biorefinery. Here, a high-concentration furfural-tolerant P. putida strain was developed via adaptive laboratory evolution (ALE). The ALE resulted in a previously engineered P. putida strain with substantially increased furfural tolerance as compared to wild-type. Whole-genome sequencing of the adapted strains and reverse engineering validation of key targets revealed for the first time that several genes and their mutations, especially for PP_RS19785 and PP_RS18130 [encoding ATP-binding cassette (ABC) transporters] as well as PP_RS20740 (encoding a hypothetical protein), play pivotal roles in the furfural tolerance and conversion of this bacterium. Finally, strains overexpressing these three striking mutations grew well in highly toxic lignocellulosic hydrolysate, with cell biomass around 9-, 3.6-, and two-fold improvement over the control strain, respectively. To our knowledge, this study first unravels the furan aldehydes tolerance mechanism of industrial workhorse P. putida, which provides a new foundation for engineering strains to enhance furfural tolerance and further facilitate the valorization of lignocellulosic biomass.

Reconstruction and optimization of a Pseudomonas putida-Escherichia coli microbial consortium for mcl-PHA production from lignocellulosic biomass.

The demand for non-petroleum-based, especially biodegradable plastics has been on the rise in the last decades. Medium-chain-length polyhydroxyalkanoate (mcl-PHA) is a biopolymer composed of 6-14 carbon atoms produced from renewable feedstocks and has become the focus of research. In recent years, researchers aimed to overcome the disadvantages of single strains, and artificial microbial consortia have been developed into efficient platforms. In this work, we reconstructed the previously developed microbial consortium composed of engineered Pseudomonas putida KT∆ABZF (p2-a-J) and Escherichia coli ∆4D (ACP-SCLAC). The maximum titer of mcl-PHA reached 3.98g/L using 10g/L glucose, 5g/L octanoic acid as substrates by the engineered P. putida KT∆ABZF (p2-a-J). On the other hand, the maximum synthesis capacity of the engineered E. coli ∆4D (ACP-SCLAC) was enhanced to 3.38g/L acetic acid and 0.67g/L free fatty acids (FFAs) using 10g/L xylose as substrate. Based on the concept of "nutrient supply-detoxification," the engineered E. coli ∆4D (ACP-SCLAC) provided nutrient for the engineered P. putida KT∆ABZF (p2-a-J) and it acted to detoxify the substrates. Through this functional division and rational design of the metabolic pathways, the engineered P. putida-E. coli microbial consortium could produce 1.30g/L of mcl-PHA from 10g/L glucose and xylose. Finally, the consortium produced 1.02g/L of mcl-PHA using lignocellulosic hydrolysate containing 10.50g/L glucose and 10.21g/L xylose as the substrate. The consortium developed in this study has good potential for mcl-PHA production and provides a valuable reference for the production of high-value biological products using inexpensive carbon sources.

Bioproduction of propionic acid using levulinic acid by engineered Pseudomonas putida.

The present study elaborates on the propionic acid (PA) production by the well-known microbial cell factory Pseudomonas putida EM42 and its capacity to utilize biomass-derived levulinic acid (LA). Primarily, the P. putida EM42 strain was engineered to produce PA by deleting the methylcitrate synthase (PrpC) and propionyl-CoA synthase (PrpE) genes. Subsequently, a LA-inducible expression system was employed to express yciA (encoding thioesterase) from Haemophilus influenzae and ygfH (encoding propionyl-CoA: succinate CoA transferase) from Escherichia coli to improve the PA production by up to 10-fold under flask scale cultivation. The engineered P. putida EM42:ΔCE:yciA:ygfH was used to optimize the bioprocess to further improve the PA production titer. Moreover, the fed-batch fermentation performed under optimized conditions in a 5 L bioreactor resulted in the titer, productivity, and molar yield for PA production of 26.8 g/L, 0.3 g/L/h, and 83%, respectively. This study, thus, successfully explored the LA catabolic pathway of P. putida as an alternative route for the sustainable and industrial production of PA from LA.