Genetic engineering of Pseudomonas putida KT2440 for rapid and high-yield production of vanillin from ferulic acid

Vanillin is one of the most important flavoring agents used today. That is why many efforts have been made on biotechnological production from natural abundant substrates. In this work, the nonpathogenic Pseudomonas putida strain KT2440 was genetically optimized to convert ferulic acid to vanillin. Deletion of the vanillin dehydrogenase gene (vdh) was not sufficiant to prevent vanillin degradation. Additional inactivation of a molybdate transporter, identified by transposon mutagenesis, led to a strain incapable to grow on vanillin as sole carbon source. The bioconversion was optimized by enhanced chromosomal expression of the structural genes for feruloyl-CoA synthetase (fcs) and enoyl-CoA hydratase/aldolase (ech) by introduction of the strong tac promoter system. Further genetic engineering led to high initial conversion rates and molar vanillin yields up to 86 % within just 3 h accompanied with very low by-product levels. To our knowledge, this represents the highest productivity and molar vanillin yield gained with a Pseudomonas strain so far. Together with its high tolerance for ferulic acid, the developed, plasmid-free P. putida strain represents a promising candidate for the biotechnological production of vanillin.     

Genetic Characterization of Accumulation of Polyhydroxyalkanoate from Styrene in Pseudomonas putida CA-3

Pseudomonas putida CA-3 is capable of accumulating medium-chain-length polyhydroxyalkanoates (MCL-PHAs) when growing on the toxic pollutant styrene as the sole source of carbon and energy. In this study, we report on the molecular characterization of the metabolic pathways involved in this novel bioconversion. With a mini-Tn5 random mutagenesis approach, acetyl-coenzyme A (CoA) was identified as the end product of styrene metabolism in P. putida CA-3. Amplified flanking-region PCR was used to clone functionally expressed phenylacetyl-CoA catabolon genes upstream from the sty operon in P. putida CA-3, previously reported to generate acetyl-CoA moieties from the styrene catabolic intermediate, phenylacetyl-CoA. However, the essential involvement of a (non-phenylacetyl-CoA) catabolon-encoded 3-hydroxyacyl-CoA dehydrogenase is also reported. The link between de novo fatty acid synthesis and PHA monomer accumulation was investigated, and a functionally expressed 3-hydroxyacyl-acyl carrier protein-CoA transacylase (phaG) gene in P. putida CA-3 was identified. The deduced PhaG amino acid sequence shared >99% identity with a transacylase from P. putida KT2440, involved in 3-hydroxyacyl-CoA MCL-PHA monomer sequestration from de novo fatty acid synthesis under inorganic nutrient-limited conditions. Similarly, with P. putida CA-3, maximal phaG expression was observed only under nitrogen limitation, with concomitant PHA accumulation. Thus, β-oxidation and fatty acid de novo synthesis appear to converge in the generation of MCL-PHA monomers from styrene in P. putida CA-3. Cloning and functional characterization of the pha locus, responsible for PHA polymerization/depolymerization is also reported and the significance and future prospects of this novel bioconversion are discussed.     

Enhanced synthesis of medium-chain-length poly(3-hydroxyalkanoates) by inactivating the tricarboxylate transport system of Pseudomonas putida KT2440 and process development using waste vegetable oil

The use of waste materials as feedstock for biosynthesis of valuable compounds has been an intensive area of research aiming at diminishing the consumption of non-renewable materials. In this study, P. putida KT2440 was employed as a cell factory for the bioconversion of waste vegetable oil into medium-chain-length Polyhydroxyalkanoates. In the presence of the waste oil this environmental strain is capable of secreting enzymes with lipase activities that enhance the bioavailability of this hydrophobic carbon substrate. It was also found that the oxygen transfer coefficient is directly correlated with high PHA levels in KT2440 cells when metabolizing the waste frying oil. By knocking out the tctA gene, encoding for an enzyme of the tripartite carboxylate transport system, an enhanced intracellular level of mcl-PHA was found in the engineered strain when grown on fatty acids. Batch bioreactors showed that the KT2440 strain produced 1.01 (g⋅L⁻¹) of PHA whereas the engineered ΔtctA P. putida strain synthesized 1.91 (g⋅L⁻¹) after 72 h cultivation on 20 (g⋅L⁻¹) of waste oil, resulting in a nearly 2-fold increment in the PHA volumetric productivity. Taken together, this work contributes to accelerate the pace of development for efficient bioconversion of waste vegetable oils into sustainable biopolymers.     

Growth of engineered Pseudomonas putida KT2440 on glucose,xylose, and arabinose: Hemicellulose hydrolysates and their major sugars as sustainable carbon sources

Lignocellulosic biomass is the most abundant bioresource on earth containing polymers mainly consisting of d ‐glucose, d ‐xylose, l ‐arabinose, and further sugars. In order to establish this alternative feedstock apart from applications in food, we engineered Pseudomonas putida KT2440 as microbial biocatalyst for the utilization of xylose and arabinose in addition to glucose as sole carbon sources. The d ‐xylose‐metabolizing strain P. putida KT2440_xylAB and l ‐arabinose‐metabolizing strain P. putida KT2440_araBAD were constructed by introducing respective operons from Escherichia coli. Surprisingly, we found out that both recombinant strains were able to grow on xylose as well as arabinose with high cell densities and growth rates comparable to glucose. In addition, the growth characteristics on various mixtures of glucose, xylose, and arabinose were investigated, which demonstrated the efficient co‐utilization of hexose and pentose sugars. Finally, the possibility of using lignocellulose hydrolysate as substrate for the two recombinant strains was verified. The recombinant P. putida KT2440 strains presented here as flexible microbial biocatalysts to convert lignocellulosic sugars will undoubtedly contribute to the economic feasibility of the production of valuable compounds derived from renewable feedstock.     

Biotransformation of corn bran derived ferulic acid to vanillic acid using engineered Pseudomonas putida KT2440

Abstract

Ferulic acid is a fraction of the phenolics present in cereals such as rice and corn as a component of the bran. Substantial amounts of waste bran are generated by the grain processing industry and this can be valorized via extraction, purification and conversion of phenolics to value added chemical products. Alkaline alcohol based extracted and purified ferulic acid from corn bran was converted to vanillic acid using engineered Pseudomonas putida KT2440. The strain was engineered by rendering the vanAB gene nonfunctional and obtaining the mutant defective in vanillic acid metabolism. Biotransformation of ferulic acid using resting Pseudomonas putida KT2440 mutant cells resulted in more than 95?±?1.4% molar yield from standard ferulic acid; while the corn bran derived ferulic acid gave 87?±?0.38% molar yield. With fermentation time of less than 24?h the mutant becomes a promising candidate for the stable biosynthesis of vanillic acid at industrial scale.     

Exploiting metagenomic diversity for novel polyhydroxyalkanoate synthases: production of a terpolymer poly(3-hydroxybutyrate-co-3-hydroxyhexanoate-co-3-hydroxyoctanoate) with a recombinant Pseudomonas putida strain

A metagenomic library of 2.1 × 10⁶ clones was constructed using oil-contaminated soil from Gujarat (India). One of the fosmid clones, 40N22, encodes a polyhydroxyalkanoate synthase showing 76% identity with an Alcaligenes sp. synthase. The corresponding gene was expressed in Pseudomonas putida KT2440 ΔphaC1 which is impaired in PHA production. The gene conferred the recombinant strain PpKT-40N22 with the ability to produce copolymers with up to 21% in medium-chain-length content. Thus, 37% and 45% of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate-co-3-hydroxyoctanoate), respectively were obtained when using sodium heptanoate and oleic acid as carbon sources. These 3-hydroxybutyrate-(3HB)-based polymers are of interest since they incorporate the properties of medium chain length polymers and thus increase the range of applications of PHAs.     

A versatile Pseudomonas putida KT2440 with new ability: selective oxidation of 5-hydroxymethylfurfural to 5-hydroxymethyl-2-furancarboxylic acid

Currently, biotransformation of 5-hydroxymethylfurfural (HMF) into a series of high-value bio-based platform chemicals is massively studied. In this study, selective biooxidation of HMF to 5-hydroxymethyl-2-furancarboxylic acid (HMFCA) by Pseudomonas putida KT2440 with superior titer, yield, and productivity was reported. The biocatalytic performances of P. putida KT2440 were optimized separately. Under optimal conditions, 100% yield of HMFCA was obtained when HMF concentration was less than 150 mM, while the maximum concentration of 155 mM was achieved from 160 mM HMF in 12 h. P. putida KT2440 was highly tolerate to HMF, up to 190 mM. Besides, it was capable of selective oxidation of other furan aldehydes to the corresponding carboxylic acids with good yield of 100%. This study further demonstrates the potential of P. putida KT2440 as a biocatalyst for biomass conversion, as this strain has been proved the capacity to convert and utilize many kinds of biomass-derived sugars and ligin-derived aromatic compounds.

     

Efficient hydroxylation of 1,8-cineole with monoterpenoid-resistant recombinant <Emphasis Type="Italic">Pseudomonas putida</Emphasis> GS1

In this work, monoterpenoid hydroxylation with Pseudomonas putida GS1 and KT2440 were investigated as host strains, and the cytochrome P450 monooxygenase CYP176A1 (P450_cin) and its native redox partner cindoxin (CinC) from Citrobacter braakii were introduced in P. putida to catalyze the stereoselective hydroxylation of 1,8-cineole to (1R)-6β-hydroxy-1,8-cineole. Growth experiments in the presence of 1,8-cineole confirmed pseudomonads’ superior resilience compared to E. coli. Whole-cell P. putida harboring P450_cin with and without CinC were capable of hydroxylating 1,8-cineole, whereas coexpression of CinC has been shown to accelerate this bioconversion. Under the same conditions, P. putida GS1 produced more than twice the amount of heterologous P450_cin and bioconversion product than P. putida KT2440. A concentration of 1.1 ± 0.1 g/L (1R)-6β-hydroxy-1,8-cineole was obtained within 55 h in shake flasks and 13.3 ± 1.9 g/L in 89 h in a bioreactor, the latter of which corresponds to a yield Y_P/S of 79 %. To the authors’ knowledge, this is the highest product titer for a P450 based whole-cell monoterpene oxyfunctionalization reported so far. These results show that solvent-tolerant P. putida GS1 can be used as a highly efficient recombinant whole-cell biocatalyst for a P450 monooxygenase-based valorization of monoterpenoids.     

Involvement of Cyclopropane Fatty Acids in the Response of Pseudomonas putida KT2440 to Freeze-Drying

Pseudomonas putida KT2440, a saprophytic soil bacterium that colonizes the plant root, is a suitable microorganism for the removal of pollutants and a stable host for foreign genes used in biotransformation processes. Because of its potential use in agriculture and industry, we investigated the conditions for the optimal preservation of the strain and its derivatives for long-term storage. The highest survival rates were achieved with cells that had reached the stationary phase and which had been subjected to freeze-drying in the presence of disaccharides (trehalose, maltose, and lactose) as lyoprotectants. Using fluorescence polarization techniques, we show that cell membranes of KT2440 were more rigid in the stationary phase than in the exponential phase of growth. This is consistent with the fact that cells grown in the stationary phase exhibited a higher proportion of C_{17:cyclopropane} as a fatty acid than cells in the exponential phase. Mutants for the cfaB gene, which encodes the main C_{17:cyclopropane} synthase, and for the cfaA gene, which encodes a minor C_{17:cyclopropane} synthase, were constructed. These mutants were more sensitive to freeze-drying than wild-type cells, particularly the mutant with a knockout in the cfaB gene that produced less than 2% of the amount of C_{17:cyclopropane} produced by the parental strain.     

Biosynthesis of zeaxanthin in recombinant Pseudomonas putida

Pseudomonas putida KT2440 strain was investigated for biosynthesis of the valuable xanthophyll zeaxanthin. A new plasmid was constructed harboring five carotenogenic genes from Pantoea ananatis and three genes from Escherichia coli under control of an l-rhamnose-inducible promoter. Pseudomonas putida KT2440 wild type hardly tolerated the plasmids for carotenoid production. Mating experiments with E. coli S17-1 strains revealed that the carotenoid products are toxic to the Pseudomonas putida cells. Several carotenoid-tolerant transposon mutants could be isolated, and different gene targets for relief of carotenoid toxicity were identified. After optimization of cultivation conditions and product processing, 51 mg/l zeaxanthin could be produced, corresponding to a product yield of 7 mg zeaxanthin per gram cell dry weight. The effect of various additives on production of hydrophobic zeaxanthin was investigated as well. Particularly, the addition of lecithin during cell cultivation increased volumetric productivity of Pseudomonas putida by a factor of 4.7 (51 mg/l vs. 239 mg/l).     

A functional<Emphasis Type="Italic"> gltB</Emphasis> gene is essential for utilization of acidic amino acids and expression of periplasmic glutaminase/asparaginase (PGA) by<Emphasis Type="Italic"> Pseudomonas putida</Emphasis> KT2440

Pseudomonas putida KT2440, a root-colonizing fluorescent pseudomonad, is capable of utilizing acidic amino acids (Asp and Glu) and their amides (Asn and Gln) as its sole source of carbon and nitrogen. The uptake of Gln and Asn is facilitated by a periplasmic glutaminase/asparaginase (PGA), which hydrolyses Asn and Gln to the respective dicarboxylates. Here, we describe transposon mutagenesis of P. putida KT2440 with a self-cloning promoter probe vector, Tn5-OT182. Transconjugants defective in Glu-mediated PGA induction were selected for further studies. In most clones the transposon was found to have integrated into the gltB gene, which encodes the major subunit of the glutamate synthase (GOGAT). The transconjugants were nonmotile, no longer showed a chemotactic response towards amino acids, and could not survive prolonged periods of starvation. The acidic amino acids and their amides supported growth of the transconjugants only when supplied together with glucose, suggesting that the gltB-mutants had lost the ability to utilize amino acids as a carbon source. To confirm that gltB inactivation was the cause of this phenotype, we constructed a mutant with a targeted disruption of gltB. This strain behaved like the clones obtained by random mutagenesis, and failed to express not only PGA but also a number of other Glu-induced proteins. In contrast to wild-type cells, the gltB ^- strain accumulated considerable amounts of both Glu and Gln during long-term incubation.Communicated by A. Kondorosi     

Unravelling the thioesterases responsible for propionate formation in engineered Pseudomonas putida KT2440

Pseudomonas putida KT2440 is becoming a new robust metabolic chassis for biotechnological applications, due to its metabolic versatility, low nutritional requirements and biosafety status. We have previously engineered P. putida KT2440 to be an efficient propionate producer from L-threonine, although the internal enzymes converting propionyl-CoA to propionate are not clear. In this study, we thoroughly investigated 13 genes annotated as potential thioesterases in the KT2440 mutant. One thioesterase encoded by locus tag PP_4975 was verified to be the major contributor to propionate production in vivo. Deletion of PP_4975 significantly decreased propionate production, whereas the performance was fully restored by gene complement. Compared with thioesterase HiYciA from Haemophilus influenza, thioesterase PP_4975 showed a faster substrate conversion rate in vitro. Thus, this study expands our knowledge on acyl-CoA thioesterases in P. putida KT2440 and may also reveal a new target for further engineering the strain to improve propionate production performance.     

Enhancement of the Potential To Utilize Octopine in the Nonfluorescent Pseudomonas sp. Strain 92

The nonfluorescent Pseudomonas sp. strain 92 requires the presence of a supplementary carbon source for growth on octopine, whereas the spontaneous mutant RB100 has acquired the capacity to utilize this opine as the sole carbon and nitrogen source. Insertional mutagenesis of RB100 with transposon Tn5 generated mutants which were unable to grow on octopine and others which grew slowly on this substrate. Both types of mutants yielded revertants that had regained the ability to utilize octopine. Some of the revertants had lost the transposon, whereas in others the transposon was retained but with rearrangements of the insertion site. Genes of octopine catabolism from strain 92 were cloned on a cosmid vector to generate pK3. The clone pK3 conferred the ability to utilize octopine as the sole carbon and nitrogen source on the host Pseudomonas putida KT2440. Although they conferred an equivalent growth phenotype, the mutant genes carried by RB100 and the cloned genes on pK3 differed in their regulation. Utilization of [¹⁴C]octopine was inducible by octopine in RB100 and was constitutive in KT2440(pK3).     

Posttranslational modification of myxobacterial carrier protein domains in Pseudomonas sp. by an intrinsic phosphopantetheinyl transferase

We demonstrate the ability of Pseudomonas putida KT2440, Pseudomonas syringae pv. tomato DC3000 and Pseudomonas stutzeri DSM10701 to posttranslationally activate carrier protein (CP) domains of various polyketide synthases, nonribosomal peptide synthetases, and fatty acid synthase by their intrinsic phosphopantetheinyl transferase. The apo-form is modified to the holo-form of the CP by attaching a phosphopantetheine moiety from coenzymeA to a conserved serine residue. The coding regions of the respective domains were cloned in order to generate C-terminal fusions with intein-chitin. The constructs were subcloned into a broad host range vector and transferred into the three pseudomonad hosts. The resulting recombinant pseudomonad strains were cultivated and each fusion protein was purified by affinity chromatography. Each purified CP was analysed using MALDI/TOF for the expected mass increase. Of the seven CPs tested, six could be purified from P. putida, which was chosen as the general host strain. Out of the six domains, five were completely activated, whereas only 5% of the protein of the sixth domain was in holo-form. Four domains were also expressed in the other hosts.     

AI-2通过甲基化趋化受体McpU调控恶臭假单胞菌KT2440的趋化运动及生物膜形成

[背景]广泛存在于革兰氏阴性菌和革兰氏阳性菌中的自诱导物autoinducer-2 (AI-2)能够介导细菌种内和种间通讯,并调节细菌的多种生理过程.然而恶臭假单胞菌KT2440能否感知AI-2信号还未见报道.[目的]挖掘介导恶臭假单胞菌KT2440对AI-2趋化反应的趋化受体,检测AI-2信号通过趋化受体对恶臭假单胞...