Engineered Pseudomonas putida KT2440 co-utilizes galactose and glucose.
ABSTRACT: Background:Efficient conversion of plant biomass to commodity chemicals is an important challenge that needs to be solved to enable a sustainable bioeconomy. Deconstruction of biomass to sugars and lignin yields a wide variety of low molecular weight carbon substrates that need to be funneled to product. Pseudomonas putida KT2440 has emerged as a potential platform for bioconversion of lignin and the other components of plant biomass. However, P. putida is unable to natively utilize several of the common sugars in hydrolysate streams, including galactose. Results:In this work, we integrated a De Ley-Doudoroff catabolic pathway for galactose catabolism into the chromosome of P. putida KT2440, using genes from several different organisms. We found that the galactonate catabolic pathway alone (DgoKAD) supported slow growth of P. putida on galactose. Further integration of genes to convert galactose to galactonate and to optimize the transporter expression level resulted in a growth rate of 0.371 h-1. Additionally, the best-performing strain was demonstrated to co-utilize galactose with glucose. Conclusions:We have engineered P. putida to catabolize galactose, which will allow future engineered strains to convert more plant biomass carbon to products of interest. Further, by demonstrating co-utilization of glucose and galactose, continuous bioconversion processes for mixed sugar streams are now possible.
Project description:Lignin is an energy-dense, heterogeneous polymer comprised of phenylpropanoid monomers used by plants for structure, water transport, and defense, and it is the second most abundant biopolymer on Earth after cellulose. In production of fuels and chemicals from biomass, lignin is typically underused as a feedstock and burned for process heat because its inherent heterogeneity and recalcitrance make it difficult to selectively valorize. In nature, however, some organisms have evolved metabolic pathways that enable the utilization of lignin-derived aromatic molecules as carbon sources. Aromatic catabolism typically occurs via upper pathways that act as a "biological funnel" to convert heterogeneous substrates to central intermediates, such as protocatechuate or catechol. These intermediates undergo ring cleavage and are further converted via the ?-ketoadipate pathway to central carbon metabolism. Here, we use a natural aromatic-catabolizing organism, Pseudomonas putida KT2440, to demonstrate that these aromatic metabolic pathways can be used to convert both aromatic model compounds and heterogeneous, lignin-enriched streams derived from pilot-scale biomass pretreatment into medium chain-length polyhydroxyalkanoates (mcl-PHAs). mcl-PHAs were then isolated from the cells and demonstrated to be similar in physicochemical properties to conventional carbohydrate-derived mcl-PHAs, which have applications as bioplastics. In a further demonstration of their utility, mcl-PHAs were catalytically converted to both chemical precursors and fuel-range hydrocarbons. Overall, this work demonstrates that the use of aromatic catabolic pathways enables an approach to valorize lignin by overcoming its inherent heterogeneity to produce fuels, chemicals, and materials.
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.
Project description:Common strategies for conversion of lignocellulosic biomass to chemical products center on deconstructing biomass polymers into fermentable sugars. Here, we demonstrate an alternative strategy, a growth-coupled, high-yield bioconversion, by feeding cells a non-sugar substrate, by-passing central metabolism, and linking a key metabolic step to generation of acetyl-CoA that is required for biomass and energy generation. Specifically, we converted levulinic acid (LA), an established degradation product of lignocellulosic biomass, to butanone (a.k.a. methyl-ethyl ketone - MEK), a widely used industrial solvent. Our strategy combines a catabolic pathway from Pseudomonas putida that enables conversion of LA to 3-ketovaleryl-CoA, a CoA transferase that generates 3-ketovalerate and acetyl-CoA, and a decarboxylase that generates 2-butanone. By removing the ability of E. coli to consume LA and supplying excess acetate as a carbon source, we built a strain of E. coli that could convert LA to butanone at high yields, but at the cost of significant acetate consumption. Using flux balance analysis as a guide, we built a strain of E. coli that linked acetate assimilation to production of butanone. This strain was capable of complete bioconversion of LA to butanone with a reduced acetate requirement and increased specific productivity. To demonstrate the bioconversion on real world feedstocks, we produced LA from furfuryl alcohol, a compound readily obtained from biomass. These LA feedstocks were found to contain inhibitors that prevented cell growth and bioconversion of LA to butanone. We used a combination of column chromatography and activated carbon to remove the toxic compounds from the feedstock, resulting in LA that could be completely converted to butanone. This work motivates continued collaboration between chemical and biological catalysis researchers to explore alternative conversion pathways and the technical hurdles that prevent their rapid deployment.
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.
Project description:In this study, we investigated the metabolism of ethylene glycol in the Pseudomonas putida strains KT2440 and JM37 by employing growth and bioconversion experiments, directed mutagenesis, and proteome analysis. We found that strain JM37 grew rapidly with ethylene glycol as a sole source of carbon and energy, while strain KT2440 did not grow within 2 days of incubation under the same conditions. However, bioconversion experiments revealed metabolism of ethylene glycol by both strains, with the temporal accumulation of glycolic acid and glyoxylic acid for strain KT2440. This accumulation was further increased by targeted mutagenesis. The key enzymes and specific differences between the two strains were identified by comparative proteomics. In P. putida JM37, tartronate semialdehyde synthase (Gcl), malate synthase (GlcB), and isocitrate lyase (AceA) were found to be induced in the presence of ethylene glycol or glyoxylic acid. Under the same conditions, strain KT2440 showed induction of AceA only. Despite this difference, the two strains were found to use similar periplasmic dehydrogenases for the initial oxidation step of ethylene glycol, namely, the two redundant pyrroloquinoline quinone (PQQ)-dependent enzymes PedE and PedH. From these results we constructed a new pathway for the metabolism of ethylene glycol in P. putida. Furthermore, we conclude that Pseudomonas putida might serve as a useful platform from which to establish a whole-cell biocatalyst for the production of glyoxylic acid from ethylene glycol.
Project description:Upgrading of furanic aldehydes to their corresponding furancarboxylic acids has received considerable interest recently. Herein we reported selective oxidation of furfural (FAL) to furoic acid (FA) with quantitative yield using whole-cells of Pseudomonas putida KT2440. The biocatalytic capacity could be substantially promoted through adding 5-hydroxymethylfurfural into media at the middle exponential growth phase. The reaction pH and cell dosage had notable impacts on both FA titer and selectivity. Based on the validation of key factors for FAL conversion, the capacity of P. putida KT2440 to produce FAL was substantially improved. In batch bioconversion, 170 mM FA was produced with selectivity nearly 100% in 2 h, whereas 204 mM FA was produced with selectivity above 97% in 3 h in fed-batch bioconversion. Particularly, the role of molybdate transporter in oxidation of FAL and 5-hydroxymethylfurfural was demonstrated for the first time. The furancarboxylic acids synthesis was repressed markedly by destroying molybdate transporter, which implied Mo-dependent enzyme/molybdoenzyme played pivotal role in such oxidation reactions. This research further highlights the potential of P. putida KT2440 as next generation industrial workhorse and provides a novel understanding of molybdoenzyme in oxidation of furanic aldehydes.
Project description: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, beta-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.
Project description:Microbial degradation of lignin and its related aromatic compounds has great potential for the sustainable production of chemicals and bioremediation of contaminated soils. We previously isolated Pseudomonas sp. strain 9.1 from historical waste deposits (forming so-called fiber banks) released from pulp and paper mills along the Baltic Sea coast. The strain accumulated vanillyl alcohol during growth on vanillin, and while reported in other microbes, this phenotype is less common in wild-type pseudomonads. As the reduction of vanillin to vanillyl alcohol is an undesired trait in Pseudomonas strains engineered to accumulate vanillin, connecting the strain 9.1 phenotype with a genotype would increase the fundamental understanding and genetic engineering potential of microbial vanillin metabolism. The genome of Pseudomonas sp. 9.1 was sequenced and assembled. Annotation identified oxidoreductases with homology to Saccharomyces cerevisiae alcohol dehydrogenase ScADH6p, known to reduce vanillin to vanillyl alcohol, in both the 9.1 genome and the model strain Pseudomonas putida KT2440. Recombinant expression of the Pseudomonas sp. 9.1 FEZ21_09870 and P. putida KT2440 PP_2426 (calA) genes in Escherichia coli revealed that these open reading frames encode aldehyde reductases that convert vanillin to vanillyl alcohol, and that P. putida KT2440 PP_3839 encodes a coniferyl alcohol dehydrogenase that oxidizes coniferyl alcohol to coniferyl aldehyde (i.e., the function previously assigned to calA). The deletion of PP_2426 in P. putida GN442 engineered to accumulate vanillin resulted in a decrease in by-product (vanillyl alcohol) yield from 17% to ?1%. Based on these results, we propose the reannotation of PP_2426 and FEZ21_09870 as areA and PP_3839 as calA-II IMPORTANCE Valorization of lignocellulose (nonedible plant matter) is of key interest for the sustainable production of chemicals from renewable resources. Lignin, one of the main constituents of lignocellulose, is a heterogeneous aromatic biopolymer that can be chemically depolymerized into a heterogeneous mixture of aromatic building blocks; those can be further converted by certain microbes into value-added aromatic chemicals, e.g., the flavoring agent vanillin. We previously isolated a Pseudomonas sp. strain with the (for the genus) unusual trait of vanillyl alcohol production during growth on vanillin. Whole-genome sequencing of the isolate led to the identification of a vanillin reductase candidate gene whose deletion in a recombinant vanillin-accumulating P. putida strain almost completely alleviated the undesired vanillyl alcohol by-product yield. These results represent an important step toward biotechnological production of vanillin from lignin using bacterial cell factories.
Project description:BACKGROUND:Marine macroalgae Gelidium amansii is a promising feedstock for production of sustainable biochemicals to replace petroleum and edible biomass. Different from terrestrial lignocellulosic biomass, G. amansii is comprised of high carbohydrate content and has no lignin. In previous studies, G. amansii biomass has been exploited to obtain fermentable sugars along with suppressing 5-hydroxymethylfurfural (HMF) formation for bioethanol production. In this study, a different strategy was addressed and verified for dual production of D-galactose and HMF, which were subsequently oxidized to D-galactonic acid and 5-hydroxymethyl-2-furancarboxylic acid (HMFCA) respectively via Pseudomonas putida. RESULTS:G. amansii biomass was hydrolyzed by dilute acid to form D-galactose and HMF. The best result was attained after pretreatment with 2% (w/w) HCl at 120 °C for 40 min. Five different Pseudomonas sp. strains including P. putida ATCC 47054, P. fragi ATCC 4973, P. stutzeri CICC 10402, P. rhodesiae CICC 21960, and P. aeruginosa CGMCC 1.10712, were screened for highly selective oxidation of D-galactose and HMF. Among them, P. putida ATCC 47054 was the outstanding suitable biocatalyst converting D-galactose and HMF to the corresponding acids without reduced or over-oxidized products. It was plausible that the pyrroloquinoline quinone-dependent glucose dehydrogenase and undiscovered molybdate-dependent enzyme(s) in P. putida ATCC 47054 individually played pivotal role for D-galactose and HMF oxidation. Taking advantage of its excellent efficiency and high selectivity, a maximum of 55.30 g/L D-galactonic acid and 11.09 g/L HMFCA were obtained with yields of 91.1% and 98.7% using G. amansii hydrolysates as substrate. CONCLUSIONS:Valorization of G. amansii biomass for dual production of D-galactonic acid and HMFCA can enrich the product varieties and improve the economic benefits. This study also demonstrates the perspective of making full use of marine feedstocks to produce other value-added products.
Project description:Levulinic acid (LA) is a building block alternative to fermentable sugars derived from cellulosic biomass. Among LA catabolic processes in Pseudomonas putida KT2440, ligation of coenzyme A (CoA) to LA by levulinyl-CoA synthetase (LvaE) is known to be an initial enzymatic step in LA metabolism. To identify the genes involved in the first step of LA metabolism in Pseudomonas citronellolis LA18T, RNA-seq-based comparative transcriptome analysis was carried out for LA18T cells during growth on LA and pyruvic acid. The two most highly upregulated genes with LA exhibited amino acid sequence homologies to cation acetate symporter and 5-aminolevulinic acid dehydratase from Pseudomonas spp. Potential LA metabolic genes (lva genes) in LA18T that clustered with these two genes and were homologous to lva genes in KT2440 were identified, including lvaE2 of LA18T, which exhibited 35% identity with lvaE of KT2440. Using Escherichia coli cells with the pCold™ expression system, LvaE2 was produced and investigated for its activity toward LA. High performance liquid chromatography analysis confirmed that crude extracts of E. coli cells expressing the lvaE2 gene could convert LA to levulinyl-CoA in the presence of both HS-CoA and ATP. Phylogenetic analysis revealed that LvaE2 and LvaE formed a cluster with medium-chain fatty acid CoA synthetase, but they fell on different branches. Superimposition of LvaE2 and LvaE homology-based model structures suggested that LvaE2 had a larger tunnel for accepting fatty acid substrates than LvaE. These results indicate that LvaE2 is a novel levulinyl-CoA synthetase.