Microbial conversion of glucose to a novel chemical building block, 2-pyrone-4,6-dicarboxylic acid

2-Pyrone-4,6-dicarboxylic acid (PDC) is a catabolic intermediate in Sphingobium sp. SYK-6 (previously characterized as Sphingomonas paucimobilis SYK-6), which is a degrader of lignin-derived aromatic compounds. Recently, PDC has been also characterized as a novel starting material for several potentially useful synthetic polymers. In a previous study, we constructed a biosynthetic system in which PDC was generated efficiently from a chemically synthesized compound, protocatechuate. In order to develop an alternative system for production of PDC, we tried to generate it from glucose, which is a low-cost sugar that can be obtained from abundant cellulosic wastes and biomass crops. We designed a metabolic bypass to PDC from the shikimate pathway in recombinant Escherichia coli cells. PDC accumulated in the medium of recombinant E. coli cells that had been transformed with genes isolated from Emericella niger, E. coli, Pseudomonas putida, and Sphingobium sp. SYK-6. The yield of PDC depended on the combination of genes that we introduced into the cells and on the specific of host strain. Under optimal conditions, the yield and titer of PDC were, respectively, 17.3% and 0.35 mg/l when the concentration of glucose was 2 g/l and the culture volume was 50 ml. Our results open up the possibility of novel utilization of biomass as the source of a useful chemical building block.     

Genetic and Biochemical Characterization of a 2-Pyrone-4,6-Dicarboxylic Acid Hydrolase Involved in the Protocatechuate 4,5-Cleavage Pathway of Sphingomonas paucimobilis SYK-6

Sphingomonas paucimobilis SYK-6 is able to grow on a wide variety of dimeric lignin compounds with guaiacyl moieties, which are converted into protocatechuate by the actions of lignin degradation enzymes in this strain. Protocatechuate is a key metabolite in the SYK-6 degradation of lignin compounds with guaiacyl moieties, and it is thought that it degrades to pyruvate and oxaloacetate via the protocatechuate 4,5-cleavage pathway. In a 10.5-kb EcoRI fragment carrying the protocatechuate 4,5-dioxygenase gene (ligAB) (Y. Noda, S. Nishikawa, K. Shiozuka, H. Kadokura, H. Nakajima, K. Yoda, Y. Katayama, N. Morohoshi, T. Haraguchi, and M. Yamasaki. J. Bacteriol. 172:2704–2709, 1990), we found the ligI gene encoding 2-pyrone-4,6-dicarboxylic acid (PDC) hydrolase. PDC hydrolase is a member of this pathway and catalyzes the interconversion between PDC and 4-carboxy-2-hydroxymuconic acid (CHM). The ligI gene is thought to be transcribed divergently from ligAB and consists of an 879-bp open reading frame encoding a polypeptide with a molecular mass of 32,737 Da. The ligI gene product (LigI), expressed in Escherichia coli, was purified to near-homogeneity and was estimated to be a monomer (31.6 kDa) by gel filtration chromatography. The isoelectric point was determined to be 4.9. The optimum pH for hydrolysis of PDC is 8.5, the optimum pH for synthesis of PDC is 6.0 to 7.5, and the K_m values for PDC and CHM are 74 and 49 μM, respectively. LigI activity was inhibited by the addition of thiol reagents, suggesting that the cysteine residue is a catalytic site. LigI is more resistant to metal ion inhibition than the PDC hydrolases of Pseudomonas ochraceae (K. Maruyama, J. Biochem. 93:557–565, 1983) and Comamonas testosteroni (P. J. Kersten, S. Dagley, J. W. Whittaker, D. M. Arciero, and J. D. Lipscomb, J. Bacteriol. 152:1154–1162, 1982). The insertional inactivation of the ligI gene in S. paucimobilis SYK-6 led to the complete loss of PDC hydrolase activity and to a growth defect on vanillic acid; it did not affect growth on syringic acid. These results indicate that the ligI gene is essential for the growth of SYK-6 on vanillic acid but is not responsible for the growth of SYK-6 on syringic acid.     

Degradation of 3-O-methylgallate in Sphingomonas paucimobilis SYK-6 by pathways involving protocatechuate 4,5-dioxygenase

Sphingomonas paucimobilis SYK-6 converts vanillate and syringate to protocatechuate and 3-O-methylgallate (3MGA), respectively. 3MGA is metabolized via multiple pathways involving 3MGA 3,4-dioxygenase, protocatechuate 4,5-dioxygenase (LigAB), and gallate dioxygenase whereas protocatechuate is degraded via the protocatechuate 4,5-cleavage pathway. Here the secondary role of LigAB in syringate metabolism is investigated. The reaction product of 3MGA catalyzed by His-tagged LigAB was identified as 4-carboxy-2-hydroxy-6-methoxy-6-oxohexa-2,4-dienoate (CHMOD) and 2-pyrone-4,6-dicarboxylate (PDC), indicating that 3MGA is transformed to CHMOD and PDC by both reactions catalyzed by DesZ and LigAB. Mutant analysis revealed that the 3MGA catabolic pathways involving LigAB are functional in SYK-6.     

Characterization of Sphingomonas paucimobilis SYK-6 genes involved in degradation of lignin-related compounds

Sphingomonas paucimobilis SYK-6 is able to grow on a wide variety of dimeric lignin compounds. These compounds are degraded via vanillate and syringate by a unique enzymatic system, composed of etherases, O demethylases, ring cleavage oxygenases and side chain cleaving enzymes. These unique and specific lignin modification enzymes are thought to be powerful tools for utilization of the most abundant aromatic biomass, lignin. Here, we focus on the genes and enzymes involved in β-aryl ether cleavage and biphenyl degradation. Two unique etherases are involved in the reductive cleavage of β-aryl ether. These two etherases have amino acid sequence similarity with the glutathione S-transferases, and use glutathione as a hydrogen donor. It was found that 5,5′-dehydrodivanillate, which is a typical lignin-related biphenyl structure, was transformed into 5-carboxyvanillate by the reaction sequence of O-demethylation, meta-ring cleavage, and hydrolysis, and the genes involved in the latter two reactions have been characterized. Vanillate and syringate are the most common intermediate metabolites in lignin catabolism. These compounds are initially O-demethylated and the resulting diol compounds, protocatechuate (PCA) and 3-O-methylgallate, respectively, are subjected to ring cleavage catalyzed by PCA 4,5-dioxygenase. The ring cleavage products generated are further degraded through the PCA 4,5-cleavage pathway. We have isolated and characterized genes for enzymes involved in this pathway. Disruption of a gene for 2-pyrone-4,6-dicarboxylate hydrolase (ligI) in this pathway suggested that an alternative route for 3-O-methylgallate degradation, in which ligI is not involved, would play a role in syringate catabolism. In this article, we describe the genetic and biochemical features of the S. paucimobilis SYK-6 genes involved in degradation of lignin-related compounds. A possible application of the SYK-6 lignin degradation system to produce a valuable chemical material is also described. Received 01 May 1999/ Accepted in revised form 29 July 1999     

Characterization of the Third Glutathione S-Transferase Gene Involved in Enantioselective Cleavage of the β-Aryl Ether by Sphingobium sp. Strain SYK-6

The glutathione S-transferases, LigF and LigE, of Sphingobium sp. strain SYK-6 respectively play a role in cleavage of the β-aryl ether of (+)-(βS)-α-(2-methoxyphenoxy)-β-hydroxypropiovanillone (MPHPV) and (?)-(βR)-MPHPV. The ligP gene, which showed 59% similarity to ligE at the amino acid level, was isolated from SYK-6. LigP produced in Escherichia coli revealed enantioselectivity for (?)-(βR)-MPHPV, and ligE and ligP alone contributed to the degradation of (?)-(βR)-MPHPV in SYK-6.     

In-planta production of the biodegradable polyester precursor 2-pyrone-4,6-dicarboxylic acid (PDC): Stacking reduced biomass recalcitrance with value-added co-product

2-Pyrone-4,6-dicarboxylic acid (PDC), a chemically stable intermediate that naturally occurs during microbial degradation of lignin by bacteria, represents a promising building block for diverse biomaterials and polyesters such as biodegradable plastics. The lack of a chemical synthesis method has hindered large-scale utilization of PDC and metabolic engineering approaches for its biosynthesis have recently emerged. In this study, we demonstrate a strategy for the production of PDC via manipulation of the shikimate pathway using plants as green factories. In tobacco leaves, we first showed that transient expression of bacterial feedback-resistant 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase (AroG) and 3-dehydroshikimate dehydratase (QsuB) produced high titers of protocatechuate (PCA), which was in turn efficiently converted into PDC upon co-expression of PCA 4,5-dioxygenase (PmdAB) and 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase (PmdC) derived from Comamonas testosteroni. We validated that stable expression of AroG in Arabidopsis in a genetic background containing the QsuB gene enhanced PCA content in plant biomass, presumably via an increase of the carbon flux through the shikimate pathway. Further, introducing AroG and the PDC biosynthetic genes (PmdA, PmdB, and PmdC) into the Arabidopsis QsuB background, or introducing the five genes (AroG, QsuB, PmdA, PmdB, and PmdC) stacked on a single construct into wild-type plants, resulted in PDC titers of ~1% and ~3% dry weight in plant biomass, respectively. Consistent with previous studies of plants expressing QsuB, all PDC producing lines showed strong reduction in lignin content in stems. This low lignin trait was accompanied with improvements of biomass saccharification efficiency due to reduced cell wall recalcitrance to enzymatic degradation. Importantly, most transgenic lines showed no reduction in biomass yields. Therefore, we conclude that engineering plants with the proposed de-novo PDC pathway provides an avenue to enrich biomass with a value-added co-product while simultaneously improving biomass quality for the supply of fermentable sugars. Implementing this strategy into bioenergy crops has the potential to support existing microbial fermentation approaches that exploit lignocellulosic biomass feedstocks for PDC production.     

QuiC2 represents a functionally distinct class of dehydroshikimate dehydratases identified in Listeria species including Listeria monocytogenes

Many Listeria species including L. monocytogenes contain the pathway for the biosynthesis of protocatechuate from shikimate and quinate. The qui1 and qui2 operons within these Listeria spp. encode enzymes for this pathway. The diversion of shikimate pathway intermediates in some Listeria species to produce protocatechuate suggests an important biological role for this compound to these organisms. A total of seven ORFs, including quiC2, were identified within qui1 and qui2, however only three proteins encoded by the operons have been functionally annotated. The final step in Listeria's protocatechuate biosynthesis involves the conversion of dehydroshikimate by a dehydroshikimate dehydratase (DSD). In this study, we demonstrate that QuiC2 functions as a DSD in Listeria spp. through biochemical and structural analyses. Moreover, we show that QuiC2 forms a phylogenetic cluster distinct from other functionally annotated DSDs. The individual phylogenetic clusters of DSD are represented by enzymes that produce protocatechuate for distinct biological processes. Similarly, QuiC2 is expected to produce protocatechuate for a novel biological process. We postulate that protocatechuate produced by DSDs found within the QuiC2 phylogenetic cluster provides an ecological niche for representative organisms.     

13C-NMR Analysis of Hydroxy-proline Arabinosides from Nicotiana tabacum

The vanillin dehydrogenase gene (ligV), which conferred the ability to transform vanillin into vanillate on Escherichia coli, was isolated from Sphingomonas paucimobilis SYK-6. The ligV gene consists of a 1,440-bp open reading frame encoding a polypeptide with a molecular mass of 50,301 Da. The deduced amino acid sequence of ligV showed about 50% identity with the known vanillin dehydrogenases of Pseudomonas vanillin degraders. The gene product of ligV (LigV) produced in E. coli preferred NAD⁺ to NADP⁺ and exhibited a broad substrate preference, including vanillin, benzaldehyde, protocatechualdehyde, m-anisaldehyde, and p-hydroxybenzaldehyde, but the activity toward syringaldehyde was less than 5% of that toward vanillin. Insertional inactivation of ligV in SYK-6 indicated that ligV is essential for normal growth on vanillin. On the other hand, growth on syringaldehyde was only slightly affected by ligV disruption, indicating the presence of a syringaldehyde dehydrogenase gene or genes in SYK-6.     

Cloning and Sequencing of the Sphingomonas (Pseudomonas) paucimobilis Gene Essential for the O Demethylation of Vanillate and Syringate

Sphingomonas (Pseudomonas) paucimobilis SYK-6 is able to grow on 5,5′-dehydrodivanillic acid (DDVA), syringate, vanillate, and other dimeric model compounds of lignin as a sole carbon source. Nitrosoguanidine mutagenesis of S. paucimobilis SYK-6 was performed, and two mutants with altered DDVA degradation pathways were isolated. The mutant strain NT-1 could not degrade DDVA, but could degrade syringate, vanillate, and 2,2′,3′-trihydroxy-3-methoxy-5,5′-dicarboxybiphenyl (OH-DDVA). Strain DC-49 could slowly assimilate DDVA, but could degrade neither vanillate nor syringate, although it could degrade protocatechuate and 3-O-methylgallate. A complementing DNA fragment of strain DC-49 was isolated from the cosmid library of strain SYK-6. The minimum DNA fragment complementing DC-49 was determined to be the 1.8-kbp insert of pKEX2.0. Sequencing analysis showed an open reading frame of 1,671 bp in this fragment, and a similarity search indicated that the deduced amino acid sequence of this open reading frame had significant similarity (60%) to the formyltetrahydrofolate synthetase of Clostridium thermoaceticum.     

Key enzymes of the protocatechuate branch of the β-ketoadipate pathway for aromatic degradation inCorynebacterium glutamicum

Although the protocatechuate branch of the β-ketoadipate pathway in Gram⁻ bacteria has been well studied, this branch is less understood in Gram⁺ bacteria. In this study,Corynebacterium glutamicum was cultivated with protocatechuate,p-cresol, vanillate and 4-hydroxybenzoate as sole carbon and energy sources for growth. Enzymatic assays indicated that growing cells on these aromatic compounds exhibited protocatechuate 3,4-dioxygenase activities. Data-mining of the genome of this bacterium revealed that the genetic locusncg12314-ncg12315 encoded a putative protocatechuate 3,4-dioxygenase. The genes,ncg12314 andncg12315, were amplified by PCR technique and were cloned into plasmid (pET21aP34D). RecombinantEscherichia coli strain harboring this plasmid actively expressed protocatechuate 3,4-dioxygenase activity. Further, when this locus was disrupted inC. glutamicum, the ability to degrade and assimilate protocatechuate,p-cresol, vanillate or 4-hydroxybenzoate was lost and protocatechuate 3,4-dioxygenase activity was disappeared. The ability to grow with these aromatic compounds and protocatechuate 3,4-dioxygenase activity ofC. glutamicum mutant could be restored by gene complementation. Thus, it is clear that the key enzyme for ring-cleavage, protocatechuate 3,4-dioxygenase, was encoded byncg12314 andncg12315. The additional genes involved in the protocatechuate branch of the β-ketoadipate pathway were identified by mining the genome data publically available in the Gen Bank. The functional identification of genes and their unique organization inC. glutamicum provided new insight into the genetic diversity of aromatic compound degradation.     

A Second 5-Carboxyvanillate Decarboxylase Gene, ligW2, Is Important for Lignin-Related Biphenyl Catabolism in Sphingomonas paucimobilis SYK-6

A lignin-related biphenyl compound, 5,5′-dehydrodivanillate (DDVA), is degraded to 5-carboxyvanillate (5CVA) by the enzyme reactions catalyzed by DDVA O-demethylase (LigX), meta-cleavage oxygenase (LigZ), and meta-cleavage compound hydrolase (LigY) in Sphingomonas paucimobilis SYK-6. 5CVA is then transformed to vanillate by a nonoxidative 5CVA decarboxylase and is further degraded through the protocatechuate 4,5-cleavage pathway. A 5CVA decarboxylase gene, ligW, was isolated from SYK-6 (X. Peng, E. Masai, H. Kitayama, K. Harada, Y, Katayama, and M. Fukuda, Appl. Environ. Microbiol. 68:4407-4415, 2002). However, disruption of ligW slightly affected the 5CVA decarboxylase activity and the growth rate on DDVA of the mutant, suggesting the presence of an alternative 5CVA decarboxylase gene. Here we isolated a second 5CVA decarboxylase gene, ligW2, which consists of a 1,050-bp open reading frame encoding a polypeptide with a molecular mass of 39,379 Da. The deduced amino acid sequence encoded by ligW2 exhibits 37% identity with the sequence encoded by ligW. Based on a gas chromatography-mass spectrometry analysis of the reaction product from 5CVA catalyzed by LigW2 in the presence of deuterium oxide, LigW2 was indicated to be a nonoxidative decarboxylase of 5CVA, like LigW. After disruption of ligW2, both the growth rate on DDVA and the 5CVA decarboxylase activity of the mutant were decreased to approximately 30% of the wild-type levels. The ligW ligW2 double mutant lost both the ability to grow on DDVA and the 5CVA decarboxylase activity. These results indicate that both ligW and ligW2 contribute to 5CVA degradation, although ligW2 plays the more important role in the growth of SYK-6 cells on DDVA.     

Shuttle vectors for hyperthermophilic archaea

Progress in understanding the basic molecular, biochemical, and physiological characteristics of archaeal hyperthermophiles has been limited by the lack of suitable expression vectors. Here, we report the construction of versatile shuttle vectors that can be maintained, and selected for, in both archaea and bacteria. The primary construct, pAG1, was produced by ligating portions of the archaeal cryptic plasmid pGT5 and the bacterial plasmid pUC19, both of which exhibit high copy numbers. A second vector construct, pAG2, was generated, with a reduced copy number in Escherichia coli, by introducing the Rom/Rop gene from pBR322 into pAG1. After transformation, both pAG1 and pAG2 were stably maintained and propagated in the euryarchaeote Pyrococcus furiosus, the crenarchaeote Sulfolobus acidocaldarius, and in Escherichia coli. An archaeal selective marker, the alcohol dehydrogenase gene from Sulfolobus solfataricus, was isolated by polymerase chain reaction (PCR) amplification and cloned into the two constructs. They were stably maintained and expressed in the two archaea and conferred resistance to butanol and benzyl alcohol. However, the vector pAG21, deriving from pAG2, proved the more stable in E. coli probably due to its lower copy number in the bacterium. Conditions are presented for the use of the vectors which, potentially, can be used for other hyperthermophilic archaea. Received: January 12, 1997 / Accepted: May 29, 1997     

Effect of the pem system on stable maintenance of plasmid R100 in various Escherichia coli hosts

Summary We cloned the pem segment of plasmid R100 containing the two genes pemI and pemK, which are responsible for stable maintenance of R100 in dividing cells, into pHS1, a temperature-sensitive replication mutant of plasmid pSC101. We then examined the effect of the pem system on the maintenance of the resultant pem ⁺ plasmid pDOM17 in various Escherichia coli host strains upon inhibition of replication of the plasmid at a high temperature. We show that the pem ⁺ plasmid was maintained stably in the cell population and efficiently in the two hosts, km1213 (polA ^ts) and KP64 (recA), but less efficiently in others, such as W3110, C600, P3478 (polA), and SH2743 (sfiA sfiC); the rate of cell growth was reduced at or after the time when the copy number of pDOM17 was supposed to be 0 in all of the hosts examined. We also show that a large fraction of the non-viable pDOM17-free segregant cells was produced in the former two hosts, while a smaller fraction of such cells was produced in the latter hosts, in which cell division was inhibited for several generations. Based on these results and other observations, we point out that the pemK gene product has the function not to kill the plasmid-free segregant cells, but primarily to inhibit division of these segregants. Inhibition of cell division secondarily leads to death of the plasmid-free segregants very efficiently in the two particular hosts, resulting in an apparently more stable maintenance of the pem ⁺ plasmid in these two hosts than in others.     

Complete genome sequence of Sphingobium sp. strain SYK-6, a degrader of lignin-derived biaryls and monoaryls

Sphingobium sp. strain SYK-6 is able to grow on an extensive variety of lignin-derived biaryls and monoaryls, and the catabolic genes for these compounds are useful for the production of industrially valuable metabolites from lignin. Here we report the complete nucleotide sequence of the SYK-6 genome which consists of the 4,199,332-bp-long chromosome and the 148,801-bp-long plasmid.     

Increased pyruvate efficiency in enzymatic production of (R)-phenylacetylcarbinol

Loss of substrate, pyruvate, a limitation for enzymatic batch production of (R)-phenylacetylcarbinol (PAC), resulted from two phenomena: temperature dependent non-enzymatic concentration decrease due to the cofactor Mg²⁺ and formation of by-products, acetaldehyde and acetoin, by pyruvate decarboxylase (PDC). In the absence of enzyme, pyruvate stabilization was achieved by lowering the Mg²⁺ concentration from 20 to 0.5 mM. With 0.5 mM Mg²⁺ Rhizopus javanicus and Candida utilis PDC produced similar levels of PAC (49 and 51 g l^–1, respectively) in 21 h at 6 °C; however C. utilis PDC formed less by-product from pyruvate and was more stable during biotransformation. The process enhancements regarding Mg²⁺ concentration and source of PDC resulted in an increase of molar yield (PAC/consumed pyruvate) from 59% (R. javanicus PDC, 20 mM Mg²⁺) to 74% (R. javanicus PDC, 0.5 mM Mg²⁺) to 89% (C. utilis PDC, 0.5 mM Mg²⁺).     

Characterization of the gallate dioxygenase gene: three distinct ring cleavage dioxygenases are involved in syringate degradation by Sphingomonas paucimobilis SYK-6

Sphingomonas paucimobilis SYK-6 converts vanillate and syringate to protocatechuate (PCA) and 3-O-methylgallate (3MGA) in reactions with the tetrahydrofolate-dependent O-demethylases LigM and DesA, respectively. PCA is further degraded via the PCA 4,5-cleavage pathway, whereas 3MGA is metabolized via three distinct pathways in which PCA 4,5-dioxygenase (LigAB), 3MGA 3,4-dioxygenase (DesZ), and 3MGA O-demethylase (LigM) are involved. In the 3MGA O-demethylation pathway, LigM converts 3MGA to gallate, and the resulting gallate appears to be degraded by a dioxygenase other than LigAB or DesZ. Here, we isolated the gallate dioxygenase gene, desB, which encodes a 418-amino-acid protein with a molecular mass of 46,843 Da. The amino acid sequences of the N-terminal region (residues 1 to 285) and the C-terminal region (residues 286 to 418) of DesB exhibited ca. 40% and 27% identity with the sequences of the PCA 4,5-dioxygenase beta and alpha subunits, respectively. DesB produced in Escherichia coli was purified and was estimated to be a homodimer (86 kDa). DesB specifically attacked gallate to generate 4-oxalomesaconate as the reaction product. The K(m) for gallate and the V(max) were determined to be 66.9 +/- 9.3 microM and 42.7 +/- 2.4 U/mg, respectively. On the basis of the analysis of various SYK-6 mutants lacking the genes involved in syringate degradation, we concluded that (i) all of the three-ring cleavage dioxygenases are involved in syringate catabolism, (ii) the pathway involving LigM and DesB plays an especially important role in the growth of SYK-6 on syringate, and (iii) DesB and LigAB are involved in gallate degradation.     

Crystal structure of an aromatic ring opening dioxygenase LigAB, a protocatechuate 4,5-dioxygenase, under aerobic conditions.

BACKGROUND: Sphingomonas paucimobilis SYK-6 utilizes an extradiol-type catecholic dioxygenase, the LigAB enzyme (a protocatechuate 4,5-dioxygenase), to oxidize protocatechuate (or 3,4-dihydroxybenzoic acid, PCA). The enzyme belongs to the family of class III extradiol-type catecholic dioxygenases catalyzing the ring-opening reaction of protocatechuate and related compounds. The primary structure of LigAB suggests that the enzyme has no evolutionary relationship with the family of class II extradiol-type catecholic dioxygenases. Both the class II and class III enzymes utilize a non-heme ferrous center for adding dioxygen to the substrate. By elucidating the structure of LigAB, we aimed to provide a structural basis for discussing the function of class III enzymes. RESULTS: The crystal structure of substrate-free LigAB was solved at 2.2 A resolution. The molecule is an alpha2beta2 tetramer. The active site contains a non-heme iron coordinated by His12, His61, Glu242, and a water molecule located in a deep cleft of the beta subunit, which is covered by the alpha subunit. Because of the apparent oxidation of the Fe ion into the nonphysiological Fe(III) state, we could also solve the structure of LigAB complexed with a substrate, PCA. The iron coordination sphere in this complex is a distorted tetragonal bipyramid with one ligand missing, which is presumed to be the O2-binding site. CONCLUSIONS: The structure of LigAB is completely different from those of the class II extradiol-type dioxygenases exemplified by the BphC enzyme, a 2,3-dihydroxybiphenyl 1,2-dioxygenase from a Pseudomonas species. Thus, as already implicated by the primary structures, no evolutionary relationship exists between the class II and III enzymes. However, the two classes of enzymes share many geometrical characteristics with respect to the nature of the iron coordination sphere and the position of a putative catalytic base, strongly suggesting a common catalytic mechanism.     

Genome analysis of the metabolically versatile Pseudomonas umsongensis GO16: the genetic basis for PET monomer upcycling into polyhydroxyalkanoates

The throwaway culture related to the single-use materials such as polyethylene terephthalate (PET) has created a major environmental concern. Recycling of PET waste into biodegradable plastic polyhydroxyalkanoate (PHA) creates an opportunity to improve resource efficiency and contribute to a circular economy. We sequenced the genome of Pseudomonas umsongensis GO16 previously shown to convert PET-derived terephthalic acid (TA) into PHA and performed an in-depth genome analysis. GO16 can degrade a range of aromatic substrates in addition to TA, due to the presence of a catabolic plasmid pENK22. The genetic complement required for the degradation of TA via protocatechuate was identified and its functionality was confirmed by transferring the tph operon into Pseudomonas putida KT2440, which is unable to utilize TA naturally. We also identified the genes involved in ethylene glycol (EG) metabolism, the second PET monomer, and validated the capacity of GO16 to use EG as a sole source of carbon and energy. Moreover, GO16 possesses genes for the synthesis of both medium and short chain length PHA and we have demonstrated the capacity of the strain to convert mixed TA and EG into PHA. The metabolic versatility of GO16 highlights the potential of this organism for biotransformations using PET waste as a feedstock.     

Mineralization of 4-sulfophthalate by aPseudomonas strain isolated from the River Elbe

The bacteriumPseudomonas sp. strain RW31 isolated from the river Elbe utilized the ammonium salt of 4-sulfophthalate (4SPA) as sole source of carbon, sulfur, nitrogen, and energy and grew also with phthalate (PA) and several other aromatic compounds as sole carbon and energy source. The xenobiotic sulfo group of 4SPA was eliminated as sulfite, which transiently accumulated in the culture supernatant up to about 10 µM and was slowly oxidized to the stoichiometrical amount of sulfate. Biodegradation routes of 4SPA as well as of PA converged into the protocatechuate pathway and from found activities for the decarboxylation of 4,5-dihydroxyphthalate we deduce this compound the first rearomaticized intermediate after initial dioxygenation. Protocatechuate then underwentmeta-cleavage mediated by a protocatechuate 4,5-dioxygenase activity which was competitively inhibited by the structurally related compound 3,4,5-trihydroxybenzoate; protocatechuate accumulated in the medium up to an about 2 mM concentration. Indications for the presence of selective transport systems are presented.