Novel reaction of succinyl coenzyme A (Succinyl-CoA) synthetase: activation of 3-sulfinopropionate to 3-sulfinopropionyl-CoA in Advenella mimigardefordensis strain DPN7T during degradation of 3,3'-dithiodipropionic acid

The sucCD gene of Advenella mimigardefordensis strain DPN7^T encodes a succinyl coenzyme A (succinyl-CoA) synthetase homologue (EC 6.2.1.4 or EC 6.2.1.5) that recognizes, in addition to succinate, the structural analogues 3-sulfinopropionate (3SP) and itaconate as substrates. Accumulation of 3SP during 3,3′-dithiodipropionic acid (DTDP) degradation was observed in Tn5::mob-induced mutants of A. mimigardefordensis strain DPN7^T disrupted in sucCD and in the defined deletion mutant A. mimigardefordensis ΔsucCD. These mutants were impaired in growth with DTDP and 3SP as the sole carbon source. Hence, it was proposed that the succinyl-CoA synthetase homologue in A. mimigardefordensis strain DPN7^T activates 3SP to the corresponding CoA-thioester (3SP-CoA). The putative genes coding for A. mimigardefordensis succinyl-CoA synthetase (SucCD_Am) were cloned and heterologously expressed in Escherichia coli BL21(DE3)/pLysS. Purification and characterization of the enzyme confirmed its involvement during degradation of DTDP. 3SP, the cleavage product of DTDP, was converted into 3SP-CoA by the purified enzyme, as demonstrated by in vitro enzyme assays. The structure of 3SP-CoA was verified by using liquid chromatography-electrospray ionization-mass spectrometry. SucCD_Am is Mg²⁺ or Mn²⁺ dependent and unspecific regarding ATP or GTP. In kinetic studies the enzyme showed highest enzyme activity and substrate affinity with succinate (V_max = 9.85 ± 0.14 μmol min⁻¹ mg⁻¹, K_m = 0.143 ± 0.001 mM). In comparison to succinate, activity with 3SP was only ca. 1.2% (V_max = 0.12 ± 0.01 μmol min⁻¹ mg⁻¹) and the affinity was 6-fold lower (K_m = 0.818 ± 0.046 mM). Based on the present results, we conclude that SucCD_Am is physiologically associated with the citric acid cycle but is mandatory for the catabolic pathway of DTDP and its degradation intermediate 3SP.     

Succinyl-CoA:3-Sulfinopropionate CoA-Transferase from Variovorax paradoxus Strain TBEA6, a Novel Member of the Class III Coenzyme A (CoA)-Transferase Family

The act gene of Variovorax paradoxus TBEA6 encodes a succinyl-CoA:3-sulfinopropionate coenzyme A (CoA)-transferase, Act_TBEA6 (2.8.3.x), which catalyzes the activation of 3-sulfinopropionate (3SP), an intermediate during 3,3′-thiodipropionate (TDP) degradation. In a previous study, accumulation of 3SP was observed in a Tn5::mob-induced mutant defective in growth on TDP. In contrast to the wild type and all other obtained mutants, this mutant showed no growth when 3SP was applied as the sole source of carbon and energy. The transposon Tn5::mob was inserted in a gene showing high homology to class III CoA-transferases. In the present study, analyses of the translation product clearly allocated Act_TBEA6 to this protein family. The predicted secondary structure indicates the lack of a C-terminal α-helix. Act_TBEA6 was heterologously expressed in Escherichia coli Lemo21(DE3) and was then purified by Ni-nitrilotriacetic acid (NTA) affinity chromatography. Analytical size exclusion chromatography revealed a homodimeric structure with a molecular mass of 96 ± 3 kDa. Enzyme assays identified succinyl-CoA, itaconyl-CoA, and glutaryl-CoA as potential CoA donors and unequivocally verified the conversion of 3SP to 3SP-CoA. Kinetic studies revealed an apparent V_max of 44.6 μmol min⁻¹ mg⁻¹ for succinyl-CoA, which corresponds to a turnover number of 36.0 s⁻¹ per subunit of Act_TBEA6. For 3SP, the apparent V_max was determined as 46.8 μmol min⁻¹ mg⁻¹, which corresponds to a turnover number of 37.7 s⁻¹ per subunit of Act_TBEA6. The apparent K_m values were 0.08 mM for succinyl-CoA and 5.9 mM for 3SP. Nonetheless, the V. paradoxus Δact mutant did not reproduce the phenotype of the Tn5::mob-induced mutant. This defined deletion mutant was able to utilize TDP or 3SP as the sole carbon source, like the wild type. Complementation of the Tn5::mob-induced mutant with pBBR1MCS5::acd_DPN7 partially restored growth on 3SP, which indicated a polar effect of the Tn5::mob transposon on acd_TBEA6, located downstream of act_TBEA6.     

Dihydrolipoamide Dehydrogenases of Advenella mimigardefordensis and Ralstonia eutropha Catalyze Cleavage of 3,3′-Dithiodipropionic Acid into 3-Mercaptopropionic Acid

The catabolism of the disulfide 3,3′-dithiodipropionic acid (DTDP) is initiated by the reduction of its disulfide bond. Three independent Tn5::mob-induced mutants of Advenella mimigardefordensis strain DPN7^T were isolated that had lost the ability to utilize DTDP as the sole source of carbon and energy and that harbored the transposon insertions in three different sites of the same dihydrolipoamide dehydrogenase gene encoding the E3 subunit of the pyruvate dehydrogenase multi-enzyme complex of this bacterium (LpdA_Am). LpdA_Am was analyzed in silico and compared to homologous proteins, thereby revealing high similarities to the orthologue in Ralstonia eutropha H16 (PdhL_Re). Both bacteria are able to cleave DTDP into two molecules of 3-mercaptopropionic acid (3MP). A. mimigardefordensis DPN7^T converted 3MP to 3-sulfinopropionic acid, whereas R. eutropha H16 showed no growth with DTDP as the sole carbon source but was instead capable of synthesizing heteropolythioesters using the resulting cleavage product 3MP. Subsequently, the genes lpdA_Am and pdhL_Re were cloned, heterologously expressed in Escherichia coli applying the pET23a expression system, purified, and assayed by monitoring the oxidation of NADH. The physiological substrate lipoamide was reduced to dihydrolipoamide with specific activities of 1,833 mkat/kg of protein (LpdA_Am) or 1,667 mkat/kg of protein (PdhL_Re). Reduction of DTDP was also unequivocally detected with the purified enzymes, although the specific enzyme activities were much lower: 0.7 and 0.5 mkat/kg protein, respectively.In Advenella mimigardefordensis strain DPN7^T (15, 42), three independent mutants with an insertion of Tn5::mob in the lpdA gene coding for the E3 component of the pyruvate dehydrogenase multi-enzyme complex revealed an interesting phenotype: these mutants were fully impaired in utilizing 3,3′-dithiodipropionic acid (DTDP) as the sole carbon and energy source, whereas the growth on no other tested carbon sources was affected (41). Our main interest in the catabolism of DTDP is to unravel the pathway and to identify the involved enzymes. Furthermore, the application of this disulfide as precursor substrate for biotechnological production of polythioesters (PTE) (22) is of interest. Since poly(3-mercaptopropionate) (PMP) biosynthesis depends hitherto on supplying the harmful thiol 3-mercaptopropionic acid (3MP) (35), an improvement of the recombinant Escherichia coli system by heterologous expression of enzymes capable of cleaving the less toxic DTDP symmetrically into two molecules of 3MP, which are then polymerized, could be an important achievement toward large-scale biotechnological production of PMP.Two different enzyme systems catalyzing the conversion of disulfides into the corresponding thiols are already known and have been described in detail. (i) Enzymes belonging to the well-characterized family of pyridine-nucleotide disulfide oxidoreductases (25) contain a redox center formed by a disulfide bridge coupled to a flavin ring. They catalyze a simultaneous two-electron transfer via the enzymatic active disulfides associated with the pyridine nucleotides and flavin, toward the substrate (39, 40). (ii) An alternative disulfide reduction is catalyzed by enzymes using iron-sulfur clusters to cleave of disulfide substrates in two one-electron reduction steps (37). The disrupted gene in A. mimigardefordensis was designated lpdA_Am (EC 1.8.1.4), and it encodes a homodimeric flavoprotein, the dihydrolipoamide dehydrogenase LpdA_Am (i.e., the E3 component of the pyruvate dehydrogenase multi-enzyme complex of A. mimigardefordensis strain DPN7^T) belonging to the above-mentioned family of pyridine nucleotide-disulfide oxidoreductases. Enzymes of this class share high sequence and structural similarities and catalyze reduction of compounds which are linked by disulfide bonds (38). Alkylhydroperoxide reductases, coenzyme A disulfide reductases, glutathione reductases, mycothione reductases, thioredoxin reductases, and trypanothione reductases also, in addition to dihydrolipoamide dehydrogenases, belong to this family (3, 38). The physiological function of LpdA_Am is most probably the conversion of lipoamide to dihydrolipoamide, but the reduction of DTDP into two molecules of 3MP (Fig. ​(Fig.1)1) is also predicted, enabling the first step in DTDP catabolism in A. mimigardefordensis strain DPN7^T (41).Open in a separate windowFIG. 1.Reactions catalyzed by LpdA_Am and PdhL_Re. Presented are the enzymatic conversions of DTDP into two molecules of 3MP (A), lipoamide into dihydrolipoamide (B), and DTNB into two molecules of NTB (C). Abbreviations: DTDP, 3,3′-dithiodipropionic acid; 3MP, 3-mercaptopropionic acid; DTNB, 5,5′-dithiobis-(2-nitrobenzoic acid); NTB, 2-nitro-5-thiobenzoic acid.Ralstonia eutropha H16 synthesizes copolymers of 3-hydroxybutyrate and 3MP, if 3MP (23) or DTDP (22) is supplied as a precursor in addition to a second utilizable carbon source. Although R. eutropha is not able to grow with DTDP as the sole carbon source, it must be capable of cleaving this organic disulfide symmetrically, because it synthesizes from it heteropolymers containing the resulting 3MP. Thus, R. eutropha must possess at least one gene encoding a DTDP-cleaving enzyme. Five genes coding for homologues of a dihydrolipoamide dehydrogenase (DHLDH), which in A. mimigardefordensis DPN7^T is obviously involved in DTDP degradation, are known to exist in the genome of R. eutropha H16 (27; M. Raberg, J. Bechmann, U. Brandt, J. Schlüter, B. Uischner, and A. Steinbüchel, unpublished data). Therefore, LpdA_Am and the five DHLDH paralogues of R. eutropha H16 were aligned and compared (Fig. ​(Fig.2).2). Subsequently, lpdA_Am and the gene encoding the DHLDH belonging to the pyruvate dehydrogenase complex of R. eutropha H16 (pdhL_Re) were cloned, heterologously expressed in Escherichia coli, purified, and assayed.Open in a separate windowFIG. 2.Phylogenetic relationships of the A. mimigardefordensis strain DPN7^T LpdA (boldface), R. eutropha H16 PdhL (boldface), and homologues. The neighbor-joining plot was derived from a CLUSTAL X alignment of amino acid sequences closely related to LpdA_Am. The amino acid sequence of the outer membrane protein P64K from Neisseria meningitidis was used as the outgroup. GenBank accession numbers are given in parentheses. Scale bar, 10% sequence divergence.     

Cloning,expression and functional analysis of nicotinate dehydrogenase gene cluster from <Emphasis Type="Italic">Comamonas testosteroni</Emphasis> JA1 that can hydroxylate 3-cyanopyridine

A nicotinate dehydrogenase (NaDH) gene cluster was cloned from Comamonas testosteroni JA1. The enzyme, termed NaDH_JA1, is composed of 21, 82, and 46 kDa subunits, respectivley containing [2Fe2S], Mo(V) and cytochrome c domains. The recombinant NaDH_JA1 can catalyze the hydroxylation of nicotinate and 3-cyanopyridine. NaDH_JA1 protein exhibits 52.8% identity to the amino acid sequence of NaDH_KT2440 from P. putida KT2440. Sequence alignment analysis showed that the [2Fe2S] domain in NaDH_JA1 had a type II [2Fe-2S] motif and a type I [2Fe-2S] motif, while the same domain in NaDH_KT2440 had only a type II [2Fe-2S] motif. NaDH_KT2440 had an additional hypoxanthine dehydrogenase motif that NaDH_JA1 does not have. When the small unit of NaDH_JA1 was replaced by the small subunit from NaDH_KT2440, the hybrid protein was able to catalyze the hydroxylation of nicotinate, but lost the ability to catalyze hydroxylation of 3-cyanopyridine. In contrast, after replacement of the small subunit of NaDH_KT2440 with the small subunit from NaDH_JA1, the resulting hybrid protein NaDH_JAS+KTL acquired the ability to hydroxylate 3-cyanopyridine. The subunits swap results indicate the [2Fe2S] motif determines the 3-cyanopyridine hydroxylation ability, which is evidently different from the previous belief that the Mo motif determines substrate specificity.     

Selective inhibition of acyl-CoA dehydrogenases by a metabolite of hypoglycin.

Extracts of liver mitochondria from donor rats given hypoglycin, the toxic amino acid from the ackee plant (Blighia sapida) showed drastically reduced levels of acyl-CoA dehydrogenase activity with butyryl-CoA as substrate. Activity with octanoyl- and palmitoyl-CoA was unaffected. Evidence that the active agent is methylenecyclopropylacetyl-CoA, a hypoglycin metabolite, was obtained by observing effects of the compound on a partially purified enzyme mixture prepared from rabbit liver. At 13 muM concentration, it strongly inhibited butyryl-CoA dehydrogenase (EC 1.3.99.2) with butyryl-CoA as substrate; it was far less effective with palmitoyl-CoA as substrate for the other similar enzymes present in the preparation. Unlike normal substrates of the acyl-CoA dehydrogenases, the compound itself, and not a reaction product, is inhibitory. The observed effect is consistent with quite general inhibition of fatty acid beta-oxidation by hypoglycin.     

A Proteomic View at the Biochemistry of Syntrophic Butyrate Oxidation in Syntrophomonas wolfei

In syntrophic conversion of butyrate to methane and CO₂, butyrate is oxidized to acetate by secondary fermenting bacteria such as Syntrophomonas wolfei in close cooperation with methanogenic partner organisms, e.g., Methanospirillum hungatei. This process involves an energetically unfavourable shift of electrons from the level of butyryl-CoA oxidation to the substantially lower redox potential of proton and/or CO₂ reduction, in order to transfer these electrons to the methanogenic partner via hydrogen and/or formate.In the present study, all prominent membrane-bound and soluble proteins expressed in S. wolfei specifically during syntrophic growth with butyrate, in comparison to pure-culture growth with crotonate, were examined by one- and two-dimensional gel electrophoresis, and identified by peptide fingerprinting-mass spectrometry. A membrane-bound, externally oriented, quinone-linked formate dehydrogenase complex was expressed at high level specifically during syntrophic butyrate oxidation, comprising a selenocystein-linked catalytic subunit with a membrane-translocation pathway signal (TAT), a membrane-bound iron-sulfur subunit, and a membrane-bound cytochrome. Soluble hydrogenases were expressed at high levels specifically during growth with crotonate. The results were confirmed by native protein gel electrophoresis, by formate dehydrogenase and hydrogenase-activity staining, and by analysis of formate dehydrogenase and hydrogenase activities in intact cells and cell extracts. Furthermore, constitutive expression of a membrane-bound, internally oriented iron-sulfur oxidoreductase (DUF224) was confirmed, together with expression of soluble electron-transfer flavoproteins (EtfAB) and two previously identified butyryl-CoA dehydrogenases.Our findings allow to depict an electron flow scheme for syntrophic butyrate oxidation in S. wolfei. Electrons derived from butyryl-CoA are transferred through a membrane-bound EtfAB:quinone oxidoreductase (DUF224) to a menaquinone cycle and further via a b-type cytochrome to an externally oriented formate dehydrogenase. Hence, an ATP hydrolysis-driven proton-motive force across the cytoplasmatic membrane would provide the energy input for the electron potential shift necessary for formate formation.     

Metabolic engineering of Cupriavidus necator H16 for heterotrophic and autotrophic production of 3-hydroxypropionic acid

3-Hydroxypropionate (3-HP) is a versatile compound for chemical synthesis and a potential building block for biodegradable polymers. Cupriavidus necator H16, a facultative chemolithoautotroph, is an attractive production chassis and has been extensively studied as a model organism for biopolymer production. Here, we engineered C. necator H16 for 3-HP biosynthesis from its central metabolism. Wild type C. necator H16 can use 3-HP as a carbon source, a highly undesirable trait for a 3-HP production chassis. However, deletion of its three (methyl-)malonate semialdehyde dehydrogenases (mmsA1, mmsA2 and mmsA3) resulted in a strain that cannot grow on 3-HP as the sole carbon source, and this strain was selected as our production host. A stepwise approach was used to construct pathways for 3-HP production via β-alanine. Two additional gene deletion targets were identified during the pathway construction process. Deletion of the 3-hydroxypropionate dehydrogenase, encoded by hpdH, prevented the re-consumption of the 3-HP produced by our engineered strains, while deletion of gdhA1, annotated as a glutamate dehydrogenase, prevented the utilization of aspartate as a carbon source, one of the key pathway intermediates. The final strain carrying these deletions was able to produce up to 8 mM 3-HP heterotrophically. Furthermore, an engineered strain was able to produce 0.5 mM 3-HP under autotrophic conditions, using CO₂ as sole carbon source. These results form the basis for establishing C. necator H16 as an efficient platform for the production of 3-HP and 3-HP-containing polymers.     

Effect of an Oxygen-Tolerant Bifurcating Butyryl Coenzyme A Dehydrogenase/Electron-Transferring Flavoprotein Complex from Clostridium difficile on Butyrate Production in Escherichia coli

The butyrogenic genes from Clostridium difficile DSM 1296^T have been cloned and expressed in Escherichia coli. The enzymes acetyl-coenzyme A (CoA) C-acetyltransferase, 3-hydroxybutyryl-CoA dehydrogenase, crotonase, phosphate butyryltransferase, and butyrate kinase and the butyryl-CoA dehydrogenase complex composed of the dehydrogenase and two electron-transferring flavoprotein subunits were individually produced in E. coli and kinetically characterized in vitro. While most of these enzymes were measured using well-established test systems, novel methods to determine butyrate kinase and butyryl-CoA dehydrogenase activities with respect to physiological function were developed. Subsequently, the individual genes were combined to form a single plasmid-encoded operon in a plasmid vector, which was successfully used to confer butyrate-forming capability to the host. In vitro and in vivo studies demonstrated that C. difficile possesses a bifurcating butyryl-CoA dehydrogenase which catalyzes the NADH-dependent reduction of ferredoxin coupled to the reduction of crotonyl-CoA also by NADH. Since the reoxidation of ferredoxin by a membrane-bound ferredoxin:NAD⁺-oxidoreductase enables electron transport phosphorylation, additional ATP is formed. The butyryl-CoA dehydrogenase from C. difficile is oxygen stable and apparently uses oxygen as a co-oxidant of NADH in the presence of air. These properties suggest that this enzyme complex might be well suited to provide butyryl-CoA for solventogenesis in recombinant strains. The central role of bifurcating butyryl-CoA dehydrogenases and membrane-bound ferredoxin:NAD oxidoreductases (Rhodobacter nitrogen fixation [RNF]), which affect the energy yield of butyrate fermentation in the clostridial metabolism, is discussed.     

Kinetic methods for the study of the enzyme systems of β-oxidation

Kinetic methods for studying the reactions of the “general” fatty acyl CoA dehydrogenase under three sets of substrate and enzyme concentration conditions have been developed. The reaction of butyryl-CoA and electron transfer flavoprotein (ETF) can be studied either under steady-state conditions with enzyme at catalytic concentration or under single-turnover conditions with enzyme in excess. Under the latter conditions, acyl-CoA dehydrogenase acts both as a catalyst and an ultimate electron-transfer acceptor. The reductive half-reaction of butyryl-CoA and enzyme can also be studied in a separate kinetic experiment. Comparison of the pH dependences of the rate constants and isotope effects of the steady-state reaction of butyryl-CoA and ETF with the same parameters for the reductive half-reaction is consistent with a mechanism involving transfer of electrons from butyryl-CoA to ETF within a ternary complex. An alternative mechanism in which the reductive half-reaction takes place prior to the binding and reaction of ETF seems unlikely because the pH 8.5 isotope effect on the reductive half-reaction is much larger than that on the complete reaction in spite of the fact that the rates of the reactions are comparable. The pH dependence of the K_m for substrate and K_I for inhibitor is consistent with a mechanism for transfer of electrons within the ternary complex which involves protonation of the C group of substrates. The protonation labilizes the C-2 proton and base catalysis of the removal of the C-2 proton results in the production of the active enzyme-substrate species, namely the C-2 anion of substrate.     

Reciprocal Isomerization of Butyrate and Isobutyrate by the Strictly Anaerobic Bacterium Strain WoG13 and Methanogenic Isobutyrate Degradation by a Defined Triculture

Isomerization of butyrate and isobutyrate was investigated with the recently isolated strictly anaerobic bacterium strain WoG13 which ferments glutarate to butyrate, isobutyrate, CO₂, and small amounts of acetate. Dense cell suspensions converted butyrate to isobutyrate and isobutyrate to butyrate. ¹³C-nuclear magnetic resonance experiments proved that this isomerization was accomplished by migration of the carboxyl group to the adjacent carbon atom. In cell extracts, both butyrate and isobutyrate were activated to their coenzyme A (CoA) esters by acyl-CoA:acetate CoA-transferases. The reciprocal rearrangement of butyryl-CoA and isobutyryl-CoA was catalyzed by a butyryl-CoA:isobutyryl-CoA mutase which depended strictly on the presence of coenzyme B₁₂. Isobutyrate was completely degraded via butyrate to acetate and methane by a defined triculture of strain WoG13, Syntrophomonas wolfei, and Methanospirillum hungatei.     

Ethylene Glycol Metabolism by Pseudomonas putida

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.     

Improved performance of Pseudomonas putida in a bioelectrochemical system through overexpression of periplasmic glucose dehydrogenase

It was recently demonstrated that a bioelectrochemical system (BES) with a redox mediator allowed Pseudomonas putida to perform anoxic metabolism, converting sugar to sugar acids with high yield. However, the low productivity currently limits the application of this technology. To improve productivity, the strain was optimized through improved expression of glucose dehydrogenase (GCD) and gluconate dehydrogenase (GAD). In addition, quantitative real‐time RT‐PCR analysis revealed the intrinsic self‐regulation of GCD and GAD. Utilizing this self‐regulation system, the single overexpression strain (GCD) gave an outstanding performance in the electron transfer rate and 2‐ketogluconic acid (2KGA) productivity. The peak anodic current density, specific glucose uptake rate and 2KGA producing rate were 0.12 mA/cm², 0.27 ± 0.02 mmol/g_CDW/hr and 0.25 ± 0.02 mmol/g_CDW/hr, which were 327%, 477%, and 644% of the values of wild‐type P. putida KT2440, respectively. This work demonstrates that expression of periplasmic dehydrogenases involved in electron transfer can significantly improve productivity in the BES.     

Metabolism of syringyl lignin-derived compounds in Pseudomonas putida enables convergent production of 2-pyrone-4,6-dicarboxylic acid

Valorization of lignin, an abundant component of plant cell walls, is critical to enabling the lignocellulosic bioeconomy. Biological funneling using microbial biocatalysts has emerged as an attractive approach to convert complex mixtures of lignin depolymerization products to value-added compounds. Ideally, biocatalysts would convert aromatic compounds derived from the three canonical types of lignin: syringyl (S), guaiacyl (G), and p-hydroxyphenyl (H). Pseudomonas putida KT2440 (hereafter KT2440) has been developed as a biocatalyst owing in part to its native catabolic capabilities but is not known to catabolize S-type lignin-derived compounds. Here, we demonstrate that syringate, a common S-type lignin-derived compound, is utilized by KT2440 only in the presence of another energy source or when vanAB was overexpressed, as syringate was found to be O-demethylated to gallate by VanAB, a two-component monooxygenase, and further catabolized via extradiol cleavage. Unexpectedly, the specificity (k_cat/K_M) of VanAB for syringate was within 25% that for vanillate and O-demethylation of both substrates was well-coupled to O₂ consumption. However, the native KT2440 gallate-cleaving dioxygenase, GalA, was potently inactivated by 3-O-methylgallate. To engineer a biocatalyst to simultaneously convert S-, G-, and H-type monomers, we therefore employed VanAB from Pseudomonas sp. HR199, which has lower activity for 3MGA, and LigAB, an extradiol dioxygenase able to cleave protocatechuate and 3-O-methylgallate. This strain converted 93% of a mixture of lignin monomers to 2-pyrone-4,6-dicarboxylate, a promising bio-based chemical. Overall, this study elucidates a native pathway in KT2440 for catabolizing S-type lignin-derived compounds and demonstrates the potential of this robust chassis for lignin valorization.     

Factors Influencing the Histochemical Demonstration of Coenzyme-Dependent Dehydrogenases and Diaphorases

Procedures for the histochemical demonstration of DPN and TPN diaphorases have been presented by other workers. These techniques rely on the coenzyme-dependent dehydrogenases present in the tissue slice to generate the substrate required by the diaphorases. In vitro studies were carried out on kidney and adrenal tissue of the rat, using NT (neotetrazolium) and INT (2-p-iodophenyl-3-p-nitrophenyl-5-phenyl tetrazolium chloride) with various substrates of DPN-dependent dehydrogenases. The solutions used for study contained alcohol and alcohol dehydrogenase, glutamate and malate, malate, glutamate, β-hydroxybutyrate, or DPNH. It has been possible to demonstrate (1) that histological distribution of dehydrogenases may differ from that of the flavoprotein oxidizing reduced coenzyme I; (2) characteristic patterns of distribution of particular dehydrogenases in the tissue proper; (3) different levels of dehydrogenase in kidney and adrenal; and (4) differences in dehydrogenase distribution in the kidneys of man and rat. The evidence presented clearly indicates the limitations inherent in the accepted procedures for the demonstration of DPN and TPN diaphorases. The possible application of the tetrazolium salts to the study of particular coenzyme-dependent dehydrogenases and the pitfalls which might occur are also discussed.     

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.     

Sulfides impair short chain fatty acid β-oxidation at acyl-CoA dehydrogenase level in colonocytes: Implications for ulcerative colitis

The disease process of ulcerative colitis (UC) is associated with a block in -oxidation of short chain fatty acid in colonic epithelial cells which can be reproduced by exposure of cells to sulfides. The aim of the current work was to assess the level in the -oxidation pathway at which sulfides might be inhibitory in human colonocytes. Isolated human colonocytes from cases without colitis (n = 12) were exposed to sulfide (1.5 mM) in the presence or absence of exogenous CoA and ATP. Short chain acyl-CoA esters were measured by a high performance liquid chromatographic assay. ¹⁴CO₂ generation was measured from [1-¹⁴C]butyrate and [6-¹⁴C]glucose. ¹⁴CO₂ from butyrate was significantly reduced (p < 0.001) by sulfide. When colonocytes were incubated with hydrogen sulfide in the presence of CoA and ATP, butyryl-CoA concentration was increased (p < 0.01), while crotonyl-CoA (p < 0.01) and acetyl-CoA (p < 0.01) concentrations were decreased. These results show that sulfides inhibit short chain acyl-CoA dehydrogenase. As oxidation of n-butyrate governs the epithelial barrier function of colonocytes the functional activity of short chain acyl-CoA dehydrogenase may be critical in maintaining colonic mucosal integrity. Maintaining the functional activity of dehydrogenases could be an important determinant in the expression of ulcerative colitis.     

Biochemical route and control of butyrate synthesis in Butyribacterium methylotrophicum

 Butyribacterium methylotrophicum produced more butyrate when grown on lactate than when grown on glucose, and only acetate was detected during growth on pyruvate. Higher levels of NADH were found in butyrate-producing than in acetate-producing cells. The addition of neutral red, an electron-flow modulator, to cells growing on pyruvate altered the carbon and electron flow from acetate plus H₂ synthesis to butyrate synthesis. Enzymatic analysis suggested that pyruvate was produced from glucose via an Embden-Meyerhof-Parnas pathway. Pyruvate was further metabolized to butyryl-CoA via, β-hydroxybutyryl-CoA and butyryl-CoA dehydrogenases. Lactate dehydrogenase, unlike butyryl-CoA dehydrogenase, was inducible and detected only in lactate-grown cells. Both of these dehydrogenases utilized 2,6-dichloroindophenol and other artificial electron acceptors but not NAD(P). Ferredoxin–NAD oxidoreductase levels were highest in lactate and lowest in pyruvate-grown cells. Cells contained both a ferredoxin–neutral-red reductase activity and a neutral-red–NAD reductase activity that coupled electron flow to butyrate synthesis. These results showed that butyrate synthesis by B. methylotrophicum was regulated by the carbon source and was dependent on the cellular NADH/NAD ratios, and the levels and direction of ferredoxin- and NAD-linked oxidoreductases. Received: 3 August 1995/Received revision: 31 October 1995/Accepted: 10 November 1995     

Energy Conservation Model Based on Genomic and Experimental Analyses of a Carbon Monoxide-Utilizing,Butyrate-Forming Acetogen,Eubacterium limosum KIST612

Eubacterium limosum KIST612 is one of the few acetogens that can produce butyrate from carbon monoxide. We have used a genome-guided analysis to delineate the path of butyrate formation, the enzymes involved, and the potential coupling to ATP synthesis. Oxidation of CO is catalyzed by the acetyl-coenzyme A (CoA) synthase/CO dehydrogenase and coupled to the reduction of ferredoxin. Oxidation of reduced ferredoxin is catalyzed by the Rnf complex and Na⁺ dependent. Consistent with the finding of a Na⁺-dependent Rnf complex is the presence of a conserved Na⁺-binding motif in the c subunit of the ATP synthase. Butyrate formation is from acetyl-CoA via acetoacetyl-CoA, hydroxybutyryl-CoA, crotonyl-CoA, and butyryl-CoA and is consistent with the finding of a gene cluster that encodes the enzymes for this pathway. The activity of the butyryl-CoA dehydrogenase was demonstrated. Reduction of crotonyl-CoA to butyryl-CoA with NADH as the reductant was coupled to reduction of ferredoxin. We postulate that the butyryl-CoA dehydrogenase uses flavin-based electron bifurcation to reduce ferredoxin, which is consistent with the finding of etfA and etfB genes next to it. The overall ATP yield was calculated and is significantly higher than the one obtained with H₂ + CO₂. The energetic benefit may be one reason that butyrate is formed only from CO but not from H₂ + CO₂.