Light‐Dependent and Aeration‐Independent Gram‐Scale Hydroxylation of Cyclohexane to Cyclohexanol by CYP450 Harboring Synechocystis sp. PCC 6803

Oxygenase‐containing cyanobacteria constitute promising whole‐cell biocatalysts for oxyfunctionalization reactions. Photosynthetic water oxidation thereby delivers the required cosubstrates, that is activated reduction equivalents and O₂, sustainably. A recombinant Synechocystis sp. PCC 6803 strain showing unprecedentedly high photosynthesis‐driven oxyfunctionalization activities is developed, and its technical applicability is evaluated. The cells functionally synthesize a heterologous cytochrome P450 monooxygenase enabling cyclohexane hydroxylation. The biocatalyst‐specific reaction rate is found to be light‐dependent, reaching 26.3 ± 0.6 U g_CDW^?1 (U = μmol min^?1 and cell dry weight [CDW]) at a light intensity of 150 µmol_photons m^?2 s^?1. In situ substrate supply via a two‐liquid phase system increases the initial specific activity to 39.2 ± 0.7 U g_CDW^?1 and stabilizes the biotransformation by preventing cell toxification. This results in a tenfold increased specific product yield of 4.5 g_cyclohexanol g_CDW^?1 as compared to the single aqueous phase system. Subsequently, the biotransformation is scaled from a shake flask to a 3 L stirred‐tank photobioreactor setup. In situ O₂ generation via photosynthetic water oxidation allows a nonaerated process operation, thus circumventing substrate evaporation as the most critical factor limiting the process performance and stability. This study for the first time exemplifies the technical applicability of cyanobacteria for aeration‐independent light‐driven oxyfunctionalization reactions involving highly toxic and volatile substrates.     

Physiological and Molecular Biological Characterization of Ammonia Oxidation of the Heterotrophic Nitrifier Pseudomonas putida

The heterotrophic nitrifier Pseudomonas putida aerobically oxidized ammonia to hydroxylamine, nitrite, and nitrate. Product formation was accompanied by a small but significant release of NO, whereas N₂O evolution could not be detected under the assay conditions employed. The isolate reduced nitrate to nitrite and partially further to NO under anaerobic conditions. Aerobically grown cells utilized γ-aminobutyrate as a carbon source and as a N-source by ammonification. The physiological experiments, in particular the inhibition pattern by C₂H₂, indicated that P. putida expressed an ammonia monooxigenase. DNA-hybridization with an amoA gene probe coding for the smaller subunit of the ammonia monooxigenase of Nitrosomonas europaea allowed us to identify, to clone, and to sequence a region with an open reading frame showing distinct sequence similarities to the amoA gene of autotrophic ammonia oxidizers. Received: 9 April 1998 / Accepted: 15 May 1998     

Fine structure of Methanomonas methanooxidans

Résumé Chez 5 bactéries anaérobies facultatives: Providencia alcalifaciens, Edwardsiella tarda, Aeromonas hydrophila, mutants catégorie 1 de Providencia stuartii et Hafnia, la nitrate-réductase B a un caractère inductible et sa biosynthèse est réprimée par O₂. Chez Micrococcus denitrificans, elle est constitutive et non répressible par NH₄ ⁺ ou par O₂. Dans le cas d'une bactérie aérobie stricte qui assimile le nitrate: Pseudomonas putida, elle est constitutive (ou faiblement inductible), non répressible par NH₄ ⁺, atteint un niveau plus élevé dans les cultures sur milieu complexe que dans les cultures sur milieu synthétique.O₂ exerce une action sur le fonctionnement de la nitrate-réductase B: l'aération inhibe réversiblement et de façon presque complète la réduction de NO₃ ^- en NO₂ ^-, aux dépens du glucose comme donneur d'électrons, par des suspensions cellulaires d'E. tarda, M. denitrificans, P. putida, et du mutant de Hafnia.

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Bacterial nitrate reductasesIV. Regulation of the biosynthesis and activity of enzyme B

Summary 5 facultative anaerobic bacteria (Providencia alcalifaciens, Edwardsiella tarda, Aeromonas hydrophila, and category 1 mutants of Providencia stuartii and Hafnia) form nitrate reductase B inducibly; the biosynthesis of the enzyme is repressed by O₂. In Micrococcus denitrificans, the enzyme is constitutive and its intracellular level is unaffected by the presence either of NH₄ ⁺ or O₂. A strict aerobe that assimilates nitrate, Pseudomonas putida, similarly forms nitrate reductase B constitutively, although the level of the enzyme is higher in cultures grown on complex than on synthetic media.O₂ affects the activity of nitrate reductase B: the reduction of NO₃ ^- to NO₂ ^-, with glucose as electron donor, by suspensions of E. tarda, M. denitrificans, P. putida and the mutant of Hafnia, is almost totally but reversibly inhibited by aeration.

     

Molybdenum-dependent degradation of quinoline by Pseudomonas putida Chin IK and other aerobic bacteria

Eighteen different aerobic bacteria were isolated which utilized quinoline as sole source of carbon, nitrogen, and energy. Attempts were unsuccessful at isolating anaerobic quinoline-degrading bacteria. The optimal concentration of quinoline for growth was in the range of 2.5 to 5 mM. Some organisms excreted 2-hydroxyquinoline as the first intermediate. Hydroxylation of quinoline was catalyzed by a dehydrogenase which was induced in the presence of quinoline or 2-hydroxyquinoline. Quinoline dehydrogenase activity was dependent on the availability of molybdate in the growth medium. Growth on quinoline was inhibited by tungstate, an antagonist of molybdate. Partially purified quinoline dehydrogenase from Pseudomonas putida Chin IK indicated the presence of flavin, iron-sulfur centers, and molybdenum-binding pterin. M _r of quinoline dehydrogenase was about 300 kDa in all isolates investigated.Abbreviations APS ammonium peroxodisulfate - DCPIP 2,6-dichlorophenol-indophenol - EEO electroendosmosis - MTT thiazolyl blue - PES phenazine ethosulfate - TEMED N,N,N,N-tetramethyl-ethylenediamine     

Xanthine dehydrogenase from Pseudomonas putida 86: specificity,oxidation–reduction potentials of its redox-active centers,and first EPR characterization

Xanthine dehydrogenase (XDH) from Pseudomonas putida 86, which was induced 65-fold by growth on hypoxanthine, was purified to homogeneity. It catalyzes the oxidation of hypoxanthine, xanthine, purine, and some aromatic aldehydes, using NAD⁺ as the preferred electron acceptor. In the hypoxanthine:NAD⁺ assay, the specific activity of purified XDH was 26.7 U (mg protein)⁻¹. Its activity with ferricyanide and dioxygen was 58% and 4%, respectively, relative to the activity observed with NAD⁺. XDH from P. putida 86 consists of 91.0 kDa and 46.2 kDa subunits presumably forming an α₄β₄ structure and contains the same set of redox-active centers as eukaryotic XDHs. After reduction of the enzyme with xanthine, electron paramagnetic resonance (EPR) signals of the neutral FAD semiquinone radical and the Mo(V) rapid signal were observed at 77 K. Resonances from FeSI and FeSII were detected at 15 K. Whereas the observable g factors for FeSII resemble those of other molybdenum hydroxylases, the FeSI center in contrast to most other known FeSI centers has nearly axial symmetry. The EPR features of the redox-active centers of P. putida XDH are very similar to those of eukaryotic XDHs/xanthine oxidases, suggesting that the environment of each center and their functionality are analogous in these enzymes. The midpoint potentials determined for the molybdenum, FeSI and FAD redox couples are close to each other and resemble those of the corresponding centers in eukaryotic XDHs.     

Shewanella oneidensis MR‐1 mutants selected for their inability to produce soluble organic‐Fe(III) complexes are unable to respire Fe(III) as anaerobic electron acceptor

Summary Recent voltammetric analyses indicate that Shewanella putrefaciens strain 200 produces soluble organic‐Fe(III) complexes during anaerobic respiration of sparingly soluble Fe(III) oxides. Results of the present study expand the range of Shewanella species capable of producing soluble organic‐Fe(III) complexes to include Shewanella oneidensis MR‐1. Soluble organic‐Fe(III) was produced by S. oneidensis cultures incubated anaerobically with Fe(III) oxides, or with Fe(III) oxides and the alternate electron acceptor fumarate, but not in the presence of O₂, nitrate or trimethylamine‐N‐oxide. Chemical mutagenesis procedures were combined with a novel MicroElectrode Screening Array (MESA) to identify four (designated Sol) mutants with impaired ability to produce soluble organic‐Fe(III) during anaerobic respiration of Fe(III) oxides. Two of the Sol mutants were deficient in anaerobic growth on both soluble Fe(III)‐citrate and Fe(III) oxide, yet retained the ability to grow on a suite of seven alternate electron acceptors. The rates of soluble organic‐Fe(III) production were proportional to the rates of iron reduction by the S. oneidensis wild‐type and Sol mutant strains, and all four Sol mutants retained wild‐type siderophore production capability. Results of this study indicate that the production of soluble organic‐Fe(III) may be an important intermediate step in the anaerobic respiration of both soluble and sparingly soluble forms of Fe(III) by S. oneidensis.     

Nitrate respiration in Pseudomonas stutzeri A15 and its involvement in rice and wheat root colonization

Unlike most bacteria, the nitrogen-fixing rice-associated Pseudomonas stutzeri A15 disposes of three different nitrate reductases that enable conversion of nitrate to nitrite through three physiologically distinct processes, called nitrate assimilation, nitrate respiration and nitrate dissimilation. To study the role of nitrate respiration in rhizosphere fitness, a Pseudomonas stutzeri narG mutant was constructed and characterized by assessing its growth characteristics and whole-cell nitrate reductase activity in different oxygen tensions. Unexpectedly, the Pseudomonas stutzeri A15 narG mutant appeared to be a better root colonizer, outcompeting the wild type strain in a wheat and rice hydroponic system.     

Active site geometry and substrate recognition of the molybdenum hydroxylase quinoline 2-oxidoreductase

The soil bacterium Pseudomonas putida 86 uses quinoline as a sole source of carbon and energy. Quinoline 2-oxidoreductase (Qor) catalyzes the first metabolic step converting quinoline to 2-oxo-1,2-dihydroquinoline. Qor is a member of the molybdenum hydroxylases. The molybdenum ion is coordinated by two ene-dithiolate sulfur atoms, two oxo-ligands, and a catalytically crucial sulfido-ligand, whose position in the active site was controversial. The 1.8 A resolution crystal structure of Qor indicates that the sulfido-ligand occupies the equatorial position at the molybdenum ion. The structural comparison of Qor with the allopurinol-inhibited xanthine dehydrogenase from Rhodobacter capsulatus allows direct insight into the mechanism of substrate recognition and the identification of putative catalytic residues. The active site protein variants QorE743V and QorE743D were analyzed to assess the catalytic role of E743.     

In silico genome-scale metabolic analysis of Pseudomonas putida KT2440 for polyhydroxyalkanoate synthesis,degradation of aromatics and anaerobic survival

Genome-scale metabolic models have been appearing with increasing frequency and have been employed in a wide range of biotechnological applications as well as in biological studies. With the metabolic model as a platform, engineering strategies have become more systematic and focused, unlike the random shotgun approach used in the past. Here we present the genome-scale metabolic model of the versatile Gram-negative bacterium Pseudomonas putida, which has gained widespread interest for various biotechnological applications. With the construction of the genome-scale metabolic model of P. putida KT2440, PpuMBEL1071, we investigated various characteristics of P. putida, such as its capacity for synthesizing polyhydroxyalkanoates (PHA) and degrading aromatics. Although P. putida has been characterized as a strict aerobic bacterium, the physiological characteristics required to achieve anaerobic survival were investigated. Through analysis of PpuMBEL1071, extended survival of P. putida under anaerobic stress was achieved by introducing the ackA gene from Pseudomonas aeruginosa and Escherichia coli.     

Nitrate respiration,denitrification, and utilization of nitrogen sources by aerobic carbon monoxide-oxidizing bacteria

We describe the ability of carboxydotrophic bacteria for nitrate respiration or denitrification. Four out of fourteen strains examined could denitrify heterotrophically forming N₂ (Pseudomonas carboxydoflava) or N₂O (Pseudomonas carboxydohydrogena, Pseudomonas compransoris, and Pseudomonas gazotropha). Three carried out a heterotrophic nitrate respiration (Arthrobacter 11/x, Azomonas B1, and Azomonas C2). P. carboxydohydrogena could use H₂ as electron donor for nitrate respiration under chemolithoautotrophic growth conditions. CO did not support denitrification or nitrate respiration of carboxydotrophic bacteria, although the free energy changes of the reactions would be sufficiently negative to allow growth. CO at 50 kPa was a weak inhibitor of N₂O-reduction in carboxydotrophic and non-carboxydotrophic bacteria and decelerated denitrifying growth. Carboxydotrophic bacteria could utilize a wide range of N-sources. Results obtained with a plasmid-cured mutant of Pseudomonas carboxydovorans OM5 showed, that genes involved in nitrogen assimilation entirely reside on the chromosome. In the presence of an suitable electron donor, most carboxydotrophic bacteria could carry out a reduction of nitrate to nitrite that did not support growth and did not lead to the formation of ammonia.This article is dedicated to Professor Hans G. Schlegel on the occasion of his 65th birthday and in admiration for his élan and eternal idealism     

Transformation of RDX and other energetic compounds by xenobiotic reductases XenA and XenB

The transformation of explosives, including hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), by xenobiotic reductases XenA and XenB (and the bacterial strains harboring these enzymes) under both aerobic and anaerobic conditions was assessed. Under anaerobic conditions, Pseudomonas fluorescens I-C (XenB) degraded RDX faster than Pseudomonas putida II-B (XenA), and transformation occurred when the cells were supplied with sources of both carbon (succinate) and nitrogen (NH₄ ⁺), but not when only carbon was supplied. Transformation was always faster under anaerobic conditions compared to aerobic conditions, with both enzymes exhibiting a O₂ concentration-dependent inhibition of RDX transformation. The primary degradation pathway for RDX was conversion to methylenedinitramine and then to formaldehyde, but a minor pathway that produced 4-nitro-2,4-diazabutanal (NDAB) also appeared to be active during transformation by whole cells of P. putida II-B and purified XenA. Both XenA and XenB also degraded the related nitramine explosives octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine and 2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazaisowurtzitane. Purified XenB was found to have a broader substrate range than XenA, degrading more of the explosive compounds examined in this study. The results show that these two xenobiotic reductases (and their respective bacterial strains) have the capacity to transform RDX as well as a wide variety of explosive compounds, especially under low oxygen concentrations.     

Cytochrome P450 Alkane Hydroxylases of the CYP153 Family Are Common in Alkane-Degrading Eubacteria Lacking Integral Membrane Alkane Hydroxylases

Several strains that grow on medium-chain-length alkanes and catalyze interesting hydroxylation and epoxidation reactions do not possess integral membrane nonheme iron alkane hydroxylases. Using PCR, we show that most of these strains possess enzymes related to CYP153A1 and CYP153A6, cytochrome P450 enzymes that were characterized as alkane hydroxylases. A vector for the polycistronic coexpression of individual CYP153 genes with a ferredoxin gene and a ferredoxin reductase gene was constructed. Seven of the 11 CYP153 genes tested allowed Pseudomonas putida GPo12 recombinants to grow well on alkanes, providing evidence that the newly cloned P450s are indeed alkane hydroxylases.     

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.     

Anaerobic growth and potential for amino acid production by nitrate respiration in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Oxygen limitation is a crucial problem in amino acid fermentation by Corynebacterium glutamicum. Toward this subject, our study was initiated by analysis of the oxygen-requiring properties of C. glutamicum, generally regarded as a strict aerobe. This organism formed colonies on agar plates up to relatively low oxygen concentrations (0.5% O₂), while no visible colonies were formed in the absence of O₂. However, in the presence of nitrate (), the organism exhibited limited growth anaerobically with production of nitrite (), indicating that C. glutamicum can use nitrate as a final electron acceptor. Assays of cell extracts from aerobic and hypoxic cultures yielded comparable nitrate reductase activities, irrespective of nitrate levels. Genome analysis revealed a narK2GHJI cluster potentially relevant to nitrate reductase and transport. Disruptions of narG and narJ abolished the nitrate-dependent anaerobic growth with the loss of nitrate reductase activity. Disruption of the putative nitrate/nitrite antiporter gene narK2 did not affect the enzyme activity but impaired the anaerobic growth. These indicate that this locus is responsible for nitrate respiration. Agar piece assays using l-lysine- and l-arginine-producing strains showed that production of both amino acids occurred anaerobically by nitrate respiration, indicating the potential of C. glutamicum for anaerobic amino acid production.     

Anaerobic expression of nitric oxide reductase from denitrifying Pseudomonas stutzeri

NO reductase synthesis was investigated immunochemically and by activity assays in cells of Pseudomonas stutzeri ZoBell grown in continuous culture at discrete aeration levels, or in O₂-limited batch cultures supplemented with N oxides as respiratory substrate. Under aerobic conditions, NO reductase was not expressed in P. stutzeri. Oxygen limitation in combination with the presence of nitrate or nitrite derepressed NO reductase synthesis. On transition from aerobic to anaerobic conditions in continuous culture, NO reductase was synthesized below 3% air saturation and reached maximum expression under anaerobic conditions. By use of mutant strains defective in nitrate respiration or nitrite respiration, the inducing effect of individual N oxides on NO reductase synthesis could be discriminated. Nitrite caused definite, concentration-dependent induction, while nitrate promoted moderate enzyme synthesis or amplified effects of nitrite. Exogenous nitric oxide (NO) in concentrations 25 M induced trace amounts of NO reductase; in higher concentrations it arrested cell growth. Nitrite reductase or NO reductase were not detected immunochemically under these conditions. NO generated as an intermediate appeared not to induce NO reductase significantly. Antiserum raised against the P. stutzeri NO reductase showed crossreaction with cell extracts from P. stutzeri JM300, but not with several other denitrifying pseudomonads or Paracoccus denitrificans.     

Transformation of indole and quinoline by Desulfobacterium indolicum (DSM 3383)

Degradation of indole and quinoline by Desulfobacterium␣indolicum was studied in batch cultures. The first step in the degradation pathway of indole and quinoline was a hydroxylation at the 2 position to oxindole and 2-hydroxyquinoline respectively. These hydroxylation reactions followed saturation kinetics. The kinetic parameters for indole were an apparent maximum specific transformation rate (V _Amax) of 263 μmol mg total protein⁻¹ day⁻¹ and an apparent half-saturation constant (K _Am) of 139 μM. The V _Amax for quinoline was 170 μmol mg total protein⁻¹ day⁻¹ and K _Am was 92 μM. Oxindole inhibited indole hydroxylation whereas 2-hydroxyquinoline stimulated quinoline hydroxylation. An adaptation period of approximately 20 days was required before transformation of 2-hydroxyquinoline in cultures previously grown on quinoline. Indole and quinoline were hydroxylated with a lag phase shorter than 4 h in a culture adapted to ethanol. Chloramphenicol inhibited the hydroxylation of indole and quinoline in ethanol-adapted cells, indicating an inducible enzyme system. Chloramphenicol had no effect on the hydroxylation of indole in quinoline-adapted cells or on the hydroxylation of quinoline in indole-adapted cells. This indicated that it was the same inducible enzyme system that hydroxylated indole and quinoline. Received: 16 July 1996 / Received revision: 23 September 1996 / Accepted: 29 September 1996     

Phosphorylation coupled to O2 and nitrate reduction inPseudomonas saccharophila grown anaerobically with succinate and nitrate

Cell-free membrane preparations fromPseudomanas saccharophila grown anterobically with succinate and nitrate catalyzed NADH oxidation by O₂ and nitrate, yielding P/O and P/NO₃ ⁻-reduced ratios of 0.76 and 0.51, respectively. Succinate oxidation yielded a P/O ratio of 0.44 and a P/NO₃ ⁻-reduced ratio of 0.08. Ascorbate oxidation by O₂ or nitrate was not coupled with ATP generation. The NADH- or succinate-linked oxidative phosphorylation was uncoupled by classical uncoupling agents: moreover, the aerobic and the anaerobic oxidation of NADH and succinate, as well as the coupled ATP synthesis, was inhibited by low concentrations of respiratory chain inhibitors. In addition, oligomycin was a potent inhibitor of ATP generation in this system.