Methylophaga Aminisulfidivorans Research Articles

A dual-enzyme cascade for production of indigo from L-tryptophan

Indigo, as a water-soluble non-azo colorant, is widely used in textile, food, pharmaceutical and other industrial fields. Currently, indigo is primarily synthesized by chemical methods, which causes environmental pollution, potential safety hazards, and other issues. Therefore, there is an urgent need to find a safer and greener synthetic method. In this study, a dual-enzyme cascade pathway was constructed with the tryptophan synthase (tryptophanase, EcTnaA) from Escherichia coli and flavin-dependent monooxygenase (flavin-dependent monooxygenase, MaFMO) from Methylophaga aminisulfidivorans to synthesize indigo with L-tryptophan as substrate. A recombinant strain EM-IND01 was obtained. The beneficial mutant MaFMOD197E was obtained by protein engineering of the rate-limiting enzyme MaFMO. MaFMOD197E showed the specific activity and kcat/Km value 2.36 times and 1.34 times higher than that of the wild type, respectively. Furthermore, MaFMOD197E was introduced into the strain EM-IND01 to construct the strain EM-IND02. After the fermentation conditions were optimized, the strain achieved the indigo titer of (1 288.59±7.50) mg/L, the yield of 0.86 mg/mg L-tryptophan, and the productivity of 26.85 mg/(L·h) in a 5 L fermenter. Protein engineering was used to obtain mutants with increased MaFMO activity in this study, which laid a foundation for industrial production of indigo.

Production of bio-indigo dye by surmounting its physical and chemical insoluble nature

Indigo is a natural blue dye that has been in use for centuries. However, its insolubility in water and most organic solvents limits its application in various fields. In this study, we aimed to enhance the indigo production by surmounting its physical and chemical insolubility. To achieve this, a combination of genetic engineering, biotransformation optimization, and solubilization techniques has been employed. First, the indole-converting enzymes, flavin-containing monooxygenase (MaFMO) from Methylophaga aminisulfidivorans and toluene monooxygenase from Pseudomonas mendocina (PmT4MO), were tested and their expression was optimized, which allowed for the efficient conversion of indole to indigo via indoxyl. Next, to overcome the insolubility of indigo and facilitate indole/indigo transport through the cell membrane, organic solvents and detergent mixing were optimized. Genetic modification was attempted by introducing caveolae complex structures into the membrane to create a permanent indole/indigo transport system. Finally, inspired by wastewater treatment and indigo solubilization during the indigo-dyeing process, indigo adsorption and reduction were applied using an Amberlite XAD4 adsorption bead and thiourea dioxide reducing agent to achieve enhanced production of indigo, respectively. These counteracting strategies against the insoluble nature of indigo resulted in a significant increase in bio-indigo production, with 1.5 g/L produced from 15 mM indole, representing an increase of more than 300% compared with that of the initial production titer. This study provides valuable insights into the optimization of indigo bioproduction and could lead to the development of more efficient methods for bio-dye production.

Increased Thermostability of an Engineered Flavin-Containing Monooxygenase to Remediate Trimethylamine in Fish Protein Hydrolysates.

Protein hydrolysates made from marine by-products are very nutritious but frequently contain trimethylamine (TMA), which has an unattractive fish-like smell. Bacterial trimethylamine monooxygenases can oxidize TMA into the odorless trimethylamine N-oxide (TMAO) and have been shown to reduce TMA levels in a salmon protein hydrolysate. To make the flavin-containing monooxygenase (FMO) Methylophaga aminisulfidivorans trimethylamine monooxygenase (mFMO) more suitable for industrial application, we engineered it using the Protein Repair One-Stop Shop (PROSS) algorithm. All seven mutant variants, containing 8 to 28 mutations, displayed increases in melting temperature of between 4.7°C and 9.0°C. The crystal structure of the most thermostable variant, mFMO_20, revealed the presence of four new stabilizing interhelical salt bridges, each involving a mutated residue. Finally, mFMO_20 significantly outperformed native mFMO in its ability to reduce TMA levels in a salmon protein hydrolysate at industrially relevant temperatures. IMPORTANCE Marine by-products are a high-quality source for peptide ingredients, but the unpleasant fishy odor caused by TMA limits their access to the food market. This problem can be mitigated by enzymatic conversion of TMA into the odorless TMAO. However, enzymes isolated from nature must be adapted to industrial requirements, such as the ability to tolerate high temperatures. This study has demonstrated that mFMO can be engineered to become more thermostable. Moreover, unlike the native enzyme, the best thermostable variant efficiently oxidized TMA in a salmon protein hydrolysate at industrial temperatures. Our results present an important next step toward the application of this novel and highly promising enzyme technology in marine biorefineries.

Identification of an indole biodegradation gene cluster from Providencia rettgeri and its contribution in selectively biosynthesizing Tyrian purple.

Tyrian purple, mainly composed of 6, 6'-dibromoindigo, is a precious dye extracted from sea snails. In this study, we found Tyrian purple can be selectively produced by a bacterial strain GS-2 when fed with 6-bromotryptophan in the presence of tryptophan. This GS-2 strain was then identified as Providencia rettgeri based on bacterial genome sequencing analysis. An indole degradation gene cluster for indole metabolism was identified from this GS-2 strain. The heterologous expression of the indole degradation gene cluster in Escherichia coli BL21 (DE3) and in vitro enzymatic reaction demonstrated that the indole biodegradation gene cluster may contribute to selectively biosynthesizing Tyrian purple. To further explore the underlying mechanism of the selectivity, we explored the intermediates in this indole biodegradation pathway using liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry (LC-ESI-QTOF-MS/MS), which indicated that the indole biodegradation pathway in Providencia rettgeri is the catechol pathway. Interestingly, the monooxygenase GS-C co-expressed with its corresponding reductase GS-D in the cluster has better activity for the biosynthesis of Tyrian purple compared with the previously reported monooxygenase from Methylophaga aminisulfidivorans (MaFMO) or Streptomyces cattleya cytochrome P450 enzyme (CYP102G4). This is the first study to show the existence of an indole biodegradation pathway in Providencia rettgeri, and the indole biodegradation gene cluster can contribute to the selective production of Tyrian purple.

Gammaproteobacteria mediating utilization of methyl-, sulfur- and petroleum organic compounds in deep ocean hydrothermal plumes.

Deep-sea hydrothermal plumes are considered natural laboratories for understanding ecological and biogeochemical interactions. Previous studies focused on interactions between microorganisms and inorganic, reduced hydrothermal inputs including sulfur, hydrogen, iron, and manganese. However, little is known about transformations of organic compounds, especially methylated, sulfur-containing compounds, and petroleum hydrocarbons. Here, we reconstructed nine gammaproteobacterial metagenome-assembled genomes, affiliated with Methylococcales, Methylophaga, and Cycloclasticus, from three hydrothermal ecosystems. We present evidence that these three groups have high transcriptional activities of genes encoding cycling of C1-compounds, petroleum hydrocarbons, and organic sulfur in hydrothermal plumes. This includes oxidation of methanethiol, the simplest thermochemically-derived organic sulfur, for energy metabolism in Methylococcales and Cycloclasticus. Together with active transcription of genes for thiosulfate and methane oxidation in Methylococcales, these results suggest an adaptive strategy of versatile and simultaneous use of multiple available electron donors. Meanwhile, the first near-complete MAG of hydrothermal Methylophaga aminisulfidivorans and its transcriptional profile point to active chemotaxis targeting small organic compounds. Petroleum hydrocarbon-degrading Cycloclasticus are abundant and active in plumes of oil spills as well as deep-sea vents, suggesting that they are indigenous and effectively respond to stimulus of hydrocarbons in the deep sea. These findings suggest that these three groups of Gammaproteobacteria transform organic carbon and sulfur compounds via versatile and opportunistic metabolism and modulate biogeochemistry in plumes of hydrothermal systems as well as oil spills, thus contributing broad ecological impact to the deep ocean globally.

Crystal Structure of Cytochrome cL from the Aquatic Methylotrophic Bacterium Methylophaga aminisulfidivorans MPT.

Cytochrome cL (CytcL) is an essential protein in the process of methanol oxidation in methylotrophs. It receives an electron from the pyrroloquinoline quinone (PQQ) cofactor of methanol dehydrogenase (MDH) to produce formaldehyde. The direct electron transfer mechanism between CytcL and MDH remains unknown due to the lack of structural information. To help gain a better understanding of the mechanism, we determined the first crystal structure of heme c containing CytcL from the aquatic methylotrophic bacterium Methylophaga aminisulfidivorans MPT at 2.13 Å resolution. The crystal structure of Ma-CytcL revealed its unique features compared to those of the terrestrial homologues. Apart from Fe in heme, three additional metal ion binding sites for Na+ , Ca+ , and Fe2+ were found, wherein the ions mostly formed coordination bonds with the amino acid residues on the loop (G93-Y111) that interacts with heme. Therefore, these ions seemed to enhance the stability of heme insertion by increasing the loop's steadiness. The basic N-terminal end, together with helix α4 and loop (G126 to Y136), contributed positive charge to the region. In contrast, the acidic C-terminal end provided a negatively charged surface, yielding several electrostatic contact points with partner proteins for electron transfer. These exceptional features of Ma-CytcL, along with the structural information of MDH, led us to hypothesize the need for an adapter protein bridging MDH to CytcL within appropriate proximity for electron transfer. With this knowledge in mind, the methanol oxidation complex reconstitution in vitro could be utilized to produce metabolic intermediates at the industry level.

Elucidating Cysteine-Assisted Synthesis of Indirubin by a Flavin-Containing Monooxygenase

Indirubin is a biologically active compound found in Danggui Longhui Wan, which is a traditional Chinese medicine for chronic myelocytic leukemia. In the biosynthesis of indirubin, the formation of indigo, which is a stereoisomer of indirubin, is a major side reaction. Recent findings have suggested that cysteine supplementation shifts product selectivity from indigo to indirubin. Here, we disclose how cysteine is involved in enhancing the product selectivity in the synthesis of indirubin using a flavin-containing monooxygenase from Methylophaga aminisulfidivorans (MaFMO). First, cysteine reacts with indoxyl to synthesize 2-cysteinylindoleninone, inhibiting the dimerization of indoxyl. Second, the reducing power of cysteine allows MaFMO to additionally hydroxylate indoxyl toward isatin, overcoming the problem in biased distribution of two different precursors. Third, cysteine activates isatin to react with 2-cysteinylindoleninone to form indirubin. Based on this revealed mechanism, indirubin derivatives with different indole ring components were synthesized.

The crystal structure of methanol dehydrogenase, a quinoprotein from the marine methylotrophic bacterium Methylophaga aminisulfidivorans MPT.

The first crystal structure of a pyrroloquinoline quinone (PQQ)-dependent methanol dehydrogenase (MDH) from a marine methylotrophic bacterium, Methylophaga aminisulfidivorans MPT (MDH Mas ), was determined at 1.7 Å resolution. The active form of MDH Mas (or MDHI Mas ) is a heterotetrameric α2β2, where each β-subunit assembles on one side of each of the α-subunits, in a symmetrical fashion, so that two β-subunits surround the two PQQ-binding pockets on the α-subunits. The active site consists of a PQQ molecule surrounded by a β-propeller fold for each α-subunit. Interestingly, the PQQ molecules are coordinated by a Mg2+ ion, instead of the Ca2+ ion that is commonly found in the terrestrial MDHI, indicating the efficiency of osmotic balance regulation in the high salt environment. The overall interaction of the β-subunits with the α-subunits appears tighter than that of terrestrial homologues, suggesting the efficient maintenance of MDHI Mas integrity in the sea water environment to provide a firm basis for complex formation with MxaJ Mas or Cyt cL. With the help of the features mentioned above, our research may enable the elucidation of the full molecular mechanism of methanol oxidation by taking advantage of marine bacterium-originated proteins in the methanol oxidizing system (mox), including MxaJ, as the attainment of these proteins from terrestrial bacteria for structural studies has not been successful.

Metabolic engineering of Escherichia coli for the production of indirubin from glucose

Indirubin is an indole alkaloid that can be used to treat various diseases including granulocytic leukemia, cancer, and Alzheimer’s disease. Microbial production of indirubin has so far been achieved by supplementation of rather expensive substrates such as indole or tryptophan. Here, we report the development of metabolically engineered Escherichia coli strain capable of producing indirubin directly from glucose. First, the Methylophaga aminisulfidivorans flavin-containing monooxygenase (FMO) and E. coli tryptophanase (TnaA) were introduced into E. coli in order to complete the biosynthetic pathway from tryptophan to indirubin. Further engineering was performed through rational strategies including disruption of the regulatory repressor gene trpR and removal of feedback inhibitions on AroG and TrpE. Then, combinatorial approach was employed by systematically screening eight genes involved in the common aromatic amino acid pathway. Moreover, availability of the aromatic precursor substrates, phosphoenolpyruvate and erythrose-4-phosphate, was enhanced by inactivating the pykF (pyruvate kinase I) and pykA (pyruvate kinase II) genes, and by overexpressing the tktA gene (encoding transketolase), respectively. Fed-batch fermentation of the final engineered strain led to production of 0.056 g/L of indirubin directly from glucose. The metabolic engineering and synthetic biology strategies reported here thus allows microbial fermentative production of indirubin from glucose.

Exploring nicotinamide cofactor promiscuity in NAD(P)H-dependent flavin containing monooxygenases (FMOs) using natural variation within the phosphate binding loop. Structure and activity of FMOs from Cellvibrio sp. BR and Pseudomonas stutzeri NF13

Flavin-containing monooxygenases (FMOs) catalyse asymmetric oxidation reactions that have potential for preparative organic synthesis, but most use the more expensive, phosphorylated nicotinamide cofactor NADPH to reduce FAD to FADH2 prior to formation of the (hydro)peroxy intermediate required for substrate oxygenation. A comparison of the structures of NADPH-dependent FMO from Methylophaga aminisulfidivorans (mFMO) and SMFMO from Stenotrophomonas maltophilia, which is able to use both NADPH and NADH, suggested that the promiscuity of the latter enzyme may be due in part to the substitution of an Arg–Thr couple in the NADPH phosphate recognition site in mFMO, for a Gln–His couple in SMFMO (Jensen et al., 2012, Chembiochem, 13, 872–878). Natural variation within the phosphate binding region, and its influence on nicotinamide cofactor promiscuity, was explored through the cloning, expression, characterisation and structural studies of FMOs from Cellvibrio sp. BR (CFMO) and Pseudomonas stutzeri NF13 (PSFMO), which possess Thr–Ser and Gln–Glu in the putative phosphate recognition positions, respectively. CFMO and PSFMO displayed 5- and 1.5-fold greater activity, respectively, than SMFMO for the reduction of FAD with NADH, and were also cofactor promiscuous, displaying a ratio of activity with NADH:NADPH of 1.7:1 and 1:1.3, respectively. The structures of CFMO and PSFMO revealed the context of the phosphate binding loop in each case, and also clarified the structure of the mobile helix–loop–helix motif that appears to shield the FAD-binding pocket from bulk solvent in this class of FMOs, a feature that was absent from the structure of SMFMO.

Mutations of an NAD(P)H-dependent flavoprotein monooxygenase that influence cofactor promiscuity and enantioselectivity

The flavoprotein monooxygenase (FPMO) from Stenotrophomonas maltophilia (SMFMO, Uniprot: B2FLR2) catalyses the asymmetric oxidation of thioethers and is unusual amongst FPMOs in its ability to use the non-phosphorylated cofactor NADH, as well as NADPH, for the reduction of the FAD coenzyme. In order to explore the basis for cofactor promiscuity, structure-guided mutation of two residues in the cofactor binding site, Gln193 and His194, in SMFMO were performed in an attempt to imitate the cofactor binding site of the NADPH-dependent FMO from Methylophaga aminisulfidivorans sp. SK1 (mFMO), in which structurally homologous residues Arg234 and Thr235 bind the NADPH 2′-ribose phosphate. Mutation of His194 to threonine proved most significant, with a switch in specificity from NADH to NADPH [(kcat/Km NADH)/kcat/Km NADPH) from 1.5:1 to 1:3.5, mostly as a result of a reduced Km for NADPH of approximately sevenfold in the His194Thr mutant. The structure of the Gln193Arg/His194Thr mutant revealed no substantial changes in the backbone of the enzyme or orientation of side chains resulting from mutation. Mutation of Phe52, in the vicinity of FAD, and which in mFMO is an asparagine thought to be responsible for flavin hydroperoxide stabilisation, is, in SMFMO, a determinant of enantioselectivity in sulfoxidation. Mutation of Phe52 to valine resulted in a mutant that transformed para-tolyl methyl sulfide into the (S)-sulfoxide with 32% e.e., compared to 25% (R)- for the wild type. These results shed further light both on the cofactor specificity of FPMOs, and their determinants of enantioselectivity, with a view to informing engineering studies of FPMOs in the future.

Enhanced indirubin production in recombinant Escherichia coli harboring a flavin-containing monooxygenase gene by cysteine supplementation

In our previous study, a batch fermentation of recombinant Escherichia coli DH5α cells harboring the fmo gene from Methylophaga aminisulfidivorans MPT produced indirubin (5.0mg/L) and indigo (920mg/L) in a 5L fermenter containing tryptophan medium (2g/L tryptophan, 5g/L yeast extract, 10g/L NaCl). In this study, it was found that indirubin production greatly increased when 0.36g/L cysteine was added to the tryptophan medium, although cysteine inhibited the growth of the recombinant E. coli harboring the fmo gene. However, the addition of cysteine did not inhibit the expression level and activity of FMO in the cell. Indigo was synthesized by the dimerization of two 3-hydroxyindole molecules under the non-enzymatic reaction. Cysteine influenced the regioselectivity of FMO and enhanced the synthesis of 2-hydroxyindole instead of 3-hydroxyindole, which might function to increase indirubin production. The optimal culture conditions for indirubin production in tryptophan medium were determined from the response surface methodology analysis: 2g/L tryptophan, 5g/L yeast extract, 10g/L NaCl, 0.36g/L (3mM) cysteine, pH 8.0 at 35°C. Under these conditions, the recombinant E. coli cells were capable of producing 223.6mg/L of indirubin from 2g/L of tryptophan. The intracellular accumulation of the indirubin crystals might stress the cell, which may be a main reason for the poor growth of the recombinant E. coli pBlue 1.7.

Comparative analysis of two types of methanol dehydrogenase from Methylophaga aminisulfidivorans MPT grown on methanol

Two types of methanol dehydrogenase (MDH) were obtained from a novel marine methylotrophic bacterium, Methylophaga aminisulfidivorans MP(T), grown on methanol. Type I MDH consisted of two identical dimers of α (65.98 kDa) and β (7.58 kDa) subunits organized to form the α(2)β(2) tetramer. Type II MDH contained an additional MxaJ protein (27.86 kDa) and had more specific activity than type I MDH. The K(m) values of type I and II MDH for methanol under cytochrome c(L) reduction assay system were estimated to be 50.3 and 13.0 μM, respectively, and the isoelectric points of type I and II MDH were determined to be 5.4 and 5.8, respectively. The average molar ratios of α:β, α:MxaJ, and β:MxaJ in type II MDH were approximately 1:0.99, 1:0.41 and 1:0.42, respectively. Based on these results, the original conformation of the MDH of M. aminisulfidivorans MP(T) is most likely the α(2)β(2)-MxaJ complex. During purification, the lysozyme and freeze-thawing cell disruption method significantly increased the amount of type II MDH in the soluble fraction compared with strong physical disruption methods such as sonication and French Press.

Purification, crystallization and preliminary X-ray crystallographic analysis of a methanol dehydrogenase from the marine bacteriumMethylophaga aminisulfidivoransMPT

Methylophaga aminisulfidivorans MP(T) is a marine methylotrophic bacterium that utilizes C(1) compounds such as methanol as a carbon and energy source. The released electron from oxidation flows through a methanol-oxidizing system (MOX) consisting of a series of electron-transfer proteins encoded by the mox operon. One of the key enzymes in the pathway is methanol dehydrogenase (MDH), which contains the prosthetic group pyrroloquinoline quinone (PQQ) and converts methanol to formaldehyde in the periplasm by transferring two electrons from the oxidation of one methanol molecule to the electron acceptor cytochrome c(L). In order to obtain molecular insights into the oxidation mechanism, a native heterotetrameric α(2)β(2) MDH complex was directly purified from M. aminisulfidivorans MP(T) grown in the presence of methanol and crystallized. The crystal diffracted to 1.7 Å resolution and belonged to the monoclinic space group P2(1) (unit-cell parameters a = 63.9, b = 109.5, c = 95.6 Å, β = 100.5°). The asymmetric unit of the crystal contained one heterotetrameric complex, with a calculated Matthews coefficient of 2.24 Å(3) Da(-1) and a solvent content of 45.0%.

Optimization of bio-indigo production by recombinant E. coli harboring fmo gene

A bacterial flavin-containing monooxygenase (FMO) gene was cloned from Methylophaga aminisulfidivorans MP T, and a plasmid pBlue 2.0 was constructed to express the bacterial fmo gene in E. coli. To increase the production of bio-indigo, upstream sequence size of fmo gene was optimized and response surface methodology was used. The pBlue 1.7 plasmid (1686 bp) was prepared by the deletion of upstream sequence of pBlue 2.0. The recombinant E. coli harboring the pBlue 1.7 plasmid produced 662 mg l −1 of bio-indigo in tryptophan medium after 24 h of cultivation in flask. The production of bio-indigo was optimized using a response surface methodology with a 2 n central composite design. The optimal combination of media constituents for the maximum production of bio-indigo was determined as tryptophan 2.4 g l −1, yeast extract 4.5 g l −1 and sodium chloride 11.4 g l −1. In addition, the optimum culture temperature and pH were 30 °C and pH 7.0, respectively. Under the optimized conditions mentioned above, the recombinant E. coli harboring pBlue 1.7 plasmid produced 920 mg of bio-indigo per liter in optimum tryptophan medium after 24 h of cultivation in fermentor. The combination of truncated insert sizes and culture optimization resulted in a 575% increase in the production of bio-indigo.

Methylophaga aminisulfidivorans sp. nov., a restricted facultatively methylotrophic marine bacterium

A novel restricted facultatively methylotrophic marine strain, MP(T), possessing the ribulose monophosphate pathway of C(1)-carbon compound assimilation was isolated from a seawater sample obtained from Mokpo, South Korea. The novel isolate is aerobic, Gram-negative, asporogenous and a non-motile short rod. It grows well on methanol, methylated amines, dimethylsulfide and DMSO. Optimal growth occurs with 3 % NaCl at 30 degrees C and pH 7.0. Fructose is utilized as a multicarbon source. Growth factors are not required and vitamin B(12) does not stimulate growth. The cellular fatty acid profile of the novel strain consists primarily of straight-chain saturated C(16 : 0) and unsaturated C(16 : 1) acids. The major ubiquinone is Q-8. The dominant phospholipids are phosphatidylethanolamine and phosphatidylglycerol. The DNA G+C content is 44.9 mol% (T(m)). Based on 16S rRNA gene sequence analysis and DNA-DNA relatedness (25-41 %) with the type strains of marine methylotrophs belonging to the genus Methylophaga, it is suggested that isolate MP(T) represents a novel species, Methylophaga aminisulfidivorans sp. nov. (type strain MP(T)=KCTC 12909(T)=VKM B-2441(T)=JCM 14647(T)).