RESUMEN
Salinipeptins belong to the type-A linaridin class of ribosomally synthesized and post-translationally modified peptides (RiPPs) comprising 22 amino acid residues with multiple D-amino acids. Although chirality of other type-A linaridins, such as grisemycin and cypemycin, has not been reported, the biosynthetic gene clusters of type-A linaridins have identical gene organization. Here, we report heterologous expression of grisemycin biosynthetic gene cluster (grm) and show that grisemycin contains multiple D-amino acids, similar to salinipeptins. The heterologous expression experiments also confirm the involvement of a novel peptide epimerase in grisemycin biosynthesis. Gene-deletion experiments indicate that grmL, a single gene with unknown function, is indispensable for grisemycin production. We also show that the presence of D-amino acids is likely a common feature of linaridin natural products by analyzing two other type-A linaridin clusters.
Asunto(s)
Procesamiento Proteico-Postraduccional , Racemasas y Epimerasas , Aminoácidos/metabolismo , Familia de Multigenes , Péptidos/química , Racemasas y Epimerasas/genética , Racemasas y Epimerasas/metabolismoRESUMEN
Plant tyrosine decarboxylase (TyrDC) is a group II pyridoxal 5'-phosphate (PLP)-dependent decarboxylase that mainly catalyzes the decarboxylation of tyrosine to tyramine. This is biologically important for diverting essential primary metabolites into secondary metabolic pathways. Intensive studies have characterized the effective of PLP-binding and the substrate specificity of mammalian 3,4-dihydroxyphenyl-l-alanine (Dopa) decarboxylases, a member of group II PLP-dependent decarboxylase. However, the characteristics of PLP binding and substrate specificity of plant TyrDCs remain unknown. In this study, we focus on the PLP binding manner, and determined the crystal structures of the apo and PLP binding form of type II TyrDC from Papaver somniferum (PsTyrDCII and PsTyrDCII-PLP). The structures showed that, unlike mammalian Dopa decarboxylase, the binding of PLP does not induce distinct conformational changes of PsTyrDCII regarding the overall structure, but the PLP binding pocket displays conformational changes at Phe124, His203 and Thr262. Combining structural comparation and the obtained biochemical findings, it is demonstrated that PsTyrDCII does not binds PLP tightly. Such characteristics of PLP binding may be required by its catalytic reaction and substrate binding. The activity of TyrDC probably regulated by the concentration of PLP in cells.
Asunto(s)
Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Fosfato de Piridoxal/metabolismo , Tirosina Descarboxilasa/química , Tirosina Descarboxilasa/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Modelos Moleculares , Papaver/enzimología , Proteínas de Plantas/genética , Conformación Proteica , Fosfato de Piridoxal/química , Tirosina Descarboxilasa/genéticaRESUMEN
To establish a reliable and practical ergothioneine (ERG) supply, we employed fermentative ERG production using Aspergillus oryzae, a fungus used for food production. We heterologously overexpressed the egt-1 and -2 genes of Neurospora crassa in A. oryzae and succeeded in producing ERG (231.0 mg/kg of media, which was 20 times higher than the wild type). Abbreviations: ERG: ergothioneine; HER: hercynine; Cys-HER: hercynylcysteine-sulfoxide; SAM: S-adenosylmethionine; SAH: S-adenosylhomocysteine; l-His: l-histidine; l-Cys: l-cysteine; LC-ESI-MS: liquid chromatography-electrospray ionization-mass spectrometry.
Asunto(s)
Aspergillus oryzae/metabolismo , Ergotioneína/biosíntesis , Antioxidantes/metabolismo , Cromatografía Liquida , Ergotioneína/genética , Fermentación , Genes Fúngicos , Neurospora crassa/genética , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and arachidonic acid (ARA) are essential fatty acids for humans. Some microorganisms biosynthesize these PUFAs through PUFA synthases composed of four subunits with multiple catalytic domains. These PUFA synthases each create a specific PUFA without undesirable byproducts, even though the multiple catalytic domains in each large subunit are very similar. However, the detailed biosynthetic pathways and mechanisms for controlling final-product profiles are still obscure. In this study, the FabA-type dehydratase domain (DHFabA ) in the C-subunit and the polyketide synthase-type dehydratase domain (DHPKS ) in the B-subunit of ARA synthase were revealed to be essential for ARA biosynthesis by in vivo gene exchange assays. Furthermore, in vitro analysis with truncated recombinant enzymes and C4 - to C8 -acyl ACP substrates showed that ARA and EPA synthases utilized two types of DH domains, DHPKS and DHFabA , depending on the carbon-chain length, to introduce either saturation or cis double bonds to growing acyl chains.
Asunto(s)
Ácido Graso Sintasas/metabolismo , Ácidos Grasos Insaturados/biosíntesis , Ácidos Grasos Insaturados/química , Cromatografía de Gases y Espectrometría de Masas , Estructura Molecular , EstereoisomerismoRESUMEN
Polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are essential fatty acids. PUFA synthases are composed of three to four subunits and each create a specific PUFA without undesirable byproducts. However, detailed biosynthetic mechanisms for controlling final product profiles have been obscure. Here, the bacterial DHA and EPA synthases were carefully dissected by in vivo and in vitro experiments. In vitro analysis with two KS domains (KSA and KSC ) and acyl-acyl carrier protein (ACP) substrates showed that KSA accepted short- to medium-chain substrates while KSC accepted medium- to long-chain substrates. Unexpectedly, condensation from C18 to C20 , the last elongation step in EPA biosynthesis, was catalyzed by KSA domains in both EPA and DHA synthases. Conversely, condensation from C20 to C22 , the last elongation step for DHA biosynthesis, was catalyzed by the KSC domain in DHA synthase. KSC domains therefore determine the chain lengths.
Asunto(s)
Carbono/metabolismo , Ácidos Grasos Insaturados/metabolismo , HumanosRESUMEN
d-Glutamate (Glu) supplied by Glu racemases or d-amino acid transaminase is utilized for peptidoglycan biosynthesis in microorganisms. Comparative genomics has shown that some microorganisms, including Xanthomonas oryzae, perhaps have no orthologues of these genes. We performed shotgun cloning experiments with a d-Glu auxotrophic Escherichia coli mutant as the host and X. oryzae as the DNA donor. We obtained complementary genes, XOO_1319 and XOO_1320, which are annotated as a hypothetical protein and MurD (UDP-MurNAc-l-Ala-d-Glu synthetase), respectively. By detailed in vitro analysis, we revealed that XOO_1320 is an enzyme to ligate l-Glu to UDP-MurNAc-l-Ala, providing the first example of MurD utilizing l-Glu, and that XOO_1319 is a novel enzyme catalyzing epimerization of the terminal l-Glu of the product in the presence of ATP and Mg2+. We investigated the occurrence of XOO_1319 orthologues and found that it exists in some categories of microorganisms, including pathogenic ones.
Asunto(s)
Gammaproteobacteria/metabolismo , Ácido Glutámico/metabolismo , Glicopéptidos/metabolismo , Peptidoglicano/biosíntesis , Racemasas y Epimerasas/metabolismo , Gammaproteobacteria/química , Ácido Glutámico/química , Glicopéptidos/química , Peptidoglicano/químicaRESUMEN
Peptide antibiotics are typically biosynthesized by one of two distinct machineries in a ribosome-dependent or ribosome-independent manner. Pheganomycin (PGM (1)) and related analogs consist of the nonproteinogenic amino acid (S)-2-(3,5-dihydroxy-4-hydroxymethyl)phenyl-2-guanidinoacetic acid (2) and a proteinogenic core peptide, making their origin uncertain. We report the identification of the biosynthetic gene cluster from Streptomyces cirratus responsible for PGM production. Unexpectedly, the cluster contains a gene encoding multiple precursor peptides along with several genes plausibly encoding enzymes for the synthesis of amino acid 2. We identified PGM1, which has an ATP-grasp domain, as potentially capable of linking the precursor peptides with 2, and validate this hypothesis using deletion mutants and in vitro reconstitution. We document PGM1's substrate permissivity, which could be rationalized by a large binding pocket as confirmed via structural and mutagenesis experiments. This is to our knowledge the first example of cooperative peptide synthesis achieved by ribosomes and peptide ligases using a peptide nucleophile.
Asunto(s)
Antibacterianos/biosíntesis , Ligasas/metabolismo , Péptidos/metabolismo , Ribosomas/metabolismo , Ligasas/genética , Modelos Moleculares , Datos de Secuencia Molecular , Familia de Multigenes , Mutagénesis , Conformación Proteica , Streptomyces/genética , Streptomyces/metabolismoRESUMEN
Polyhydroxyalkanoates (PHAs) incorporating vinyl-bearing 3-hydroxyalkanoates were prepared in 8.5-12.9 g L(-1) yield. The molar ratios (0-16 mol%) of the vinyl-bearing 3-hydroxyalkanoate derivatives were controlled by the continuous feeding of undecylenate at various concentrations. Subsequently, the PHAs were functionalized by UV-initiated thiol-ene click reaction and chemical modification. (1)H NMR spectra suggested that 3-mercaptopropionic acid and 2-aminoethanethiol were successfully introduced into the vinyl-bearing PHA. Subsequently, chemical modification using fluorescein or a fibronectin active fragment (GRGDS) was attempted. The former yielded a PHA derivative capable of emitting fluorescence under UV irradiation, which was useful for determining the miscibility of PHA in a composite film comprising poly-Ê-lactic acid (PLLA) and PHA. In the latter case, PHA bearing GRGDS peptides exhibited cell adhesiveness, suggesting that its biocompatibility was improved upon peptide introduction. Taken together, the UV-initiated thiol-ene click reaction was demonstrated to be useful in PHA modification.
Asunto(s)
Química Clic/métodos , Polihidroxialcanoatos/química , Rayos Ultravioleta , Ácido 3-Mercaptopropiónico/química , Cisteamina/química , Fibronectinas/química , Fluoresceína/química , Células Endoteliales de la Vena Umbilical Humana , Humanos , Espectroscopía de Resonancia Magnética , Peso Molecular , Poliésteres/químicaRESUMEN
Tyrosol (4-hydroxyphenylethanol) is a phenolic compound used in the pharmaceutical and chemical industries. However, current supply methods, such as extraction from natural resources and chemical synthesis, have disadvantages from the viewpoint of cost and environmental protection. Here, we developed a tyrosol-producing Escherichia coli cell factory from a high-tyrosine-producing strain by expressing selected tyrosine decarboxylase-, tyramine oxidase (TYO)-, and medium-chain dehydrogenase/reductase (YahK)-encoding genes. The genes were controlled by the strong T7 promoter and integrated into the chromosome because of the advantages over plasmid-based systems. The strain produced a melanin-like pigment as a by-product, which is suggested to be formed from 4-hydroxyphenylacetaldehyde (a TYO product/YahK substrate). By using a culture medium containing a high concentration of glycerol, which was reported to enhance NADH supply required for YahK activity, the final titer of tyrosol reached 2.42 g/L in test tube-scale cultivation with a concomitant decrease in the amount of pigment. These results indicate that chromosomally integrated and T7 promoter-controlled gene expression system in E. coli is useful for high production of heterologous enzymes and might be applied for industrial production of useful compounds including tyrosine and tyrosol.
Asunto(s)
Escherichia coli , Alcohol Feniletílico/análogos & derivados , Tirosina , Escherichia coli/genética , Escherichia coli/metabolismo , Tirosina/metabolismo , Tirosina Descarboxilasa/genética , Tirosina Descarboxilasa/metabolismo , Monoaminooxidasa/genética , Monoaminooxidasa/metabolismo , Ingeniería MetabólicaRESUMEN
The cellulose synthesizing terminal complex consisting of subunits A, B, C, and D in Acetobacter xylinum spans the outer and inner cell membranes to synthesize and extrude glucan chains, which are assembled into subelementary fibrils and further into a ribbon. We determined the structures of subunit D (AxCeSD/AxBcsD) with both N- and C-terminal His(6) tags, and in complex with cellopentaose. The structure of AxCeSD shows an exquisite cylinder shape (height: â¼65 Å, outer diameter: â¼90 Å, and inner diameter: â¼25 Å) with a right-hand twisted dimer interface on the cylinder wall, formed by octamer as a functional unit. All N termini of the octamer are positioned inside the AxCeSD cylinder and create four passageways. The location of cellopentaoses in the complex structure suggests that four glucan chains are extruded individually through their own passageway along the dimer interface in a twisted manner. The complex structure also shows that the N-terminal loop, especially residue Lys6, seems to be important for cellulose production, as confirmed by in vivo assay using mutant cells with axcesD gene disruption and N-terminus truncation. Taking all results together, a model of the bacterial terminal complex is discussed.
Asunto(s)
Gluconacetobacter xylinus/enzimología , Glucosiltransferasas/química , Modelos Moleculares , Conformación Proteica , Dispersión de Radiación , Difracción de Rayos XRESUMEN
BACKGROUND: Aromatic compounds derived from tyrosine are important and diverse chemicals that have industrial and commercial applications. Although these aromatic compounds can be obtained by extraction from natural producers, their growth is slow, and their content is low. To overcome these problems, many of them have been chemically synthesized from petroleum-based feedstocks. However, because of the environmental burden and depleting availability of feedstock, microbial cell factories are attracting much attention as sustainable and environmentally friendly processes. RESULTS: To facilitate development of microbial cell factories for producing tyrosine derivatives, we developed simple and convenient tyrosine-producing Escherichia coli platforms with a bacterial phenylalanine hydroxylase, which converted phenylalanine to tyrosine with tetrahydromonapterin as a cofactor, using a synthetic biology approach. By introducing a tetrahydrobiopterin-regeneration system, the tyrosine titer of the plasmid-based engineered strain was 4.63 g/L in a medium supplemented with 5.00 g/L phenylalanine with a test tube. The strains were successfully used to produce industrially attractive compounds, such as tyrosol with a yield of 1.58 g/L by installing a tyrosol-producing module consisting of genes encoding tyrosine decarboxylase and tyramine oxidase on a plasmid. Gene integration into E. coli chromosomes has an advantage over the use of plasmids because it increases genetic stability without antibiotic feeding to the culture media and enables more flexible pathway engineering by accepting more plasmids with artificial pathway genes. Therefore, we constructed a plasmid-free tyrosine-producing platform by integrating five modules, comprising genes encoding the phenylalanine hydroxylase and tetrahydrobiopterin-regeneration system, into the chromosome. The platform strain could produce 1.04 g/L of 3,4-dihydroxyphenylalanine, a drug medicine, by installing a gene encoding tyrosine hydroxylase and the tetrahydrobiopterin-regeneration system on a plasmid. Moreover, by installing the tyrosol-producing module, tyrosol was produced with a yield of 1.28 g/L. CONCLUSIONS: We developed novel E. coli platforms for producing tyrosine from phenylalanine at multi-gram-per-liter levels in test-tube cultivation. The platforms allowed development and evaluation of microbial cell factories installing various designed tyrosine-derivative biosynthetic pathways at multi-grams-per-liter levels in test tubes.
RESUMEN
In this study, Corynebacterium glutamicum was engineered to produce ergothioneine, an amino acid derivative with high antioxidant activity. The ergothioneine biosynthesis genes, egtABCDE, from Mycolicibacterium smegmatis were introduced into wild-type and l-cysteine-producing strains of C. glutamicum to evaluate their ergothioneine production. In the l-cysteine-producing strain, ergothioneine production reached approximately 40 mg L-1 after 2 weeks, and the amount was higher than that in the wild-type strain. As C. glutamicum possesses an ortholog of M. smegmatis egtA, which encodes an enzyme responsible for γ-glutamyl-l-cysteine synthesis, the effect of introducing egtBCDE genes on ergothioneine production in the l-cysteine-producing strain was evaluated, revealing that a further increase to more than 70 mg L-1 was achieved. As EgtBs from Methylobacterium bacteria are reported to use l-cysteine as a sulfur donor in ergothioneine biosynthesis, egtB from Methylobacterium was expressed with M. smegmatis egtDE in the l-cysteine-producing strain. As a result, ergothioneine production was further improved to approximately 100 mg L-1. These results indicate that utilization of the l-cysteine-producing strain and introduction of heterologous biosynthesis pathways from M. smegmatis and Methylobacterium bacteria are effective for improved ergothioneine production by C. glutamicum.
Asunto(s)
Corynebacterium glutamicum , Ergotioneína , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Cisteína/metabolismo , Antioxidantes/metabolismo , Ingeniería Metabólica/métodosRESUMEN
The hydroxylation of tyrosine is an important reaction in the biosynthesis of many natural products. The use of bacteria for this reaction has not been very successful due to either the over-oxidation to ortho-quinone when using tyrosinases from bacteria or plants, or the lack of the native cofactor, tetrahydrobiopterin (BH4), needed for the activity of tyrosine hydroxylases (TH). Here, we demonstrate that an Escherichia coli cofactor, tetrahydromonapterin (MH4), can be used as an alternative cofactor for TH in presence of the BH4 regeneration pathway, and tyrosine hydroxylation is performed without over-oxidation. We used this platform for biosynthesis of one of the most powerful antioxidants, hydroxytyrosol. An endogenous aromatic aldehyde oxidase was identified and knocked out to prevent formation of the side product, and this resulted in nearly exclusive production of hydroxytyrosol in engineered E. coli. Finally, hydroxytyrosol production from a simple sugar as a sole carbon source was demonstrated.
Asunto(s)
Escherichia coli/fisiología , Glucosa/metabolismo , Alcohol Feniletílico/análogos & derivados , Ingeniería de Proteínas/métodos , Tirosina 3-Monooxigenasa/fisiología , Tirosina/metabolismo , Animales , Clonación Molecular , Ratones , Oxidación-Reducción , Alcohol Feniletílico/aislamiento & purificación , Alcohol Feniletílico/metabolismo , Proteínas Recombinantes/metabolismoRESUMEN
Recently, we succeeded in isolating a thermotolerant bacterium, Pseudomonas sp. SG4502, which is capable of accumulating polyhydroxyalkanoate (PHA) even at 55 °C, as a source of thermostable enzymes. In this study, we cloned a pha locus from the bacterium and identified two genes encoding PHA synthases (PhaC1(SG) and PhaC2(SG)). Two mutations, Ser324Thr and Gln480Lys, corresponding to those of a lactate (LA)-polymerizing enzyme (LPE) from mesophilic Pseudomonas sp. 61-3 were introduced into PhaC1(SG) to evaluate the potential of the resulting protein as a "thermostable LPE". The mutated PhaC1(SG) [PhaC1(SG)(STQK)] showed high thermal stability in synthesizing P(LA-co-3HB) in an in vitro reaction system under a range of high temperatures. Requirement of 3HBCoA as a priming unit for LA polymerization by the LPE has been suggested in both of the in vitro and in vivo experiments. Based on the finding, the PhaC1(SG)(STQK)-mediated synthesis of a LA-based copolymer with a block sequence was achieved in the in vitro system by sequential feeding of the corresponding two substrates. This in vitro reaction system using the thermostable LPE provides us with a versatile way to synthesize the various types of LA-based copolymers with desired sequence patterns, random or block, depending on the way of supplying hydroxyalkanoates (mixed or sequential feeding).
Asunto(s)
Aciltransferasas/metabolismo , Ácido Láctico/metabolismo , Polihidroxialcanoatos/metabolismo , Pseudomonas/enzimología , Aciltransferasas/química , Aciltransferasas/genética , Aciltransferasas/aislamiento & purificación , Sustitución de Aminoácidos , Clonación Molecular , Estabilidad de Enzimas , Calor , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/aislamiento & purificación , Proteínas Mutantes/metabolismo , Mutación Puntual , Pseudomonas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
A previously established improved two-phase reaction system has been applied to analyze the substrate specificities and polymerization activities of polyhydroxyalkanoate (PHA) synthases. We first analyzed the substrate specificity of propionate coenzyme A (CoA) transferase and found that 2-hydroxybutyrate (2HB) was converted into its CoA derivative. Then, the synthesis of PHA incorporating 2HB was achieved by a wild-type class I PHA synthase from Ralstonia eutropha. The PHA synthase stereoselectively polymerized (R)-2HB, and the maximal molar ratio of 2HB in the polymer was 9 mol%. The yields and the molecular weights of the products were decreased with the increase of the (R)-2HB concentration in the reaction mixture. The weight-average molecular weight of the polymer incorporating 9 mol% 2HB was 1.00 × 10(5), and a unimodal peak with polydispersity of 3.1 was observed in the GPC chart. Thermal properties of the polymer incorporating 9 mol% 2HB were analyzed by DSC and TG-DTA. T (g), T (m), and T (d) (10%) were observed at -1.1°C, 158.8°C, and 252.7°C, respectively. In general, major components of PHAs are 3-hydroxyalkanoates, and only engineered class II PHA synthases have been reported as enzymes having the ability to polymerize HA with the hydroxyl group at C2 position. Thus, this is the first report to demonstrate that wild-type class I PHA synthase was able to polymerize 2HB.
Asunto(s)
Aciltransferasas/aislamiento & purificación , Aciltransferasas/metabolismo , Cupriavidus necator/enzimología , Hidroxibutiratos/metabolismo , Polihidroxialcanoatos/metabolismo , Aciltransferasas/química , Coenzima A/metabolismo , Hidroxibutiratos/química , Cinética , Peso Molecular , Polihidroxialcanoatos/química , Propionatos/metabolismo , Estereoisomerismo , Especificidad por Sustrato , TemperaturaRESUMEN
Polylactate (PLA) is synthesized as a representative bio-based polyester by the chemo-bio process on the basis of metal catalyst-mediated chemical polymerization of lactate (LA) supplied by microbial fermentation. To establish the one-step microbial process for synthesis of LA-based polyesters, we explored whether polyhydroxyalkanoate (PHA) synthase would exhibit polymerizing activity toward a LA-coenzyme A (CoA), based on the fact that PHA monomeric constituents, especially 3-hydroxybutyrate (3HB), are structurally analogous to LA. An engineered PHA synthase was discovered as a candidate by a two-phase in vitro polymerization system previously developed. An LA-CoA producing Escherichia coli strain with a CoA transferase gene was constructed, and the generation of LA-CoA was demonstrated by capillary electrophoresis/MS analysis. Next, when the engineered PHA synthase gene was introduced into the resultant recombinant strain, we confirmed the one-step biosynthesis of the LA-incorporated copolyester, P(6 mol% LA-co-94 mol% 3HB), with a number-average molecular weight of 1.9 x 10(5), as revealed by gel permeation chromatography, gas chromatography/MS, and NMR.
Asunto(s)
Aciltransferasas/metabolismo , Coenzima A/metabolismo , Escherichia coli/metabolismo , Ácido Láctico/biosíntesis , Aciltransferasas/genética , Cromatografía de Gases , Electroforesis Capilar , Escherichia coli/genética , Espectrometría de Masas , Resonancia Magnética Nuclear Biomolecular , Poliésteres , Polímeros , Ingeniería de ProteínasRESUMEN
Polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid, eicosapentaenoic acid, and arachidonic acid are essential fatty acids for humans. PUFAs are biosynthesized by either desaturases/elongases from oleic acid or PUFA synthases from acetyl units. PUFA synthases are composed of three or four subunits, and each creates a specific PUFA even though the multiple catalytic domains in each subunit are very similar. We recently dissected these PUFA synthases by in vivo and in vitro experiments and elucidated how the enzymes control PUFA profiles. Moreover, for the first time, we converted a practical microalgal docosahexaenoic acid synthase into an eicosapentaenoic acid synthase based on the results.
Asunto(s)
Ácido Graso Sintasas/metabolismo , Ácidos Grasos Insaturados/metabolismo , Animales , Vías Biosintéticas , Ácido Graso Sintasas/genética , Ácidos Grasos Insaturados/genética , Regulación de la Expresión Génica , Humanos , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismoRESUMEN
Marine microorganisms de novo biosynthesize polyunsaturated fatty acids such as docosahexaenoic acid and eicosapentaenoic acid by polyunsaturated fatty acid (PUFA) synthases composed of three or four polypeptides in a manner similar to fatty acid synthases (FASs). FASs usually possess thioesterase (TE) domains to release free fatty acids from acyl carrier protein (ACP)-tethered intermediates. Here, we investigated the off-loading mechanism with microalgal and bacterial PUFA synthases through in vivo and in vitro experiments. The in vitro experiments with acyltransferase (AT)-like domains and acyl-ACP substrates clearly demonstrated that the AT-like domains catalyzed the hydrolysis of acyl-ACPs to yield free fatty acids.
Asunto(s)
Ácido Graso Sintasas/metabolismo , Ácidos Grasos Insaturados/química , Ácidos Grasos/química , Proteínas Recombinantes/metabolismo , Proteína Transportadora de Acilo/metabolismo , Secuencia de Aminoácidos , Catálisis , Ácidos Docosahexaenoicos/química , Ácido Eicosapentaenoico/química , Escherichia coli/genética , Ácido Graso Sintasas/genética , Hidrólisis , Palmitoil-CoA Hidrolasa/metabolismo , Dominios Proteicos , Proteínas Recombinantes/genética , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
We previously constructed a heterologous production system for ergothioneine (ERG) in Escherichia coli using five ERG biosynthesis genes (egtABCDE) from Mycobacterium smegmatis. However, significant amounts of hercynine (HER), an intermediate of ERG, as ERG were accumulated, suggesting that the reaction of EgtB catalyzing the attachment of γ-glutamylcysteine (γGC) to HER to yield hercynyl-γ-glutamylcysteine sulfoxide was a bottleneck. In this study, we searched for other EgtBs and found many egtB orthologs in diverse microorganisms. Among these, Methylobacterium strains possessed EgtBs that catalyze the direct conversion of HER into hercynylcysteine sulfoxide with l-cysteine (l-Cys) as a sulfur donor, in a manner similar to those of acidobacterial CthEgtB and fungal Egt1. An in vitro study with recombinant EgtBs from Methylobacterium brachiatum and Methylobacterium pseudosasicola clearly showed that both enzymes accepted l-Cys but not γGC. We reconstituted the ERG production system in E. coli with egtB from M. pseudosasicola; ERG productivity reached 657 mg L-1.
Asunto(s)
Proteínas Bacterianas/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Methylobacterium/enzimología , Sulfóxidos/metabolismo , Proteínas Bacterianas/metabolismo , Betaína/análogos & derivados , Betaína/metabolismo , Vías Biosintéticas , Dipéptidos/metabolismo , Ergotioneína/biosíntesis , Histidina/análogos & derivados , Histidina/metabolismo , Ingeniería Metabólica , Methylobacterium/genéticaRESUMEN
We recently revealed that a previously unknown pathway for peptidoglycan biosynthesis operates in some microorganisms, including Xanthomonas oryzae. It involves two enzymes, MurD2 and MurL, which catalyze the ligation of l-glutamate (l-Glu) to UDP- N-acetylmuramic acid-l-alanine and the epimerization of the terminal l-Glu of the product, respectively. MurD2 of X. oryzae possesses a 26% identity with MurD of Escherichia coli (MurDec), which ligates d-Glu to UDP- N-acetylmuramic acid-l-alanine. To understand how X. oryzae MurD2 recognizes the isomer substrate, we estimated its structure based on that of MurDec during docking simulations. Several amino acid residues, which may be responsible for l-Glu recognition, were replaced with their corresponding amino acid residues in MurDec. Consequently, we obtained a mutated MurD2 enzyme that contained two amino acid substitutions and accepted only d-Glu as the substrate. We next tried to convert the substrate specificity of MurDec using the same strategy, but the mutant enzyme still accepted only d-Glu. Then, MurD of Streptococcus mutans (MurDsm), which possesses the key amino acid residue for l-Glu recognition identified in MurD2, was used for random screenings of mutant enzymes accepting l-Glu. We obtained a mutated MurDsm that had one amino acid substitution and slightly accepted l-Glu. A mutated MurDec possessing the corresponding one amino acid substitution also accepted l-Glu. Thus, we revealed that a few amino acid residues in MurD/MurD2 might control the acceptability of substrates with different stereochemistries.