RESUMO
Aromatic amino acids and their derivatives are diverse primary and secondary metabolites with critical roles in protein synthesis, cell structure and integrity, defense and signaling. All de novo aromatic amino acid production relies on a set of ancient and highly conserved chemistries. Here we introduce a new enzymatic transformation for L-tyrosine synthesis by demonstrating that the ß-subunit of tryptophan synthase-which natively couples indole and L-serine to form L-tryptophan-can act as a latent 'tyrosine synthase'. A single substitution of a near-universally conserved catalytic residue unlocks activity toward simple phenol analogs and yields exclusive para carbon-carbon bond formation to furnish L-tyrosines. Structural and mechanistic studies show how a new active-site water molecule orients phenols for a nonnative mechanism of alkylation, with additional directed evolution resulting in a net >30,000-fold rate enhancement. This new biocatalyst can be used to efficiently prepare valuable L-tyrosine analogs at gram scales and provides the missing chemistry for a conceptually different pathway to L-tyrosine.
Assuntos
Triptofano Sintase , Tirosina , Triptofano Sintase/metabolismo , Triptofano Sintase/química , Tirosina/química , Tirosina/metabolismo , Domínio Catalítico , Modelos Moleculares , Tirosina Fenol-Liase/metabolismo , Tirosina Fenol-Liase/química , Tirosina Fenol-Liase/genética , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Biocatálise , Triptofano/química , Triptofano/metabolismoRESUMO
Pyridoxal-5'-phosphate (PLP) Schiff's bases of 2-aminoacrylate are intermediates in ß-elimination and ß-substitution reaction of PLP-dependent enzymes. These enzymes are found in two major families, the α-, or aminotransferase, superfamily, and the ß-family. While the α-family enzymes primarily catalyze ß-eliminations, the ß-family enzymes catalyze both ß-elimination and ß-substitution reactions. Tyrosine phenol-lyase (TPL), which catalyzes the reversible elimination of phenol from l-tyrosine, is an example of an α-family enzyme. Tryptophan synthase catalyzes the irreversible formation of l-tryptophan from l-serine and indole, and is an example of a ß-family enzyme. The identification and characterization of aminoacrylate intermediates in the reactions of both of these enzymes is discussed. The use of UV-visible absorption and fluorescence spectroscopy, X-ray and neutron crystallography, and NMR spectroscopy to identify aminoacrylate intermediates in these and other PLP enzymes is presented.
Assuntos
Triptofano Sintase , Tirosina Fenol-Liase , Humanos , Triptofano Sintase/química , Triptofano Sintase/metabolismo , Tirosina Fenol-Liase/química , Tirosina Fenol-Liase/metabolismo , Fosfato de Piridoxal/metabolismo , Catálise , Fosfatos , CinéticaRESUMO
L-DOPA is an amino acid that is used as a treatment for Parkinson's disease. A simple enzymatic synthesis method of L-DOPA had been developed using bacterial L-tyrosine phenol-lyase (Tpl). This review describes research on screening of bacterial strains, culture conditions, properties of the enzyme, reaction mechanism of the enzyme, and the reaction conditions for the production of L-DOPA. Furthermore, molecular bleeding of constitutively Tpl-overproducing strains is described, which were developed based on mutations in a DNA binding protein, TyrR, which controls the induction of tpl gene expression.
Assuntos
Tirosina Fenol-Liase , Tirosina Fenol-Liase/genética , Tirosina Fenol-Liase/metabolismo , Levodopa , BactériasRESUMO
The M379A mutant of Citrobacter freundii tyrosine phenol-lyase (TPL) has been prepared. M379A TPL is a robust catalyst to prepare a number of tyrosines substituted at the 3-position with bulky groups that cannot be made with wild type TPL. The three dimensional structures of M379A TPL complexed with L-methionine and 3-bromo-DL-phenylalanine have been determined by X-ray crystallography. Methionine is bound as a quinonoid complex in a closed active site in 3 of 4 chains of homotetrameric M379A TPL. M379A TPL reacts with L-methionine about 8-fold slower than wild type TPL. The temperature dependence shows that the slower reaction is due to less positive activation entropy. The structure of the M379A TPL complex of 3-bromo-DL-phenylalanine has a quinonoid complex in two subunits, with an open active site conformation. The effects of the M379A mutation on TPL suggest that the mutant enzyme has altered the conformational dynamics of the active site.
Assuntos
Tirosina Fenol-Liase , Domínio Catalítico , Citrobacter freundii/genética , Citrobacter freundii/metabolismo , Cinética , Metionina , Mutagênese Sítio-Dirigida , Fenilalanina/metabolismo , Tirosina/metabolismo , Tirosina Fenol-Liase/química , Tirosina Fenol-Liase/genética , Tirosina Fenol-Liase/metabolismoRESUMO
Tyrosine phenol-lyase (TPL) exhibits great potential in industrial biosynthesis of l-tyrosine and its derivates. To uncover and screen TPLs with excellent catalytic properties, there is unmet demand for development of facile and reliable screening system for TPL. Here we presented a novel assay format for the detection of TPL activity based on catechol 2,3-dioxygenase (C23O)-catalyzed reaction. Catechol released from TPL-catalyzed cleavage of 3,4-dihydroxy-l-phenylalanine (l-DOPA) was further oxidized by C23O to form 2-hydroxymuconate semialdehyde, which could be readily detected by spectrophotometric measurements at 375 nm. The assay achieved a unique balance between the ease of operation and superiority of analytical performances including linearity, sensitivity and accuracy. In addition, this assay enabled real-time monitoring of TPL activity with high efficiency and reliability. As C23O is highly specific towards catechol, a non-natural product of microorganism, the assay was therefore accessible to both crude cell extracts and the whole-cell system without elaborate purification steps of enzymes, which could greatly expedite discovery and engineering of TPLs. This study provided fundamental principle for high-throughput screening of other enzymes consuming or producing catechol derivatives.
Assuntos
Tirosina Fenol-LiaseRESUMO
OBJECTIVE: To solve the bottleneck of plasmid instability during microbial fermentation of L-DOPA with recombinant Escherichia coli expressing heterologous tyrosine phenol lyase. RESULTS: The tyrosine phenol lyase from Fusobacterium nucleatum was constitutively expressed in E. coli and a fed-batch fermentation process with temperature down-shift cultivation was performed. Efficient strategies including replacing the original ampicillin resistance gene, as well as inserting cer site that is active for resolving plasmid multimers were applied. As a result, the plasmid stability was increased. The co-use of cer site on plasmid and kanamycin in culture medium resulted in proportion of plasmid containing cells maintained at 100% after fermentation for 35 h. The specific activity of tyrosine phenol lyase reached 1493 U/g dcw, while the volumetric activity increased from 2943 to 14,408 U/L for L-DOPA biosynthesis. CONCLUSIONS: The established strategies for plasmid stability is not only promoted the applicability of the recombinant cells for L-DOPA production, but also provides important guidance for industrial fermentation with improved microbial productivity.
Assuntos
Escherichia coli/crescimento & desenvolvimento , Fusobacterium nucleatum/enzimologia , Levodopa/metabolismo , Plasmídeos/genética , Tirosina Fenol-Liase/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Técnicas de Cultura Celular por Lotes , Meios de Cultura/química , Escherichia coli/genética , Fermentação , Fusobacterium nucleatum/genética , Engenharia de Proteínas , Proteínas Recombinantes/metabolismo , Tirosina Fenol-Liase/metabolismoRESUMO
L-Tyrosine is a versatile compound used in the fine chemical, pharmaceutical, and functional food industries. Here, we report a bi-enzymatic cascade involving alanine racemase (ALR) and D-amino acid oxidase (DAAO) to produce pyruvate, as co-substrate for L-tyrosine production, from the cheap substrate L-alanine. The BpALR (ALR from Bacillus pseudofirmus) was used as a whole-cell biocatalyst, converting L-alanine to D, L-alanine. The FsDAAO (DAAO from Fusarium solani) was immobilized to oxidize the D-alanine generated in the first step to pyruvate. Both systems were combined as a continuous-flow reactor for maximized L-alanine-to-pyruvate conversion rates. The optimal parameters and appropriate conditions for FsDAAO immobilization were investigated. The pyruvate concentration of 86.6 g/L was achieved within 17 h. Subsequently, a whole-cell biocatalyst system for L-tyrosine production, catalyzed by the tyrosine phenol-lyase (TPL) from Erwinia herbicola (EhTPL), was developed, and a fed-batch approach was applied with phenol and the pyruvate produced with the ALR/DAAO system mentioned above. The concentration of phenol and pyruvate in the reactor should not exceed 7.5 g/L and 10 g/L, respectively. Significantly, the L-tyrosine concentration of 152.5 g/L was achieved within 10 h, demonstrating the great potential for high-efficiency production of L-tyrosine through the approach we established in this paper. Graphical abstract KEY POINTS: ⢠A specific bioreactor system for pyruvate produced from l-alanine was developed ⢠The appropriate condition for immobilization of FsDAAO was investigated ⢠A fed-batch process was established to produce l-tyrosine with recombinant E. coli ⢠The bi-enzymatic cascade was successfully used for l-tyrosine production at low cost.
Assuntos
Tirosina Fenol-Liase , Tirosina , Bacillus , Escherichia coli/genética , Fusarium , Ácido Pirúvico , Tirosina Fenol-Liase/genéticaRESUMO
The formation of inclusion bodies (IBs) without enzyme activity in bacterial research is generally undesirable. Researchers have attempted to recovery the enzyme activities of IBs, which are commonly known as active IBs. Tyrosine phenol-lyase (TPL) is an important enzyme that can convert pyruvate and phenol into 3,4-dihydroxyphenyl-L-alanine (L-DOPA) and IBs of TPL can commonly occur. To induce the correct folding and recover the enzyme activity of the IBs, peptides, such as ELK16, DKL6, L6KD, ELP10, ELP20, L6K2, EAK16, 18A, and GFIL16, were fused to the carboxyl terminus of TPL. The results showed that aggregate particles of TPL-DKL6, TPL-ELP10, TPL-EAK16, TPL-18A, and TPL-GFIL16 improved the enzyme activity by 40.9%, 50.7%, 48.9%, 86.6%, and 97.9%, respectively. The peptides TPL-DKL6, TPL-EAK16, TPL-18A, and TPL-GFIL16 displayed significantly improved thermostability compared with TPL. L-DOPA titer of TPL-ELP10, TPL-EAK16, TPL-18A, and TPL-GFIL16, with cells reaching 37.8 g/L, 53.8 g/L, 37.5 g/L, and 29.1 g/L, had an improvement of 111%, 201%, 109%, and 63%, respectively. A higher activity and L-DOPA titer of the TPL-EAK16 could be valuable for its industrial application to biosynthesize L-DOPA.
Assuntos
Escherichia coli/enzimologia , Corpos de Inclusão/metabolismo , Levodopa/biossíntese , Peptídeos/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Tirosina Fenol-Liase/metabolismo , Escherichia coli/genética , Engenharia Metabólica , Peptídeos/química , Dobramento de Proteína , Proteínas Recombinantes de Fusão/química , Tirosina Fenol-Liase/genéticaRESUMO
Pyridoxal 5'-phosphate (PLP) is an essential cofactor that participates in â¼4% enzymatic activities cataloged by the Enzyme Commission. The intracellular level of PLP is usually lower than that demanded in industrial catalysis. To realize the self-supply of PLP cofactor in whole-cell biotransformation, the de novo ribose 5-phosphate (R5P)-dependent PLP synthesis pathway was constructed. The pdxST genes from Bacillus subtilis 168 were introduced into the tyrosine phenol-lyase (TPL)-overexpressing Escherichia coli BL21(DE3) strain. TPL and PdxST were co-expressed with a double-promoter or a compatible double-plasmid system. The 3,4-dihydroxyphenylacetate-L-alanine (L-DOPA) titer did not increase with the increase in the intracellular PLP concentration in these strains with TPL and PdxST co-expression. Therefore, it is necessary to optimize the intracellular PLP metabolism level so as to achieve a higher L-DOPA titer and avoid the formation of L-DOPA-PLP cyclic adducts. The thi riboswitch binds to PLP and forms a complex such that the ribosome cannot have access to the Shine-Dalgarno (SD) sequence. Therefore, this metabolite-sensing regulation system was applied to regulate the translation of pdxST mRNA. Riboswitch was introduced into pET-TPL-pdxST-2 to downregulate the expression of PdxST and biosynthesis of PLP at the translation level by sequestering the ribosome-binding site. As a result, the titer and productivity of L-DOPA using the strain BL21-TPLST-Ribo1 improved to 69.8â¯g/L and 13.96â¯g/L/h, respectively, with a catechol conversion of 95.9% and intracellular PLP accumulation of 24.8 µM.
Assuntos
Escherichia coli/genética , Levodopa , Fosfato de Piridoxal , Riboswitch/genética , Biotransformação , Escherichia coli/metabolismo , Levodopa/análise , Levodopa/genética , Levodopa/metabolismo , Fosfato de Piridoxal/biossíntese , Fosfato de Piridoxal/química , Fosfato de Piridoxal/genética , Fosfato de Piridoxal/metabolismo , Tirosina Fenol-Liase/química , Tirosina Fenol-Liase/genética , Tirosina Fenol-Liase/metabolismoRESUMO
Genetic circuit-based biosensors have emerged as an effective analytical tool in synthetic biology; these biosensors can be applied to high-throughput screening of new biocatalysts and metabolic pathways. Sigma 54 (σ54)-dependent transcription factor (TF) can be a valuable component of these biosensors owing to its intrinsic silent property compared to most of the housekeeping sigma 70 (σ70) TFs. Here, we show that these unique characteristics of σ54-dependent TFs can be used to control the host cell state to be more appropriate for high-throughput screening. The acclimation of cell state was achieved by using guanosine (penta)tetraphosphate ((p)ppGpp)-related genes (relA, spoT) and nutrient conditions, to link the σ54 TF-based reporter expression with the target enzyme activity. By controlling stringent programmed responses and optimizing assay conditions, catalytically improved tyrosine phenol lyase (TPL) enzymes were successfully obtained using a σ54-dependent DmpR as the TF component, demonstrating the practical feasibility of this biosensor. This combinatorial strategy of biosensors using σ factor-dependent TFs will allow for more effective high-throughput enzyme engineering with broad applicability.
Assuntos
Proteínas de Bactérias/genética , Ensaios de Triagem em Larga Escala/métodos , Engenharia de Proteínas/métodos , Transativadores/genética , Ativação Transcricional , Tirosina Fenol-Liase/genética , Aclimatação , Técnicas Biossensoriais/métodos , Escherichia coli , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , GTP Pirofosfoquinase/genética , GTP Pirofosfoquinase/metabolismo , Regiões Promotoras Genéticas , Pseudomonas putida , Pirofosfatases/genética , Pirofosfatases/metabolismo , Fator sigma/genética , Fator sigma/metabolismo , Tirosina Fenol-Liase/metabolismoRESUMO
Tyrosine phenol-lyase (TPL) is a valuable and cost-effective biocatalyst for the biosynthesis of L-tyrosine and its derivatives, which are valuable intermediates in the pharmaceutical industry. A TPL from Morganella morganii (Mm-TPL) was overexpressed in Escherichia coli and characterized. Mm-TPL was determined as a homotetramer with molecular weight of 52 kDa per subunit. Its optimal temperature and pH for ß-elimination of L-tyrosine were 45 °C and pH 8.5, respectively. Mm-TPL manifested strict substrate specificity for the reverse reaction of ß-elimination and ortho- and meta-substituted phenols with small steric size were preferred substrates. The enzyme showed excellent catalytic performance for synthesis of L-tyrosine, 3-fluoro-L-tyrosine, and L-DOPA with a yield of 98.1%, 95.1%, and 87.2%, respectively. Furthermore, the fed-batch bioprocess displayed space-time yields of 9.6 g L-1 h-1 for L-tyrosine and 4.2 g L-1 h-1 for 3-fluoro-L-tyrosine with a yield of 67.4 g L-1 and 29.5 g L-1, respectively. These results demonstrated the great potential of Mm-TPL for industrial application.
Assuntos
Proteínas de Bactérias/química , Morganella morganii/enzimologia , Tirosina Fenol-Liase/química , Catálise , Células Imobilizadas/enzimologia , Citrobacter freundii/enzimologia , Escherichia coli , Concentração de Íons de Hidrogênio , Íons , Cinética , Levodopa/química , Peso Molecular , Domínios Proteicos , Estrutura Secundária de Proteína , Especificidade por Substrato , Temperatura , Tirosina/análogos & derivados , Tirosina/químicaRESUMO
3,4-Dihydroxyphenyl-L-alanine (L-DOPA) is the most important antiparkinsonian drug, and tyrosine phenol-lyase (TPL)-based enzyme catalysis process is one of the most adopted methods on industrial scale production. TPL activity and stability represent the rate-limiting step in L-DOPA synthesis. Here, 25â¯TPL mutants were predicted, and two were confirmed as exhibiting the highest L-DOPA production and named E313W and E313M. The L-DOPA production from E313W and E313M was 47.5â¯g/L and 62.1â¯g/L, which was 110.2 % and 174.8 % higher, respectively, than that observed from wild-type (WT) TPL. The Km of E313W and E313M showed no apparent decrease, whereas the kcat of E313W and E313M improved by 45.5 % and 36.4 %, respectively, relative to WT TPL. Additionally, E313W and E313M displayed improved thermostability, a higher melting temperature, and enhanced affinity between for pyridoxal-5'-phosphate. Structural analysis of the mutants suggested increased stability of the N-terminal region via enhanced interactions between the mutated residues and H317. Application of these mutants in a substrate fed-batch strategy as whole-cell biocatalysts allows realization of a cost-efficient short fermentation period resulting in high L-DOPA yield.
Assuntos
Proteínas de Bactérias , Mutagênese Sítio-Dirigida , Tirosina Fenol-Liase , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Citrobacter freundii/enzimologia , Citrobacter freundii/genética , Estabilidade Enzimática , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Tirosina Fenol-Liase/química , Tirosina Fenol-Liase/genéticaRESUMO
L-DOPA (L-dihydroxyphenylalanine) is a promising drug for Parkinson's disease and thereby has a growing annual demand. Tyrosine phenol-lyase (TPL)-based catalysis is considered to be a low-cost yet efficient route for biosynthesis of L-DOPA. TPL is a tetrameric enzyme that catalyzes the synthesis of L-DOPA from pyrocatechol, sodium pyruvate, and ammonium acetate. The implementation of TPL for L-DOPA production has been hampered and the need for the most efficient TPL source with higher L-DOPA production and substrate conversion rate is prevailing. This study involves identifying a novel TPL from Kluyvera intermedia (Ki-TPL) and displayed a robust expression in Escherichia coli. The recombinant strain YW000 carrying Ki-TPL proved strong catalytic activity with a highest L-DOPA yield compared with 16 other TPLs from different organisms. With a further aim to improve this efficiency, random mutagenesis of Ki-TPL was performed and a mutant namely YW021 was obtained. The whole cells of YW021 as biocatalyst yielded 150.4 g L-1 of L-DOPA with a 99.99 % of pyrocatechol conversion at the optimum condition of pH 8.0 at 25 °C, which is the highest level reported to date. Further, the homology modeling and structural analysis revealed the mutant residues responsible for the extensive L-DOPA biosynthesis.
Assuntos
Biocatálise , Escherichia coli/metabolismo , Kluyvera/enzimologia , Levodopa/química , Tirosina Fenol-Liase/genética , Acetatos/química , Catecóis/química , Clonagem Molecular , Escherichia coli/genética , Concentração de Íons de Hidrogênio , Mutagênese , Mutação , Fosfato de Piridoxal/química , Ácido Pirúvico/química , Sódio/química , TemperaturaRESUMO
Herein we report the development of an efficient cellular system for the in vivo biosynthesis of Tyr-analogs and their concurrent incorporation into target proteins by the residue-specific approach. This system makes use of common phenol derivatives and the tyrosine phenol lyase machinery to create various tyrosine analogues that impart desired properties on the target proteins. Biosynthesized 2-fluorotyrosine was incorporated into three industrially important enzymes which resulted in enhanced thermostability.
Assuntos
Engenharia de Proteínas , Tirosina Fenol-Liase/metabolismo , Tirosina/biossíntese , Biocatálise , Fluorometria , Oxirredutases/genética , Oxirredutases/metabolismo , Transaminases/genética , Transaminases/metabolismo , Tirosina/análogos & derivados , Tirosina Fenol-Liase/genéticaRESUMO
L-tyrosine is an amino acid that has been widely used in the food, agriculture and pharmaceutical industries. In order to screen a tyrosine phenol-lyase (TPL) with excellent catalytic performance for L-tyrosine production, TPL genes from Citrobacter freundii (CfTPL), Erwinia herbicola (EhTPL) and Rhodobacter capsulatus (TutA) were codon-optimized and overexpressed in Escherichia coli. The results showed that EhTPL had the highest whole cell catalysis activity and tyrosine yield (3-fold that of CfTPL). The results of RT-qPCR and a stability analysis also revealed that EhTPL had a higher transcriptional level in whole cell catalysis, while CfTPL possessed greater stability. Conditions for the production by whole cell transformation were optimized in terms of reaction conditions and fed-batch strategy. Finally, the maximum production was obtained with a titer of 48.5â¯g·L-1 by intermittent feeding with a conversion ratio of 75%.
Assuntos
Escherichia coli/metabolismo , Proteínas Recombinantes/metabolismo , Tirosina Fenol-Liase/metabolismo , Tirosina/metabolismo , Biotransformação , Citrobacter freundii/enzimologia , Erwinia/enzimologia , Escherichia coli/genética , Perfilação da Expressão Gênica , Proteínas Recombinantes/genética , Rhodobacter capsulatus/enzimologia , Transcrição Gênica , Tirosina Fenol-Liase/genéticaRESUMO
L-DOPA is a key pharmaceutical agent for treating Parkinson's, and market demand has exploded due to the aging population. There are several challenges associated with the chemical synthesis of L-DOPA, including complicated operation, harsh conditions, and serious pollution. A biocatalysis route for L-DOPA production is promising, especially via a route catalyzed by tyrosine phenol lyase (TPL). In this study, using TPL derived from Erwinia herbicola (Eh-TPL), a mutant Eh-TPL was obtained by integrating enzyme evolution and high-throughput screening methods. L-DOPA production using recombinant Escherichia coli BL21 (DE3) cells harbouring mutant Eh-TPL was enhanced by 36.5% in shake flasks, and the temperature range and alkali resistance of the Eh-TPL mutant were promoted. Sequence analysis revealed two mutated amino acids in the mutant (S20C and N161S), which reduced the length of a hydrogen bond and generated new hydrogen bonds. Using a fed-batch mode for whole-cell catalysis in a 5 L bioreactor, the titre of L-DOPA reached 69.1 g L-1 with high productivity of 11.52 g L-1 h-1, demonstrating the great potential of Eh-TPL variants for industrial production of L-DOPA.
Assuntos
Levodopa/biossíntese , Tirosina Fenol-Liase/metabolismo , Biocatálise , Reatores Biológicos , Escherichia coli/genética , Escherichia coli/metabolismoRESUMO
D-danshensu (D-DSS), extracted from the plant Salvia miltiorrhiza (Danshen), is widely used to treat cardiovascular and cerebrovascular diseases. Here we engineered Escherichia coli strains to produce D-DSS from catechol, pyruvate and ammonia by one-pot biotransformation. Tyrosin-phenol lyase (TPL), L-amino acid deaminase (aadL), D-lactate dehydrogenase (ldhD) and glucose dehydrogenase (gdh) genes were overexpressed in Escherichia coli strain. First, the expression of genes was regulated by different copy number plasmids combination, the result of E. coli TALG6, with strong overexpression of TPL, aadL, ldhD and moderate overexpression of gdh, exhibited 253.7% increase D-DSS production compared to E. coli TALG1. Second, the optimum concentration of catechol was found to be 50 mM. Finally, a fed-batch biotransformation strategy was proposed, namely the amount of catechol was added to 50 mM every 2 h. The total production of D-DSS reached 55.35 mM within 14 h, which was 1.7 times that without feeding.
Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , Lactatos/metabolismo , Engenharia Metabólica , Amônia/metabolismo , Técnicas de Cultura Celular por Lotes , Biotransformação , Catecóis/metabolismo , Escherichia coli/enzimologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Glucose 1-Desidrogenase/genética , Glucose 1-Desidrogenase/metabolismo , L-Aminoácido Oxidase/genética , L-Aminoácido Oxidase/metabolismo , L-Lactato Desidrogenase/genética , L-Lactato Desidrogenase/metabolismo , Plasmídeos/genética , Ácido Pirúvico/metabolismo , Tirosina Fenol-Liase/genética , Tirosina Fenol-Liase/metabolismoRESUMO
Diabetic kidney disease is a major cause of renal failure that urgently necessitates a breakthrough in disease management. Here we show using untargeted metabolomics that levels of phenyl sulfate, a gut microbiota-derived metabolite, increase with the progression of diabetes in rats overexpressing human uremic toxin transporter SLCO4C1 in the kidney, and are decreased in rats with limited proteinuria. In experimental models of diabetes, phenyl sulfate administration induces albuminuria and podocyte damage. In a diabetic patient cohort, phenyl sulfate levels significantly correlate with basal and predicted 2-year progression of albuminuria in patients with microalbuminuria. Inhibition of tyrosine phenol-lyase, a bacterial enzyme responsible for the synthesis of phenol from dietary tyrosine before it is metabolized into phenyl sulfate in the liver, reduces albuminuria in diabetic mice. Together, our results suggest that phenyl sulfate contributes to albuminuria and could be used as a disease marker and future therapeutic target in diabetic kidney disease.
Assuntos
Albuminúria/etiologia , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Tipo 1/complicações , Diabetes Mellitus Tipo 2/complicações , Nefropatias Diabéticas/sangue , Microbioma Gastrointestinal/fisiologia , Ésteres do Ácido Sulfúrico/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Albuminúria/sangue , Albuminúria/tratamento farmacológico , Albuminúria/patologia , Animais , Animais Geneticamente Modificados , Estudos de Coortes , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/urina , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 2/sangue , Nefropatias Diabéticas/etiologia , Nefropatias Diabéticas/patologia , Cães , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Feminino , Humanos , Células Madin Darby de Rim Canino , Masculino , Metabolômica/métodos , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Transportadores de Ânions Orgânicos/genética , Podócitos/metabolismo , Podócitos/patologia , Ratos , Estreptozocina/toxicidade , Ésteres do Ácido Sulfúrico/sangue , Tirosina Fenol-Liase/antagonistas & inibidores , Tirosina Fenol-Liase/metabolismo , Adulto JovemRESUMO
Tyrosine phenol lyase (TPL) is a robust biocatalyst for the production of L-dihydroxyphenylalanine (L-DOPA). The improvement of TPL production is conducive to the industrial potential. In this study, the optimization of culture medium of recombinant Escherichia coli harboring TPL from Fusobacterium nucleatum (Fn-TPL) was carried out. Sucrose and combination of yeast extract and peptone were selected as carbon and nitrogen source, respectively. Their optimal concentrations were determined by Box-Behnken design and the synergistic effect between yeast extract and peptone was found to be significant, with p-value < 0.05. The DO-STAT fed-batch fermentation under optimized culture condition was established and the oxygen level was fixed at 20%. Both the biomass and Fn-TPL activity were significantly increased, which were 35.6 g dcw/L and 12292 U/L, respectively. The results obtained significantly promote the industrial production of L-DOPA production.
Assuntos
Proteínas de Bactérias/metabolismo , Técnicas de Cultura Celular por Lotes/métodos , Meios de Cultura/metabolismo , Escherichia coli/metabolismo , Fusobacterium nucleatum/metabolismo , Levodopa/metabolismo , Tirosina Fenol-Liase/metabolismo , Carbono/metabolismo , Fermentação , Microbiologia Industrial/métodos , Nitrogênio/metabolismo , Peptonas/metabolismo , Proteínas Recombinantes/metabolismoRESUMO
The radical S-adenosylmethionine (SAM) superfamily is currently the largest known enzyme family. These enzymes reductively cleave SAM to produce a highly reactive 5'-deoxyadenosyl (dAdo) radical, which abstracts a hydrogen from the substrate and initiates diverse reactions. The canonic dAdo radical-mediated hydrogen abstraction can be changed to radical addition reactions by using olefin-containing substrate analogues, which result in adenosylation reactions. Here we report investigation of the adenosylation reactions catalyzed by four radical SAM l-Tyr lyases (RSTLs), including HydG, FbiC, and two ThiH enzymes from different organisms. We show RSTLs have diverse substrate specificity, and ThiH from E. coli exhibits the highest substrate tolerance toward the tested substrates. We also show ThiH from Clostridium berjerinckii does not act on 4-amino-l-phenylalanine, but catalyzes adenosylation of the corresponding olefin-containing analogue, suggesting adenosylation may occur more easily than the canonic radical SAM reactions. Our study highlights the remarkable catalytic promiscuity of radical SAM enzyme and the potential in using these enzymes for the synthesis of nucleotide-containing compounds.