Tyrosine Phenol-lyase Research Articles

A computational strategy to improve the activity of tyrosine phenol-lyase for the synthesis of L-DOPA

Enzymes with high catalytic activity and stability are essential for industrial production, yet most natural enzymes do not meet these requirements. Therefore, efficient strategies for enzyme engineering are crucial. In this study, we developed a cost-effective computational design strategy to enhance the activity of tyrosine phenol-lyase (TPL) for the production of L-DOPA. By integrating structural analysis with computational design, and guided by our understanding of conformational flexibility of TPL, we identified a region where enhanced stability is most likely to facilitate enzyme activity. We screened stabilizing mutations by Cartesian_ddg in Rosetta. After identifying single stabilizing mutations, we grouped the nearby positions harboring multiple stabilizing mutations and calculated the energy of combinatorial variants. We found two promising groups where most variants exhibited lower calculated energy than the wild-type. Experimental validation showed five variants in these groups exhibit increased activity, with the two best variants showing catalytic activity enhancements of 1.8-fold and 1.6-fold compared to the wild-type enzyme.

Lignin-based monophenolic model compounds in L-tyrosine derivative synthesis via tyrosine phenol lyase

Tyrosine phenol lyase (TPL) synthesises L-tyrosine derivatives from monophenols, pyruvate and ammonia. Production of such high-value aromatic chemicals from biomass-derived raw materials is of great interest. In this study, six monophenols (guaiacol, phenol, o-cresol, m-cresol, catechol and syringol) were chosen based on the structure of lignin and were studied as substrates in the enzymatic reaction. Single monophenol reactions (SMR) and binary monophenol reactions (BMR) with guaiacol were carried out. TPL-M379V was found to be selective towards guaiacol (84.5 % conv.). The highest single activity was measured towards phenol (93.9 % conv.). However, the enzyme preferred guaiacol over phenol in the BMRs. Syringol was found to be inert in the reaction, whereas catechol had an inhibitory effect on the enzymatic reaction, in addition to causing degradation of all the substrates in the medium. Doubling the guaiacol concentration in the SMR did not significantly increase the production of 3-O-methyldopa (conv. 45.9 %). However, in the binary reaction systems the total monophenol conversions were higher with guaiacol and phenol (total 62.4 %) or o-cresol (total 57.1 %). This indicates possible substrate/product specific inhibition. The study provides new data on activity, selectivity and inhibitory effects of monophenols in the synthetic reaction catalysed by TPL-M379V, especially in mixed-substrate reactions.

Tyrosine phenol-lyase inhibitor quercetin reduces fecal phenol levels in mice.

Tyrosine phenol-lyase (TPL), which is expressed in intestinal bacteria, catalyzes the formation of phenol from the substrate L-Tyr. Bacterial metabolite phenol and the sulfate conjugate (phenyl sulfate) are known as a type of uremic toxins, some of which exert cytotoxicity. Therefore, pathologically elevated phenol and phenyl sulfate levels are strongly implicated in the etiology and outcome of uremia. In this study, we explored the inhibitory effects of dietary polyphenols on TPL-catalyzed phenol production using a TPL activity assay. Quercetin, one of the most popular polyphenols, exhibited the strongest inhibitory activity (Ki = 19.9 µM). Quercetin competitively inhibited TPL, and its activity was stronger than that of a known TPL inhibitor (Tyr analog; 2-aza-Tyr, Ki = 42.0 µM). Additionally, quercetin significantly inhibited phenol production in TPL-expressing bacterial cultures (Morganella morganii and Citrobacter koseri) and Tyr-rich (5%) diet-fed C57BL/6J mouse feces. Our findings suggest that quercetin is the most promising polyphenol for reducing phenol levels. Because quercetin has a low gastrointestinal absorption rate, TPL inhibition in the intestinal tract by quercetin may be an effective strategy for treating uremia.

Abstract 1375 Enzymatic Synthesis of Naphthol-Based Unnatural Amino Acids using Tyrosine Phenol Lyase

Abstract 1375 Enzymatic Synthesis of Naphthol-Based Unnatural Amino Acids using Tyrosine Phenol Lyase

High-level phenol bioproduction by engineered Pichia pastoris in glycerol fed-batch fermentation using an efficient pertraction system

This study aimed to establish a high-level phenol bioproduction system from glycerol through metabolic engineering of the yeast Pichia pastoris (Komagataella phaffii). Introducing tyrosine phenol-lyase to P. pastoris led to a production of 59 mg/L of phenol in flask culture. By employing a strain of P. pastoris that overproduces tyrosine—a precursor to phenol—we achieved a phenol production of 1052 mg/L in glycerol fed-batch fermentation. However, phenol concentrations exceeding 1000 mg/L inhibited P. pastoris growth. A phenol pertraction system utilizing a hollow fiber membrane contactor and tributyrin as the organic solvent was developed to reduce phenol concentration in the culture medium. Integrating this system with glycerol fed-batch fermentation resulted in a 214 % increase in phenol titer (3304 mg/L) compared to glycerol fed-batch fermentation alone. These approaches offer a significant framework for the microbial production of chemicals and materials that are highly toxic to microorganisms.

Therapy for CKD and DKD

Diabetic kidney disease is a major cause of renal failure that urgently necessitates a breakthrough in disease management. Specific remedies are needed for preventing Type 2 diabetes which causes significant changes in an array of plasma metabolites. By untargeted metabolome analysis, phenyl sulfate (PS) increased with the progression of diabetes. In experimental diabetes models, PS administration induces albuminuria and podocyte damage due to the mitochondrial dysfunction. By clinical diabetic kidney disease (DKD) cohort analysis, it was also confirmed that the PS levels significantly correlate with basal and predicted 2-year progression of albuminuria. Phenol is synthesized from dietary tyrosine by gut bacterial-specific tyrosine phenol-lyase (TPL), and absorbed phenol is metabolized into PS in the liver. Inhibition of TPL reduces not only the circulating PS level but also albuminuria in diabetic mice. TPL inhibitor did not significantly alter the major composition, showing the non-lethal inhibition of microbial-specific enzymes has a therapeutic advantage, with lower selective pressure for the development of drug resistance. Clinically, 362 patients in a multi-center clinical study in diabetic nephropathy cohort (U-CARE) were analyzed with full data. The basal plasma PS level significantly correlated with ACR, eGFR, age, duration, HbA1c and uric acid, but not with suPAR. Multiple regression analysis revealed that ACR was the only factor that significantly correlated with PS. By stratified logistic regression analysis, in the microalbuminuria group, PS was the only factor related to the amount of change in the 2-year ACR in all models. PS is not only an early diagnosis marker, but also a modifiable cause and therefore a target for the treatment of DKD. Reduction of microbiota-derived phenol by the inhibitor should represent another aspect for developing drugs of DKD prevention.

A process for p-hydroxystyrene production from glycerol based on cell-free biosynthesis system

As an essential chemical, p-hydroxystyrene has attracted much attention and is widely used in the field of semiconductors. However, the current production methods have some disadvantages, such as low molar conversion yield, p-hydroxystyrene toxicity, and high substrate cost. To overcome these problems, an five enzymes cell-free cascade, consisting of alditol oxidase from Streptomyces coelicolor A3 (ScALDO), dehydratase from Paralcaligenes ureilyticus (PuDHT), tyrosine phenol lyase from Citrobacter freundii (CfTPL), tyrosine ammonia lyase from Rhodotorula glutinis (RgTAL) and phenylacrylate decarboxylase from Enterobacter sp. (EsPAD), was constructed for the de novo biosynthesis of p-hydroxystyrene from glycerol. Through process strategy optimization, a molar conversion yield of more than 84.2% was finally obtained from 10 mM crude glycerol. It is worth mentioning that the reaction system performed an excellent titer using 50 mM crude glycerol, and the p-hydroxystyrene concentration reached 30.32 mM, which exceeded the maximum titer of fermentation. This showed that our system is robust to p-hydroxystyrene and crude glycerol. In addition, a ‘two-step purification process’ was designed to produce pure p-hydroxystyrene. This is the first study focused on the cell-free biosynthesis of p-hydroxystyrene, and the highest conversion yield and titer thus far was obtained for p-hydroxystyrene de novo biosynthesis.

Synthesis of l-DOPA catalyzed by recombinant Escherichia coli expressing tyrosine phenol lyase gene from Desulfitobacterium hafniense

L-DOPA (L-dihydroxyphenylalanine) is a potential drug for the treatment of Parkinson’s disease, and the demand is increasing every year. Tyrosine phenol-lyase (TPL) is a valuable biocatalyst for the biosynthesis of L-tyrosine and its derivatives, which are valuable intermediates in the pharmaceutical industry. In this study, a new TPL gene (Dh-TPL) was cloned from Desulfitobacterium hafniense, ligated into pJJDuet30 vector, and successfully expressed in Escherichia coli. To increase the yield and stability of L-DOPA, as well as decrease the by-product formation, the enzyme production conditions and the catalytic reaction conditions were studied. The optimal TPL production conditions were as follows: the concentration of IPTG was 0.2 mmol/L and the induction temperature was controlled at 18°C. The optimum medium composition for recombinant bacteria includes yeast extract 64 g/L, tryptone 36 g/L, glycerol 4 g/L, KH2PO4 2.31 g/L and K2HPO4 9.4 g/L. The best biosynthesis of L-DOPA was performed in a reaction mixture containing 10.0 g/L catechol, 20 g/L sodium pyruvate and 20 g/L recombinant E. coli resting cells. The optimal reaction temperature and pH were determined to be 18°C and pH 8.5, respectively. Under these conditions, the yield of L-DOPA was 0.21 mol/L (41.7 g/L) after 3 hours reaction.

Predictive markers for efficiency of the amino-acid deprivation therapies in cancer

Amino acid deprivation therapy (AADT) is a promising strategy for developing novel anticancer treatments, based on variations in metabolism of healthy and malignant cells. L-asparaginase was the first amino acid-degrading enzyme that received FDA approval for the treatment of acute lymphoblastic leukemia (ALL). Arginase and arginine deiminase were effective in clinical trials for the treatment of metastatic melanomas and hepatocellular carcinomas. Essential dependence of certain cancer cells on methionine explains the anticancer efficacy of methionine-g-lyase. Along with significant progress in identification of metabolic vulnerabilities of cancer cells, new amino acid-cleaving enzymes appear as promising agents for cancer treatment: lysine oxidase, tyrosine phenol-lyase, cysteinase, and phenylalanine ammonia-lyase. However, sensitivity of specific cancer cell types to these enzymes differs. Hence, search for prognostic and predictive markers for AADT and introduction of the markers into clinical practice are of great importance for translational medicine. As specific metabolic pathways in cancer cells are determined by the enzyme expression, some of these enzymes may define the sensitivity to AADT. This review considers the known predictors for efficiency of AADT, emphasizing the importance of knowledge on cancer-specific amino acid significance for such predictions.

Development of a single culture E. coli expression system for the enzymatic synthesis of fluorinated tyrosine and its incorporation into proteins

Current experiments that rely on biosynthetic metabolic protein labeling with 19F often require fluorinated amino acids, which in the case of 2- and 3-fluorotyrosine can be expensive. However, using these amino acids has provided valuable insight into protein dynamics, structure, and function. Here, we develop a new in-cell method for fluorinated tyrosine generation from readily available substituted phenols and subsequent metabolic labeling of proteins in a single bacterial expression culture. This approach uses a dual-gene plasmid encoding for a model protein BRD4(D1) and a tyrosine phenol lyase from Citrobacter freundii, which catalyzes the formation of tyrosine from phenol, pyruvate, and ammonium. Our system demonstrated both enzymatic fluorotyrosine production and expression of 19F-labeled proteins as analyzed by 19F NMR and LC-MS methods. Further optimization of our system should provide a cost-effective alternative to a variety of traditional protein-labeling strategies.

M379A Mutant Tyrosine Phenol‐lyase from Citrobacter freundii Has Altered Conformational Dynamics

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.

High-throughput assay of tyrosine phenol-lyase activity using a cascade of enzymatic reactions

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.

Rapid production of l-DOPA by Vibrio natriegens, an emerging next-generation whole-cell catalysis chassis.

Summary3, 4‐Dihydroxyphenyl‐l‐alanine (l‐DOPA) is a compound of high medical value and is considered effective as a treatment for Parkinson’s disease. Currently, bioproduction of l‐DOPA is mainly carried out by whole‐cell catalysis mediated by recombinant Escherichia coli carrying heterogeneous tyrosine phenol lyase. Vibrio natriegens is increasingly attracting attention owing to its superiority, including extremely rapid growth and high soluble protein expression capacity. In this study, we attempt to develop an efficient whole‐cell catalyst for l‐DOPA production using V. natriegens as the chassis. The maximum soluble protein expression by V. natriegens was accomplished in 4 h at 37°C, which was equivalent to that achieved by E. coli in 16 h at 16°C. Furthermore, the maximum productivity reached over 10.0 g l−1 h−1 in the early stage of biocatalysis, nearly two‐fold higher than previously reported. Approximately 54.0 g l−1 l‐DOPA was obtained with a catechol conversion rate greater than 95%. In conclusion, V. natriegens displays advantages, including rapid protein expression and catalytic rate in the catalysis process for l‐DOPA production. These findings strongly suggest that V. natriegens has remarkable potential as a whole‐cell catalysis chassis for the production of valuable chemicals.

Constitutive expression of tyrosine phenol-lyase from Erwinia herbicola in Escherichia coli for l-DOPA production

l-DOPA is a major drug for the treatment of Parkinson’s disease, and microbial enzymatic synthesis is an essential way for its industrial production. The expression of tyrosine phenol-lyase (TPL) derived from Erwinia herbicola under six different constitutive promoters in Escherichia coli was studied. The recombinant strain 5 can express TPL well with growth. The optimal medium composition of this strain was as follows: tryptone 18 g/L, yeast extract 28 g/L, glycerol 10 g/L. After the cell culture is completed, the collected cells were used to catalyze the production of l-DOPA, and the concentration can reach 67.9 g/L.

Expanding the Use of Tyrosine Phenol Lyase in the Enzymatic Synthesis of Unnatural Amino Acids

The use of enzymes in the synthesis of pharmaceuticals and biochemical tools offers an attractive approach to generating complex structures, as enzymatic reactions often lead to the formation of specific products in high yields. Tyrosine phenol lyase (TPL), an enzyme biologically involved in the degradation of tyrosine, has been used to synthesize a variety of substituted tyrosines for biochemical studies. Expanding the scope of substrates to naphthol-based compounds or substituted tyrosine-based structures offers an attractive target for enzymatic synthesis of complex molecules which can then be used for in potential pharmaceuticals. Using Citrobacter freundii TPL, we enzymatically synthesized the naphthol-analog of tyrosine, but product was in low yield. The simplest model for the low conversion is that this result suggests the larger substrate is poorly accommodated by the enzyme active site and this poor binding leads to the low yield in the enzymatic reaction. To test this hypothesis, we mutated several bulky residues near the substrate-binding site (M288, M397, F448) to open space for substrate binding and facilitate synthesis of the unnatural amino acid. A series of mutant plasmids were generated using site-directed mutagenesis and sequenced to confirm the mutation. The wild type and mutant enzymes were recombinantly expressed in E. coli and subjected to affinity and ion-exchange chromatography. Although gel electrophoresis of the samples showed a single band and initial enzyme assays with tyrosine showed that the wild type and mutants are active, subsequent size-exclusion chromatography assays suggested the samples contain potential different multimeric forms of the enzyme. As previous studies suggested TPL is tetrameric enzyme, we are currently developing additional purification steps to isolate the tetrameric form of the wild type and mutants. The purified enzyme will then be used to evaluate the effects of the mutations on enzyme activity and substrate specificity. While a goal of this study is aimed at the more efficient production of tyrosine derivatives, a larger goal is to better understand the features important for substrate binding in enzymatic reactions. This understanding may aid in the development of enzymes with a broad range of substrates efficiently converted to products in the biochemical generation of complex molecules.

Efficient strategies to enhance plasmid stability for fermentation of recombinant Escherichia coli harboring tyrosine phenol lyase.

To solve the bottleneck of plasmid instability during microbial fermentation of L-DOPA with recombinant Escherichia coli expressing heterologous tyrosine phenol lyase. 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 35h. The specific activity oftyrosine phenol lyase reached 1493U/g dcw, while the volumetric activity increased from 2943 to 14,408U/L for L-DOPA biosynthesis. 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.

Antagonistic Control of Genetic Circuit Performance for Rapid Analysis of Targeted Enzyme Activity in Living Cells.

Genetic circuits have been developed for quantitative measurement of enzyme activity, metabolic engineering of strain development, and dynamic regulation of microbial cells. A genetic circuit consists of several bio-elements, including enzymes and regulatory cassettes, that can generate the desired output signal, which is then used as a precise criterion for enzyme screening and engineering. Antagonists and inhibitors are small molecules with inhibitory effects on regulators and enzymes, respectively. In this study, an antagonist and an inhibitor were applied to a genetic circuit for a dynamic detection range. We developed a genetic circuit relying on regulators and enzymes, allowing for straightforward control of its output signal without additional genetic modification. We used para-nitrophenol and alanine as an antagonist of DmpR and inhibitor of tyrosine phenol-lyase, respectively. We show that the antagonist resets the detection range of the genetic circuit similarly to a resistor in an electrical logic circuit. These biological resistors in genetic circuits can be used as a rapid and precise controller of variable outputs with minimal circuit configuration.

Synthesis of l-Tyrosine by Pichia pastoris Displaying Tyrosine Phenol Lyase

l-Tyrosine, which is widely used in the medical field and the food and chemical industries, is a high-value-added compound. In this research, we explored the ability to synthesize l-tyrosine from p...

A bi-enzymatic cascade to yield pyruvate as co-substrate for L-tyrosine production.

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 86.6g/L was achieved within 17h. 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 7.5g/L and 10g/L, respectively. Significantly, the L-tyrosine concentration of 152.5g/L was achieved within 10h, 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.

Active tyrosine phenol-lyase aggregates induced by terminally attached functional peptides in Escherichia coli

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.