One-Pot Biocatalytic In Vivo Methylation-Hydroamination of Bioderived Lignin Monomers to Generate a Key Precursor to L-DOPA.

Electron-rich phenolic substrates can be derived from the depolymerisation of lignin feedstocks. Direct biotransformations of the hydroxycinnamic acid monomers obtained can be exploited to produce high-value chemicals, such as α-amino acids, however the reaction is often hampered by the chemical autooxidation in alkaline or harsh reaction media. Regioselective O-methyltransferases (OMTs) are ubiquitous enzymes in natural secondary metabolic pathways utilising an expensive co-substrate S-adenosyl-l-methionine (SAM) as the methylating reagent altering the physicochemical properties of the hydroxycinnamic acids. In this study, we engineered an OMT to accept a variety of electron-rich phenolic substrates, modified a commercial E. coli strain BL21 (DE3) to regenerate SAM in vivo, and combined it with an engineered ammonia lyase to partake in a one-pot, two whole cell enzyme cascade to produce the l-DOPA precursor l-veratrylglycine from lignin-derived ferulic acid.

Sorting for secreted molecule production using a biosensor-in-microdroplet approach.

Sorting large libraries of cells for improved small molecule secretion is throughput limited. Here, we combine producer/secretor cell libraries with whole-cell biosensors using a microfluidic-based screening workflow. This approach enables a mix-and-match capability using off-the-shelf biosensors through either coencapsulation or pico-injection. We demonstrate the cell type and library agnostic nature of this workflow by utilizing single-guide RNA, transposon, and ethyl-methyl sulfonate mutagenesis libraries across three distinct microbes (Escherichia coli, Saccharomyces cerevisiae, and Yarrowia lipolytica), biosensors from two organisms (E. coli and S. cerevisiae), and three products (triacetic acid lactone, naringenin, and L-DOPA) to identify targets improving production/secretion.

Cost-Effective Production of L-DOPA by Tyrosinase-Immobilized Polyhydroxyalkanoate Nanogranules in Engineered Halomonas bluephagenesis TD01.

3,4-dihydroxyphenyl-L-alanine (L-DOPA) is a preferred drug for Parkinson's disease, with an increasing demand worldwide that mainly relies on costly and environmentally problematic chemical synthesis. Yet, biological L-DOPA production is unfeasible at the industrial scale due to its low L-DOPA yield and high production cost. In this study, low-cost Halomonas bluephagenesis TD01 was engineered to produce tyrosinase TyrVs-immobilized polyhydroxyalkanoate (PHA) nanogranules in vivo, with the improved PHA content and increased immobilization efficiency of TyrVs accounting for 6.85% on the surface of PHA. A higher L-DOPA-forming monophenolase activity of 518.87 U/g PHA granules and an L-DOPA concentration of 974.36 mg/L in 3 h catalysis were achieved, compared to those of E. coli. Together with the result of L-DOPA production directly by cell lysates containing PHA-TyrVs nanogranules, our study demonstrated the robust and cost-effective production of L-DOPA by H. bluephagenesis, further contributing to its low-cost industrial production based on next-generation industrial biotechnology (NGIB).

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.

Integrating enzyme evolution and high-throughput screening for efficient biosynthesis of L-DOPA.

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.

Vector-mediated l-3,4-dihydroxyphenylalanine delivery reverses motor impairments in a primate model of Parkinson's disease.

Ever since its introduction 40 years ago l-3,4-dihydroxyphenylalanine (l-DOPA) therapy has retained its role as the leading standard medication for patients with Parkinson's disease. With time, however, the shortcomings of oral l-DOPA treatment have become apparent, particularly the motor fluctuations and troublesome dyskinetic side effects. These side effects, which are caused by the excessive swings in striatal dopamine caused by intermittent oral delivery, can be avoided by delivering l-DOPA in a more continuous manner. Local gene delivery of the l-DOPA synthesizing enzymes, tyrosine hydroxylase and guanosine-tri-phosphate-cyclohydrolase-1, offers a new approach to a more refined dopaminergic therapy where l-DOPA is delivered continuously at the site where it is needed i.e. the striatum. In this study we have explored the therapeutic efficacy of adeno-associated viral vector-mediated l-DOPA delivery to the putamen in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated rhesus monkeys, the standard non-human primate model of Parkinson's disease. Viral vector delivery of the two enzymes, tyrosine hydroxylase and guanosine-5'-tri-phosphate-cyclohydrolase-1, bilaterally into the dopamine-depleted putamen, induced a significant, dose-dependent improvement of motor behaviour up to a level identical to that obtained with the optimal dose of peripheral l-DOPA. Importantly, this improvement in motor function was obtained without any adverse dyskinetic effects. These results provide proof-of-principle for continuous vector-mediated l-DOPA synthesis as a novel therapeutic strategy for Parkinson's disease. The constant, local supply of l-DOPA obtained with this approach holds promise as an efficient one-time treatment that can provide long-lasting clinical improvement and at the same time prevent the appearance of motor fluctuations and dyskinetic side effects associated with standard oral dopaminergic medication.

Highly efficient biocatalytic synthesis of L-DOPA using in situ immobilized Verrucomicrobium spinosum tyrosinase on polyhydroxyalkanoate nano-granules.

L-DOPA (3,4-dihydroxyphenyl-L-alanine) is a preferred drug for Parkinson's disease, and is currently in great demand every year worldwide. Biocatalytic conversion of L-tyrosine by tyrosinases is the most promising method for the low-cost production of L-DOPA in both research and industry. Yet, it has been hampered by low productivity, low conversion rate, and low stability of the biocatalyst, tyrosinase. An alternative tyrosinase TyrVs from Verrucomicrobium spinosum with more efficient expression in heterologous host and better stability than the commercially available Agaricus bisporus tyrosinase was identified in this study. Additionally, it was prepared as a novel nano-biocatalyst based on the distinct one-step in situ immobilization on the surface of polyhydroxyalkanoate (PHA) nano-granules. The resulting PHA-TyrVs nano-granules demonstrated improved L-DOPA-forming monophenolase activity of 9155.88 U/g (Tyr protein), which was 3.19-fold higher than that of free TyrVs. The nano-granules also exhibited remarkable thermo-stability, with an optimal temperature of 50 °C, and maintained more than 70% of the initial activity after incubation at 55 °C for 24 h. And an enhanced affinity of copper ion was observed in the PHA-TyrVs nano-granules, making them even better biocatalysts for L-DOPA production. Therefore, a considerable productivity of L-DOPA, amounting to 148.70 mg/L h, with a conversion rate of L-tyrosine of 90.62% can be achieved by the PHA-TyrVs nano-granules after 3 h of biocatalysis under optimized conditions, without significant loss of enzyme activity or L-DOPA yield after 8 cycles of repeated use. Our study provides an excellent and robust nano-biocatalyst for the cost-effective production of L-DOPA.

Metabolic engineering of Escherichia coli BL21 (DE3) for de novo production of L-DOPA from D-glucose.

BACKGROUND: Production of L-tyrosine is gaining grounds as the market size of 3,4-dihydroxyphenyl-L-alanine (L-DOPA) is expected to increase due to increasing cases of Parkinson's disease a neurodegenerative disease. Attempts to overproduce L-tyrosine for conversion to L-DOPA has stemmed on the overexpressing of critical pathway enzymes, an introduction of feedback-resistant enzymes, and deregulation of transcriptional regulators. RESULTS: An E. coli BL21 (DE3) was engineered by deleting tyrR, ptsG, crr, pheA and pykF while directing carbon flow through the overexpressing of galP and glk. TktA and PpsA were also overexpressed to enhance the accumulation of E4P and PEP. Directed evolution was then applied on HpaB to optimize its activity. Three mutants, G883R, G883A, L1231M, were identified to have improved activity as compared to the wild-type hpaB showing a 3.03-, 2.9- and 2.56-fold increase in L-DOPA production respectively. The use of strain LP-8 resulted in the production of 691.24 mg/L and 25.53 g/L of L-DOPA in shake flask and 5 L bioreactor, respectively. CONCLUSION: Deletion of key enzymes to channel flux towards the shikimate pathway coupled with the overexpression of pathway enzymes enhanced the availability of L-tyrosine for L-DOPA production. Enhancing the activity of HpaB increased L-DOPA production from glucose and glycerol. This work demonstrates that increasing the availability of L-tyrosine and enhancing enzyme activity ensures maximum L-DOPA productivity.

Iterative screening methodology enables isolation of strains with improved properties for a FACS-based screen and increased L-DOPA production.

Optimizing microbial hosts for the large-scale production of valuable metabolites often requires multiple mutations and modifications to the host's genome. We describe a three-round screen for increased L-DOPA production in S. cerevisiae using FACS enrichment of an enzyme-coupled biosensor for L-DOPA. Multiple rounds of screening were enabled by a single build of a barcoded in vitro transposon-mediated disruption library. New background strains for screening were built for each iteration using results from previous iterations. The same in vitro transposon-mediated disruption library was integrated by homologous recombination into new background strains in each round of screening. Compared with creating new transposon insertions in each round, this method takes less time and saves the cost of additional sequencing to characterize transposon insertion sites. In the first two rounds of screening, we identified deletions that improved biosensor compartmentalization and, consequently, improved our ability to screen for L-DOPA production. In a final round, we discovered that deletion of heme oxygenase (HMX1) increases total heme concentration and increases L-DOPA production, using dopamine measurement as a proxy. We further demonstrated that deleting HMX1 may represent a general strategy for P450 function improvement by improving activity of a second P450 enzyme, BM3, which performs a distinct reaction.

Simultaneous synthesis of l-DOPA and oxidation of d-amino acid by specific coupling of a peroxidase to d-amino acid oxidase.

3,4 Dihydroxy phenyl l-alanine (L-DOPA) is the gold standard Parkinson's disease therapy. A heme-dependent peroxidase (HDP) catalyzes the ortho-hydroxylation of l-tyrosine to l-DOPA using H2O2 as the co-substrate. d-amino acid oxidase (DAAO) catalyzes the oxidative deamination of d-amino acids (e.g. d-alanine), and H2O2 is evolved. However, both the enzymes DAAO and HDP can be inactivated by H2O2 during the catalysis. In situ generation and utilization of H2O2 can siginificatly reduce the inactivation by H2O2. HDP exists as a monomer and DAAO is a dimeric enzyme. Herein, the C-terminus of HDP was specifically ligated to the N-terminus of the DAAO subunit with native peptide through the in vivo monomer-subunit splicing. In the splicing product HDP&DAAO, HDP is close to the DAAO subunit at a molecular distance, and the transfer of H2O2 from DAAO to HDP is facilitated. In addition, HDP&DAAO exhibited a higher stability than HDP. Kinetics analysis showed that both the substrates l-tyrosine and d-alanine obey the Michaelis-Menten kinetics. For the deamination of d-alanine, the catalytic efficiency of HDP&DAAO is 3.05 times that of DAAO. For the sybthesis of l-DOPA from l-tyrosine, the catalytic efficiency of HDP&DAAO is 1.58 times that of HDP. Furthermore, HDP&DAAO was encapsulated within a Znic-based coordination polymer (Zn-CP). The morphorogy of HDP&DAAO/Zn-CP can be regulated by the enzyme concentration, the catalytic efficiency of the conjugates was found to be dependent on the morphorogy. The conjugates HDP&DAAO/Zn-CP exhibited a higher catalytic efficiency than free HDP&DAAO.

Biochemical characterization and spatio-temporal analysis of the putative L-DOPA pathway in Mucuna pruriens.

MAIN CONCLUSION: Transcriptome analysis and biochemical characterization of the putative l-3,4-dihydroxyphenylalanine (l-DOPA) pathway in Mucuna pruriens (L.) DC have been performed. Spatio-temporal quantification of the putative l-DOPA biosynthetic pathway genes and its correlation with respective metabolites was established. l-tyrosine, l-DOPA, and dopamine from all plant parts were quantified. The de novo transcriptome analysis was performed using leaves of the selected M. pruriens mutant T-IV-9 during maturity. The putative L-DOPA pathway and its regulatory genes were retrieved from transcriptome data and the L-DOPA pathway was biochemically characterized. The spatial and temporal gene expression for the L-DOPA pathway was identified with respect to the chemical constituents. L-tyrosine, L-DOPA, and dopamine contents were highest in leaves during maturity (about 170-day-old plants). The polyphenol oxidase (PPO) was highly expressed in tender stems (230-fold higher as compared to seeds) as well as a high L-DOPA content. The PPO gene was highly expressed in leaves (3367.93 in FPKM) with a 79-fold increase compared to control plants during maturity. L-DOPA was found in every part with varied levels. The highest L-DOPA content was found in mature dried seed (3.18-5.8%), whereas the lowest amount was recorded in mature and dried leaves. The reproductive parts of the plant had a higher amount of L-DOPA content (0.9-5.8%) compared to the vegetative parts (0.2-0.91%). Various amino acid transporters and permeases were expressed in M. pruriens. The transcripts of dopa decarboxylase (DDC) were found in almost all parts of the plant, but its higher content was limited to the leaf.

Metabolic engineering of Escherichia coli W3110 strain by incorporating genome-level modifications and synthetic plasmid modules to enhance L-Dopa production from glycerol.

L-Tyrosine which is one of the terminal metabolites of highly regulated aromatic amino-acid biosynthesis pathway in Escherichia coli is a precursor for synthesis of L-Dopa. In this study, we report over production of L-Dopa by enhancing expression of rate limiting isoenzyme of shikimate kinase (aroL), chorismate synthase (aroC), aromatic-amino-acid aminotransferase (tyrB) and 3-phosphoshikimate 1-carboxyvinyltransferase (aroA) form a plasmid module harboring five enzymes under two inducible promoters converting shikimate to tyrosine. 4-hydroxyphenylacetate-3-hydrolase (hpaBC) which converts L-Tyrosine to L-Dopa was expressed constitutively from a separate plasmid module. Feedback deregulated expression of 3-Deoxy-D-arabinoheptulosonate-7-phosphate (DAHP) synthase (aroG*) replacing wild type aroG under its natural promoter led to enhancement of L-Dopa production. Deletion of transcriptional repressor tyrR and links to other competing pathways improved titers of L-Dopa. We focused on having a balanced flux by constitutive expression of pathway enzymes from plasmid constructs rather than achieving higher amounts of catalytic protein by induction. We observed glycerol when used as a carbon source for the final strain led to low acid production. The best performing strain led to decoupling of acid production and product formation in bioreactor. Fed batch analysis of the final strain led to 12.5 g/L of L-Dopa produced in bioreactor.

Process development for efficient biosynthesis of L-DOPA with recombinant Escherichia coli harboring tyrosine phenol lyase from Fusobacterium nucleatum.

The tyrosine phenol lyase (TPL) catalyzed synthesis of L-DOPA was regarded as one of the most economic route for L-DOPA synthesis. In our previous study, a novel TPL from Fusobacterium nucleatum (Fn-TPL) was exploited for efficient biosynthesis of L-DOPA. However, the catalytic efficiency decreased when the reaction system expanded from 100 mL to 1 L. As such, the bioprocess for scale-up production of L-DOPA was developed in this study. To increase the stability of substrate and product, as well as decrease the by-product formation, the optimum temperature and pH were determined to be 15 °C and pH 8.0, respectively. The initial concentration of pyrocatechol, pyruvate and ammonium acetate was fixed at 8, 5 and 77 g/L and a fed-batch approach was applied with sodium pyruvate, pyrocatechol and ammonium acetate fed in a concentration of 5, 5 and 3.5 g/L, respectively. In addition, L-DOPA crystals were exogenously added to inhibit cell encapsulation by the precipitated product. The final L-DOPA concentration reached higher than 120 g/L with pyrocatechol conversion more than 96% in a 15-L stirred tank, demonstrating the great potential of Fn-TPL for industrial production of L-DOPA.

New insight into the allosteric effect of L-tyrosine on mushroom tyrosinase during L-dopa production.

Kinetics studies of L-tyrosine (LTy) ortho-hydroxylation by mushroom tyrosinase (MT) confirmed that MT was severely, but not completely, inhibited at higher concentrations of LTy. Despite the availability of the crystal structure reports, no allosteric site has been identified on MT. To examine the assumption that a non-specific binding site works as a regulatory site, docking simulations were run for the second molecule of L-tyrosine (LTy2) on the complexes of the first L-tyrosine molecule (LTy1) with the heavy chain (H) of MT (LTy1/HMT) and its dimer with the light chain (Ty1/LHMT). In both, LTy2 occupied a non-specific binding site (MTPc). MD simulations revealed LTy2/HMT/LTy1 and LTy2/LHMT/LTy1 were stable. Binding free-energy analysis supported the formation of LTy2/HMT/LTy1 and LTy2/LHMT/LTy1 at higher concentrations of LTy and disclosed the importance of ΔEelec and ΔGpolar during binding of LTy2 to MTPc. Upon LTy2 binding to MTPc, the Cu-Cu distance remained unchanged while the spatial position of LTy1 in the active site (MTPa) changed so that it would not be able to participate in ortho-hydroxylation. This study suggests a tuning role for L chain during binding of the ligands to MTPa and MTPc. Given these results, a plausible mechanism was proposed for the MT substrate inhibition.

Biochemical characterization of a novel tyrosine phenol-lyase from Fusobacterium nucleatum for highly efficient biosynthesis of l-DOPA.

Tyrosine phenol-lyase (TPL) catalyzes the reversible cleavage of l-tyrosine to phenol, pyruvate and ammonia. When pyrocatechol is substituted for phenol, l-dihydroxyphenylalanine (l-DOPA) is produced. The TPL-catalyzed route was regarded as the most economic process for l-DOPA production. In this study, a novel TPL from Fusobacterium nucleatum (Fn-TPL) was successfully overexpressed in Escherichia coli and screened for l-DOPA synthesis with a specific activity of 2.69Umg-1. Fn-TPL was found to be a tetramer, and the optimal temperature and pH for α, ß-elimination of l-tyrosine was 60°C and pH 8.5, respectively. The enzyme showed broad substrate specificity toward natural and synthetic l-amino acids. Kinetic analysis suggested that the kcat/Km value for l-tyrosine decomposition was much higher than that for l-DOPA decomposition, while Fn-TPL exhibited similar catalytic efficiency for synthesis of l-tyrosine and l-DOPA. With whole cells of recombinant E. coli as biocatalyst, l-DOPA yield reached 110gL-1 with a pyrocatechol conversion of 95%, which was comparable to the reported highest level. The results demonstrated the great potential of Fn-TPL for industrial production of l-DOPA.

Tamoxifen activates hypothalamic l-dopa synthesis to stimulate ovarian estrogen production in chicken.

Estrogen is the primary sex hormone responsible for the development and modulation of the female reproductive system in all vertebrates including avian species. The actions of estrogen are mediated by the estrogen receptor, which could be modulated by the selective estrogen receptor modulator tamoxifen (TAM). In this study, we administered TAM into the actively laying chicken to investigate the ovarian and hypothalamic responses to the estrogen action blockage. The laying was disrupted and the development of the pre-ovulatory hierarchical follicles was arrested. However, the TAM treatment caused an increase of estrogen level in both serum and ovary. Among the main estrogen targeted tissues, the hypothalamus showed specific dopaminergic activation as indicated by gene expression analysis. In the ovary, l-dopa, the precursor of dopamine, could stimulate the estrogen synthesis in undifferentiated follicles but not in the differentiated pre-ovulatory follicles. Thus, we established a feedback loop links ovarian estrogen production with hypothalamic l-dopa synthesis and we propose that the dopamine is involved in estrogen action to regulate the ovarian follicle development and ovulation.

Cooperative Synthesis of Dopamine in Rat Mediobasal Hypothalamus as a Compensatory Mechanism in Hyperprolactinemia.

Dopamine (DA), synthesized in the mediobasal hypothalamus by dopaminergic neurons containing two enzymes of DA synthesis - tyrosine hydroxylase and decarboxylase of aromatic L-amino acids, or by monoenzymatic non-dopaminergic neurons containing one DA synthesis enzyme in cooperation, is known to have an inhibitory effect on prolactin secretion. Deterioration of this inhibitory control leads to an increase in prolactin concentration in the blood and to the development of hyperprolactinemia syndrome. In a rat model of hyperprolactinemia induced by administration of a neurotoxin causing degeneration of dopaminergic and noradrenergic neurons, the level of DA first decreases, leading to an increase in prolactin level (decompensation stage), while later both levels are restored to normal (compensation stage). However, the mechanism of such compensation is still not clear. The aim of the present study was to analyze whether the increase in cooperative synthesis of DA by monoenzymatic neurons during hyperprolactinemia is a manifestation of a compensatory mechanism representing a particular case of neuroplasticity. The level of cooperative synthesis in the hyperprolactinemia model and in the control was estimated as the level of synthesis of DA and L-dihydroxyphenylalanine (L-DOPA) - an intermediate product of DA synthesis, when L-DOPA transfer from neurons containing tyrosine hydroxylase into neurons containing aromatic L-amino acid decarboxylase is inhibited. The level of DA synthesis during the decompensation stage was not changed, while during the compensation stage it was lower than the control. Along with a reduction in DA level, during the compensation stage an increase in the extracellular L-DOPA level in the medium was detected. Thus, the compensation of DA deficiency after degeneration of dopaminergic neurons in the mediobasal hypothalamus is due to the increase in cooperative synthesis of DA by monoenzymatic neurons containing one of the complementary enzymes of the DA synthesis pathway.

Inducing mutations through γ-irradiation in seeds of Mucuna pruriens for developing high L-DOPA-yielding genotypes.

PURPOSE: To develop elite genotypes in Mucuna pruriens (L.) DC with high L-DOPA (L-3, 4 dihydroxyphenylalanine) yields, with non-itching characteristics and better adaptability by applying γ-irradiation. Molecular and chemical analysis was performed for screening based on specific characteristics desired for developing suitable genotypes. MATERIALS AND METHODS: Developed, mutant populations were analyzed for L-DOPA % in seeds through TLC (thin layer chromatography), and the results obtained were validated with the HPLC (High performance liquid chromatography). The DNA (Deoxyribonucleic acid) was isolated from the leaf at the initial stage and used for DNA polymorphism. RNA (Ribonucleic acid) was isolated from the leaf during maturity and used for expression analysis. RESULTS: The selected mutant T-I-7 showed 5.7% L-DOPA content compared to 3.18% of parent CIM-Ajar. The total polymorphism obtained was 57% with the molecular marker analysis. The gene expression analysis showed higher fold change expression of the dopadecarboxylase gene (DDC) in control compared to selected mutants (T-I-7, T-II-23, T-IV-9, T-VI-1). CONCLUSION: DNA polymorphism was used for the screening of mutants for efficient screening at an early stage. TLC was found suitable for the large-scale comparative chemical analysis of L-DOPA. The expression profile of DDC clearly demonstrated the higher yields of L-DOPA in selected mutants developed by γ-irradiation in the seeds of the control.

Molecular Cloning and Characteristic Features of a Novel Extracellular Tyrosinase from Aspergillus niger PA2.

Aspergillus niger PA2, a novel strain isolated from waste effluents of food industry, is a potential extracellular tyrosinase producer. Enzyme activity and L-DOPA production were maximum when glucose and peptone were employed as C source and nitrogen source respectively in the medium and enhanced notably when the copper was supplemented, thus depicting the significance of copper in tyrosinase activity. Tyrosinase-encoding gene from the fungus was cloned, and amplification of the tyrosinase gene yielded a 1127-bp DNA fragment and 374 amino acid residue long product that encoded for a predicted protein of 42.3 kDa with an isoelectric point of 4.8. Primary sequence analysis of A. niger PA2 tyrosinase had shown that it had approximately 99% identity with that of A. niger CBS 513.88, which was further confirmed by phylogenetic analysis. The inferred amino acid sequence of A. niger tyrosinase contained two putative copper-binding sites comprising of six histidines, a characteristic feature for type-3 copper proteins, which were highly conserved in all tyrosinases throughout the Aspergillus species. When superimposed onto the tertiary structure of A. oryzae tyrosinase, the conserved residues from both the organisms occupied same spatial positions to provide a di-copper-binding peptide groove.

Genome engineering Escherichia coli for L-DOPA overproduction from glucose.

Genome engineering has become a powerful tool for creating useful strains in research and industry. In this study, we applied singleplex and multiplex genome engineering approaches to construct an E. coli strain for the production of L-DOPA from glucose. We first used the singleplex genome engineering approach to create an L-DOPA-producing strain, E. coli DOPA-1, by deleting transcriptional regulators (tyrosine repressor tyrR and carbon storage regulator A csrA), altering glucose transport from the phosphotransferase system (PTS) to ATP-dependent uptake and the phosphorylation system overexpressing galactose permease gene (galP) and glucokinase gene (glk), knocking out glucose-6-phosphate dehydrogenase gene (zwf) and prephenate dehydratase and its leader peptide genes (pheLA) and integrating the fusion protein chimera of the downstream pathway of chorismate. Then, multiplex automated genome engineering (MAGE) based on 23 targets was used to further improve L-DOPA production. The resulting strain, E. coli DOPA-30N, produced 8.67 g/L of L-DOPA in 60 h in a 5 L fed-batch fermentation. This titer is the highest achieved in metabolically engineered E. coli having PHAH activity from glucose.