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.

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.

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).

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Overview on the biotechnological production of L-DOPA.

L-DOPA (3,4-dihydroxyphenyl-L-alanine) has been widely used as a drug for Parkinson's disease caused by deficiency of the neurotransmitter dopamine. Since Monsanto developed the commercial process for L-DOPA synthesis for the first time, most of currently supplied L-DOPA has been produced by the asymmetric method, especially asymmetric hydrogenation. However, the asymmetric synthesis shows critical limitations such as a poor conversion rate and a low enantioselectivity. Accordingly, alternative biotechnological approaches have been researched for overcoming the shortcomings: microbial fermentation using microorganisms with tyrosinase, tyrosine phenol-lyase, or p-hydroxyphenylacetate 3-hydroxylase activity and enzymatic conversion by immobilized tyrosinase. Actually, Ajinomoto Co. Ltd commercialized Erwinia herbicola fermentation to produce L-DOPA from catechol. In addition, the electroenzymatic conversion system was recently introduced as a newly emerging scheme. In this review, we aim to not only overview the biotechnological L-DOPA production methods, but also to briefly compare and analyze their advantages and drawbacks. Furthermore, we suggest the future potential of biotechnological L-DOPA production as an industrial process.

Bioconversion of L-tyrosine to L-DOPA by a novel bacterium Bacillus sp. JPJ.

L-DOPA is an amino acid derivative and most potent drug used against Parkinson's disease, generally obtained from Mucuna pruriens seeds. In present communication, we have studied the in vitro production of L-DOPA from L-tyrosine by novel bacterium Bacillus sp. JPJ. This bacterium produced 99.4% of L-DOPA from L-tyrosine in buffer (pH 8) containing 1 mg ml(-1) cell mass incubated at 40°C for 60 min. The combination of CuSO(4) and L-ascorbic acid showed the inducing effect at concentrations of 0.06 and 0.04 mg ml(-1), respectively. The activated charcoal 2 mg ml(-1) was essential for maximum bioconversion of L-tyrosine to L-DOPA and the crude tyrosinase activity was 2.7 U mg(-1) of tyrosinase. Kinetic studies showed significant values of Y (p/s) (0.994), Q (s) (0.500) and q (s) (0.994) after optimization of the process. The production of L-DOPA was confirmed by analytical techniques such as HPTLC, HPLC and GC-MS. This is the first report on rapid and efficient production of L-DOPA from L-tyrosine by bacterial source which is more effective than the plant, fungal and yeast systems.

Metabolic engineering of Escherichia coli for improving L-3,4-dihydroxyphenylalanine (L-DOPA) synthesis from glucose.

L-3,4-dihydroxyphenylalanine (L-DOPA) is an aromatic compound employed for the treatment of Parkinson's disease. Metabolic engineering was applied to generate Escherichia coli strains for the production of L-DOPA from glucose by modifying the phosphoenolpyruvate:sugar phosphotransferase system (PTS) and aromatic biosynthetic pathways. Carbon flow was directed to the biosynthesis of L-tyrosine (L-Tyr), an L-DOPA precursor, by transforming strains with compatible plasmids carrying genes encoding a feedback-inhibition resistant version of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase, transketolase, the chorismate mutase domain from chorismate mutase-prephenate dehydratase from E. coli and cyclohexadienyl dehydrogenase from Zymomonas mobilis. The effects on L-Tyr production of PTS inactivation (PTS(-) gluc(+) phenotype), as well as inactivation of the regulatory protein TyrR, were evaluated. PTS inactivation caused a threefold increase in the specific rate of L-Tyr production (q( L-Tyr)), whereas inactivation of TyrR caused 1.7- and 1.9-fold increases in q( L-Tyr) in the PTS(+) and the PTS(-) gluc(+) strains, respectively. An 8.6-fold increase in L-Tyr yield from glucose was observed in the PTS(-) gluc(+) tyrR (-) strain. Expression of hpaBC genes encoding the enzyme 4-hydroxyphenylacetate 3-hydroxylase from E. coli W in the strains modified for L-Tyr production caused the synthesis of L-DOPA. One of such strains, having the PTS(-) gluc(+) tyrR (-) phenotype, displayed the best production parameters in minimal medium, with a specific rate of L-DOPA production of 13.6 mg/g/h, L-DOPA yield from glucose of 51.7 mg/g and a final L-DOPA titer of 320 mg/l. In a batch fermentor culture in rich medium this strain produced 1.51 g/l of L-DOPA in 50 h.

Comparative genetic analysis of trichome-less and normal pod genotypes of Mucuna pruriens (Fabaceae).

Velvet bean (Mucuna pruriens) seeds contain the catecholic amino acid L-DoPA (L-3,4-dihydroxyphenylalanine), which is a neurotransmitter precursor and used for the treatment of Parkinson's disease and mental disorders. The great demand for L-DoPA is largely met by the pharmaceutical industry through extraction of the compound from wild populations of this plant; commercial exploitation of this compound is hampered because of its limited availability. The trichomes present on the pods can cause severe itching, blisters and dermatitis, discouraging cultivation. We screened genetic stocks of velvet bean for the trichome-less trait, along with high seed yield and L-DoPA content. The highest yielding trichome-less elite strain was selected and indentified on the basis of a PCR-based DNA fingerprinting method (RAPD), using deca-nucleotide primers. A genetic similarity index matrix was obtained through multivariant analysis using Nei and Li's coefficient. The similarity coefficients were used to generate a tree for cluster analysis using the UPGMA method. Analysis of amplification spectra of 408 bands obtained with 56 primers allowed us to distinguish a trichome-less elite strain of M. pruriens.

Production of 3,4-dihydroxy L-phenylalanine by a newly isolated Aspergillus niger and parameter significance analysis by Plackett-Burman design.

BACKGROUND: The amino acid derivative 3,4-dihydroxy L-phenylalanine (L-dopa) is gaining interest as a drug of choice for Parkinson's disease. Aspergillus oryzae is commonly used for L-dopa production; however, a slower growth rate and relatively lower tyrosinase activity of mycelia have led to an increasing interest in exploiting alternative fungal cultures. In the present investigation, we report on the microbiological transformation of L-tyrosine to L-dopa accomplished by a newly isolated filamentous fungus Aspergillus niger. RESULTS: The culture A. niger (isolate GCBT-8) was propagated in 500 ml Erlenmeyer flasks and the pre-grown mycelia (48 h old) were used in the reaction mixture as a source of enzyme tyrosinase. Grinded mycelia gave 1.26 fold higher L-dopa production compared to the intact at 6% glucose (pH 5.5). The rate of L-tyrosine consumption was improved from 0.198 to 0.281 mg/ml. Among the various nitrogen sources, 1.5% peptone, 1% yeast extract and 0.2% ammonium chloride were optimized. The maximal L-dopa was produced (0.365 mg/ml) at 0.3% potassium dihydrogen phosphate with L-tyrosine consumption of 0.403 mg/ml. CONCLUSION: Over ~73% yield was achieved (degree of freedom 3) when the process parameters were identified using 2k-Plackett-Burman experimental design. The results are highly significant (p ≤ 0.05) and mark the commercial utility (LSD 0.016) of the mould culture which is perhaps the first ever report on L-dopa production from A. niger.

L-Ascorbate, a strong inducer of L-dopa (3,4-dihydroxy-L-phenylalanine) production from pre-grown mycelia of Aspergillus oryzae NRRL-1560.

The inductive effect of L-ascorbate on the microbiological production of L-dopa (3,4-dihydroxy-L-phenylalanine) from Aspergillus oryzae NRRL-1560 was investigated. All biochemical reactions were performed aerobically using mould mycelia as a source of enzyme tyrosinase and acetate buffer (pH 3.0) as an extractant. L-Tyrosine as a substrate was added at a level of 2.5 mg/ml. Maximal L-dopa production (1.876 mg/ml) was achieved when L-ascorbate (5.0 mg/ml) was added 6 min after the initiation of the biochemical reaction at 50 degrees C, consuming 2.144 mg/ml L-tyrosine. The performance of fuzzy-logic control of the reaction was found to be highly promising for improvement of the substrate conversion rate (~80%). After optimizing the reaction conditions, particularly the addition of L-ascorbate, an increase in L-dopa yield of 22.96% was achieved compared with the control (without ascorbate addition) when the process variables, namely buffer pH, L-tyrosine concentration and reaction temperature, were further identified using a two-factorial Plackett-Burman design.