Optimization of tyrosol-producing pathway with tyrosine decarboxylase and tyramine oxidase in high-tyrosine-producing Escherichia coli.

Tyrosol (4-hydroxyphenylethanol) is a phenolic compound used in the pharmaceutical and chemical industries. However, current supply methods, such as extraction from natural resources and chemical synthesis, have disadvantages from the viewpoint of cost and environmental protection. Here, we developed a tyrosol-producing Escherichia coli cell factory from a high-tyrosine-producing strain by expressing selected tyrosine decarboxylase-, tyramine oxidase (TYO)-, and medium-chain dehydrogenase/reductase (YahK)-encoding genes. The genes were controlled by the strong T7 promoter and integrated into the chromosome because of the advantages over plasmid-based systems. The strain produced a melanin-like pigment as a by-product, which is suggested to be formed from 4-hydroxyphenylacetaldehyde (a TYO product/YahK substrate). By using a culture medium containing a high concentration of glycerol, which was reported to enhance NADH supply required for YahK activity, the final titer of tyrosol reached 2.42 g/L in test tube-scale cultivation with a concomitant decrease in the amount of pigment. These results indicate that chromosomally integrated and T7 promoter-controlled gene expression system in E. coli is useful for high production of heterologous enzymes and might be applied for industrial production of useful compounds including tyrosine and tyrosol.

Association Between Microbial Tyrosine Decarboxylase Gene and Levodopa Responsiveness in Patients With Parkinson Disease.

BACKGROUND AND OBJECTIVES: Interindividual variability in levodopa efficacy is a challenge for the personalized treatment of Parkinson disease (PD). Gut microbiota might represent a new approach for personalized medicine. Recently, a novel microbial levodopa metabolism pathway was identified, which is mediated by tyrosine decarboxylase mainly encoded by tyrosine decarboxylase gene (tyrDC) in Enterococcus faecalis. In this study, we aimed to identify whether the abundance of microbial tyrDC gene and E faecalis is associated with levodopa responsiveness and could predict the drug response. METHODS: This cross-sectional study enrolled patients with PD between December 2019 and January 2022 and evaluated levodopa responsiveness using a levodopa challenge test. Patients were stratified into moderate and good responders based on levodopa responsiveness. The tyrDC gene and E. faecalis abundance in fecal samples were measured using quantitative real-time PCR. Plasma levodopa concentrations were measured using liquid chromatography-tandem mass spectrometry analysis. The predictive models for levodopa responsiveness were constructed and verified through cross-validation and external validation. RESULTS: A total of 101 patients with PD were enrolled in the primary cohort and 43 were enrolled in the external validation cohort. Moderate responders had higher abundances of the tyrDC gene (3.6 [3.1-4.3] vs 2.6 [2.1-2.9], p < 0.001) and E faecalis (3.2 [2.5-4.4] vs 2.6 [2.1-3.6], p = 0.010) than good responders. The tyrDC gene abundance was independently associated with levodopa responsiveness (OR: 5.848; 95% CI: 2.664-12.838; p < 0.001). Notably, tyrDC gene abundance showed certain discriminative power for levodopa responsiveness in primary cohort (sensitivity: 80.0%; specificity: 84.3%; area under the curve [AUC]: 0.85; 95% CI: 0.77-0.93; p < 0.001) and external validation cohort (sensitivity: 85.0%; specificity: 95.7%; AUC: 0.95; 95% CI: 0.89-1.02; p < 0.001). The prediction of levodopa responsiveness based on tyrDC gene abundance had good calibration and discrimination in cross-validation (C-index in training and test sets: 0.856 and 0.851, respectively) and external validation (C-index: 0.952). DISCUSSION: The microbial tyrDC gene abundance could serve as a potential biomarker of levodopa responsiveness. Novel strategies targeting the tyrDC gene may provide new approaches for personalized levodopa treatment.

Functional characterization of tyrosine decarboxylase genes that contribute to acteoside biosynthesis in Rehmannia glutinosa.

MAIN CONCLUSION: The RgTyDCs possess typical decarboxylase functional activity in vitro and in vivo and participate in acteoside biosynthesis in R. glutinosa, positively controlling its production via activated acteoside/tyrosine-derived pathways. Acteoside is an important ingredient in Rehmannia glutinosa and an active natural component that contributes to human health. Tyrosine decarboxylase (TyDC) is thought to play an important role in acteoside biosynthesis. Several plant TyDC family genes have been functionally characterized and shown to play roles in some bioactive metabolites' biosynthesis by mediating the decarboxylation of L-tyrosine and L-dihydroxyphenylalanine (L-DOPA); however, one TyDC (named RgTyDC1) in R. glutinosa has been identified to date, but the family genes that contribute to acteoside biosynthesis remain largely characterized. Here, by in silico and experimental analyses, we isolated and identified three RgTyDCs (RgTyDC2 to RgTyDC4) in this species; these genes' sequences showed 50.92-82.55% identity, included highly conserved domains with homologues in other plants, classified into two subsets, and encoded proteins that localized to the cytosol. Enzyme kinetic analyses of RgTyDC2 and RgTyDC4 indicated that they both efficiently catalysed L-tyrosine and L-dopa. The overexpression of RgTyDC2 and RgTyDC4 in R. glutinosa, which was associated with enhanced TyDC activity, significantly increased tyramine and dopamine contents, which was positively correlated with improved acteoside production; moreover, the overexpression of RgTyDCs led to upregulated expression of some other genes-related to acteoside biosynthesis. This result suggested that the overexpression of RgTyDCs can positively activate the molecular networks of acteoside pathways, enhancing the accumulation of tyramine and dopamine, and promoting end-product acteoside biosynthesis. Our findings provide an evidence that RgTyDCs play vital molecular roles in acteoside biosynthesis pathways, contributing to the increase in acteoside yield in R. glutinosa.

Overexpression of the tyrosine decarboxylase gene MdTyDC in apple enhances long-term moderate drought tolerance and WUE.

Drought stress affects the apple yield and quality. Tyrosine decarboxylase (TyDC) plays a fundamental role in many secondary metabolite reactions in higher plants (including those involving dopamine). Our aims of this study are: 1) to identify the role of TyDC in dopamine derivative biosynthesis and its function in long-term moderate drought conditions; and 2) to explore the role of MdTyDC in plant growth and development as well as the drought stress response. Wild type and three independently apple plants overexpression of MdTyDC were treated for long-term moderate drought stress. The growth and physiological parameters of apple plant, photosynthetic capacity, antioxidant enzymes activity, water use efficiency (WUE), stomatal behavior, amino acid content and dopamine content were detected under long-term moderate drought stress. Overexpression of MdTyDC (OE) in apple showed better growth performance, higher photosynthetic capacity and higher capacity for photochemical reactions than wild type lines (WT). Under long-term moderate drought stress, OE lines showed higher WUE, increased ABA content, decreased stomatal aperture, higher antioxidant activity, lower accumulation of ROS and increases in amino acids, such as proline, phenylalanine and lysine. In addition, qRT-PCR revealed higher gene expression of MdTyDC and dopamine content in OE compared with WT lines under long-term moderate drought stress. These results indicate that MdTyDC confers long-term moderate drought tolerance by improving photosynthetic capacity, WUE, antioxidant activity, dopamine content and changing the contents of amino acids (such as proline accumulation).

Cloning and characterization of tyrosine decarboxylase (TDC) from Litopenaeus vannamei, and its roles in biogenic amines synthesis, immune regulation, and resistance to Vibrio alginolyticus by RNA interference.

The biogenic amines, tyramine and octopamine, in the octopaminergic synthesis pathway play critical roles in regulating physiological and immunological homeostasis in Litopenaeus vannamei. Tyrosine decarboxylase (TDC) is an enzyme catalyzing the first decarboxylation step in the biosynthesis of tyramine and octopamine. The full-length gene sequence of TDC cloned from the brain of L. vannamei (LvTDC) was predicted to encode a 779-amino acid protein with a pyridoxal-dependent decarboxylase-conserved domain in close phylogenetic relationship with arthropod TDCs. LvTDC gene expression was found to be abundant in nervous thoracic ganglia. RNA interference was used to assess the immune and physiological function of LvTDC. The LvTDC knockdown shrimp revealed significant decreases in the total haemocyte count, hyaline cells, antimicrobial peptides, respiratory bursts, gene expression, respiratory bursts of haemocytes per unit of haemolymph, and phagocytic activity and clearance efficiency toward Vibrio alginolyticus. Furthermore, LvTDC knockdown was accompanied by decreases in octopamine deficiency. In the V. alginolyticus challenge test, the survival rate of LvTDC knockdown shrimp was lower than the shrimp injected with DEPC-water or GAPDH-dsRNA. In conclusion, the cloned LvTDC was responsible for octopaminergic synthesis, which then regulated physiological and immune responses in L. vannamei.

Crystallographic Snapshots of the Dunathan and Quinonoid Intermediates provide Insights into the Reaction Mechanism of Group II Decarboxylases.

PLP-dependent enzymes catalyze a plethora of chemical reactions affecting diverse physiological functions. Here we report the structural determinants of the reaction mechanism in a Group II PLP-dependent decarboxylase by assigning two early intermediates. The in-crystallo complexes of the PLP bound form, and the Dunathan and quinonoid intermediates, allowed direct observation of the active site interactions. The structures reveal that a subtle rearrangement of a conserved Arg residue in concert with a water-mediated interaction with the carboxylate of the Dunathan intermediate, appears to directly stabilize the alignment and facilitate the release of CO2 to yield the quinonoid. Modeling indicates that the conformational change of a dynamic catalytic loop to a closed form controls a conserved network of hydrogen bond interactions between catalytic residues to protonate the quinonoid. Our results provide a structural framework to elucidate mechanistic roles of residues that govern reaction specificity and catalysis in PLP-dependent decarboxylation.

A neurotransmitter produced by gut bacteria modulates host sensory behaviour.

Animals coexist in commensal, pathogenic or mutualistic relationships with complex communities of diverse organisms, including microorganisms1. Some bacteria produce bioactive neurotransmitters that have previously been proposed to modulate nervous system activity and behaviours of their hosts2,3. However, the mechanistic basis of this microbiota-brain signalling and its physiological relevance are largely unknown. Here we show that in Caenorhabditis elegans, the neuromodulator tyramine produced by commensal Providencia bacteria, which colonize the gut, bypasses the requirement for host tyramine biosynthesis and manipulates a host sensory decision. Bacterially produced tyramine is probably converted to octopamine by the host tyramine ß-hydroxylase enzyme. Octopamine, in turn, targets the OCTR-1 octopamine receptor on ASH nociceptive neurons to modulate an aversive olfactory response. We identify the genes that are required for tyramine biosynthesis in Providencia, and show that these genes are necessary for the modulation of host behaviour. We further find that C. elegans colonized by Providencia preferentially select these bacteria in food choice assays, and that this selection bias requires bacterially produced tyramine and host octopamine signalling. Our results demonstrate that a neurotransmitter produced by gut bacteria mimics the functions of the cognate host molecule to override host control of a sensory decision, and thereby promotes fitness of both the host and the microorganism.

Crystal structures clarify cofactor binding of plant tyrosine decarboxylase.

Plant tyrosine decarboxylase (TyrDC) is a group II pyridoxal 5'-phosphate (PLP)-dependent decarboxylase that mainly catalyzes the decarboxylation of tyrosine to tyramine. This is biologically important for diverting essential primary metabolites into secondary metabolic pathways. Intensive studies have characterized the effective of PLP-binding and the substrate specificity of mammalian 3,4-dihydroxyphenyl-l-alanine (Dopa) decarboxylases, a member of group II PLP-dependent decarboxylase. However, the characteristics of PLP binding and substrate specificity of plant TyrDCs remain unknown. In this study, we focus on the PLP binding manner, and determined the crystal structures of the apo and PLP binding form of type II TyrDC from Papaver somniferum (PsTyrDCII and PsTyrDCII-PLP). The structures showed that, unlike mammalian Dopa decarboxylase, the binding of PLP does not induce distinct conformational changes of PsTyrDCII regarding the overall structure, but the PLP binding pocket displays conformational changes at Phe124, His203 and Thr262. Combining structural comparation and the obtained biochemical findings, it is demonstrated that PsTyrDCII does not binds PLP tightly. Such characteristics of PLP binding may be required by its catalytic reaction and substrate binding. The activity of TyrDC probably regulated by the concentration of PLP in cells.

Discovery and inhibition of an interspecies gut bacterial pathway for Levodopa metabolism.

The human gut microbiota metabolizes the Parkinson's disease medication Levodopa (l-dopa), potentially reducing drug availability and causing side effects. However, the organisms, genes, and enzymes responsible for this activity in patients and their susceptibility to inhibition by host-targeted drugs are unknown. Here, we describe an interspecies pathway for gut bacterial l-dopa metabolism. Conversion of l-dopa to dopamine by a pyridoxal phosphate-dependent tyrosine decarboxylase from Enterococcus faecalis is followed by transformation of dopamine to m-tyramine by a molybdenum-dependent dehydroxylase from Eggerthella lenta These enzymes predict drug metabolism in complex human gut microbiotas. Although a drug that targets host aromatic amino acid decarboxylase does not prevent gut microbial l-dopa decarboxylation, we identified a compound that inhibits this activity in Parkinson's patient microbiotas and increases l-dopa bioavailability in mice.

Improving Soluble Expression of Tyrosine Decarboxylase from Lactobacillus brevis for Tyramine Synthesis with High Total Turnover Number.

The soluble expression of tyrosine decarboxylase (TDC) in heterologous host is often challenging. Here, acidic condition was found to be favorable for improving the soluble expression of TDC from Lactobacillus brevis in Escherichia coli, while addition of carbohydrates (such as glucose, arabinose, and fructose) was vital for decreasing the insoluble fraction. By simple pH control and addition of glucose, the specific activity of TDC in crude extract was enhanced to 46.3 U mg-1, 3.67-fold of that produced from LB medium. Optimization of the reaction conditions revealed that Tween-80 was effective in improving the tyramine production catalyzed by TDC, especially at high tyrosine loadings. As much as 400 mM tyrosine could be completely converted into tyramine with a substrate to catalyst ratio of 29.0 g g-1 and total turnover number of 23,300. This study provides efficient strategies for the highly soluble expression of TDC and biocatalytic production of tyramine.

Spatiotemporal oscillations of morphinan alkaloids in opium poppy.

Here, a comprehensive endeavor is made to simultaneously scrutinize spatiotemporal oscillations of three imperative morphinan alkaloids (i.e. thebaine, codeine, and morphine) alongside dynamic transcriptional patterns of TYDC, SalAT, COR, T6ODM, and CODM genes in different tissues of Papaver somniferum (i.e. root, bottom part of stem, upper part of stem, leaf, capsule wall, and capsule content) over five distinguished ontogenic stages (i.e. rosette, bud initiation, pendulous bud, flowering, and lancing). Apart from bottom stem and leaf, the maximum thebaine content occurred in lancing stage, while its minimum content did not follow a systematic rhythm, either among six tissues or five various sampling times. Regarding codeine, excepting upper stem, the highest ratios of codeine were observed at flowering and lacing stages, while negligible amounts were overall detected at early stages of plant growth like rosette. Considering morphine, apart from upper stem, it appears that late ontogeneic times including lancing and flowering are the most appropriate phases to achieve high amounts of morphine, while at early stages the aforesaid alkaloid possessed lower accumulation. Furthermore, all the five genes under study, overall, exhibited a variety of transcript levels either among six tissues or five various sampling times. Interestingly, a connection occurred between transcript ratio of SalAT and thebaine content, suggesting that thebaine biosynthesis is coordinated tightly by the enzymatic function of SalAT enzyme. Meanwhile, despite low magnitudes of T6ODM and CODM transcripts in the root-harvested samples at pendulous bud and flowering stages, both codeine and morphine were surprisingly in acceptable quantities, plausibly owing to the translocation of both alkaloids from the producing (source) tissues to the roots (sink), known as a phenomenon of 'source-to-sink transportation'. The results, altogether, could provide us enough information in acquiring new insights towards potential impacts of spatiotemporal oscillations on the magnitudes of all the above-mentioned alkaloids alongside transcription ratios of the key genes in opium poppy.

Tyramine and tyrosine decarboxylase gene contributes to the formation of cyanic blotches in the petals of pansy (Viola × wittrockiana).

Tyrosine decarboxylase (TYDC) can catalyze tyrosine into tyramine. Several studies demonstrated its roles in the acidity, salidroside and defense response. Here we found that TYDC from Viola × wittrockiana Gam (VwTYDC) may contribute to the formation of cyaninc blotches in the petal. VwTYDC gene were cloned from Viola × wittrockiana and the cDNA full-length sequences were 1634 bp encoding 494 amino acids. Gene expression of VwTYDC in different tissues and developmental stages showed that they were significantly higher expressed in flowers than stems, leaves and roots. In addition, VwTYDC expression were higher in cyanic blotches than those observed in acyanic blotches of petal. Metabolites analysis showed the contents of tyramine in cyanic blotches were also higher than that in acyanic areas. Furthermore, in vitro assay revealed the absorption peak of anthocyanins had a red shift and an increase when fed tyramine. We speculated that tyramine might contribute to flower color expression of pansy as co-pigment. Our study demonstrated for the first time that the contents of tyramine led to flower blotches formation in cyanic blotches of the petals in plant flowers, and this may due to the higher expression of VwTYDC gene.

Fungal endophyte-induced salidroside and tyrosol biosynthesis combined with signal cross-talk and the mechanism of enzyme gene expression in Rhodiola crenulata.

Endophyte is a factor that affects the physiology and metabolism of plant. However, limited information is available on the mechanism of interaction between endophyte and plant. To investigate the effects of endophytic fungus ZPRs-R11, that is, Trimmatostroma sp., on salidroside and tyrosol accumulations in Rhodiola crenulata, signal transduction, enzyme gene expression, and metabolic pathway were investigated. Results showed that hydrogen peroxide (H2O2), nitric oxide (NO), and salicylic acid (SA) involved in fungus-induced salidroside and tyrosol accumulations. NO acted as an upstream signal of H2O2 and SA. No up- or down-stream relationship was observed, but mutual coordination existed between H2O2 and SA. Rate-limiting enzyme genes with the maximum expression activities were UDP-glucosyltransferase, tyrosine decarboxylase (TYDC), monoamine oxidase, phenylalanine ammonialyase (PAL), and cinnamic-4-hydroxylase sequentially. Nevertheless, the genes of tyrosine transaminase and pyruvate decarboxylase only indicated slightly higher activities than those in control. Thus, TYDC and PAL branches were the preferential pathways in ZPRs-R11-induced salidroside and tyrosol accumulation. Trimmatostroma sp. was a potential fungus for promoting salidroside and tyrosol accumulations. The present data also provided scientific basis for understanding complex interaction between endophytic fungus and R. crenulata.

Growth, biogenic amine production and tyrDC transcription of Enterococcus faecalis in synthetic medium containing defined amino acid concentrations.

AIMS: The tyraminogenic potential of the strains Enterococcus faecalis EF37 and ATCC 29212 was investigated in a synthetic medium containing defined amounts of tyrosine and phenylalanine at different temperatures. METHODS AND RESULTS: Enterococci growth and the production of biogenic amines (BA) were evaluated in relation to their pre-growth in medium containing tyrosine. Significant differences between the two strains were evidenced at metabolic level. Both the pre-adapted strains grew faster in all the tested conditions, independently of the presence of the precursor. Temperatures of 30 and 40°C positively affected the growth parameters. The tyrosine decarboxylase (tyrDC) activity of the strain EF37 was positively affected by pre-adaptation, while ATCC 29212 showed a faster and higher tyramine accumulation with not-adapted cells. The expression analysis of the gene tyrDC confirmed the influence of the growth conditions on gene transcription. CONCLUSIONS: The small differences found between the two strains in the maximum transcript level reached rapidly after the inoculum and the different behaviour in the tyramine accumulation suggested the possible involvement of complex regulation mechanisms on the tyrDC or on the membrane transport systems, which could affect the different BA accumulation trend. SIGNIFICANCE AND IMPACT OF THE STUDY: This study gives deeper insight into the metabolic regulation of tyrDC activity of enterococci.

Safety assessment of Tetragenococcus halophilus isolates from doenjang, a Korean high-salt-fermented soybean paste.

We assessed the safety of 49 Tetragenococcus halophilus strains isolated from doenjang in Korea. Minimum inhibitory concentration assays showed that all strains can be considered as susceptible to ampicillin, erythromycin, penicillin G, tetracycline, and vancomycin, but resistant to ciprofloxacin based on the Enterococcus breakpoint values provided by the European Committee on Antimicrobial Susceptibility testing in 2015. Ciprofloxacin resistance was sufficiently high to consider the potential for acquisition of transmissible determinants. Two strains exhibiting potentially acquired resistance to chloramphenicol and gentamicin, and chloramphenicol alone, were identified. None of the strains exhibited α-hemolytic activity or biofilm formation; two strains exhibited weak ß-hemolytic activity. Doenjang isolates produced an average of 3338.6 ppm of tyramine in the laboratory, considerably higher than the levels produced by two reference strains. All of the test strains exhibited similar cadaverine, histamine, and putrescine production patterns. Most T. halophilus strains could grow at a NaCl concentration >18%, exhibited acid production at 15% NaCl, and expressed strain-specific protease and lipase activities. The potential acquisition of transmissible determinants for antibiotic resistance and tyramine production identified in this study necessitate the need for a thorough safety assessment of T. halophilus before it can be considered for use in food fermentation processes.

Octopamine controls starvation resistance, life span and metabolic traits in Drosophila.

The monoamines octopamine (OA) and tyramine (TA) modulate numerous behaviours and physiological processes in invertebrates. Nevertheless, it is not clear whether these invertebrate counterparts of norepinephrine are important regulators of metabolic and life history traits. We show that flies (Drosophila melanogaster) lacking OA are more resistant to starvation, while their overall life span is substantially reduced compared with control flies. In addition, these animals have increased body fat deposits, reduced physical activity and a reduced metabolic resting rate. Increasing the release of OA from internal stores induced the opposite effects. Flies devoid of both OA and TA had normal body fat and metabolic rates, suggesting that OA and TA act antagonistically. Moreover, OA-deficient flies show increased insulin release rates. We inferred that the OA-mediated control of insulin release accounts for a substantial proportion of the alterations observed in these flies. Apparently, OA levels control the balance between thrifty and expenditure metabolic modes. Thus, changes in OA levels in response to external and internal signals orchestrate behaviour and metabolic processes to meet physiological needs. Moreover, chronic deregulation of the corresponding signalling systems in humans may be associated with metabolic disorders, such as obesity or diabetes.

Tyrosine decarboxylase activity of Enterococcus mundtii: new insights into phenotypic and genetic aspects.

Few information is available about the tyraminogenic potential of the species Enterococcus mundtii. In this study, two plant-derived strains of E. mundtii were selected and investigated to better understand the phenotypic behaviour and the genetic mechanisms involved in tyramine accumulation. Both the strains accumulated tyramine from the beginning of exponential phase of growth, independently on the addition of tyrosine to the medium. The strains accumulated also 2-phenylethylamine, although with lower efficiency and in greater extent when tyrosine was not added. Accordingly, the tyrosine decarboxylase (tyrDC) gene expression level increased during the exponential phase with tyrosine added, while it remained constant and high without precursor. The genetic organization as well as sequence identity levels of tyrDC and tyrosine permease (tyrP) genes indicated a correlation with those of phylogenetically closer enterococcal species, such as E. faecium, E. hirae and E. durans; however, the gene Na+/H+ antiporter (nhaC) that usually follow tyrP is missing. In addition, BLAST analysis revealed the presence of additional genes encoding for decarboxylase and permease in the genome of several E. mundtii strains. It is speculated the occurrence of a duplication event and the acquisition of different specificity for these enzymes that deserves further investigations.

Crystal structure of tyrosine decarboxylase and identification of key residues involved in conformational swing and substrate binding.

Tyrosine decarboxylase (TDC) is a pyridoxal 5-phosphate (PLP)-dependent enzyme and is mainly responsible for the synthesis of tyramine, an important biogenic amine. In this study, the crystal structures of the apo and holo forms of Lactobacillus brevis TDC (LbTDC) were determined. The LbTDC displays only 25% sequence identity with the only reported TDC structure. Site-directed mutagenesis of the conformationally flexible sites and catalytic center was performed to investigate the potential catalytic mechanism. It was found that H241 in the active site plays an important role in PLP binding because it has different conformations in the apo and holo structures of LbTDC. After binding to PLP, H241 rotated to the position adjacent to the PLP pyridine ring. Alanine scanning mutagenesis revealed several crucial regions that determine the substrate specificity and catalytic activity. Among the mutants, the S586A variant displayed increased catalytic efficiency and substrate affinity, which is attributed to decreased steric hindrance and increased hydrophobicity, as verified by the saturation mutagenesis at S586. Our results provide structural information about the residues important for the protein engineering of TDC to improve catalytic efficiency in the green manufacturing of tyramine.

Fast-Scan Cyclic Voltammetry (FSCV) Detection of Endogenous Octopamine in Drosophila melanogaster Ventral Nerve Cord.

Octopamine is an endogenous biogenic amine neurotransmitter, neurohormone, and neuromodulator in invertebrates and has functional analogy with norepinephrine in vertebrates. Fast-scan cyclic voltammetry (FSCV) can detect rapid changes in neurotransmitters, but FSCV has not been optimized for octopamine detection in situ. The goal of this study was to characterize octopamine release in the ventral nerve cord of Drosophila larvae for the first time. A FSCV waveform was optimized so that the potential for octopamine oxidation would not be near the switching potential where interferences can occur. Endogenous octopamine release was stimulated by genetically inserting either the ATP sensitive channel, P2X2, or the red-light sensitive channelrhodopsin, CsChrimson, into cells expressing tyrosine decarboxylase (TDC), an octopamine synthesis enzyme. To ensure that release is due to octopamine and not the precursor tyramine, the octopamine synthesis inhibitor disulfiram was applied, and the signal decreased by 80%. Stimulated release was vesicular, and a 2 s continuous light stimulation of CsChrimson evoked 0.22 ± 0.03 µM of octopamine release in the larval ventral nerve cord. Repeated stimulations were stable with 2 or 5 min interstimulation times. With pulsed stimulations, the release was dependent on the frequency of applied light pulse. An octopamine transporter has not been identified, and blockers of the dopamine transporter and serotonin transporter had no significant effect on the clearance time of octopamine, suggesting that they do not take up octopamine. This study shows that octopamine can be monitored in Drosophila, facilitating future studies of how octopamine release functions in the insect brain.

[Cloning and expression analysis of a tyrosine decarboxylase gene from Rehmannia glutinosa].

Tyrosine decarboxylase (TyrDC) is an important enzyme in the secondary metabolism of several plant species, and was hypothesized to play a key role in the biosynthesis of phenylethanoid glycosides. Based on the transcriptome data, we cloned the full-length cDNA (GenBank accession NO. KU640395) of RgTyDC gene from Rehmannia glutinosa, and then performed bioinformatic analysis of the sequence. Further, we detected the expression pattern in different organs and hair roots treated with four elicitors by qRT-PCR. The results showed that the full length of RgTyDC cDNA was 1 530 bp encoding 509 amino acids. The molecular weight of the putative RgTyDC protein was about 56.6 kDa and the theoretical isoelectric point was 6.25. The RgTyDC indicated the highest homology with Sesamum indicum SiTyDC and Erythranthe guttata EgTyDC, both of them were reached 88%. RgTyDC highly expressed in R. glutinosa leaf, especially in senescing leaf, and rarely expressed in tuberous root. After the treatment of SA and MeJA, the relative expression level of RgTyDC mRNA was substantially increased. The results provide a foundation for exploring the molecular function of RgTyDC involved in phenylethanoid glycosides biosynthesis.