Metabolic engineering study on biosynthesis of 4-hydroxybenzyl alcohol from L-tyrosine in Escherichia coli / 中国中药杂志

As an important active ingredient in the rare Chinese herb Gastrodiae Rhizoma and also the main precursor for gastrodin biosynthesis, 4-hydroxybenzyl alcohol has multiple pharmacological activities such as anti-inflammation, anti-tumor, and anti-cerebral ischemia. The pharmaceutical products with 4-hydroxybenzyl alcohol as the main component have been increasingly favored. At present, 4-hydroxybenzyl alcohol is mainly obtained by natural extraction and chemical synthesis, both of which, however, exhibit some shortcomings that limit the long-term application of 4-hydroxybenzyl alcohol. The wild and cultivated Gastrodia elata resources are limited. The chemical synthesis requires many steps, long time, and harsh reaction conditions. Besides, the resulting by-products are massive and three reaction wastes are difficult to treat. Therefore, how to artificially prepare 4-hydroxybenzyl alcohol with high yield and purity has become an urgent problem facing the medical researchers. Guided by the theory of microbial metabolic engineering, this study employed the genetic engineering technologies to introduce three genes ThiH, pchF and pchC into Escherichia coli for synthesizing 4-hydroxybenzyl alcohol with L-tyrosine. And the fermentation conditions of engineering strain for producing 4-hydroxybenzyl alcohol in shake flask were also discussed. The experimental results showed that under the conditions of 0.5 mmol·L~(-1) IPTG, 15 ℃ induction temperature, and 40 ℃ transformation temperature, M9 Y medium containing 200 mg·L~(-1) L-tyrosine could be transformed into(69±5)mg·L~(-1) 4-hydroxybenzyl alcohol, which has laid a foundation for producing 4-hydroxybenzyl alcohol economically and efficiently by further expanding the fermentation scale in the future.

Miopatía nemalínica tratada con L-tirosina para aliviar los síntomas en un recién nacido / Nemaline rod myopathy treated with L-tyrosine to relieve symptoms in a neonate

La miopatía nemalínica es un trastorno heterogéneo definido por la presencia de estructuras con forma de bastones, conocidas como cuerpos nemalínicos (o bastones de nemalina). El diagnóstico se funda en la debilidad muscular, además de la visualización de cuerpos nemalínicos en la biopsia muscular. La miopatía nemalínica no tiene cura. Las estrategias terapéuticas para este trastorno son sintomáticas y empíricas. En este artículo, presentamos el caso de una recién nacida con insuficiencia respiratoria grave y debilidad muscular generalizada, a la que se le diagnosticó miopatía nemalínica a través de la biopsia muscular. La paciente tuvo una notable disminución de la sialorrea y una mejora de los movimientos espontáneos después del tratamiento con L-tirosina. Este caso se presenta para destacar la importancia de la biopsia muscular en el diagnóstico diferencial de la hipotonía grave durante el período neonatal y el posible beneficio del aporte suplementario de L-tirosina para disminuir la sialorrea y restaurar la fuerza muscular.

Nemaline myopathy (NM) is a heterogeneous disorder defined by the presence of rod-shaped structures known as nemaline bodies or rods. The diagnosis is based on muscle weakness, combined with visualization of nemaline bodies on muscle biopsy. There is no curative treatment for nemaline myopathy. Therapeutic strategies for this condition are symptomatic and empirical. Herein, we present a newborn with severe respiratory failure and generalized muscle weakness, who was diagnosed as NM by muscle biopsy. The patient experienced remarkable decrease in sialorrhea and improvement of spontaneous movements after L-tyrosine treatment. This case is presented to emphasize the importance of muscle biopsy in the differential diagnosis of severe hypotonia during neonatal period and a possible benefit of L-tyrosine supplementation for decreasing sialorrhea and restoring muscle strength.

Study on synergistic mechanism of pangolin in high temperature sand-fried processing / 中草药

Objective Traditional raw pangolin products are not used as medicine, which can only be used as medicine after processing. Therefore, the processing mechanism of high temperature sand-fried pangolin was studied. Methods The changes of liposolubility and protein composition of pangolin before and after processing were analyzed by TLC and Nano LC-Q Exactive Orbitrap MS. Meanwhile, the simulation processing of cyclic dipeptides, which were significantly increased during processing, was performed. The activity of L-serine-L-tyrosine cyclic dipeptide was screened. Results The results showed that there was no significant change in fat-soluble components, significant decrease in polypeptides and significant increase in cyclic dipeptides after the sand-fried processing of pangolin. The formation of cyclic dipeptides was mainly related to the heating of the processing. At low temperature, the N-terminal of the linear peptide could be cycled to form L-shaped cyclic dipeptides. At high temperature, the N-terminal and C-terminal of the linear peptide could be rapidly cycled to form cyclic dipeptides. L-serine-L-tyrosine cyclic dipeptide could prolong coagulation time and increase the proliferation rate of mammary epithelial cells and the expression of genes related to milk protein synthesis in dairy cows. It also had significant analgesic activity, which was consistent with the traditional efficacy of pangolin. Conclusion These results suggested that large amounts of L-serine-L-tyrosine cyclic dipeptide produced by the processing of pangolin may be one of the material bases for enhancing the processing efficiency of pangolin. It was of great significance for revealing the material basis of pharmacodynamics of pangolin, searching for alternative resources and protecting pangolin.

Effect of gene knockout of L-tyrosine transport system on L-tyrosine production in Escherichia coli / 生物工程学报

L-tyrosine is one of three aromatic amino acids that are widely used in food, pharmaceutical and chemical industries. The transport system engineering provides an important research strategy for the metabolic engineering of Escherichia coli to breed L-tyrosine producing strain. The intracellular transport of L-tyrosine in E. coli is mainly regulated by two distinct permeases encoded by aroP and tyrP genes. The aroP and tyrP gene knockout mutants were constructed by CRISPR-Cas technique on the basis of L-tyrosine producing strain HGXP, and the effects of regulating transport system on L-tyrosine production were investigated by fermentation experiments. The fermentation results showed that the aroP and tyrP knockout mutants produced 3.74 and 3.45 g/L L-tyrosine, respectively, which were 19% and 10% higher than that of the original strain. The optimum induction temperature was determined to be 38 °C. Fed-batch fermentation was carried out on a 3-L fermentor. The L-tyrosine yields of aroP and tyrP knockout mutants were further increased to 44.5 and 35.1 g/L, respectively, which were 57% and 24% higher than that of the original strain. The research results are of great reference value for metabolic engineering of E. coli to produce L-tyrosine.

Evaluation of melanin production by Sporothrix luriei

There is a paucity of studies on the cell biology of Sporothrix luriei, the less common of the pathogenic Sporothrix species worldwide. The production of DHN-melanin, eumelanin, and pyomelanin were evaluated on the mycelial and yeast forms of the S. luriei ATCC 18616 strain. The mycelial form of this species produced only pyomelanin, which protected the fungus against environmental stressors such as ultraviolet light, heat, and cold. The yeast form was unable to produce any of the tested melanin types. The lack of melanin in the parasitic form of S. luriei may be an explanation for its low frequency in human infections.

Evaluation of the effect of red guava (Psidium guajava) fruit extract on tyrosinase (EC 1.14.18.1) activity by spectrophotometry.

Tyrosinase is one of the most important enzymes in melanin biosynthesis. Inhibition of tyrosinase activity will cause a decrease in melanin production. Tyrosinase inhibitory activity by ascorbic acid has been studied before. Based on reported experiments, ascorbic acid can inhibit tyrosinase activity in enzymatic reaction competitively. As an effort to find out a skin whitening agent which is effective, safe and have minimum adverse effect, the inhibitory activities of Psidium guajava extract was studied on tyrosinase activity. This is one of the fruits that contains high amount of ascorbic acid. To determine the efficacy of tyrosinase inhibition, L-tyrosine was used as the substrate, P. guajava ex-tract as inhibitor and ascorbic acid as positive control that was measured by spectrophotometry. The optimization of method was also performed. The inhibitory kinetics was determined by measuring the absorbance of dopachrome as the end product of the tyrosinase reaction. Michaelis-Menten constant (Km) and maximum velocity (Vmax) of the ty-rosinase were determined by Lineweaver-Burk’s plots. The Km and Vmax without the fruit extract were 0.315mM and 0.0265μmol/min. The Km value with the fruit extract of 1%, 2% & 3% w/v were 0.4824, 0.698 & 0.543mM, while the Vmax value were 0.0269, 0.0283 & 0.0255 μmol/min, respectively. Lineweaver-Burk’s plots in presence of P. guajava fruit extract showed that the extract inhibited tyrosinase competitively. From the plots, the IC50 of the fruit extract was determined as 0.26mM; the control in 0.26mM concentration inhibited 56.523%. Finally, P. guajava fruit extract showed higher effect than ascorbic acid on Tyrosinase activity.

Evaluation of the effect of red guava (Psidium guajava) fruit extract on tyrosinase (EC 1.14.18.1) activity by spectrophotometry.

Tyrosinase is one of the most important enzymes in melanin biosynthesis. Inhibition of tyrosinase activity will cause a decrease in melanin production. Tyrosinase inhibitory activity by ascorbic acid has been studied before. Based on reported experiments, ascorbic acid can inhibit tyrosinase activity in enzymatic reaction competitively. As an effort to find out a skin whitening agent which is effective, safe and have minimum adverse effect, the inhibitory activities of Psidium guajava extract was studied on tyrosinase activity. This is one of the fruits that contains high amount of ascorbic acid. To determine the efficacy of tyrosinase inhibition, L-tyrosine was used as the substrate, P. guajava ex-tract as inhibitor and ascorbic acid as positive control that was measured by spectrophotometry. The optimization of method was also performed. The inhibitory kinetics was determined by measuring the absorbance of dopachrome as the end product of the tyrosinase reaction. Michaelis-Menten constant (Km) and maximum velocity (Vmax) of the ty-rosinase were determined by Lineweaver-Burk’s plots. The Km and Vmax without the fruit extract were 0.315mM and 0.0265μmol/min. The Km value with the fruit extract of 1%, 2% & 3% w/v were 0.4824, 0.698 & 0.543mM, while the Vmax value were 0.0269, 0.0283 & 0.0255 μmol/min, respectively. Lineweaver-Burk’s plots in presence of P. guajava fruit extract showed that the extract inhibited tyrosinase competitively. From the plots, the IC50 of the fruit extract was determined as 0.26mM; the control in 0.26mM concentration inhibited 56.523%. Finally, P. guajava fruit extract showed higher effect than ascorbic acid on Tyrosinase activity.

Effect of L - tyrosine on Proliferation and Melanization in Cultured Normal Human Melanocytes / 대한피부과학회지

BACKGROUND: The biosynthesis of melanin is initiated by the enzymatic oxidation of L-tyrosine to L-dopa by tyrosinase. Some precursors of melanin are cytotoxic, and melanoma cells are killed as a risk of exposare to excess tyrosine or dopa in the culture medium. However, there have been few observations of the effects of L-tyrosine on cultured normal human melanocyte. OBJECTIVE: In order to investigate whether exogenous tyrosine induces cytotoxicity in cultured normal human melanocytes as in melanoma cells, we examined the effects of L-tyrosine on proliferation and melanization in normal human melanocytes. METHODS: A melanocyte culture was produced with a modified TIC medium. L-tyrosine was added to the culture medium, 100, 200, 400, and 800uM. After 2 days of incubation, the proliferation was measured by methylthiazol tetrazolium(MTT) assay and sulforhodamine B(SRB) assay. The melanin contenis were also measured by the modified Whittaker's method. RESULTS: On MTT assay, the proliferation of melanocytes had been stirnulated significantly (p< 0.05) in all L-tyrosine added groups. On SRB assay, the proliferation of melanocytes had heen stimulated significantly (p<005) in 200, 400, 800uM of L-tyrosine added groups. The melanin contents had increased in all L-tyrosine added groups, and had increased significantly (p<0.05) in 400uM of L-tyrosine added group. CONCLUSION: L-tyrosine is not toxic to normal melanocytes, It stimulates the proliferation and melnization of cultured normal human melanocytes.

Effect of L - tyrosine on Proliferation and Melanization in Cultured Normal Human Melanocytes / 대한피부과학회지

BACKGROUND: The biosynthesis of melanin is initiated by the enzymatic oxidation of L-tyrosine to L-dopa by tyrosinase. Some precursors of melanin are cytotoxic, and melanoma cells are killed as a risk of exposare to excess tyrosine or dopa in the culture medium. However, there have been few observations of the effects of L-tyrosine on cultured normal human melanocyte. OBJECTIVE: In order to investigate whether exogenous tyrosine induces cytotoxicity in cultured normal human melanocytes as in melanoma cells, we examined the effects of L-tyrosine on proliferation and melanization in normal human melanocytes. METHODS: A melanocyte culture was produced with a modified TIC medium. L-tyrosine was added to the culture medium, 100, 200, 400, and 800uM. After 2 days of incubation, the proliferation was measured by methylthiazol tetrazolium(MTT) assay and sulforhodamine B(SRB) assay. The melanin contenis were also measured by the modified Whittaker's method. RESULTS: On MTT assay, the proliferation of melanocytes had been stirnulated significantly (p< 0.05) in all L-tyrosine added groups. On SRB assay, the proliferation of melanocytes had heen stimulated significantly (p<005) in 200, 400, 800uM of L-tyrosine added groups. The melanin contents had increased in all L-tyrosine added groups, and had increased significantly (p<0.05) in 400uM of L-tyrosine added group. CONCLUSION: L-tyrosine is not toxic to normal melanocytes, It stimulates the proliferation and melnization of cultured normal human melanocytes.