Characterization of aspartate kinase and homoserine dehydrogenase from <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> IWJ001 and systematic investigation of <Emphasis Type="SmallCaps">l</Emphasis>-isoleucine biosynthesis

Previously we have characterized a threonine dehydratase mutant TD^F383V (encoded by ilvA1) and an acetohydroxy acid synthase mutant AHAS^{P176S, D426E, L575W} (encoded by ilvBN1) in Corynebacterium glutamicum IWJ001, one of the best l-isoleucine producing strains. Here, we further characterized an aspartate kinase mutant AK^A279T (encoded by lysC1) and a homoserine dehydrogenase mutant HD^G378S (encoded by hom1) in IWJ001, and analyzed the consequences of all these mutant enzymes on amino acids production in the wild type background. In vitro enzyme tests confirmed that AK^A279T is completely resistant to feed-back inhibition by l-threonine and l-lysine, and that HD^G378S is partially resistant to l-threonine with the half maximal inhibitory concentration between 12 and 14 mM. In C. glutamicum ATCC13869, expressing lysC1 alone led to exclusive l-lysine accumulation, co-expressing hom1 and thrB1 with lysC1 shifted partial carbon flux from l-lysine (decreased by 50.1 %) to l-threonine (4.85 g/L) with minor l-isoleucine and no l-homoserine accumulation, further co-expressing ilvA1 completely depleted l-threonine and strongly shifted carbon flux from l-lysine (decreased by 83.0 %) to l-isoleucine (3.53 g/L). The results demonstrated the strongly feed-back resistant TD^F383V might be the main driving force for l-isoleucine over-synthesis in this case, and the partially feed-back resistant HD^G378S might prevent the accumulation of toxic intermediates. Information exploited from such mutation-bred production strain would be useful for metabolic engineering.     

<Emphasis Type="SmallCaps">l</Emphasis>-carnosine induces apoptosis/cell cycle arrest via suppression of NF-κB/STAT1 pathway in HCT116 colorectal cancer cells

l-carnosine, a dipeptide of the amino acids β-alanine and histidine, is found in various tissues, such as the central nervous system and skeletal muscles. Recently, l-carnosine has been reported to possess anti-tumor activity; however, the molecular mechanism underlying its activity in colorectal cancer is still unknown. Therefore, we investigated the effect of l-carnosine using a human colorectal cancer cell line, HCT116. Treatment with l-carnosine (0, 100, or 200 mM) for 24 h gradually reduced cellular proliferation according to immunochemistry and 7-aminoactinomycin D (7-AAD) analyses and induced G0/G1 phase arrest. In the RT-PCR analysis, l-carnosine decreased the mRNA levels of cell cycle-related genes in HCT116 cells. In the Western blot analysis, levels of the cyclin D1, BAX/Bcl-2, cleaved caspase-3, p21, and p53 proteins were significantly increased in cells treated with l-carnosine. We next determined whether STAT1/NF-κB pathway is involved in regulation of cell cycle arrest- and cell death-associated gene in HCT116. The l-carnosine treatment significantly inhibited the phosphorylation of STAT1 on Tyr701 and NF-κB p65 on Ser276 and Ser536, and then, we exogenously blocked the NF-κB phosphorylation using Bay 11-7082. Based on our findings, l-carnosine induces cell cycle arrest and apoptosis in human colorectal cancer cells by suppressing of NF-κB/STAT1 signaling.     

<Emphasis Type="SmallCaps">l</Emphasis>-Serine overproduction with minimization of by-product synthesis by engineered <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

The direct fermentative production of l-serine by Corynebacterium glutamicum from sugars is attractive. However, superfluous by-product accumulation and low l-serine productivity limit its industrial production on large scale. This study aimed to investigate metabolic and bioprocess engineering strategies towards eliminating by-products as well as increasing l-serine productivity. Deletion of alaT and avtA encoding the transaminases and introduction of an attenuated mutant of acetohydroxyacid synthase (AHAS) increased both l-serine production level (26.23 g/L) and its productivity (0.27 g/L/h). Compared to the parent strain, the by-products l-alanine and l-valine accumulation in the resulting strain were reduced by 87 % (from 9.80 to 1.23 g/L) and 60 % (from 6.54 to 2.63 g/L), respectively. The modification decreased the metabolic flow towards the branched-chain amino acids (BCAAs) and induced to shift it towards l-serine production. Meanwhile, it was found that corn steep liquor (CSL) could stimulate cell growth and increase sucrose consumption rate as well as l-serine productivity. With addition of 2 g/L CSL, the resulting strain showed a significant improvement in the sucrose consumption rate (72 %) and the l-serine productivity (67 %). In fed-batch fermentation, 42.62 g/L of l-serine accumulation was achieved with a productivity of 0.44 g/L/h and yield of 0.21 g/g sucrose, which was the highest production of l-serine from sugars to date. The results demonstrated that combined metabolic and bioprocess engineering strategies could minimize by-product accumulation and improve l-serine productivity.     

Production of <Emphasis Type="SmallCaps">d</Emphasis>-alanine from <Emphasis Type="SmallCaps">dl</Emphasis>-alanine using immobilized cells of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> HLZ-68

Immobilized cells of Bacillus subtilis HLZ-68 were used to produce d-alanine from dl-alanine by asymmetric degradation. Different compounds such as polyvinyl alcohol and calcium alginate were employed for immobilizing the B. subtilis HLZ-68 cells, and the results showed that cells immobilized using a mixture of these two compounds presented higher l-alanine degradation activity, when compared with free cells. Subsequently, the effects of different concentrations of polyvinyl alcohol and calcium alginate on l-alanine consumption were examined. Maximum l-alanine degradation was exhibited by cells immobilized with 8% (w/v) polyvinyl alcohol and 2% (w/v) calcium alginate. Addition of 400 g of dl-alanine (200 g at the beginning of the reaction and 200 g after 30 h of incubation) into the reaction solution at 30 °C, pH 6.0, aeration of 1.0 vvm, and agitation of 400 rpm resulted in complete l-alanine degradation within 60 h, leaving 185 g of d-alanine in the reaction solution. The immobilized cells were applied for more than 15 cycles of degradation and a maximum utilization rate was achieved at the third cycle. d-alanine was easily extracted from the reaction solution using cation-exchange resin, and the chemical and optical purity of the extracted d-alanine was 99.1 and 99.6%, respectively.     

Increased productivity of <Emphasis Type="SmallCaps">l</Emphasis>-2-aminobutyric acid and total turnover number of NAD<Superscript>+</Superscript>/NADH in a one-pot system through enhanced thermostability of <Emphasis Type="SmallCaps">l</Emphasis>-threonine deaminase

Objective

To strengthen NADH regeneration in the biosynthesis of l-2-aminobutyric acid (l-ABA).

Results

l-Threonine deaminase (l-TD) from Escherichia coli K12 was modified by directed evolution and rational design to improve its endurance to heat treatment. The half-life of mutant G323D/F510L/T344A at 42 °C increased from 10 to 210 min, a 20-fold increase compared to the wild-type l-TD, and the temperature at which the activity of the enzyme decreased by 50% in 15 min increased from 39 to 53 °C. The mutant together with thermostable l-leucine dehydrogenase from Bacillus sphaericus DSM730 and formate dehydrogenase from Candida boidinii constituted a one-pot system for l-ABA biosynthesis. Employing preheat treatment in the one-pot system, the biosynthesis of l-ABA and total turnover number of NAD⁺/NADH were 0.993 M and 16,469, in contrast to 0.635 M and 10,531 with wild-type l-TD, respectively.

Conclusions

By using the engineered l-TD during endured preheat treatment, the one-pot system has achieved a higher productivity of l-ABA and total turnover number of coenzyme.

     

Engineering rTCA pathway and C4-dicarboxylate transporter for <Emphasis Type="SmallCaps">l</Emphasis>-malic acid production

l-Malic acid is an important component of a vast array of food additives, antioxidants, disincrustants, pharmaceuticals, and cosmetics. Here, we presented a pathway optimization strategy and a transporter modification approach to reconstruct the l-malic acid biosynthesis pathway and transport system, respectively. First, pyruvate carboxylase (pyc) and malate dehydrogenase (mdh) from Aspergillus flavus and Rhizopus oryzae were combinatorially overexpressed to construct the reductive tricarboxylic acid (rTCA) pathway for l-malic acid biosynthesis. Second, the l-malic acid transporter (Spmae) from Schizosaccharomyces pombe was engineered by removing the ubiquitination motification to enhance the l-malic acid efflux system. Finally, the l-malic acid pathway was optimized by controlling gene expression levels, and the final l-malic acid concentration, yield, and productivity were up to 30.25 g L^?1, 0.30 g g^?1, and 0.32 g L^?1 h^?1 in the resulting strain W4209 with CaCO₃ as a neutralizing agent, respectively. In addition, these corresponding parameters of pyruvic acid remained at 30.75 g L^?1, 0.31 g g^?1, and 0.32 g L^?1 h^?1, respectively. The metabolic engineering strategy used here will be useful for efficient production of l-malic acid and other chemicals.     

Rational design and analysis of an <Emphasis Type="Italic">Escherichia coli</Emphasis> strain for high-efficiency tryptophan production

l-tryptophan (l-trp) is a precursor of various bioactive components and has great pharmaceutical interest. However, due to the requirement of several precursors and complex regulation of the pathways involved, the development of an efficient l-trp production strain is challenging. In this study, Escherichia coli (E. coli) strain KW001 was designed to overexpress the l-trp operator sequences (trpEDCBA) and 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase (aroG ^fbr ). To further improve the production of l-trp, pyruvate kinase (pykF) and the phosphotransferase system HPr (ptsH) were deleted after inactivation of repression (trpR) and attenuation (attenuator) to produce strain KW006. To overcome the relatively slow growth and to increase the transport rate of glucose, strain KW018 was generated by combinatorial regulation of glucokinase (galP) and galactose permease (glk) expression. To reduce the production of acetic acid, strain KW023 was created by repressive regulation of phosphate acetyltransferase (pta) expression. In conclusion, strain KW023 efficiently produced 39.7 g/L of l-trp with a conversion rate of 16.7% and a productivity of 1.6 g/L/h in a 5 L fed-batch fermentation system.     

The modulation of NMDA receptors and <Emphasis Type="SmallCaps">l</Emphasis>-arginine/nitric oxide pathway is implicated in the anti-immobility effect of creatine in the tail suspension test

The modulation of N-methyl-D-aspartate receptor (NMDAR) and l-arginine/nitric oxide (NO) pathway is a therapeutic strategy for treating depression and neurologic disorders that involves excitotoxicity. Literature data have reported that creatine exhibits antidepressant and neuroprotective effects, but the implication of NMDAR and l-arginine/nitric oxide (NO) pathway in these effects is not established. This study evaluated the influence of pharmacological agents that modulate NMDAR/l-arginine-NO pathway in the anti-immobility effect of creatine in the tail suspension test (TST) in mice. The NOx levels and cellular viability in hippocampal and cerebrocortical slices of creatine-treated mice were also evaluated. The anti-immobility effect of creatine (10 mg/kg, po) in the TST was abolished by NMDA (0.1 pmol/mouse, icv), d-serine (30 µg/mouse, icv, glycine-site NMDAR agonist), arcaine (1 mg/kg, ip, polyamine site NMDAR antagonist), l-arginine (750 mg/kg, ip, NO precursor), SNAP (25 μg/mouse, icv, NO donor), L-NAME (175 mg/kg, ip, non-selective NOS inhibitor) or 7-nitroindazole (50 mg/kg, ip, neuronal NOS inhibitor), but not by DNQX (2.5 µg/mouse, icv, AMPA receptor antagonist). The combined administration of sub-effective doses of creatine (0.01 mg/kg, po) and NMDAR antagonists MK-801 (0.001 mg/kg, po) or ketamine (0.1 mg/kg, ip) reduced immobility time in the TST. Creatine (10 mg/kg, po) increased cellular viability in hippocampal and cerebrocortical slices and enhanced hippocampal and cerebrocortical NO _x levels, an effect potentiated by l-arginine or SNAP and abolished by 7-nitroindazole or L-NAME. In conclusion, the anti-immobility effect of creatine in the TST involves NMDAR inhibition and enhancement of NO levels accompanied by an increase in neural viability.     

An optimized fed-batch culture strategy integrated with a one-step fermentation improves <Emphasis Type="SmallCaps">l</Emphasis>-lactic acid production by <Emphasis Type="Italic">Rhizopus oryzae</Emphasis>

In previous work, we proposed a novel modified one-step fermentation fed-batch strategy to efficiently generate l-lactic acid (l-LA) using Rhizopus oryzae. In this study, to further enhance efficiency of l-LA production through one-step fermentation in fed-batch cultures, we systematically investigated the initial peptone- and glucose-feeding approaches, including different initial peptone and glucose concentrations and maintained residual glucose levels. Based on the results of this study, culturing R. oryzae with initial peptone and glucose concentrations of 3.0 and 50.0 g/l, respectively, using a fed-batch strategy is an effective approach of producing l-LA through one-step fermentation. Changing the residual glucose had no obvious effect on the generation of l-LA. We determined the maximum LA production and productivity to be 162 g/l and 6.23 g/(l·h), respectively, during the acid production stage. Compared to our previous work, there was almost no change in l-LA production or yield; however, the productivity of l-LA increased by 14.3%.     

Enhanced biocatalytic production of <Emphasis Type="SmallCaps">l</Emphasis>-cysteine by <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. B-3 with in situ product removal using ion-exchange resin

Bioconversion of dl-2-amino-Δ²-thiazoline-4-carboxylic acid (dl-ATC) catalyzed by whole cells of Pseudomonas sp. was successfully applied for the production of l-cysteine. It was found, however, like most whole-cell biocatalytic processes, the accumulated l-cysteine produced obvious inhibition to the activity of biocatalyst and reduced the yield. To improve l-cysteine productivity, an anion exchange-based in situ product removal (ISPR) approach was developed. Several anion-exchange resins were tested to select a suitable adsorbent used in the bioconversion of dl-ATC for the in situ removal of l-cysteine. The strong basic anion-exchange resin 201 × 7 exhibited the highest adsorption capacity for l-cysteine and low adsorption for dl-ATC, which is a favorable option. With in situ addition of 60 g L^?1 resin 201 × 7, the product inhibition can be reduced significantly and 200 mmol L^?1 of dl-ATC was converted to l-cysteine with 90.4 % of yield and 28.6 mmol L^?1 h^?1 of volumetric productivity. Compared to the bioconversion without the addition of resin, the volumetric productivity of l-cysteine was improved by 2.27-fold using ISPR method.     

Genome shuffling and high-throughput screening of <Emphasis Type="Italic">Brevibacterium flavum</Emphasis> MDV1 for enhanced <Emphasis Type="SmallCaps">l</Emphasis>-valine production

l-valine is an essential branched-amino acid that is widely used in multiple areas such as pharmaceuticals and special dietary products and its use is increasing. As the world market for l-valine grows rapidly, there is an increasing interest to develop an efficient l-valine-producing strain. In this study, a simple, sensitive, efficient, and consistent screening procedure termed 96 well plate-PC-HPLC (96-PH) was developed for the rapid identification of high-yield l-valine strains to replace the traditional l-valine assay. l-valine production by Brevibacterium flavum MDV1 was increased by genome shuffling. The starting strains were obtained using ultraviolet (UV) irradiation and binary ethylenimine treatment followed by preparation of protoplasts, UV irradiation inactivation, multi-cell fusion, and fusion of the inactivated protoplasts to produce positive colonies. After two rounds of genome shuffling and the 96-PH method, six l-valine high-yielding mutants were selected. One genetically stable mutant (MDVR2-21) showed an l-valine yield of 30.1 g/L during shake flask fermentation, 6.8-fold higher than that of MDV1. Under fed-batch conditions in a 30 L automated fermentor, MDVR2-21 accumulated 70.1 g/L of l-valine (0.598 mol l-valine per mole of glucose; 38.9% glucose conversion rate). During large-scale fermentation using a 120 m³ fermentor, this strain produced?>?66.8 g/L l-valine (36.5% glucose conversion rate), reflecting a very productive and stable industrial enrichment fermentation effect. Genome shuffling is an efficient technique to improve production of l-valine by B. flavum MDV1. Screening using 96-PH is very economical, rapid, efficient, and well-suited for high-throughput screening.     

<Emphasis Type="Italic">C7</Emphasis>-prenylation of tryptophanyl and <Emphasis Type="Italic">O</Emphasis>-prenylation of tyrosyl residues in dipeptides by an <Emphasis Type="Italic">Aspergillus terreus</Emphasis> prenyltransferase

During our search for novel prenyltransferases, a putative gene ATEG_04218 from Aspergillus terreus raised our attention and was therefore amplified from strain DSM 1958 and expressed in Escherichia coli. Biochemical investigations with the purified recombinant protein and different aromatic substrates in the presence of dimethylallyl diphosphate revealed the acceptance of all the tested tryptophan-containing cyclic dipeptides. Structure elucidation of the main enzyme products by NMR and MS analyses confirmed the attachment of the prenyl moiety to C-7 of the indole ring, proving the identification of a cyclic dipeptide C7-prenyltransferase (CdpC7PT). For some substrates, reversely C3- or N1-prenylated derivatives were identified as minor products. In comparison to the known tryptophan-containing cyclic dipeptide C7-prenyltransferase CTrpPT from Aspergillus oryzae, CdpC7PT showed a much higher substrate flexibility. It also accepted cyclo-l-Tyr-l-Tyr as substrate and catalyzed an O-prenylation at the tyrosyl residue, providing the first example from the dimethylallyltryptophan synthase (DMATS) superfamily with an O-prenyltransferase activity towards dipeptides. Furthermore, products with both C7-prenyl at tryptophanyl and O-prenyl at tyrosyl residue were detected in the reaction mixture of cyclo-l-Trp-l-Tyr. Determination of the kinetic parameters proved that (S)-benzodiazepinedione consisting of a tryptophanyl and an anthranilyl moiety was accepted as the best substrate with a K _M value of 204.1 μM and a turnover number of 0.125 s^?1. Cyclo-l-Tyr-l-Tyr was accepted with a K _M value of 1,411.3 μM and a turnover number of 0.012 s^?1.     

Safety of long-term dietary supplementation with <Emphasis Type="SmallCaps">l</Emphasis>-arginine in rats

This study was conducted with rats to determine the safety of long-term dietary supplementation with l-arginine. Beginning at 6 weeks of age, male and female rats were fed a casein-based semi-purified diet containing 0.61 % l-arginine and received drinking water containing l-arginine-HCl (0, 1.8, or 3.6 g l-arginine/kg body-weight/day; n = 10/group). These supplemental doses of l-arginine were equivalent to 0, 286, and 573 mg l-arginine/kg body-weight/day, respectively, in humans. After a 13-week supplementation period, blood samples were obtained from rats for biochemical analyses. Supplementation with l-arginine increased plasma concentrations of arginine, ornithine, proline, homoarginine, urea, and nitric oxide metabolites without affecting those for lysine, histidine, or methylarginines, while reducing plasma concentrations of ammonia, glutamine, free fatty acids, and triglycerides. l-Arginine supplementation enhanced protein gain and reduced white-fat deposition in the body. Based on general appearance, feeding behavior, and physiological parameters, all animals showed good health during the entire experimental period; Plasma concentrations of all measured hormones (except leptin) did not differ between control and arginine-supplemented rats. l-Arginine supplementation reduced plasma levels of leptin. Additionally, l-arginine supplementation increased l-arginine:glycine amidinotransferase activity in kidneys but not in the liver or small intestine, suggesting tissue-specific regulation of enzyme expression by l-arginine. Collectively, these results indicate that dietary supplementation with l-arginine (e.g., 3.6 g/kg body-weight/day) is safe in rats for at least 91 days. This dose is equivalent to 40 g l-arginine/kg body-weight/day for a 70-kg person. Our findings help guide clinical studies to determine the safety of long-term oral administration of l-arginine to humans.     

Enhancement of NAD(H) pool for formation of oxidized biochemicals in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The NAD⁺/NADH ratio and the total NAD(H) play important roles for whole-cell biochemical redox transformations. After the carbon source is exhausted, the degradation of NAD(H) could contribute to a decline in the rate of a desired conversion. In this study, methods to slow the native rate of NAD(H) degradation were examined using whole-cell Escherichia coli with two model oxidative NAD⁺-dependent biotransformations. A high phosphate concentration (50 mM) was observed to slow NAD(H) degradation. We also constructed E. coli strains with deletions in genes coding several enzymes involved in NAD⁺ degradation. In shake-flask experiments, the total NAD(H) concentration positively correlated with conversion of xylitol to l-xylulose by xylitol 4-dehydrogenase, and the greatest conversion (80%) was observed using MG1655 nadR nudC mazG/pZE12-xdh/pCS27-nox. Controlled 1-L batch processes comparing E. coli nadR nudC mazG with a wild-type background strain demonstrated a 30% increase in final l-xylulose concentration (5.6 vs. 7.9 g/L) and a 25% increase in conversion (0.53 vs. 0.66 g/g). MG1655 nadR nudC mazG was also examined for the conversion of galactitol to l-tagatose by galactitol 2-dehydrogenase. A batch process using 15 g/L glycerol and 10 g/L galactitol generated over 9.4 g/L l-tagatose, corresponding to 90% conversion and a yield of 0.95 g l-tagatose/g galactitol consumed. The results demonstrate the value of minimizing NAD(H) degradation as a means to improve NAD⁺-dependent biotransformations.     

Functional expression of a human GDP-<Emphasis Type="SmallCaps">l</Emphasis>-fucose transporter in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Objectives

To investigate the translocation of nucleotide-activated sugars from the cytosol across a membrane into the endoplasmatic reticulum or the Golgi apparatus which is an important step in the synthesis of glycoproteins and glycolipids in eukaryotes.

Results

The heterologous expression of the recombinant and codon-adapted human GDP-l-fucose antiporter gene SLC35C1 (encoding an N-terminal OmpA-signal sequence) led to a functional transporter protein located in the cytoplasmic membrane of Escherichia coli. The in vitro transport was investigated using inverted membrane vesicles. SLC35C1 is an antiporter specific for GDP-l-fucose and depending on the concomitant reverse transport of GMP. The recombinant transporter FucT1 exhibited an activity for the transport of ³H-GDP-l-fucose with a V_max of 8 pmol/min mg with a K_m of 4 µM. The functional expression of SLC35C1 in GDP-l-fucose overproducing E. coli led to the export of GDP-l-fucose to the culture supernatant.

Conclusions

The export of GDP-l-fucose by E. coli provides the opportunity for the engineering of a periplasmatic fucosylation reaction in recombinant bacterial cells.

     

Enhanced cadaverine production from <Emphasis Type="SmallCaps">l</Emphasis>-lysine using recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> co-overexpressing CadA and CadB

The effect of fusing the PelB signal sequence to lysine/cadaverine antiporter (CadB) on the bioconversion of l-lysine to cadaverine was investigated. To construct a whole-cell biocatalyst for cadaverine production, four expression plasmids were constructed for the co-expression of lysine decarboxylase (CadA) and lysine/cadaverine antiporter (CadB) in Escherichia coli. Expressing CadB with the PelB signal sequence increased cadaverine production by 12 %, and the optimal expression plasmid, pETDuet-pelB-CadB-CadA, contained two T7 promoter-controlled genes, CadA and the PelB-CadB fusion protein. Based on pETDuet-pelB-CadB-CadA, a whole-cell system for the bioconversion of l-lysine to cadaverine was constructed, and three strategies for l-lysine feeding were evaluated to eliminate the substrate inhibition problem. A cadaverine titer of 221 g l^?1 with a molar yield of 92 % from lysine was obtained.     

Comparative Effects of Hydrogen Sulfide-Releasing Compounds on [<Superscript>3</Superscript>H]D-Aspartate Release from Bovine Isolated Retinae

We investigated the pharmacological actions of a slow-releasing H₂S donor, GYY 4137; a substrate for the biosynthesis of H₂S, l-cysteine and its precursor, N-acetylcysteine on potassium (K⁺; 50 mM)-evoked [³H]D-aspartate release from bovine isolated retinae using the Superfusion Method. GYY 4137 (10 nM–10 µM), l-cysteine (100 nM–10 µM) and N-acetylcysteine (10 µM–1 mM) elicited a concentration-dependent decrease in K⁺-evoked [³H]D-aspartate release from isolated bovine retinae without affecting basal tritium efflux. At equimolar concentration of 10 µM, the rank order of activity was as follows: l-cysteine?>?GYY 4137?>?N-acetylcysteine. A dual inhibitor of the biosynthetic enzymes for H₂S, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), amino-oxyacetic acid (AOA; 3 mM) reversed the inhibitory responses caused by GYY 4137, l-cysteine and N-acetylcysteine on K⁺-evoked [³H]D-aspartate release. Glibenclamide (300 µM), an inhibitor of K_ATP channels blocked the inhibitory action of GYY 4137 and l-cysteine but not that elicited by N-acetylcysteine on K⁺-induced [³H]D-aspartate release. The inhibitory effect of GYY 4137 and l-cysteine on K⁺-evoked [³H]D-aspartate release was reversed by the non-specific inhibitor of nitric oxide synthase (NOS), l-NAME (300 µM). Furthermore, a specific inhibitor of inducible NOS (iNOS), aminoguanidine (10 µM) blocked the inhibitory action of l-cysteine on K⁺-evoked [³H]D-aspartate release. We conclude that both donors and substrates for H₂S production can inhibit amino acid neurotransmission in bovine isolated retinae, an effect that is dependent, at least in part, upon the intramural biosynthesis of this gas, and on the activity of K_ATP channels and NO synthase.     

Isolation and characterization of a novel <Emphasis Type="SmallCaps">l</Emphasis>-glutamate oxidase with strict substrate specificity from <Emphasis Type="Italic">Streptomyces diastatochromogenes</Emphasis>

Objectives

To find an l-glutamate oxidase (LGox), to be used for the quantitative analysis of l-glutamic acid, an lgox gene encoding LGox from Streptomyces diastatochromogenes was isolated, cloned and characterized.

Results

The gene had an ORF of 1974 bp encoding a protein of 657 amino acid residues. In comparison to the LGox precursor, the proteinase K-treated enzyme exhibited improved affinity to substrate and with a K _m of 0.15 mM and V _max of 62 μmol min^?1 mg^?1. The 50% thermal inactivation temperature of the proteinase K treated enzyme was increased from 50 to 70 °C. The enzyme exhibited strict specificity for l-glutamate.

Conclusions

LGox treated by proteinase K exhibited strict specificity for l-glutamate, good thermostability and high substrate affinity.

     

An <Emphasis Type="SmallCaps">l</Emphasis>-Glutamine Transporter Isoform for Neurogenesis Facilitated by <Emphasis Type="SmallCaps">l</Emphasis>-Theanine

l-Theanine (=γ-glutamylethylamide) is an amino acid ingredient in green tea with a structural analogy to l-glutamine (l-GLN) rather than l-glutamic acid (l-GLU), with regards to the absence of a free carboxylic acid moiety from the gamma carbon position. l-theanine markedly inhibits [³H]l-GLN uptake without affecting [³H]l-GLU uptake in cultured neurons and astroglia. In neural progenitor cells with sustained exposure to l-theanine, upregulation of the l-GLN transporter isoform Slc38a1 expression and promotion of both proliferation and neuronal commitment are seen along with marked acceleration of the phosphorylation of mammalian target of rapamycin (mTOR) and relevant downstream proteins. Stable overexpression of Slc38a1 leads to promotion of cellular growth with facilitated neuronal commitment in pluripotent embryonic carcinoma P19 cells. In P19 cells stably overexpressing Slc38a1, marked phosphorylation is seen with mTOR and downstream proteins in a fashion insensitive to the additional stimulation by l-theanine. The green tea amino acid l-theanine could thus elicit pharmacological actions to up-regulate Slc38a1 expression for activation of the mTOR signaling pathway required for cell growth together with accelerated neurogenesis after sustained exposure in undifferentiated neural progenitor cells. In this review, I summarize a novel pharmacological property of the green tea amino acid l-theanine for embryonic and adult neurogenesis with a focus on the endogenous amino acid analog l-GLN. A possible translational strategy is also discussed on the development of dietary supplements and nutraceuticals enriched of l-theanine for the prophylaxis of a variety of untoward impairments and malfunctions seen in patients with different neurodegenerative and/or neuropsychiatric disorders.     

Combinatorial analysis of enzymatic bottlenecks of <Emphasis Type="SmallCaps">l</Emphasis>-tyrosine pathway by <Emphasis Type="Italic">p</Emphasis>-coumaric acid production in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Objective

To identify new enzymatic bottlenecks of l-tyrosine pathway for further improving the production of l-tyrosine and its derivatives.

Result

When ARO4 and ARO7 were deregulated by their feedback resistant derivatives in the host strains, the ARO2 and TYR1 genes, coding for chorismate synthase and prephenate dehydrogenase were further identified as new important rate-limiting steps. The yield of p-coumaric acid in the feedback-resistant strain overexpressing ARO2 or TYR1, was significantly increased from 6.4 to 16.2 and 15.3 mg l^?1, respectively. Subsequently, we improved the strain by combinatorial engineering of pathway genes increasing the yield of p-coumaric acid by 12.5-fold (from 1.7 to 21.3 mg l^?1) compared with the wild-type strain. Batch cultivations revealed that p-coumaric acid production was correlated with cell growth, and the formation of by-product acetate of the best producer NK-M6 increased to 31.1 mM whereas only 19.1 mM acetate was accumulated by the wild-type strain.

Conclusion

Combinatorial metabolic engineering provides a new strategy for further improvement of l-tyrosine or other metabolic biosynthesis pathways in S. cerevisiae.