Cloning and expression of <Emphasis Type="Italic">mel</Emphasis> gene from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Previous work from our laboratory has shown that most of Bacillus thuringiensis strains possess the ability to produce melanin in the presence of l-tyrosine at elevated temperatures (42 °C). Furthermore, it was shown that the melanin produced by B. thuringiensis was synthesized by the action of tyrosinase, which catalyzed the conversion of l-tyrosine, via l-DOPA, to melanin. In this study, the tyrosinase-encoding gene (mel) from B. thuringiensis 4D11 was cloned using PCR techniques and expressed in Escherichia coli DH5 . A DNA fragment with 1179 bp which contained the intact mel gene in the recombinant plasmid pGEM1179 imparted the ability to synthesize melanin to the E. coli recipient strain. The nucleotide sequence of this DNA fragment revealed an open reading frame of 744 bp, encoding a protein of 248 amino acids. The novel mel gene from B.thuringiensis expressed in E. coli DH5 conferred UV protection on the recipient strain.     

Mutational analysis of the active site residues of a <Emphasis Type="SmallCaps">d</Emphasis>-psicose 3-epimerase from <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

d-Psicose 3-epimerase from Agrobacterium tumefacience catalyzes the conversion of d-fructose to d-psicose. According to mutational analysis, the ring at position 112, the negative charge at position 156, and the positive charge at position 215 were essential components for enzyme activity and for binding fructose and psicose. The surface contact area and distance to the bound substrate by molecular modeling suggest that the positive charge of Arg215 was involved in stabilization of cis-endiol intermediate. The distances between the catalytic residues (Glu150 and Glu244) and Mn²⁺ are critical to the catalysis, and the negative charges of the metal-binding residues are important for interaction with metal ion. The kinetic parameters of the D183E and H209A mutants for metal-binding residues with substrate and the near-UV circular dichroism spectra indicate that the metal ion bound to Asp183 and His209 is involved not only in catalysis but also in substrate binding.     

<Emphasis Type="SmallCaps">L</Emphasis>-Tyrosine production by deregulated strains of <Emphasis Type="Italic">Escherichia coli</Emphasis>

The excretion of the aromatic amino acid l-tyrosine was achieved by manipulating three gene targets in the wild-type Escherichia coli K12: The feedback-inhibition-resistant (fbr) derivatives of aroG and tyrA were expressed on a low-copy-number vector, and the TyrR-mediated regulation of the aromatic amino acid biosynthesis was eliminated by deleting the tyrR gene. The generation of this l-tyrosine producer, strain T1, was based only on the deregulation of the aromatic amino acid biosynthesis pathway, but no structural genes in the genome were affected. A second tyrosine over-producing strain, E. coli T2, was generated considering the possible limitation of precursor substrates. To enhance the availability of the two precursor substrates phosphoenolpyruvate and erythrose-4-phosphate, the ppsA and the tktA genes were over-expressed in the strain T1 background, increasing l-tyrosine production by 80% in 50-ml batch cultures. Fed-batch fermentations revealed that l-tyrosine production was tightly correlated with cell growth, exhibiting the maximum productivity at the end of the exponential growth phase. The final l-tyrosine concentrations were 3.8 g/l for E. coli T1 and 9.7 g/l for E. coli T2 with a yield of l-tyrosine per glucose of 0.037 g/g (T1) and 0.102 g/g (T2), respectively.     

Gradually accumulating beneficial mutations to improve the thermostability of <Emphasis Type="Italic">N</Emphasis>-carbamoyl-<Emphasis Type="SmallCaps">d</Emphasis>-amino acid amidohydrolase by step-wise evolution

To further enhance repeated batch reactions with immobilized N-carbamoyl-d-amino acid amidohydrolase (DCase), which can be used for the industrial production of d-amino acids, the stability of high soluble mutant DCase-M3 from Ralstonia pickettii CGMCC1596 was improved by step-wise evolution. In our previous report, six thermostability-related sites were identified by error-prone PCR. Based on the above result, an improved mutant B5 (Q12L/Q23L/H248Q/T262A/T263S) was obtained through two rounds of DNA shuffling, showing a 10°C increase in the T ₅₀ (defined as the temperature at which heat treatment for 15 min reduced the initial activity by 50%) compared with the parental enzyme DCase-M3. Furthermore, several thermostability-related sites (Met³¹, Asn⁹³, Gln²⁰⁷, Asn²⁴², Glu²⁶⁶, Thr²⁷¹, Ala²⁷³) on B5 were identified using amino acid consensus approach based on sequence alignment of homologous DCases. These sites were further investigated by iterative saturation mutagenesis (ISM), and a combinational mutant D1 (Q12L/Q23L/Q207E/N242G/H248Q/T262A/T263S/E266D/T271I/A273P) that enhanced the T ₅₀ by about 16°C over DCase-M3 was obtained. Oxidative stability assay showed that the most heat-resisting mutant displayed only a slight increase in resistance to hydrogen peroxide. Comparative characterization showed that D1 not only maintained its characteristic high solubility but also shared similar k _cat and K _m values and optimum reaction pHs with the parental enzyme. The significantly improved mutants in the immobilized form are expected to be applied in the industrial production of d-p-hydroxyphenylglycine.     

A mutant leucine aminopeptidase from <Emphasis Type="Italic">Streptomyces cinnamoneus</Emphasis> with enhanced <Emphasis Type="SmallCaps">l</Emphasis>-aspartyl <Emphasis Type="SmallCaps">l</Emphasis>-amino acid methyl ester synthetic activity

l-Aspartyl l-amino acid methyl ester was synthesized using a mutant of a thermostable leucine aminopeptidase from Streptomyces cinnamoneus, D198 K SSAP, obtained in previously. A peptide of high-intensity sweetener, l-aspartyl-l-phenylalanine methyl ester, was selected as a model for demonstrating the synthesis of l-aspartyl l-amino acid methyl ester. The hydrolytic activities of D198 K SSAP toward l-aspartyl-l-phenylalanine and its methyl ester were, respectively, 74-fold and fourfold higher than those of wild type. Similarly, the initial rate of the enzyme for l-aspartyl-l-phenylalanine methyl ester synthesis was over fivefold higher than that of wild-type SSAP in 90% methanol (v/v) in a one-pot reaction. Furthermore, other l-aspartyl l-amino acid methyl esters were synthesized efficiently using D198 K SSAP. Results show that the substitution of Asp198 of SSAP with Lys is effective for synthesizing l-aspartyl l-amino acid methyl ester.     

Probing the catalytically essential residues of a recombinant dipeptidyl carboxypeptidase from <Emphasis Type="Italic">Escherichia coli</Emphasis>

In studying the structure and function of Escherichia coli dipeptidyl carboxypeptidase (EcDCP), we have employed in vitro mutagenesis and subsequent protein expression to genetically dissect the enzyme in order to gain insight into the catalytic mechanism. Comparison of the amino acid sequence of EcDCP with other homologues indicates that the active site of the enzyme exhibits an HEXXH motif, a common feature of zinc metalloenzymes. The third metal binding ligand, presumed to coordinate directly to the active-site zinc ion in concert with His470 and His474 has been proposed as Glu499. Alterations to these residues completely abolished the catalytic activity against N-benzoyl-l-glycyl-l-histidyl-l-leucine. A significant loss of the enzymatic activity was also observed in F472V and F500V mutant enzymes. Intrinsic tryptophan fluorescence revealed the significant alterations of the microenvironment of aromatic amino acid residues in all mutant enzymes, whereas circular dichroism spectra were nearly identical for the tested proteins. Computer modeling suggests that residues His470, Glu471, His474, Glu499, and Phe500 are essential for EcDCP in maintaining the stable active-site environment. Taken together, these studies contribute to a more comprehensive understanding of the catalytic mechanism of the enzyme.     

The alternative<Emphasis Type="SmallCaps"> d</Emphasis>-galactose degrading pathway of<Emphasis Type="Italic"> Aspergillus nidulans</Emphasis> proceeds via<Emphasis Type="SmallCaps"> l</Emphasis>-sorbose

The catabolism of d-galactose in yeast depends on the enzymes of the Leloir pathway. In contrast, Aspergillus nidulans mutants in galactokinase (galE) can still grow on d-galactose in the presence of ammonium—but not nitrate—ions as nitrogen source. A. nidulans galE mutants transiently accumulate high (400 mM) intracellular concentrations of galactitol, indicating that the alternative d-galactose degrading pathway may proceed via this intermediate. The enzyme degrading galactitol was identified as l-arabitol dehydrogenase, because an A. nidulans loss-of-function mutant in this enzyme (araA1) did not show NAD⁺-dependent galactitol dehydrogenase activity, still accumulated galactitol but was unable to catabolize it thereafter, and a double galE/araA1 mutant was unable to grow on d-galactose or galactitol. The product of galactitol oxidation was identified as l-sorbose, which is a substrate for hexokinase, as evidenced by a loss of l-sorbose phosphorylating activity in an A. nidulans hexokinase (frA1) mutant. l-Sorbose catabolism involves a hexokinase step, indicated by the inability of the frA1 mutant to grow on galactitol or l-sorbose, and by the fact that a galE/frA1 double mutant of A. nidulans was unable to grow on d-galactose. The results therefore provide evidence for an alternative pathway of d-galactose catabolism in A. nidulans that involves reduction of the d-galactose to galactitol and NAD⁺-dependent oxidation of galactitol by l-arabitol dehydrogenase to l-sorbose.     

Substrate specificity of a recombinant ribose-5-phosphate isomerase from <Emphasis Type="Italic">Streptococcus pneumoniae</Emphasis> and its application in the production of <Emphasis Type="SmallCaps">l</Emphasis>-lyxose and <Emphasis Type="SmallCaps">l</Emphasis>-tagatose

A putative ribose-5-phosphate isomerase (RpiB) from Streptococcus pneumoniae was purified with a specific activity of 26.7 U mg⁻¹ by Hi-Trap Q HP anion exchange and Sephacryl S-300 HR 16/60 gel filtration chromatographies. The native enzyme existed as a 96-kDa tetramer with activity maxima at pH 7.5 and 35°C. The RpiB exhibited isomerization activity with l-lyxose, l-talose, d-gulose, d-ribose, l-mannose, d-allose, l-xylulose, l-tagatose, d-sorbose, d-ribulose, l-fructose, and d-psicose and exhibited particularly high activity with l-form monosaccharides such as l-lyxose, l-xylulose, l-talose, and l-tagatose. With l-xylulose (500 g l⁻¹) and l-talose (500 g l⁻¹) substrates, the optimum concentrations of RpiB were 300 and 600 U ml⁻¹, respectively. The enzyme converted 500 g l⁻¹ l-xylulose to 350 g l⁻¹ l-lyxose after 3 h, and yielded 450 g l⁻¹ l-tagatose from 500 g l⁻¹ l-talose after 5 h. These results suggest that RpiB from S. pneumoniae can be employed as a potential producer of l-form monosaccharides.     

Synthesis of <Emphasis Type="SmallCaps">dl</Emphasis>-tryptophan by modified broad specificity amino acid racemase from <Emphasis Type="Italic">Pseudomonas putida</Emphasis> IFO 12996

Broad specificity amino acid racemase (E.C. 5.1.1.10) from Pseudomonas putida IFO 12996 (BAR) is a unique racemase because of its broad substrate specificity. BAR has been considered as a possible catalyst which directly converts inexpensive l-amino acids to dl-amino acid racemates. The gene encoding BAR was cloned to utilize BAR for the synthesis of d-amino acids, especially d-Trp which is an important intermediate of pharmaceuticals. The substrate specificity of cloned BAR covered all of the standard amino acids; however, the activity toward Trp was low. Then, we performed random mutagenesis on bar to obtain mutant BAR derivatives with high activity for Trp. Five positive mutants were isolated after the two-step screening of the randomly mutated BAR. After the determination of the amino acid substitutions in these mutants, it was suggested that the substitutions at Y396 and I384 increased the Trp specific racemization activity and the racemization activity for overall amino acids, respectively. Among the positive mutants, I384M mutant BAR showed the highest activity for Trp. l-Trp (20 mM) was successfully racemized, and the proportion of d-Trp was reached 43% using I384M mutant BAR, while wild-type BAR racemized only 6% of initial l-Trp.     

Purification and characterization of <Emphasis Type="SmallCaps">l</Emphasis>-arabinose isomerase from <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> producing <Emphasis Type="SmallCaps">d</Emphasis>-tagatose

l-arabinose isomerase (EC5.3.1.4. AI) mediates the isomerization of d-galactose into d-tagatose as well as the conversion of l-arabinose into l-ribulose. The AI from Lactobacillus plantarum SK-2 was purified to an apparent homogeneity giving a single band on SDS–PAGE with a molecular mass of 59.6 kDa. Optimum activity was observed at 50°C and pH 7.0. The enzyme was stable at 50°C for 2 h and held between pH 4.5 and 8.5 for 1 h. AI activity was stimulated by Mn²⁺, Fe³⁺, Fe²⁺, Ca²⁺ and inhibited by Cu²⁺, Ag⁺, Hg²⁺, Pb²⁺. d-galactose and l-arabinose as substrates were isomerized with high activity. l-arabitol was the strongest competitive inhibitor of AI. The apparent Michaelis–Menten constant (K _m), for galactose, was 119 mM. The first ten N-terminal amino acids of the enzyme were determined as MLSVPDYEFW, which is identical to L. plantarum (Q88S84). Using the purified AI, 390 mg tagatose could be converted from 1,000 mg galactose in 96 h, and this production corresponds to a 39% equilibrium.     

<Emphasis Type="Italic">Physcomitrella patens</Emphasis> arabinogalactan proteins contain abundant terminal 3-<Emphasis Type="Italic">O</Emphasis>-methyl-<Emphasis Type="SmallCaps">l</Emphasis>-rhamnosyl residues not found in angiosperms

A biochemical investigation of arabinogalactan proteins (AGPs) in Physcomitrella patens was undertaken with particular emphasis on the glycan chains. Following homogenization and differential centrifugation of moss gametophytes, AGPs were obtained by Yariv phenylglycoside-induced precipitation from the soluble, microsomal membrane, and cell wall fractions. Crossed-electrophoresis indicated that each of these three AGP fractions was a mixture of several AGPs. The soluble AGP fraction was selected for further separation by anion-exchange and gel-permeation chromatography. The latter indicated molecular masses of ∼100 and 224 kDa for the two major soluble AGP subfractions. The AGPs in both of these subfractions contained the abundant (1,3,6)-linked galactopyranosyl residues, terminal arabinofuranosyl residues, and (1,4)-linked glucuronopyranosyl residues that are typical of many angiosperm AGPs. Unexpectedly, however, the moss AGP glycan chains contained about 15 mol% terminal 3-O-methyl-l-rhamnosyl residues, which have not been found in angiosperm AGPs. This unusual and relatively nonpolar sugar, also called l-acofriose, is likely to have considerable effects on the overall polarity of Physcomitrella AGPs. A review of the literature indicates that the capacity to synthesize polymers containing 3-O-methyl-l-rhamnosyl residues is present in a variety of bacteria, algae and lower land plants but became less common through evolution to the extent that this sugar has been found in only a few species of angiosperms where it occurs as a single residue on steroidal glycosides.     

Characterization of NADP<Superscript>+</Superscript>-specific <Emphasis Type="SmallCaps">l</Emphasis>-rhamnose dehydrogenase from the thermoacidophilic Archaeon <Emphasis Type="Italic">Thermoplasma acidophilum</Emphasis>

Thermoplasma acidophilum utilizes l-rhamnose as a sole carbon source. To determine the metabolic pathway of l-rhamnose in Archaea, we identified and characterized l-rhamnose dehydrogenase (RhaD) in T. acidophilum. Ta0747P gene, which encodes the putative T. acidophilum RhaD (Ta_RhaD) enzyme belonging to the short-chain dehydrogenase/reductase family, was expressed in E. coli as an active enzyme catalyzing the oxidation of l-rhamnose to l-rhamnono-1,4-lactone. Analysis of catalytic properties revealed that Ta_RhaD oxidized l-rhamnose, l-lyxose, and l-mannose using only NADP⁺ as a cofactor, which is different from NAD⁺/NADP⁺-specific bacterial RhaDs and NAD⁺-specific eukaryal RhaDs. Ta_RhaD showed the highest activity toward l-rhamnose at 60 °C and pH 7. The K _m and k _cat values were 0.46 mM, 1,341.3 min⁻¹ for l-rhamnose and 0.1 mM, 1,027.2 min⁻¹ for NADP⁺, respectively. Phylogenetic analysis indicated that branched lineages of archaeal RhaD are quite distinct from those of Bacteria and Eukarya. This is the first report on the identification and characterization of NADP⁺-specific RhaD.     

Increased <Emphasis Type="SmallCaps">d</Emphasis>-allose production by the R132E mutant of ribose-5-phosphate isomerase from <Emphasis Type="Italic">Clostridium thermocellum</Emphasis>

Ribose-5-phosphate isomerase from Clostridium thermocellum converted d-psicose to d-allose, which may be useful as a pharmaceutical compound, with no by-product. The 12 active-site residues, which were obtained by molecular modeling on the basis of the solved three-dimensional structure of the enzyme, were substituted individually with Ala. Among the 12 Ala-substituted mutants, only the R132A mutant exhibited an increase in d-psicose isomerization activity. The R132E mutant showed the highest activity when the residue at position 132 was substituted with Ala, Gln, Ile, Lys, Glu, or Asp. The maximal activity of the wild-type and R132E mutant enzymes for d-psicose was observed at pH 7.5 and 80°C. The half-lives of the wild-type enzyme at 60°C, 65°C, 70°C, 75°C, and 80°C were 11, 7.0, 4.2, 1.5, and 0.6 h, respectively, whereas those of the R132E mutant enzymes were 13, 8.2, 5.1, 3.1, and 0.9 h, respectively. The specific activity and catalytic efficiency (k _cat/K _m) of the R132E mutant for d-psicose were 1.4- and 1.5-fold higher than those of the wild-type enzyme, respectively. When the same amount of enzyme was used, the conversion yield of d-psicose to d-allose was 32% for the R132E mutant enzyme and 25% for the wild-type enzyme after 80 min.     

New ubiquitous translocators: amino acid export by<Emphasis Type="Italic"> Corynebacterium glutamicum</Emphasis> and<Emphasis Type="Italic"> Escherichia coli</Emphasis>

Molecular access to amino acid excretion by Corynebacterium glutamicum and Escherichia coli led to the identification of structurally novel carriers and novel carrier functions. The exporters LysE, RhtB, ThrE and BrnFE each represent the protoype of new transporter families, which are in part distributed throughout all of the kingdoms of life. LysE of C. glutamicum catalytes the export of basic amino acids. The expression of the carrier gene is regulated by the cell-internal concentration of basic amino acids. This serves, for example, to maintain homoeostasis if an excess of l-lysine or l-arginine inside the cell should arise during growth on complex media. RhtB is one of five paralogous systems in E. coli, of which at least two are relevant for l-threonine production. A third system is relevant for l-cysteine production. It is speculated that the physiological function of these paralogues is related to quorum sensing. ThrE of C. glutamicum exports l-threonine and l-serine. However, a ThrE domain with a putative hydrolytic function points to an as yet unknown role of this exporter. BrnFE in C. glutamicum is a two-component permease exporting branched-chained amino acids from the cell, and an orthologue in B. subtilis exports 4-azaleucine.     

<Emphasis Type="SmallCaps">l</Emphasis>-Proline nutrition and catabolism in <Emphasis Type="Italic">Staphylococcus saprophyticus</Emphasis>

Staphylococcus saprophyticus strains ATCC 15305, ATCC 35552, and ATCC 49907 were found to require l-proline but not l-arginine for growth in a defined culture medium. All three strains could utilize l-ornithine as a proline source and contained l-ornithine aminotransferase and Δ¹-pyrroline-5-carboxylate reductase activities; strains ATCC 35552 and ATCC 49907 could use l-arginine as a proline source and had l-arginase activity. The proline requirement also could be met by l-prolinamide, l-proline methyl ester, and the dipeptides l-alanyl-l-proline and l-leucyl-l-proline. The bacteria exhibited l-proline degradative activity as measured by the formation of Δ¹-pyrroline-5-carboxylate. The specific activity of proline degradation was not affected by addition of l-proline or NaCl but was highest in strain ATCC 49907 after growth in Mueller–Hinton broth. A membrane fraction from this strain had l-proline dehydrogenase activity as detected both by reaction of Δ¹-pyrroline-5-carboxylate with 2-aminobenzaldehyde (0.79 nmol min⁻¹ mg⁻¹) and by the proline-dependent reduction of p-iodonitrotetrazolium (20.1 nmol min⁻¹ mg⁻¹). A soluble fraction from this strain had Δ¹-pyrroline-5-carboxylate dehydrogenase activity (88.8 nmol min⁻¹ mg⁻¹) as determined by the NAD⁺-dependent oxidation of dl-Δ¹-pyrroline-5-carboxylate. Addition of l-proline to several culture media did not increase the growth rate or final yield of bacteria but did stimulate growth during osmotic stress. When grown with l-ornithine as the proline source, S. saprophyticus was most susceptible to the proline analogues L-azetidine-2-carboylate, 3,4-dehydro-dl-proline, dl-thiazolidine-2-carboxylate, and l-thiazolidine-4-carboxylate. These results indicate that proline uptake and metabolism may be a potential target of antimicrobial therapy for this organism.     

A novel <Emphasis Type="SmallCaps">l</Emphasis>-aspartate dehydrogenase from the mesophilic bacterium <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PAO1: molecular characterization and application for <Emphasis Type="SmallCaps">l</Emphasis>-aspartate production

l-aspartate dehydrogenase (EC 1.4.1.21; l-AspDH) is a rare member of amino acid dehydrogenase superfamily and so far, two thermophilic enzymes have been reported. In our study, an ORF PA3505 encoding for a putative l-AspDH in the mesophilic bacterium Pseudomonas aeruginosa PAO1 was identified, cloned, and overexpressed in Escherichia coli. The homogeneously purified enzyme (PaeAspDH) was a dimeric protein with a molecular mass of about 28 kDa exhibiting a very high specific activity for l-aspartate (l-Asp) and oxaloacetate (OAA) of 127 and 147 U mg⁻¹, respectively. The enzyme was capable of utilizing both nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) as coenzyme. PaeAspDH showed a T _m value of 48°C for 20 min that was improved to approximately 60°C by the addition of 0.4 M NaCl or 30% glycerol. The apparent K _m values for OAA, NADH, and ammonia were 2.12, 0.045, and 10.1 mM, respectively; comparable results were observed with NADPH. The l-Asp production system B consisting of PaeAspDH, Bacillus subtilis malate dehydrogenase and E. coli fumarase, achieved a high level of l-Asp production (625 mM) from fumarate in fed-batch process with a molar conversion yield of 89.4%. Furthermore, the fermentative production system C released 33 mM of l-Asp after 50 h by using succinate as carbon source. This study represented an extensive characterization of the mesophilic AspDH and its potential applicability for efficient and attractive production of l-Asp. Our novel production systems are also hopeful for developing the new processes for other compounds production.     

Metabolic engineering of <Emphasis Type="Italic">Escherichia coli</Emphasis> for improving <Emphasis Type="SmallCaps">l</Emphasis>-3,4-dihydroxyphenylalanine (<Emphasis Type="SmallCaps">l</Emphasis>-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.     

Double deletion of <Emphasis Type="Italic">dtsR1</Emphasis> and <Emphasis Type="Italic">pyc</Emphasis> induce efficient <Emphasis Type="SmallCaps">l</Emphasis>-glutamate overproduction in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Corynebacterium glutamicum strains are used for the fermentative production of l-glutamate. Five C. glutamicum deletion mutants were isolated by two rounds of selection for homologous recombination and identified by Southern blot analysis. The growth, glucose consumption and glutamate production of the mutants were analyzed and compared with the wild-type ATCC 13032 strain. Double disruption of dtsR1 (encoding a subunit of acetyl-CoA carboxylase complex) and pyc (encoding pyruvate carboxylase) caused efficient overproduction of l-glutamate in C. glutamicum; production was much higher than that of the wild-type strain and ΔdtsR1 strain under glutamate-inducing conditions. In the absence of any inducing conditions, the amount of glutamate produced by the double-deletion strain ΔdtsR1Δpyc was more than that of the mutant ΔdtsR1. The activity of phosphoenolpyruvate carboxylase (PEPC) was found to be higher in the ΔdtsR1Δpyc strain than in the ΔdtsR1 strain and the wild-type strain. Therefore, PEPC appears to be an important anaplerotic enzyme for glutamate synthesis in ΔdtsR1 derivatives. Moreover, this conclusion was confirmed by overexpression of ppc and pyc in the two double-deletion strains (ΔdtsR1Δppc and ΔdtsR1Δpyc), respectively. Based on the data generated in this investigation, we suggest a new method that will improve glutamate production strains and provide a better understanding of the interaction(s) between the anaplerotic pathway and fatty acid synthesis.     

The outer membrane TolC is involved in cysteine tolerance and overproduction in <Emphasis Type="Italic">Escherichia coli</Emphasis>

l-Cysteine is an important amino acid in terms of its industrial applications. We previously found marked production of l-cysteine directly from glucose in recombinant Escherichia coli cells by the combination of enhancing biosynthetic activity and weakening the degradation pathway. Further improvements in l-cysteine production are expected to use the amino acid efflux system. Here, we identified a novel gene involved in l-cysteine export using a systematic and comprehensive collection of gene-disrupted E. coli K-12 mutants (the Keio collection). Among the 3,985 nonessential gene mutants, tolC-disrupted cells showed hypersensitivity to l-cysteine relative to wild-type cells. Gene expression analysis revealed that the tolC gene encoding the outer membrane channel is essential for l-cysteine tolerance in E. coli cells. However, l-cysteine tolerance is not mediated by TolC-dependent drug efflux systems such as AcrA and AcrB. It also appears that other outer membrane porins including OmpA and OmpF do not participate in TolC-dependent l-cysteine tolerance. When a low-copy-number plasmid carrying the tolC gene was introduced into E. coli cells with enhanced biosynthesis, weakened degradation, and improved export of l-cysteine, the transformants exhibited more l-cysteine tolerance and production than cells carrying the vector only. We concluded that TolC plays an important role in l-cysteine tolerance probably due to its export ability and that TolC overexpression is effective for l-cysteine production in E. coli. Natthawut Wiriyathanawudhiwong and Iwao Ohtsu contributed equally to this work.