Effects of a Feedback-Resistant Aspartokinase III Gene on L-Isoleucine Production in Escherichia coli K-12

An L-isoleucine-overproducing recombinant strain of E. coli, TVD5, was also found to overproduce L-valine. The L-isoleucine productivity of TVD5 was markedly decreased by addition of L-lysine to the medium. Introduction of a gene encoding feedback-resistant aspartokinase III increased L-isoleucine productivity and decreased L-valine by-production. The resulting strain accumulated 12 g/l L-isoleucine from 40 g/l glucose, and suppression of L-isoleucine productivity by L-lysine was relieved.     

S-Adenosyl-L-methionine Activates Actinorhodin Biosynthesis by Increasing Autophosphorylation of the Ser/Thr Protein Kinase AfsK in Streptomyces coelicolor A3(2)

S-Adenosyl-L-methionine (SAM) is one of the major methyl donors in all living organisms. The exogenous treatment with SAM leads to increased actinorhodin production in Streptomyces coelicolor A3(2). In this study, mutants from different stages of the AfsK-AfsR signal transduction cascade were used to test the possible target of SAM. SAM had no significant effect on actinorhodin production in afsK, afsR, afsS, or actII-open reading frame 4 (ORF4) mutant. This confirms that afsK plays a critical role in delivering the signal generated by exogenous SAM. The afsK-pHJL-KN mutant did not respond to SAM, suggesting the involvement of the C-terminal of AfsK in binding with SAM. SAM increased the in vitro autophosphorylation of kinase AfsK in a dose-dependent manner, and also abolished the effect of decreased actinorhodin production by a Ser/Thr kinase inhibitor, K252a. In sum, our results suggest that SAM activates actinorhodin biosynthesis in S. coelicolor M130 by increasing the phosphorylation of protein kinase AfsK.     

Conversion of L-Galactono-1,4-lactone to L-Ascorbate Is Regulated by the Photosynthetic Electron Transport Chain in Arabidopsis

In this study we focused on the effects of light irradiation and the addition of L-galactono-1,4-lactone (L-GalL) on the conversion of exogenous L-GalL to L-ascorbate (AsA) and the total AsA pool size in detached leaves of Arabidopsis plants and transgenic plants expressing the rat L-gulono-1,4-lactone oxidase gene. Increases in the total AsA level in L-GalL-treated leaves depended entirely on light irradiation. Treatment with an inhibitor of photosynthetic electron transport together with L-GalL reduced the increase in total AsA under light. Light, particularly the redox state of photosynthetic electron transport, appeared to play an important role in the regulation of the conversion of L-GalL to AsA in the mitochondria, reflecting the cellular level of AsA in plants.     

Mutant of Escherichia coli Tolerant to the Lysis Induced by Cephalexin

2,3-Diaminopropionate ammonia-lyase (DAPAL), which catalyzes α,β-elimination of 2,3-diaminopropionate regardless of its stereochemistry, was purified from Salmonella typhimurium. We cloned the Escherichia coli ygeX gene encoding a putative DAPAL and purified the gene product to homogeneity. The protein obtained contained pyridoxal 5′-phosphate and was composed of two identical subunits with a calculated molecular weight of 43,327. It catalyzed the α,β-elimination of both D- and L-2,3-diaminopropionate. The results confirmed that ygeX encoded DAPAL. The enzyme acted on D-serine, but its catalytic efficiency was only 0.5% that with D-2,3-diaminopropionate. The enzymologic properties of E. coli DAPAL resembled those of Salmonella DAPAL, except that L-serine, D- and L-β-Cl-alanine were inert as substrates of the enzyme from E. coli. DAPAL had significant sequence similarity with the catalytic domain of L-threonine dehydratase, which is a member of the fold-type II group of pyridoxal phosphate enzymes, together with D-serine dehydratase and mammalian serine racemase.     

N-Carbamyl-L-Amino Acid Amidohydrolase of Pseudomonas sp. Strain NS671: Purification and Some Properties of the Enzyme Expressed in Escherichia coli

An N-carbamyl-L-amino acid amidohydrolase was purified from cells of Escherichia coli in which the gene for N-carbamyl-L-amino acid amidohydrolase of Pseudomonas sp. strain NS671 was expressed. The purified enzyme was homogeneous by the criterion of SDS–polyacrvlamide gel electrophoresis. The results of gel filtration chromatography and SDS–polyacrylamide gel electrophoresis suggested that the enzyme was a dimeric protein with 45-kDa identical subunits. The enzyme required Mn²⁺ ion (above 1 mM) for the activity. The optimal pH and temperature were 7.5 and around 40°C, respectively, with N-carbamyl-L-methionine as the substrate. The enzyme activity was inhibited by ATP and was iost completely with p-chloromercuribenzoate (1 mM). The enzyme was strictly L-specific and showed a broad substrate specificity for N-carbamyl-L-α-amino acids.     

Production of d-lactate using a pyruvate-producing Escherichia coli strain

To generate an organism capable of producing d-lactate, NAD⁺-dependent d-lactate dehydrogenase was expressed in our pyruvate-producing strain, Escherichia coli strain LAFCPCPt-accBC-aceE. After determining the optimal culture conditions for d-lactate production, 18.4 mM d-lactate was produced from biomass-based medium without supplemental mineral or nitrogen sources. Our results show that d-lactate can be produced in simple batch fermentation processes.     

Characterization of Mesorhizobium loti L-Rhamnose Isomerase and Its Application to L-Talose Production

The L-rhamnose isomerase gene (rhi) of Mesorhizobium loti was cloned and expressed in Escherichia coli, and then characterized. The enzyme exhibited activity with respect to various aldoses, including D-allose and L-talose. Application of it in L-talose production from galactitol was achieved by a two-step reaction, indicating that it can be utilized in the large-scale production of L-talose.     

Preparation of Phgsphatioyll2-3Hjinositgl from Yeast Grown in Medium Containing N;Yol2-3Hjinositol

Phosphatidy[2-³]jinositol was prepared from Saccharoniycts cerevisiae (YSC-2), grown in synthetic meaiurn containing myo[2-³H]inositol. Over 44 μCi (or 81 %) of the racio-labeleo inositol was taken up by the organism, with 34 yCi incorporated into phospnatiaylinositol. Upon purification d) silicic acia-meaium pressure liquia chrcnatography (MPLC), a final yield of 24 to 2b μCi of phosphatiayl[2-³h]inositot with a specific radioactivity of 40 ± 10³ apm/nmoie wäs obtained. The purified phosphatiuyl[2-³H] inositol was founo to be a suitable substrate for phospholipase C from human platelets     

Enzymatic Quantification of L-Tartrate in Wines and Grapes by Using the Secondary Activity of D-Malate Dehydrogenase

L-Tartrate in wines and grapes was enzymatically quantified by using the secondary activity of D-malate dehydrogenase (D-MDH). NADH formed by the D-MDH reaction was monitored spectrophotometrically. Under the optimal conditions, L-tartrate (a 1.0 mM sample solution) was fully oxidized by D-MDH in 30 min. A linear relationship was obtained between the absorbance difference and the L-tartrate concentration in the range of a 0.02-1.0 mM sample solution with a correlation coefficient of 0.9991. The relative standard deviation from ten measurements was 1.71% at the 1.0 mM sample solution level. The proposed method was compared with HPLC, and the values determined by both methods were in good agreement.     

Gene Cloning and Characterization of α-Amino Acid Ester Acyl Transferase in Empedobacter brevis ATCC14234 and Sphingobacterium siyangensis AJ2458

The gene encoding α-amino acid ester acyl transferase (AET), the enzyme that catalyzes the peptide-forming reaction from amino acid methyl esters and amino acids, was cloned from Empedobacter brevis ATCC14234 and Sphingobacterium siyangensis AJ2458 and expressed in Escherichia coli. This is the first report on the aet gene. It encodes a polypeptide composed of 616 (ATCC14234) and 619 (AJ2458) amino acids residues. The V _max values of these recombinant enzymes during the catalysis of L-alanyl-L-glutamine formation from L-alanine methylester and L-glutamine were 1,010 U/mg (ATCC14234) and 1,154 U/mg (AJ2458). An amino acid sequence similarity search revealed 35% (ATCC14234) and 36% (AJ2458) identity with an α-amino acid ester hydrolase from Acetobacter pasteurianus, which contains an active-site serine in the consensus serine enzyme motif, GxSYxG. In the deduced amino acid sequences of AET from both bacteria, the GxSYxG motif was conserved, suggesting that AET is a serine enzyme.     

Structural Analysis of the Fundamental Polymer of the Sheath Constructed by Sphaerotilus natans

The sheath of Sphaerotilus natans is composed of cysteine-rich peptide and polysaccharide moieties. The polysaccharide was prepared by treating the sheath with hydrazine, and was determined to be a mucopolysaccharide containing β-D-GlcA, β-D-Glc, α-D-GalN, and β-D-GalN. To elucidate the structure of the peptide, the sheath was labeled with a thiol-selective fluorogenic reagent, 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole. Enantiomeric determination of the S-derivatized Cys in the fluorescent sheath suggested that it contained L-Cys mainly. Fluorescent cysteinylglycine was detected in the partial acid hydrolysate of the fluorescent sheath. The sheath-degrading enzyme secreted by Paenibacillus koleovorans produced a fluorescent disaccharide-dipeptide composed of GalN, Gly, and N-acetylated Cys from the fluorescent sheath. The disaccharide and dipeptide moieties were found to be connected by an amide bond. Based on these results, the sheath was deduced to be formed by association of a mucopolysaccharide modified with N-acetyl-L-cysteinylglycine.     

A single membrane-bound enzyme catalyzes the conversion of 2,5-diketo-d-gluconate to 4-keto-d-arabonate in d-glucose oxidative fermentation by Gluconobacter oxydans NBRC 3292

4-Keto-d-arabonate synthase (4KAS), which converts 2,5-diketo-d-gluconate (DKGA) to 4-keto-d-arabonate (4KA) in d-glucose oxidative fermentation by some acetic acid bacteria, was solubilized from the Gluconobacter oxydans NBRC 3292 cytoplasmic membrane, and purified in an electrophoretically homogenous state. A single membrane-bound enzyme was found to catalyze the conversion from DKGA to 4KA. The 92-kDa 4KAS was a homodimeric protein not requiring O₂ or a cofactor for the conversion, but was stimulated by Mn²⁺. N-terminal amino acid sequencing of 4KAS, followed by gene homology search indicated a 1,197-bp open reading frame (ORF), corresponding to the GLS_c04240 locus, GenBank accession No. CP004373, encoding a 398-amino acid protein with a calculated molecular weight of 42,818 Da. An Escherichia coli transformant with the 4kas plasmid exhibited 4KAS activity. Furthermore, overexpressed recombinant 4KAS was purified in an electrophoretically homogenous state and had the same molecular size as the natural enzyme.     

The Mode of Binding of Carbohydrate in Ricin D

Dehydro-L-ascorbic acid (DAA) exists mainly in its C2 hydrated bicyclic form (5) in an aqueous solution, and monocyclic DAA (3), which is the expected reaction product immediately after the oxidation of AA, has not been observed by NMR spectroscopy. The formation mechanism for 5 from 3 and the stability of 5 were examined by the semi-empirical molecular orbital method (MOPAC). It was indicated that the protonation reaction was the key step in the formation of 5, therefore, the formation of 5 is thought to be more difficult under physiological conditions which mostly involve in the neutral or slightly alkaline state. However, by NMR, it was confirmed that, even in a neutral or slightly alkaline state very close to physiological conditions, the predominant form of DAA existing in an aqueous solution immediately after the enzymatic oxidation of AA was confirmed to be 5, although the possible existence of other forms of DAA at very low concentrations could not be completely excluded.     

Plasmid-determined Transformation of cis-Abienol and Sclareol in Nocardia restricta JTS-162

The cis-abienol-transformable bacterium JTS-162 was identified as Nocardia restricta. This bacterium has three plasmids (pCA1, pCA2 and pCA3). Two types of cured strains were obtained by mitomicin C treatment or growth at the maximum temperature; one type had two plasmids (pCA2 and pCA3) and the other type had no plasmid. These two types of cured strains lost the ability to oxidize C-18 methyl and to split the A-ring of cis-abienol. These two types of cured strains were also devoid of the. ability to oxidize the C-18 methyl of sclareol. From these results, it was considered that C-18 methyl oxidation and A-ring splitting of these labdanes were determined by plasmid pCA1 in N. restricta JTS-162.     

The Contribution of Arabidopsis Homologs of L-Gulono-1,4-lactone Oxidase to the Biosynthesis of Ascorbic Acid

To clarify the involvement of seven Arabidopsis homologs of rat L-gulono-1,4-lactone (L-GulL) oxidase, AtGulLOs, in the biosynthesis of L-ascorbic acid (AsA), transgenic tobacco cells overexpressing the various AtGulLOs were generated. Under treatment with L-GulL, the levels of total AsA in three transgenic tobacco cell lines, overexpressing AtGulLO2, 3, or 5, were significantly increased as compared with those in control cells.     

Production of L-xylose from L-xylulose using Escherichia coli L-fucose isomerase

l-Xylulose was used as a raw material for the production of l-xylose with a recombinantly produced Escherichia colil-fucose isomerase as the catalyst. The enzyme had a very alkaline pH optimum (over 10.5) and displayed Michaelis-Menten kinetics for l-xylulose with a K_m of 41 mM and a V_max of 0.23 μmol/(mg min). The half-lives determined for the enzyme at 35 °C and at 45 °C were 6 h 50 min and 1 h 31 min, respectively. The reaction equilibrium between l-xylulose and l-xylose was 15:85 at 35 °C and thus favored the formation of l-xylose. Contrary to the l-rhamnose isomerase catalyzed reaction described previously [14]l-lyxose was not detected in the reaction mixture with l-fucose isomerase. Although xylitol acted as an inhibitor of the reaction, even at a high ratio of xylitol to l-xylulose the inhibition did not reach 50%.     

Purification and Properties of Two Chitinases from Streptomyces sp. J-13-3

Two chitinases (Chi-A and Chi-B) purified from Streptomyces sp. J-13-3 had the same molecular weights (31,000) and enzymatic properties (optimum pH and temperature of pH 6.0 and 45°C) but had significantly different isoelectric points (3.9 for Chi-A, 3.5 for Chi-B). Chi-A and -B had identical N-terminal amino acid sequences (ADXAAAWNASSVYTGGGSASYNGHN), similar amino acid compositions, and immunological cross-reactivities. A concomitant decrease of Chi-A and increase of Chi-B was observed in their productions during cultivation.     

Effective enhancement of polylactic acid-degrading enzyme production by Amycolatopsis sp. strain SCM_MK2-4 using statistical and one-factor-at-a-time approaches

This study aims to find the optimal medium and conditions for polylactic acid (PLA)-degrading enzyme production by Amycolatopsis sp. SCM_MK2-4. Screening of the most effective components in the enzyme production medium by Plackett–Burman design revealed that the silk cocoon and PLA film were the most significant variables enhancing the PLA-degrading enzyme production. After an response surface methodology, a maximum amount of PLA-degrading enzyme activity at 0.74?U?mL^?1 was predicted and successfully validated at 95% after 0.39% (w/v) silk cocoon and 1.62% (w/v) PLA film were applied to the basal medium. The optimal initial pH value, temperature, and inoculum size were evaluated by a method considering one-factor-at-a-time. The values were recorded at an initial pH in the range of 7.5–9.0, a temperature of 30–32°C, and an inoculum size of 4–10%. The highest activity of approximately 0.95?U?mL^?1 was achieved after 4 days of cultivation using the optimized medium and under optimized conditions in a shake flask. Upscaling to the use of a 3-L stirred tank fermenter was found to be successful with a PLA-degrading activity of 5.53?U?mL^?1; which represents a 51-fold increase in the activity compared with that obtained from the nonoptimized medium and conditions in the shake flask.     

Genetic Engineering of Candida utilis Yeast for Efficient Production of L-Lactic Acid

Polylactic acid is receiving increasing attention as a renewable alternative for conventional petroleum-based plastics. In the present study, we constructed a metabolically-engineered Candida utilis strain that produces L-lactic acid with the highest efficiency yet reported in yeasts. Initially, the gene encoding pyruvate decarboxylase (CuPDC1) was identified, followed by four CuPDC1 disruption events in order to obtain a null mutant that produced little ethanol (a by-product of L-lactic acid). Two copies of the L-lactate dehydrogenase (L-LDH) gene derived from Bos taurus under the control of the CuPDC1 promoter were then integrated into the genome of the CuPdc1-null deletant. The resulting strain produced 103.3 g/l of L-lactic acid from 108.7 g/l of glucose in 33 h, representing a 95.1% conversion. The maximum production rate of L-lactic acid was 4.9 g/l/h. The optical purity of the L-lactic acid was found to be more than 99.9% e.e.