Isolation of proline-based cyclic dipeptides from Bacillus sp. N strain associated with rhabitid entomopathogenic nematode and its antimicrobial properties

Entomopathogenic nematodes (EPN) are well-known as biological control agents and are found to have associated bacteria which can produce a wide range of bioactive secondary metabolites. We report herewith isolation of six proline containing cyclic dipeptides cyclo(d-Pro-l-Leu), cyclo(l-Pro-l-Met), cyclo(d-Pro-l-Phe), cyclo(l-Pro-l-Phe), cyclo(l-Pro-l-Tyr) and cyclo(l-Pro-d-Tyr) from ethyl acetate extract of the Luria Broth (LB) cell free culture filtrate of Bacillus sp. strain N associated with a new EPN Rhabditis sp. from sweet potato weevil grubs collected from Central Tuber Crops Research Institute farm. Antimicrobial studies of these 2,5-diketopiperazines (DKPs) against both medicinally and agriculturally important bacterium and fungi showed potent inhibitory values in the range of μg/mL. Cyclic dipeptides showed significantly higher activity than the commercial fungicide bavistin against agriculturally important fungi, viz., Fusarium oxysporum, Rhizoctonia solani, and Pencillium expansum. The highest activity of 2 μg/mL by cyclo(l-Pro-l-Phe) was recorded against P. expansum, a plant pathogen responsible for causing post harvest decay of stored apples and oranges. To our knowledge, this is the first report on the isolation of these DKPs from Rhabditis EPN bacterial strain Bacillus sp.     

Cyclic dipeptides from rhabditid entomopathogenic nematode-associated Bacillus cereus have antimicrobial activities

The cell free culture filtrate of Bacillus cereus associated with an entomopathogenic nematode, Rhabditis (Oscheius) sp. exhibited strong antimicrobial activity. The ethyl acetate extract of the bacterial culture filtrate was purified by silica gel column chromatography to obtain four bioactive compounds. The structure and absolute stereochemistry of these compounds were determined based on extensive spectroscopic analyses (FABMS, ¹H NMR, ¹³C NMR, ¹H–¹H COSY, ¹H–¹³C HMBC) and Marfey’s method. The compounds were identified as cyclic dipeptides (CDPs): cyclo(l-Pro-l-Trp), cyclo(l-Leu-l-Val), cyclo(d-Pro-d-Met), and cyclo(d-Pro-d-Phe), respectively. Compounds recorded significant antibacterial activity against all the test bacteria (Staphylococcus epidermidis, Staphylococcus aureus, Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa and methicillin-resistant S. aureus) except cyclo(l-Leu-l-Val). Cyclo(l-Leu-l-Val) recorded activity only against Gram positive bacteria. Best antibacterial activity was recorded by cyclo(l-Pro-l-Trp) against S. aureus (4 μg/ml). The four compounds were active against all the five fungi tested (Trichophyton rubrum, Aspergillus flavus, Candida albicans, Candida tropicalis and Cryptococcus neoformans) and the activity was compared with amphotericin B, the standard fungicide. The highest activity of 1 μg/ml by cyclo(l-Pro-l-Trp) was recorded against T. rubrum, a human pathogen responsible for causing athlete’s foot, jock itch, and ringworm. The activity of cyclo(l-Pro-l-Trp) against T. rubrum, C. neoformans and C. albicans were better than amphotericin B, the standard antifungal agent. To our knowledge, this is the first report of antifungal activity of CDPs against the human pathogenic fungi T. rubrum and C. neoformans. The four CDPs are nontoxic to healthy human cell line up to 200 μg/ml. We conclude that the bacterium associated with entomopathogenic nematode is promising sources of natural antimicrobial secondary metabolites, which may receive greater benefit as potential sources of new drugs in the pharmaceutical industry.     

Quorum-sensing inhibitory compounds from extremophilic microorganisms isolated from a hypersaline cyanobacterial mat

In this study, extremely halophilic and moderately thermophilic microorganisms from a hypersaline microbial mat were screened for their ability to produce antibacterial, antidiatom, antialgal, and quorum-sensing (QS) inhibitory compounds. Five bacterial strains belonging to the genera Marinobacter and Halomonas and one archaeal strain belonging to the genus Haloterrigena were isolated from a microbial mat. The strains were able to grow at a maximum salinity of 22–25 % and a maximum temperature of 45–60 °C. Hexanes, dichloromethane, and butanol extracts from the strains inhibited the growth of at least one out of nine human pathogens. Only butanol extracts of supernatants of Halomonas sp. SK-1 inhibited growth of the microalga Dunaliella salina. Most extracts from isolates inhibited QS of the acyl homoserine lactone producer and reporter Chromobacterium violaceum CV017. Purification of QS inhibitory dichloromethane extracts of Marinobacter sp. SK-3 resulted in isolation of four related diketopiperazines (DKPs): cyclo(l-Pro-l-Phe), cyclo(l-Pro-l-Leu), cyclo(l-Pro-l-isoLeu), and cyclo(l-Pro-d-Phe). QS inhibitory properties of these DKPs were tested using C. violaceum CV017 and Escherichia coli-based QS reporters (pSB401 and pSB1075) deficient in AHL production. Cyclo(l-Pro-l-Phe) and cyclo(l-Pro-l-isoLeu) inhibited QS-dependent production of violacein by C. violaceum CV017. Cyclo(l-Pro-l-Phe), cyclo(l-Pro-l-Leu), and cyclo(l-Pro-l-isoLeu) reduced QS-dependent luminescence of the reporter E. coli pSB401 induced by 3-oxo-C6-HSL. Our study demonstrated the ability of halophilic and moderately thermophilic strains from a hypersaline microbial mat to produce biotechnologically relevant compounds that could be used as antifouling agents.     

In vitro synergistic activity of diketopiperazines alone and in combination with amphotericin B or clotrimazole against Candida albicans

The synergistic anticandidal activity of three diketopiperazines [cyclo-(l-Pro-l-Leu) (1), cyclo-(d-Pro-l-Leu) (2), and cyclo-(d-Pro-l-Tyr) (3)] purified from a Bacillus sp. N strain associated with entomopathogenic nematode Rhabditis (Oscheius) in combination with amphotericin B and clotrimazole was investigated using the macrodilution method. The minimum inhibitory concentration and minimum fungicidal concentration of the diketopiperazines was compared with that of the standard antibiotics. The synergistic anticandidal activities of diketopiperazines with amphotericin B or clotrimazole were assessed using the checkerboard and time-kill methods. The results of the present study showed that the combined effects of diketopiperazines with amphotericin B or clotrimazole predominantly recorded synergistic (<0.5). Time-kill study showed that the growth of the Candida was completely attenuated after 12–24 h of treatment with 50:50 ratios of diketopiperazines and antibiotics. These results suggest that diketopiperazines combined with antibiotics may be microbiologically beneficial and not antagonistic. These findings have potential implications in delaying the development of resistance as the anticandidal effect is achieved with lower concentrations of both drugs (diketopiperazines and antibiotics). The cytotoxicity of diketopiperazines was also tested against two normal human cell lines (L231 lung epithelial and FS normal fibroblast) and no cytotoxicity was recorded for diketopiperazines up to 200 μg/mL. The in vitro synergistic activity of diketopiperazines with antibiotics against Candida albicans is reported here for the first time.     

Improved Protease Stability of the Antimicrobial Peptide Pin2 Substituted with d-Amino Acids

Cationic antimicrobial peptides (AMPs) have attracted a great interest as novel class of antibiotics that might help in the treatment of infectious diseases caused by pathogenic bacteria. However, some AMPs with high antimicrobial activities are also highly hemolytic and subject to proteolytic degradation from human and bacterial proteases that limit their pharmaceutical uses. In this work a d-diastereomer of Pandinin 2, d-Pin2, was constructed to observe if it maintained antimicrobial activity in the same range as the parental one, but with the purpose of reducing its hemolytic activity to human erythrocytes and improving its ability to resist proteolytic cleavage. Although, the hydrophobic and secondary structure characteristics of l- and d-Pin2 were to some extent similar, an important reduction in d-Pin2 hemolytic activity (30–40 %) was achieved compared to that of l-Pin2 over human erythrocytes. Furthermore, d-Pin2 had an antimicrobial activity with a MIC value of 12.5 μM towards Staphylococcus aureus, Escherichia coli, Streptococcus agalactiae and two strains of Pseudomonas aeruginosa in agar diffusion assays, but it was half less potent than that of l-Pin2. Nevertheless, the antimicrobial activity of d-Pin2 was equally effective as that of l-Pin2 in microdilution assays. Yet, when d- and l-Pin2 were incubated with trypsin, elastase and whole human serum, only d-Pin2 kept its antimicrobial activity towards all bacteria, but in diluted human serum, l- and d-Pin2 maintained similar peptide stability. Finally, when l- and d-Pin2 were incubated with proteases from P. aeruginosa DFU3 culture, a clinical isolated strain, d-Pin2 kept its antibiotic activity while l-Pin2 was not effective.     

Principal component analysis of the relationship between the d-amino acid concentrations and the taste of the sake

We performed sensory evaluations on 141 bottles of sake and analyzed the relationship between the d-amino acid concentrations, and the taste of the sake using principal component analysis, which yielded seven principal components (PC1–7) that explained 100 % of the total variance in the data. PC1, which explains 33.6 % of the total variance, correlates most positively with strong taste and most negatively with balanced tastes. PC2, which explains 54.4 % of the total variance, correlates most positively with a sweet taste and most negatively with bitter and sour tastes. Sakes brewed with “Kimoto yeast starter” and “Yamahaimoto” had high scores for PC1 and PC2, and had strong taste in comparison with sakes brewed with “Sokujo-moto”. When present at concentrations below 50 μM, d-Ala did not affect the PC1 score, but all the sakes showed a high PC1 score, when the d-Ala was above 100 μM. Similar observations were found for the d-Asp and d-Glu concentrations with regard to PC1, and the threshold concentrations of d-Asp and d-Glu that affected the taste were 33.8 and 33.3 μM, respectively. Certain bacteria present in sake, especially lactic acid bacteria, produce d-Ala, d-Asp and d-Glu during storage, and these d-amino acids increased the PC1 score and produced a strong taste (Nojun). When d- and l-Ala were added to the sakes, the value for the umami taste in the sensory evaluation increased, with the effect of d-Ala being much stronger than that of l-Ala. The addition of 50–5,000 μM dl-Ala did not effect on the aroma of the sakes at all.     

Characterization of a recombinant l-fucose isomerase from Caldicellulosiruptor saccharolyticus that isomerizes l-fucose, d-arabinose, d-altrose, and l-galactose

A recombinant l-fucose isomerase from Caldicellulosiruptor saccharolyticus was purified as a single 68 kDa band with an activity of 76 U mg^?1. The molecular mass of the native enzyme was 204 kDa as a trimer. The maximum activity for l-fucose isomerization was at pH 7 and 75°C in the presence of 1 mM Mn²⁺. Its half-life at 70°C was 6.1 h. For aldose substrates, the enzyme displayed activity in decreasing order for l-fucose, with a k _cat of 11,910 min^?1 and a K _m of 140 mM, d-arabinose, d-altrose, and l-galactose. These aldoses were converted to the ketoses l-fuculose, d-ribulose, d-psicose, and l-tagatose, respectively, with 24, 24, 85, 55% conversion yields after 3 h.     

Detection of d-amino acids in purified proteins synthesized in Escherichia coli

It has long been believed that amino acids comprising proteins of all living organisms are only of the l-configuration, except for Gly. However, peptidyl d-amino acids were observed in hydrolysates of soluble high molecular weight fractions extracted from cells or tissues of various organisms. This strongly suggests that significant amounts of d-amino acids are naturally present in usual proteins. Thus we analyzed the d-amino acid contents of His-tag-purified β-galactosidase and human urocortin, which were synthesized by Escherichia coli grown in controlled synthetic media. After acidic hydrolysis for various times at 110°C, samples were derivatized with 4-fluoro-7-nitro-2, 1, 3-benzoxadiazole (NBD-F) and separated on a reverse-phase column followed by a chiral column into d- and l-enantiomers. The contents of d-enantiomers of Ala, Leu, Phe, Val, Asp, and Glu were determined by plotting index d/(d + l) against the incubation time for hydrolysis and extrapolating the linear regression line to 0 h to eliminate the effect of racemization of amino acids during the incubation. Significant contents of d-amino acids were reproducibly detected, the d-amino acid profile being specific to an individual protein. This finding indicated the likelihood that d-amino acids are in fact present in the purified proteins. On the other hand, the d-amino acid contents of proteins were hardly influenced by the addition of d- or l-amino acids to the cultivation medium, whereas intracellular free d-amino acids sensitively varied according to the extracellular conditions. The origin of these d-amino acids detected in proteins was discussed.     

Backbone and side-chain 1H, 15N and 13C assignment of apo- and imipenem-acylated l,d-transpeptidase from Bacillus subtilis

The d,d-transpeptidase activity of Penicillin Binding Proteins (PBPs) is essential to maintain cell wall integrity. PBPs catalyze the final step of the peptidoglycan synthesis by forming 4 → 3 cross-links between two peptide stems. Recently, a novel β-lactam resistance mechanism involving l,d-transpeptidases has been identified in Enterococcus faecium and Mycobacterium tuberculosis. In this resistance pathway, the classical 4 → 3 cross-links are replaced by 3 → 3 cross-links, whose formation are catalyzed by the l,d-transpeptidases. To date, only one class of the entire β-lactam family, the carbapenems, is able to inhibit the l,d-transpeptidase activity. Nevertheless, the specificity of this inactivation is still not understood. Hence, the study of this new transpeptidase family is of considerable interest in order to understand the mechanism of the l,d-transpeptidases inhibition by carbapenems. In this context, we present herein the backbone and side-chain ¹H, ¹⁵N and ¹³C NMR assignment of the l,d-transpeptidase from Bacillus subtilis (Ldt_Bs) in the apo and in the acylated form with a carbapenem, the imipenem.     

Enantioselective N-acetylation of 2-phenylglycine by an unusual N-acetyltransferase from Chryseobacterium sp.

The demand for d-2-phenylglycine used to synthesize semisynthetic antibiotics and pesticides is increasing. We have isolated a Chryseobacterium sp. that selectively transformed the l-form of racemic d,l-2-phenylglycine to (2S)-2-acetylamide-2-phenylacetic acid with a molar yield of 50 % and an enantiomer excess of >99.5 % under optimal culture conditions, consequently resulting in 99 % pure d-2-phenylglycine remaining in the culture. The enantioselective N-acetylation was catalyzed by an acetyl-CoA-dependent N-acetyltransferase whose synthesis was induced by l-2-phenylglycine. The enzyme differed from previously reported bacterial arylamine N-acetyltransferases in molecular mass and substrate specificity. The relative activity ratio of the enzyme with the substrates l-2-phenylglycine, d-2-phenylglycine, 2-(2-chlorophenyl)glycine, and 5-aminosalicylic acid (a good substrate of arylamine N-acetyltransferase) was 100:0:56.9:5.49, respectively. The biotransformation by the N-acetyltransferase-producing bacterium reported here could constitute a new preparative route for the enzymatic resolution of d,l-2-phenylglycine.     

Characterization of a recombinant l-rhamnose isomerase from Bacillus subtilis and its application on production of l-lyxose and l-mannose

The gene of an l-rhamnose isomerase (RhaA) from Bacillus subtilis was cloned to the pET28a(+) and then expressed in the E. coli ER2566. The expressed enzyme was purified with a specific activity of 3.58 U/mg by His-Trap affinity chromatography. The recombinant enzyme existed as a 194 kDa tetramer and the maximal activity was observed at pH 8.0 and 60°C. The RhaA displayed activity for l-rhamnose, l-lyxose, l-mannose, d-allose, d-gulose, d-ribose, and l-talose, among all aldopentoses and aldohexoses and it showed enzyme activity for l-form monosaccharides such as l-rhamnose, l-lyxose, l-mannose, and l-talose. The catalytic efficiency (k _cat/K _m) of the recombinant enzyme for l-rhamnose, l-lyxose, and l-mannose were 7,460, 1,013, and 258 M/sec. When l-xylulose 100 g/L and l-fructose 100 g/L were used as substrates, the optimum concentrations of RpiB were determined with 6 and 15 U/mL, respectively. The l-lyxose 40 g/L was produced from l-xylulose 100 g/L by the enzyme during 60 min, while l-mannose 25 g/L was produced from l-fructose 100 g/L for 80 min. The results suggest that RhaA from B. subtilis is a potential producer of l-form monosaccharides.     

l-Arabinose/d-galactose 1-dehydrogenase of Rhizobium leguminosarum bv. trifolii characterised and applied for bioconversion of l-arabinose to l-arabonate with Saccharomyces cerevisiae

Four potential dehydrogenases identified through literature and bioinformatic searches were tested for l-arabonate production from l-arabinose in the yeast Saccharomyces cerevisiae. The most efficient enzyme, annotated as a d-galactose 1-dehydrogenase from the pea root nodule bacterium Rhizobium leguminosarum bv. trifolii, was purified from S. cerevisiae as a homodimeric protein and characterised. We named the enzyme as a l-arabinose/d-galactose 1-dehydrogenase (EC 1.1.1.-), Rl AraDH. It belongs to the Gfo/Idh/MocA protein family, prefers NADP⁺ but uses also NAD⁺ as a cofactor, and showed highest catalytic efficiency (k _cat/K _m) towards l-arabinose, d-galactose and d-fucose. Based on nuclear magnetic resonance (NMR) and modelling studies, the enzyme prefers the α-pyranose form of l-arabinose, and the stable oxidation product detected is l-arabino-1,4-lactone which can, however, open slowly at neutral pH to a linear l-arabonate form. The pH optimum for the enzyme was pH 9, but use of a yeast-in-vivo-like buffer at pH 6.8 indicated that good catalytic efficiency could still be expected in vivo. Expression of the Rl AraDH dehydrogenase in S. cerevisiae, together with the galactose permease Gal2 for l-arabinose uptake, resulted in production of 18 g of l-arabonate per litre, at a rate of 248 mg of l-arabonate per litre per hour, with 86 % of the provided l-arabinose converted to l-arabonate. Expression of a lactonase-encoding gene from Caulobacter crescentus was not necessary for l-arabonate production in yeast.     

Function of aspartic acid residues in optimum pH control of l-arabinose isomerase from Lactobacillus fermentum

l-Arabinose isomerase (l-AI) catalyzes the isomerization of l-arabinose to l-ribulose and d-galactose to d-tagatose. Most reported l-AIs exhibit neutral or alkaline optimum pH, which is less beneficial than acidophilic ones in industrial d-tagatose production. Lactobacillus fermentum l-AI (LFAI) is a thermostable enzyme that can achieve a high conversion rate for d-galactose isomerization. However, its biocatalytic activity at acidic conditions can still be further improved. In this study, we report the single- and multiple-site mutagenesis on LFAI targeting three aspartic acid residues (D268, D269, and D299). Some of the lysine mutants, especially D268K/D269K/D299K, exhibited significant optimum pH shifts (from 6.5 to 5.0) and enhancement of pH stability (half-life time increased from 30 to 62 h at pH 6.0), which are more favorable for industrial applications. With the addition of borate, d-galactose was isomerized into d-tagatose by D268K/D269K/D299K at pH 5.0, resulting in a high conversion rate of 62 %. Based on the obtained 3.2-Å crystal structure of LFAI, the three aspartic acid residues were found to be distant from the active site and possibly did not participate in substrate catalysis. However, they were proven to possess similar optimum pH control ability in other l-AI, such as that derived from Escherichia coli. This study sheds light on the essential residues of l-AIs that can be modified for desired optimum pH and better pH stability, which are useful in d-tagatose bioproduction.     

An l-glucitol oxidizing dehydrogenase from Bradyrhizobium japonicum USDA 110 for production of d-sorbose with enzymatic or electrochemical cofactor regeneration

A gene in Bradyrhizobium japonicum USDA 110, annotated as a ribitol dehydrogenase (RDH), had 87 % sequence identity (97 % positives) to the N-terminal 31 amino acids of an l-glucitol dehydrogenase from Stenotrophomonas maltophilia DSMZ 14322. The 729-bp long RDH gene coded for a protein consisting of 242 amino acids with a molecular mass of 26.1 kDa. The heterologously expressed protein not only exhibited the main enantio selective activity with d-glucitol oxidation to d-fructose but also converted l-glucitol to d-sorbose with enzymatic cofactor regeneration and a yield of 90 %. The temperature stability and the apparent K _m value for l-glucitol oxidation let the enzyme appear as a promising subject for further improvement by enzyme evolution. We propose to rename the enzyme from the annotated RDH gene (locus tag bll6662) from B. japonicum USDA as a d-sorbitol dehydrogenase (EC 1.1.1.14).     

Transportable and Non-transportable Inhibitors of L-glutamate Uptake Produce Astrocytic Stellation and Increase EAAT2 Cell Surface Expression

Astrocytic excitatory amino acid transporters (EAATs) regulate excitatory transmission and limit excitotoxicity. Evidence for a functional interface between EAATs and glial fibrillary acidic protein (GFAP) relevant to astrocytic morphology led to investigations of actions of transportable (d-Aspartate (d-Asp) and (2S,3S,4R)-2-(carboxycyclopropyl)glycine (l-CCG-III)) and non-transportable (dl-threo-β-benzyloxyaspartate (dl-TBOA)) inhibitors of Glu uptake in murine astrocytes. d-Asp (1 mM), l-CCG-III (0.5 mM) and dl-TBOA (0.5 mM) produced time-dependent (24–72 h) reductions in ³[H]d-Asp uptake (approximately 30–70%) with little or no gliotoxicity. All drugs induced a profound change in phenotype from cobblestone to stellate morphology and image analysis revealed increases in the intensity of GFAP immunolabelling for l-CCG-III and dl-TBOA. Cytochemistry indicated localized changes in F-actin distribution. Cell surface expression of EAAT2, but not EAAT1, was elevated at 72 h. Blockade of Glu uptake by both types of EAAT inhibitor exerts longer-term effects on astrocytic morphology and a compensatory homeostatic rise in EAAT2 abundance.     

d-Tagatose production in the presence of borate by resting Lactococcus lactis cells harboring Bifidobacterium longum l-arabinose isomerase

Bifidobacterium longum NRRL B-41409 l-arabinose isomerase (l-AI) was overexpressed in Lactococcus lactis using a phosphate depletion inducible expression system. The resting L. lactis cells harboring the B. longum l-AI were used for production of d-tagatose from d-galactose in the presence of borate buffer. Multivariable analysis suggested that high pH, temperature and borate concentration favoured the conversion of d-galactose to d-tagatose. Almost quantitative conversion (92 %) was achieved at 20 g L^?1 substrate and at 37.5 °C after 5 days. The d-tagatose production rate of 185 g L^?1 day^?1 was obtained at 300 g L^?1 galactose, at 1.15 M borate, and at 41 °C during 10 days when the production medium was changed every 24 h. There was no significant loss in productivity during ten sequential 24 h batches. The initial d-tagatose production rate was 290 g L^?1 day^?1 under these conditions.     

A combinatorial approach to the structure elucidation of a pyoverdine siderophore produced by a Pseudomonas putida isolate and the use of pyoverdine as a taxonomic marker for typing P. putida subspecies

The structure of a pyoverdine produced by Pseudomonas putida, W15Oct28, was elucidated by combining mass spectrometric methods and bioinformatics by the analysis of non-ribosomal peptide synthetase genes present in the newly sequenced genome. The only form of pyoverdine produced by P. putida W15Oct28 is characterized to contain α-ketoglutaric acid as acyl side chain, a dihydropyoverdine chromophore, and a 12 amino acid peptide chain. The peptide chain is unique among all pyoverdines produced by Pseudomonas subspecies strains. It was characterized as –l-Asp-l-Ala-d-AOHOrn-l-Thr-Gly-c[l-Thr(O-)-l-Hse-d-Hya-l-Ser-l-Orn-l-Hse-l-Ser-O-]. The chemical formula and the detected and calculated molecular weight of this pyoverdine are: C₆₅H₉₃N₁₇O₃₂, detected mass 1624.6404 Da, calculated mass 1624.6245. Additionally, pyoverdine structures from both literature reports and bioinformatics prediction of the genome sequenced P. putida strains are summarized allowing us to propose a scheme based on pyoverdines structures as tool for the phylogeny of P. putida. This study shows the strength of the combination of in silico analysis together with analytical data and literature mining in determining the structure of secondary metabolites such as peptidic siderophores.     

Metabolic engineering of Escherichia coli for biosynthesis of d-galactonate

d-galactose is an attractive substrate for bioconversion. Herein, Escherichia coli was metabolically engineered to convert d-galactose into d-galactonate, a valuable compound in the polymer and cosmetic industries. d-galactonate productions by engineered E. coli strains were observed in shake flask cultivations containing 2 g L^?1 d-galactose. Engineered E. coli expressing gld coding for galactose dehydrogenase from Pseudomonas syringae was able to produce 0.17 g L^?1 d-galactonate. Inherent metabolic pathways for assimilating both d-galactose and d-galactonate were blocked to enhance the production of d-galactonate. This approach finally led to a 7.3-fold increase with d-galactonate concentration of 1.24 g L^?1 and yield of 62.0 %. Batch fermentation in 20 g L^?1 d-galactose of E. coli ?galK?dgoK mutant expressing the gld resulted in 17.6 g L^?1 of d-galactonate accumulation and highest yield of 88.1 %. Metabolic engineering strategy developed in this study could be useful for industrial production of d-galactonate.     

d-Amino Acid-Induced Expression of d-Amino Acid Oxidase in the Yeast Schizosaccharomyces pombe

We investigated d-amino acid oxidase (DAO) induction in the popular model yeast Schizosaccharomyces pombe. The product of the putative DAO gene of the yeast expressed in E.?coli displayed oxidase activity to neutral and basic d-amino acids, but not to an l-amino acid or acidic d-amino acids, showing that the putative DAO gene encodes catalytically active DAO. DAO activity was weakly detected in yeast cells grown on a culture medium without d-amino acid, and was approximately doubled by adding d-alanine. The elimination of ammonium chloride from culture medium induced activity by up to eight-fold. l-Alanine also induced the activity, but only by about half of that induced by d-alanine. The induction by d-alanine reached a maximum level at 2?h cultivation; it remained roughly constant until cell growth reached a stationary phase. The best inducer was d-alanine, followed by d-proline and then d-serine. Not effective were N-carbamoyl-d,l-alanine (a better inducer of DAO than d-alanine in the yeast Trigonopsis variabilis), and both basic and acidic d-amino acids. These results showed that S. pombe DAO could be a suitable model for analyzing the regulation of DAO expression in eukaryotic organisms.