Isolation of the DLD gene of Saccharomyces cerevisiae encoding the mitochondrial enzyme D-lactate ferricytochrome c oxidoreductase

In Saccharomyces cerevisiae the utilization of lactate occurs via specific oxidation of l- and d-lactate to pyruvate catalysed by l-lactate ferricytochrome c oxidoreductase (L-LCR) (EC 1.1.2.3) encoded by the CYB2 gene, and d-lactate ferricytochrome c oxidoreductase (D-LCR) (EC 1.1.2.4), respectively. We selected several lactate^? pyruvate⁺ mutants in a cyb2 genetic background. Two of them were devoid of D -LCR activity (dld mutants, belonging to the same complementation group). The mutation mapped in the structural gene. This was demonstrated by a gene dosage effect and by the thermosensitivity of the enzyme activity of thermosensitive revertants. The DLD gene was cloned by complementation for growth on d-, l-lactate in the strain WWF18-3D, carrying both a CYB2 disruption and the dld mutation. The minimal complete complementing sequence was localized by subcloning experiments. From the sequence analysis an open reading frame (ORF) was identified that could encode a polypeptide of 576 amino-acids, corresponding to a calculated molecular weight of 64000 Da. The deduced protein sequence showed significant homology with the previously described microsomal flavoprotein l-gulono-γ-lactone oxidase isolated from Rattus norvegicus, which catalyses the terminal step of l-ascorbic acid biosynthesis. These results are discussed together with the role of L-LCR and D-LCR in lactate metabolism of S. cerevisiae.     

Properties of the sugar carrier in Baker's yeast

Incubation ofSaccharomyces cerevisiae cells withd-galactose induced the formation of galactose-utilizing enzymes, among them a monosaccharide carrier, apparently synthesized as a proteinde novo. The synthesis of the carrier preceded that of galactokinase by as much as 2 h. The inducible carrier shows a preference for monosaccharides with an axial hydroxyl group at carbon 4 of theC1 chair conformation or at carbon 2 of the1C chair conformation. Through its mediation, some sugars normally poorly transported (d-galactose,d-fucose,l-xylose,l-arabinose) can enter into the entire cell water, occupying then one more kinetic (and morphological ?) compartment than before induction. Some other monosaccharides, readily transported even by a constitutive carrier system (e.g.l-sorbose,d-xylose,d-arabinose) share the newly synthesized carrier.     

Reactions upstream of glycerate-1,3-bisphosphate drive Corynebacterium glutamicum d-lactate productivity under oxygen deprivation

We previously demonstrated the simplicity of oxygen-deprived Corynebacterium glutamicum to produce d-lactate, a primary building block of next-generation biodegradable plastics, at very high optical purity by introducing heterologous D-ldhA gene from Lactobacillus delbrueckii. Here, we independently evaluated the effects of overexpressing each of genes encoding the ten glycolytic enzymes on d-lactate production in C. glutamicum. We consequently show that while the reactions catalyzed by glucokinase (GLK), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), phosphofructokinase (PFK), triosephosphate isomerase (TPI), and bisphosphate aldolase had positive effects on d-lactate productivity by increasing 98, 39, 15, 13, and 10 %, respectively, in 10 h reactions in minimal salts medium, the reaction catalyzed by pyruvate kinase had large negative effect by decreasing 70 %. The other glycolytic enzymes did not affect d-lactate productivity when each of encoding genes was overexpressed. It is noteworthy that all reactions associated with positive effects are located upstream of glycerate-1,3-bisphosphate in the glycolytic pathway. The d-lactate yield also increased by especially overexpressing TPI encoding gene up to 94.5 %. Interestingly, overexpression of PFK encoding gene reduced the yield of succinate, one of the main by-products of d-lactate production, by 52 %, whereas overexpression of GAPDH encoding gene increased succinate yield by 26 %. Overexpression of GLK encoding gene markedly increased the yield of dihydroxyacetone and glycerol by 10- and 5.8-fold in exchange with decreasing the d-lactate yield. The effect of overexpressing glycolytic genes was also evaluated in 80 h long-term reactions. The variety of effects of overexpressing each of genes encoding the ten glycolytic enzymes on d-lactate production is discussed.     

On the structure of the peptidoglycan of cell walls from Myxobacter AL-1 (Myxobacterales)

Basically the peptidoglycan of Myxobater AL-1 consists of alternating β-1,4-linked N-acetylglucosamic-N-acetylmuramic acid chains. After splitting the aminosugar backbone with a specific algal enzyme three subunits arise: a monomer, a dimer and a trimer. Investigation of the monomer with specific enzymes and comparison of the degradation products to standards derived from other bacterial peptidoglycans suggest the following structure of the monomer peptide: l-alanyl-d-glutamic-l-meso-diaminopimelic-d-alanine. A d-alanyl-d-meso-diaminopimelic acid bond is the bridgebond between the peptides of the subunits.     

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.     

High Yield Recombinant Expression, Characterization and Homology Modeling of Two Types of Cis-epoxysuccinic Acid Hydrolases

The cis-epoxysuccinate hydrolases (CESHs), members of epoxide hydrolase, catalyze cis-epoxysuccinic acid hydrolysis to form d(?)-tartaric acid or l(+)-tartaric acid which are important chemicals with broad scientific and industrial applications. Two types of CESHs (CESH[d] and CESH[l], producing d(?)- and l(+)-tartaric acids, respectively) have been reported with low yield and complicated purification procedure in previous studies. In this paper, the two CESHs were overexpressed in Escherichia coli using codon-optimized genes. High protein yields by one-step purifications were obtained for both recombinant enzymes. The optimal pH and temperature were measured for both recombinant CESHs, and the properties of recombinant enzymes were similar to native enzymes. Kinetics parameters measured by Lineweaver?CBurk plot indicates both enzymes exhibited similar affinity to cis-epoxysuccinic acid, but CESH[l] showed much higher catalytic efficiency than CESH[d], suggesting that the two CESHs have different catalytic mechanisms. The structures of both CESHs constructed by homology modeling indicated that CESH[l] and CESH[d] have different structural folds and potential active site residues. CESH[l] adopted a typical ??/??-hydrolase fold with a cap domain and a core domain, whereas CESH[d] possessed a unique TIM barrel fold composed of 8 ??-helices and 8 ??-strands, and 2 extra short ??-helices exist on the top and bottom of the barrel, respectively. A divalent metal ion, preferred to be zinc, was found in CESH[d], and the ion was proved to be crucial to the enzymatic activity. These results provide structural insight into the different catalytic mechanisms of the two CESHs.     

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.     

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.     

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.     

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.     

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.     

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.     

Effects of l-lysine and d-lysine on ɛ-Poly-l-lysine biosynthesis and their metabolites by Streptomyces ahygroscopicus GIM8

?-Poly-l-lysine (?-PL), produced by Streptomyces or Kitasatospora strains, is a homo-poly-amino acid of l-lysine, which is used as a safe food preservative. In this study, the effects of l-lysine and its isomer, d-lysine, on ?-PL biosynthesis and their metabolites by the ?-PL-producing strain Streptomyces ahygroscopicus GIM8 were determined. The results indicated that l-lysine added into the fermentation medium in the production phase mainly served as a precursor for ?-PL biosynthesis during the flask culture phase, leading to greater ?-PL production. At an optimum level of 3 mM l-lysine, a ?-PL yield of 1.16 g/L was attained, with a 41.4% increment relative to the control of 0.78 g/L. Regarding d-lysine, the production of ?-PL increased by increasing its concentrations up to 6 mM in the initial fermentation medium. Interestingly, ?-PL production (1.20 g/L) with the addition of 3 mM d-lysine into the initial fermentation medium in flasks was higher than that of the initial addition of 3 mM L-lysine (1.06 g/L). The mechanism by which d-lysine improves ?-PL biosynthesis involves its utilization that leads to greater biomass. After S. ahygroscopicus GIM8 was cultivated in the defined medium with L-lysine, several key metabolites, including 5-aminovalerate, pipecolate, and l-2-aminoadipate formed in the cells, whereas only l-2-aminoadipate was observed after d-lysine metabolism. This result indicates that l-lysine and d-lysine undergo different metabolic pathways in the cells. Undoubtedly, the results of this study are expected to aid the understanding of ?-PL biosynthesis and serve as reference for the formulation of an alternative approach to improve ?-PL productivity using l-lysine as an additional substrate in the fermentation medium.     

Serine racemase: an unconventional enzyme for an unconventional transmitter

The discovery of large amounts of d-serine in the brain challenged the dogma that only l-amino acids are relevant for eukaryotes. The levels of d-serine in the brain are higher than many l-amino acids and account for as much as one-third of l-serine levels. Several studies in the last decades have demonstrated a role of d-serine as an endogenous agonist of N-methyl-d-aspartate receptors (NMDARs). d-Serine is required for NMDAR activity during normal neurotransmission as well as NMDAR overactivation that takes place in neurodegenerative conditions. Still, there are many unanswered questions about d-serine neurobiology, including regulation of its synthesis, release and metabolism. Here, we review the mechanisms of d-serine synthesis by serine racemase and discuss the lessons we can learn from serine racemase knockout mice, focusing on the roles attributed to d-serine and its cellular origin.     

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.     

Effect of sugars on the morphogenesis ofAureobasidium pullulans

The dominance of individual elements of the vegetative fructification of five selected strains of the polymorphic organismAureobasidium pullulans (de Baby)Arnaud was studied in media with basic assimilable sugars,d-glucose,d-galactose,d-xylose, maltose, sucrose, lactose and a mixture ofl -arabinose andd-mannitol. Pronounced differences between cultures grown in the presence of monosaccharides and those cultivated in the presence of disaccharides were detected.     

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.     

Improvement of l-lactic acid production by osmotic-tolerant mutant of Lactobacillus casei at high temperature

l-Lactic acid production by Lactobacillus casei was used as a model to study the mechanism of substrate inhibition and the strategy for enhancing l-lactic acid production. It was found that the concentration of cell growth and l-lactate decreased with the increase of glucose concentration and fermentation temperature. To enhance the osmotic stress resistance of the strain at high temperature, a mutant G-03 was screened and selected with 360?g/L glucose at 45°C as the selective criterion. To further increase the cell growth for lactic acid production, 3?g/L of biotin was supplemented to the medium. As a result, l-lactate concentration by the mutant G-03 reached 198.2?g/L (productivity of 5.5?g?L^?1?h^?1) at 41°C in a 7-L fermentor with 210?g/L glucose as carbon source. l-Lactate concentration and productivity of mutant G-03 were 115.2% and 97.8% higher than those of the parent strain, respectively. The strategy for enhancing l-lactic acid production by increasing osmotic stress resistance at high temperature may provide an alternative approach to enhance organic acid production with other strains.