The Aminolysis Reaction of Streptomyces S9 Aminopeptidase Promotes the Synthesis of Diverse Prolyl Dipeptides

Prolyl dipeptide synthesis by S9 aminopeptidase from Streptomyces thermocyaneoviolaceus (S9AP-St) has been demonstrated. In the synthesis, S9AP-St preferentially used l-Pro-OBzl as the acyl donor, yielding synthesized dipeptides having an l-Pro-Xaa structure. In addition, S9AP-St showed broad specificity toward the acyl acceptor. Furthermore, S9AP-St produced cyclo (l-Pro-l-His) with a conversion ratio of substrate to cyclo (l-Pro-l-His) higher than 40%.Some proline-containing dipeptides and their cyclic analogs exhibit biological activity. For example, cyclo (l-arginyl-d-proline) [c(lR-dP)] is known to act as a specific inhibitor of family 18 chitinase (4, 10). A cyclic peptide, c(lP-lH), produced by the cleavage of thyrotropin-releasing hormone protects against oxidative stress, promotes cytoprotection (6, 7), and exhibits antihyperglycemic activity (11).Some serine peptidases exhibit peptide bond formation (i.e., aminolysis of esters, thioesters, and amides) in accordance with their hydrolytic activity (2, 14). The exchange of catalytic Ser for Cys to engineer the serine endopeptidase into “transpeptidase” for peptide bond formation has been well characterized (3, 5). Our recent approach confirmed the wide distribution of family S9 aminopeptidases that have catalytic Ser in actinomycetes (12). Of them, we obtained S9 aminopeptidase from Streptomyces thermocyaneoviolaceus NBRC14271 (S9AP-St). The enzyme was engineered into “transaminopeptidase” by exchange of catalytic Ser for Cys, and its aminolytic activity was evaluated (13). The engineered enzyme, designated as aminolysin-S, can synthesize hydrophobic dipeptides through an aminolysis reaction. However, aminolysin-S was unable to synthesize peptides containing proline. Although the report of aminolysin-S demonstrated that S9AP-St shows no aminolysis reaction toward limited substrates, details of its characteristics remain unknown. This study verified the peptide synthetic activity of S9AP-St, demonstrating that S9AP-St can synthesize widely varied prolyl dipeptides through an aminolysis reaction. The report also shows that S9AP-St is applicable to the synthesis of a biologically active peptide—c(lP-lH).     

Isolation and Characterization of a Novel Lysine Racemase from a Soil Metagenomic Library

A lysine racemase (lyr) gene was isolated from a soil metagenome by functional complementation for the first time by using Escherichia coli BCRC 51734 cells as the host and d-lysine as the selection agent. The lyr gene consisted of a 1,182-bp nucleotide sequence encoding a protein of 393 amino acids with a molecular mass of about 42.7 kDa. The enzyme exhibited higher specific activity toward lysine in the l-lysine-to-d-lysine direction than in the reverse reaction.Amino acids are the building blocks of proteins and play an important role in the regulation of the metabolism of living organisms. Among two enantiomers of naturally occurring amino acids, l-amino acids are predominant in living organisms, while d-amino acids are found in both free and bound states in various organisms like bacteria (36), yeasts (35), plants (47), insects (11), mammals (17), bivalves (39), and fish (28). The d-amino acids are mostly endogenous and produced by racemization from their counterparts by the action of a racemase. Thus, the amino acid racemases are involved in d-amino acid metabolism (29, 46). Since the discovery of alanine racemase in 1951 (42), several racemases toward amino acids, such as those for glutamate, threonine, serine, aspartate, methionine, proline, arginine, and phenylalanine, have been reported in bacteria, archaea, and eukaryotes, including mammals (1, 2, 15, 30, 31, 44). They are classified into two groups: pyridoxal 5′-phosphate (PLP)-dependent and PLP-independent enzymes (9, 36).Lysine racemase (Lyr, EC 5.1.1.5) was first reported in Proteus vulgaris ATCC 4669 (19) and proposed to be involved in the lysine degradation of bacterial cells (5, 19). Catabolism of lysine occurs via two parallel pathways. In one of the pathways, δ-aminovalerate is the key metabolite, whereas in the other l-lysine is racemized to d-lysine, and l-pipecolate and α-aminoadipate (AMA) are the key metabolites (5). d-Lysine catabolism proceeds through a series of cyclized intermediates which are necessary to regenerate an α-amino acid and comprise the following metabolites (AMA pathway): d-lysine→α-keto-ɛ-amino caproate→Δ¹-piperideine-2-carboxylate→pipecolate→Δ¹-piperideine-6-carboxylate→α-amino-δ-formylcaproate→α-AMA→α-ketoadipate (6, 7, 12, 27). The final product is converted to α-ketoglutarate via a series of coenzyme A derivatives and subsequently participates as an intermediate in the Krebs cycle. This pathway suggests that the biological function of d-lysine in the bacteria is that of a carbon or nitrogen source. Racemization of added l-lysine to d-lysine by whole cells of Proteus spp. and Escherichia spp. (19) and by the cell extract of Pseudomonas putida ATCC 15070 (5, 20) has been found. However, the enzyme has not been purified to homogeneity, and thus, its molecular and catalytic characteristics, including its gene structure, have not been elucidated. In this study, we explored a metagenomic library constructed from a garden soil to isolate a novel Lyr enzyme. After expression in Escherichia coli, the purified enzyme was characterized in terms of optimal pH and temperature, thermal stability, and racemization activity.     

Glycosylation of the Collagen Adhesin EmaA of Aggregatibacter actinomycetemcomitans Is Dependent upon the Lipopolysaccharide Biosynthetic Pathway

The human oropharyngeal pathogen Aggregatibacter actinomycetemcomitans synthesizes multiple adhesins, including the nonfimbrial extracellular matrix protein adhesin A (EmaA). EmaA monomers trimerize to form antennae-like structures on the surface of the bacterium, which are required for collagen binding. Two forms of the protein have been identified, which are suggested to be linked with the type of O-polysaccharide (O-PS) of the lipopolysaccharide (LPS) synthesized (G. Tang et al., Microbiology 153:2447-2457, 2007). This association was investigated by generating individual mutants for a rhamnose sugar biosynthetic enzyme (rmlC; TDP-4-keto-6-deoxy-d-glucose 3,5-epimerase), the ATP binding cassette (ABC) sugar transport protein (wzt), and the O-antigen ligase (waaL). All three mutants produced reduced amounts of O-PS, and the EmaA monomers in these mutants displayed a change in their electrophoretic mobility and aggregation state, as observed in sodium dodecyl sulfate (SDS)-polyacrylamide gels. The modification of EmaA with O-PS sugars was suggested by lectin blots, using the fucose-specific Lens culinaris agglutinin (LCA). Fucose is one of the glycan components of serotype b O-PS. The rmlC mutant strain expressing the modified EmaA protein demonstrated reduced collagen adhesion using an in vitro rabbit heart valve model, suggesting a role for the glycoconjugant in collagen binding. These data provide experimental evidence for the glycosylation of an oligomeric, coiled-coil adhesin and for the dependence of the posttranslational modification of EmaA on the LPS biosynthetic machinery in A. actinomycetemcomitans.The Gram-negative, nonmotile, microaerophilic, and oropharyngeal bacterium Aggregatibacter actinomycetemcomitans preferentially colonizes the subgingival region of the human oral cavity. This microorganism is implicated as the etiological agent of localized aggressive periodontitis (9, 13) and causes extraoral infections, including pneumonia, osteitis (30), and infective endocarditis (6). Recent studies also link this periodontal pathogen to cardiovascular diseases, such as atherosclerosis (20).Typical of Gram-negative bacteria, the outer membrane of A. actinomycetemcomitans possesses an asymmetric lipid-protein bilayer. The inner leaflet of the outer membrane is mainly phospholipids, and the outer leaflet consists of lipopolysaccharide (LPS), phospholipids, and proteins (4). LPS molecules are ubiquitously distributed on the outer membrane and are essential for maintaining the membrane integrity (3). Intact LPS molecules are also required for the assembly of some large outer membrane proteins (3, 18, 41). A typical LPS molecule is composed of hydrophobic lipid A, a nonrepeat core oligosaccharide, and a repeating O-antigen or O-polysaccharide (O-PS). The distal O-PS is a major antigen, stimulating the host immune response, and the basis for serotyping Gram-negative bacteria (36), including A. actinomycetemcomitans (32, 50).Six different serotypes (a to f) and the corresponding genetic loci have been identified for A. actinomycetemcomitans (19, 22, 27, 44, 50, 54, 55). Serotype b remains one of the common serotypes found in the human oral cavity (9, 13, 51). The serotype b O-PS of A. actinomycetemcomitans is encoded by an operon composed of 21 genes, which are responsible for the biosynthesis of the repeating trisaccharide unit of this particular serotype (53, 55). Each O-PS unit of serotype b contains a disaccharide backbone composed of d-fucose (d-Fuc) and l-rhamnose (l-Rha), linked by a nonreducing d-N-acetylgalactosamine (d-GalNAc) at the O-3 position of l-Rha (33) (Fig. ​(Fig.1A1A).Open in a separate windowFIG. 1.(A) O-PS structure of serotype b A. actinomycetemcomitans. (B) Silver-stained 5 to 15% polyacrylamide-SDS gel of serotype b LPS. A total of 1.0 ml of mid-logarithmic-phase cells were collected and lysed. Three lysates from each strain were combined and treated with proteinase K at 60°C for 60 min before electrophoresis, followed by silver staining. C, control: whole-cell lysate without proteinase K digestion; WT, wild type (VT1169); emaA, extracellular matrix protein adhesin A mutant; rmlC, rhamnose epimerase mutant; wzt, ATP-binding cassette sugar transport mutant. The dark brown staining of the high molecular weight (75,000 to 250,000) corresponds to polymerized O-PS.The assembly of LPS molecules in Gram-negative bacteria involve diverse enzymes and pathways due to the variation of the O-PS structures among different bacteria (36). RmlC (previously RfbD), Wzt (previously AbcA or RfbB), and WaaL are three enzymes involved in different stages of the LPS synthesis of some Gram-negative bacteria (7, 36, 37). A homologue of RmlC, TDP-4-keto-6-deoxy-d-glucose 3,5-epimerase, which is required for l-Rha synthesis, has been identified in A. actinomycetemcomitans (53, 55). Wzt is an ATP binding cassette (ABC) transporter that exports saccharide polymers from the cytoplasm to the periplasmic space (7, 36). A homologue of wzt was originally identified from a serotype b strain of A. actinomycetemcomitans, based on protein sequence identity with Aeromonas salmonicida (55). Kaplan et al. (19) later showed that a serotype f wzt mutant strain of A. actinomycetemcomitans produces less O-PS. WaaL, an O-antigen ligase found in Escherichia coli and Pseudomonas aeruginosa, ligates an undecaprenol pyrophosphate-linked oligo- or polysaccharide onto the lipid A-core oligosaccharide in the periplasm (1, 36). A putative O-antigen ligase is located in the chromosome of a serotype b A. actinomycetemcomitans strain (HK1651), based on sequence homology (Oralgen, Los Alamos, NM).Our earlier work suggested a correlation between the type of LPS molecule and the form of EmaA synthesized by A. actinomycetemcomitans (46). The EmaA of serotype b A. actinomycetemcomitans is a 202-kDa protein that forms the antennae-like appendages found on the surface of A. actinomycetemcomitans and is required for collagen binding (40). The appendages are composed of three EmaA monomers that oligomerize to form an ellipsoidal structure required for the collagen binding activity (56, 57). The ellipsoidal structure corresponds to the amino termini of the proteins and is located at the distal end of a long stalk domain that is attached to the outer membrane by the carboxyl termini (57). The carboxyl termini of the proteins assume β-barrel structures required for pore formation and translocation of the molecules through the outer membrane, similar to those of other type V_c autotransporter proteins (14). Recently, we have demonstrated that EmaA is important in the initiation of infective endocarditis in a rabbit model of infectious endocarditis (45).Two transposon mutant strains (rmlC and wzt) and a waaL mutant strain generated by site-directed insertional mutagenesis have been developed and characterized in this study. The rmlC mutant did not synthesize l-Rha and did not produce detectable O-PS. The wzt and waaL mutant strains synthesized less O-PS than the wild-type strain. Complementation of the mutant strains restored the production of the serotype b O-PS to wild-type levels. An increase in the electrophoretic mobility of the EmaA monomer was observed in all three mutants, which suggests the presence of carbohydrate. The EmaA mobility reverted to wild-type mobility upon complementation. The presence of carbohydrate associated with EmaA was confirmed by lectin blotting, and in vitro collagen binding assessment demonstrated that the glycoconjugant is important for the full function of this adhesin. The experimental data suggest that EmaA contains carbohydrate similar to that present in O-PS and is a substrate for the O-antigen ligase of the LPS biosynthetic pathway of A. actinomycetemcomitans.     

The Ultramicrobacterium “Elusimicrobium minutum” gen. nov., sp. nov., the First Cultivated Representative of the Termite Group 1 Phylum

Insect intestinal tracts harbor several novel, deep-rooting clades of as-yet-uncultivated bacteria whose biology is typically completely unknown. Here, we report the isolation of the first representative of the termite group 1 (TG1) phylum from sterile-filtered gut homogenates of a humivorous scarab beetle larva. Strain Pei191^T is a mesophilic, obligately anaerobic ultramicrobacterium with a gram-negative cell envelope. Cells are typically rod shaped, but cultures are pleomorphic in all growth phases (0.3 to 2.5 μm long and 0.17 to 0.3 μm wide). The isolate grows heterotrophically on sugars and ferments d-galactose, d-glucose, d-fructose, d-glucosamine, and N-acetyl-d-glucosamine to acetate, ethanol, hydrogen, and alanine as major products but only if amino acids are present in the medium. PCR-based screening and comparative 16S rRNA gene sequence analysis revealed that strain Pei191^T belongs to the “intestinal cluster,” a lineage of hitherto uncultivated bacteria present in arthropod and mammalian gut systems. It is only distantly related to the previously described so-called “endomicrobia” lineage, which comprises mainly uncultivated endosymbionts of termite gut flagellates. We propose the name “Elusimicrobium minutum” gen. nov., sp. nov. (type strain, Pei191^T = ATCC BAA-1559^T = JCM 14958^T) for the first isolate of this deep-branching lineage and the name “Elusimicrobia” phyl. nov. for the former TG1 phylum.Insect intestinal tracts harbor an enormous diversity of as-yet-uncultivated bacteria that are characterized only by their 16S rRNA gene sequences and whose biology is typically completely obscure (9, 17, 49). As in other environments (45), many of these sequences form deep-branching phylogenetic lineages that do not contain a single isolate (18, 28). One of these lineages is the termite group 1 (TG1), which was originally discovered by Ohkuma and Kudo (37) and recognized as a phylum-level group (candidate division) by Hugenholtz et al. (20). TG1 bacteria form a major proportion of the microbial community in the hindgut of lower termites (17, 69), where they inhabit the cytoplasm of the intestinal flagellates (38, 53). These so-called “endomicrobia” are specific for the respective flagellate species (21) and, at least in the case of “Candidatus Endomicrobium trichonymphae,” are cospeciating with their flagellate host (22).However, the TG1 phylum also comprises several other deep-rooting lineages (>15% 16S rRNA gene sequence divergence). They are present in a variety of environments, including soils, sediments, and intestinal tracts (14). One of these lineages, the “intestinal cluster,” comprises sequences originating exclusively from intestinal habitats, including the termite gut, but is only distantly related to the lineage comprising the “endomicrobia” (14). Here, we report the isolation of a member of the intestinal cluster from the hindgut of a humivorous scarab beetle larva and its physiological and ultrastructural characterization. We propose a new species, “Elusimicrobium minutum” gen. nov. sp. nov., and define the phylogenetic framework for the first cultivated representative of the TG1 phylum.     

Two Kdo-Heptose Regions Identified in Hafnia alvei 32 Lipopolysaccharide: the Complete Core Structure and Serological Screening of Different Hafnia O Serotypes

Hafnia alvei, a gram-negative bacterium, is an opportunistic pathogen associated with mixed hospital infections, bacteremia, septicemia, and respiratory diseases. Various 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo)-containing fragments different from known structures of core oligosaccharides were previously found among fractions obtained by mild acid hydrolysis of some H. alvei lipopolysaccharides (LPSs). However, the positions of these segments in the LPS structure were not known. Analysis of de-N,O-acylated LPS by nuclear magnetic resonance spectroscopy and mass spectrometry allowed the determination of the location of a Kdo-containing trisaccharide in the structure of H. alvei PCM 32 LPS. It was established that the trisaccharide {l-α-d-Hepp-(1→4)-[α-d-Galp6OAc-(1→7)]-α-Kdop-(2→} is an integral part of the outer-core oligosaccharide of H. alvei 32 LPS. The very labile ketosidic linkage between →4,7)-α-Kdop and →2)-Glcp in the core oligosaccharide was identified. Screening for this Kdo-containing trisaccharide was performed on the group of 37 O serotypes of H. alvei LPSs using monospecific antibodies recognizing the structure. It was established that this trisaccharide is a characteristic component of the outer-core oligosaccharides of H. alvei 2, 32, 600, 1192, 1206, and 1211 LPSs. The weaker cross-reactions with LPSs of strains 974, 1188, 1198, 1204, and 1214 suggest the presence of similar structures in these LPSs, as well. Thus, we have identified new examples of endotoxins among those elucidated so far. This type of core oligosaccharide deviates from the classical scheme by the presence of the structural Kdo-containing motif in the outer-core region.Lipopolysaccharide (LPS) (endotoxin) is the main surface antigen and an important virulence factor of most of the gram-negative bacteria that are pathogenic for humans and animals (46). LPS contributes greatly to the structural integrity of bacteria and constitutes a “pathogen-associated molecular pattern” for host infection (46). As one of the most potent natural activators of the innate immune system, LPS is recognized by different classes of receptors present on macrophages, monocytes, B and T cells, neutrophils, endothelial cells, and epithelial cells (46). Endotoxins stimulate these cells to produce multiple inflammatory mediators responsible for immunotoxicity (e.g., tumor necrosis factor alpha, interleukin 1 [IL-1], IL-6, IL-8, gamma/alpha interferon, NO, platelet-activating factor, and endorphins). Interaction of LPS with the CD14/Toll-like receptor 4/MD-2 receptor complex constitutes a major mechanism responsible for the innate immune response to infection by gram-negative bacteria (1, 46). A large amount of LPS released into the bloodstream triggers the excessive inflammatory response of the innate immune system, leading to sepsis and septic shock (6). High levels of inflammatory mediators have profound effects on the cardiovascular system, kidneys, lungs, liver, central nervous system, and coagulation system. Following their action, renal failure, myocardial dysfunction, acute respiratory distress syndrome, hepatic failure, and disseminated intravascular coagulation can occur, which may result in death (6). Despite intense research on the etiology and treatment of sepsis, its severe form still carries a high mortality rate (6, 46).Hafnia alvei has been reported to be an opportunistic human pathogen. This gram-negative bacterium and its LPS are among the identified causative agents of bacteremia and septicemia in humans and animals (19). For the years 2001 to 2003, up to 42 cases of H. alvei bacteremia were reported annually in the United Kingdom. Most of them were monomicrobic infections, and in ∼33% of the cases, H. alvei was isolated, not only from the blood, but also from hepatic abscesses, pancreatic pseudocyst fluid, sputum, feces, and central venous catheters (19). Besides bacteremia and sepsis, which seem to be the most common syndromes reported, H. alvei is also associated with respiratory diseases and mixed hospital infections in humans. Since the gastrointestinal and respiratory tracts represent very common habitats for hafniae, most cases of H. alvei bacteremia originate there. H. alvei sepsis is also a serious clinical problem in the animal production industry, as infections of H. alvei can be severe, causing septicemia in commercial laying hens, pullets, and rainbow trout (19).Our knowledge of the pathogenicity of H. alvei is limited. LPS is the major virulence factor in cases of H. alvei septicemia and bacteremia (19). Studies of other virulence factors of H. alvei have reported only on the iron-scavenging mechanism, mannose-sensitive/mannose-resistant hemagglutinins, and plasmids encoding bacteriocins and alveicins (19).Most of the elucidated structures of H. alvei LPS are smooth-type molecules built up of O-specific polysaccharide, core oligosaccharide (OS), and lipid A. The O antigens of H. alvei are subdivided into 40 O serotypes (2, 28, 42). The structures of the O-specific polysaccharides from 30 serologically different H. alvei strains have been elucidated (15, 24, 26, 28, 42).So far, four types of core OS have been identified for H. alvei LPSs (9, 17, 25, 27, 30, 43). The most common core OS, isolated by mild acidic hydrolysis from LPSs of smooth H. alvei strains, is a hexasaccharide composed of two d-Glc, three l,d-Hep, and one 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) residues. Two l,d-Hep residues are substituted by phosphoethanolamine (PEtn) and phosphoryl (P) groups (9, 17, 25). In LPSs isolated from H. alvei PCM 1185 and 1204, core OSs are terminated with d-Galp instead of d-Glcp (27). LPSs of H. alvei containing nontypical core OSs, identical with those found in LPSs of Escherichia coli R4 (strains 23 and 1222) and Salmonella enterica Ra (strain 39), were also identified (43).The chemical structures of H. alvei O-specific chains and core OSs were elucidated using fractions obtained by mild acid hydrolysis of LPS. The procedure was optimized for the delipidation of LPS, exploiting the susceptibility of a ketosidic linkage between the inner core and lipid A to acid. However, other acid-labile linkages within the LPS could also be affected, leading to partial degradation of the isolated molecules.The presence of Kdo-containing OSs among fractions obtained by mild acid hydrolysis of LPSs, other than previously identified core OSs, makes the structural analysis of entire H. alvei LPSs difficult. Two types of trisaccharides were previously identified: (i) l-α-d-Hepp-(1→4)-[α-d-Galp-(1→7)]-α-Kdop (l-α-d-Hep is α-l-glycero-d-manno-heptose) for strains 2, 1211, 32, and 1192 (16, 23) and (ii) α-d-Galp-(1→2)-l-α-d-Hepp-(1→4)-α-Kdop for strains 1188 and 1196 (22). These Kdo-containing motifs were never located in any of the LPS segments. Thus, it is of interest to complete the structure of H. alvei LPSs with the locations of such acid-labile motifs in the structures of LPSs isolated from these bacteria.We now report on structural studies of de-N,O-acylated LPS of H. alvei 32 containing a carbohydrate backbone of lipid A, core OS, an additional trisaccharide in the outer region of the core OS, and all of the acid-labile substituents. Additionally, data obtained previously for a trisaccharide isolated from H. alvei 32 LPS (16) was complemented with detailed ¹H and ¹³C nuclear magnetic resonance (NMR) analyses and the assignment of all proton and carbon signals. Screening for the presence of these acid-labile trisaccharides, identical with those found in the strain 32 LPS, was performed on 37 different O serotypes of H. alvei LPSs with antibodies against the conjugate of the de-N,O-acylated H. alvei 32 endotoxin fragment with bovine serum albumin (BSA), specific for the isolated trisaccharide.(Part of this work was presented at the 21st International Carbohydrate Symposium, Cairns, Australia, 7 to 12 July 2002, and the 3rd German-Polish-Russian Meeting on Bacterial Carbohydrates, Wroclaw, Poland, 6 to 9 October 2004.)     

Identification and Characterization of gshA,a Gene Encoding the Glutamate-Cysteine Ligase in the Halophilic Archaeon Haloferax volcanii

Halophilic archaea were found to contain in their cytoplasm millimolar concentrations of γ-glutamylcysteine (γGC) instead of glutathione. Previous analysis of the genome sequence of the archaeon Halobacterium sp. strain NRC-1 has indicated the presence of a sequence homologous to sequences known to encode the glutamate-cysteine ligase GshA. We report here the identification of the gshA gene in the extremely halophilic archaeon Haloferax volcanii and show that H. volcanii gshA directs in vivo the synthesis and accumulation of γGC. We also show that the H. volcanii gene when expressed in an Escherichia coli strain lacking functional GshA is able to restore synthesis of glutathione.Many organisms contain millimolar concentrations of low-molecular-weight thiol compounds that participate in a number of important biological functions involving thiol-disulfide exchanges (7). In particular, they serve to maintain an intracellular reducing environment, to provide reducing power for key reductive enzymes, to combat the effects of oxidative and disulfide stress, and to detoxify xenobiotic compounds (7). Glutathione (GSH), a cysteine-containing tripeptide, l-γ-glutamyl-l-cysteinylglycine, is the best-characterized low-molecular-weight thiol (7, 19, 21). GSH is made in a highly conserved two-step ATP-dependent process by two unrelated peptide bond-forming enzymes (3, 21). The γ-carboxyl group of l-glutamate and the amino group of l-cysteine are ligated by the enzyme glutamylcysteine (GC) ligase EC 6.3.2.2 (GshA, encoded by gshA), which is then condensed with glycine in a reaction catalyzed by GSH synthetase (GshB, encoded by gshB) to form GSH (10, 38). GSH is found primarily in gram-negative bacteria and eukaryotes and only rarely in gram-positive bacteria (26). Fahey and coworkers showed that GSH is absent from the high-GC gram-positive actinomycetes which produce, as the major low-molecular-weight thiol, mycothiol, 1-d-myo-inosityl-2-(N-acetyl-l-cysteinyl)-amido-2-deoxy-α-d-glucopyranoside (13, 26-28, 35). GSH is also absent in Archaea. In Pyrococcus furiosus, coenzyme A SH (CoASH) is the main thiol (11), whereas in Halobacterium salinarum, γGC is the predominant thiol and the organism possesses bis-γGC reductase activity (30, 36). Similarly, Leuconostoc kimchi and Leuconostoc mesenteroides, gram-positive lactic acid bacterial species, were recently found to contain γGC rather than GSH (15). To date, these are the sole procaryotic species reported to naturally produce γGC but not GSH (6, 30). In this report, we describe the identification of the gshA gene in the extremely halophilic archaeon Haloferax volcanii. Copley and Dhillon (6) previously identified, using bioinformatic tools, an open reading frame (ORF) (gene VNG1397C) in Halobacterium sp. strain NRC-1 with limited sequence relatedness to known GshA proteins (6). However, no genetic or biochemical evidence was presented to substantiate their conclusion. Here, we show that Haloferax volcanii strain DS2 (1, 25) contains an ORF that directs in vivo the synthesis and accumulation of γGC. We also show that the H. volcanii ORF, when expressed in Escherichia coli lacking functional GshA, is able to restore synthesis of GSH.     

Substrate Specificity of a Mannose-6-Phosphate Isomerase from Bacillus subtilis and Its Application in the Production of l-Ribose

The uncharacterized gene previously proposed as a mannose-6-phosphate isomerase from Bacillus subtilis was cloned and expressed in Escherichia coli. The maximal activity of the recombinant enzyme was observed at pH 7.5 and 40°C in the presence of 0.5 mM Co²⁺. The isomerization activity was specific for aldose substrates possessing hydroxyl groups oriented in the same direction at the C-2 and C-3 positions, such as the d and l forms of ribose, lyxose, talose, mannose, and allose. The enzyme exhibited the highest activity for l-ribulose among all pentoses and hexoses. Thus, l-ribose, as a potential starting material for many l-nucleoside-based pharmaceutical compounds, was produced at 213 g/liter from 300-g/liter l-ribulose by mannose-6-phosphate isomerase at 40°C for 3 h, with a conversion yield of 71% and a volumetric productivity of 71 g liter⁻¹ h⁻¹.l-Ribose is a potential starting material for the synthesis of many l-nucleoside-based pharmaceutical compounds, and it is not abundant in nature (5, 19). l-Ribose has been produced mainly by chemical synthesis from l-arabinose, l-xylose, d-glucose, d-galactose, d-ribose, or d-mannono-1,4-lactone (2, 17, 23). Biological l-ribose manufacture has been investigated using ribitol or l-ribulose. Recently, l-ribose was produced from ribitol by a recombinant Escherichia coli containing an NAD-dependent mannitol-1-dehydrogenase (MDH) with a 55% conversion yield when 100 g/liter ribitol was used in a 72-h fermentation (18). However, the volumetric productivity of l-ribose in the fermentation is 28-fold lower than that of the chemical method synthesized from l-arabinose (8). l-Ribulose has been biochemically converted from l-ribose using an l-ribose isomerase from an Acinetobacter sp. (9), an l-arabinose isomerase mutant from Escherichia coli (4), a d-xylose isomerase mutant from Actinoplanes missouriensis (14), and a d-lyxose isomerase from Cohnella laeviribosi (3), indicating that l-ribose can be produced from l-ribulose by these enzymes. However, the enzymatic production of l-ribulose is slow, and the enzymatic production of l-ribose from l-ribulose has been not reported.Sugar phosphate isomerases, such as ribose-5-phosphate isomerase, glucose-6-phosphate isomerase, and galactose-6-phosphate isomerase, work as general aldose-ketose isomerases and are useful tools for producing rare sugars, because they convert the substrate sugar phosphates and the substrate sugars without phosphate to have a similar configuration (11, 12, 21, 22). l-Ribose isomerase from an Acinetobacter sp. (9) and d-lyxose isomerase from C. laeviribosi (3) had activity with l-ribose, d-lyxose, and d-mannose. Thus, we can apply mannose-6-phosphate (EC 5.3.1.8) isomerase to the production of l-ribose, because there are no sugar phosphate isomerases relating to l-ribose and d-lyxose. The production of the expensive sugar l-ribose (bulk price, $1,000/kg) from the rare sugar l-ribulose by mannose-6-phosphate isomerase may prove to be a valuable industrial process, because we have produced l-ribulose from the cheap sugar l-arabinose (bulk price, $50/kg) using the l-arabinose isomerase from Geobacillus thermodenitrificans (20) (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Schematic representation for the production of l-ribulose from l-arabinose by G. thermodenitrificans l-arabinose isomerase and the production of l-ribose from l-ribulose by B. subtilis mannose-6-phosphate isomerase.In this study, the gene encoding mannose-6-phosphate isomerase from Bacillus subtilis was cloned and expressed in E. coli. The substrate specificity of the recombinant enzyme for various aldoses and ketoses was investigated, and l-ribulose exhibited the highest activity among all pentoses and hexoses. Therefore, mannose-6-phosphate isomerase was applied to the production of l-ribose from l-ribulose.     

Efficient Production of Optically Pure d-Lactic Acid from Raw Corn Starch by Using a Genetically Modified l-Lactate Dehydrogenase Gene-Deficient and α-Amylase-Secreting Lactobacillus plantarum Strain

In order to achieve direct and efficient fermentation of optically pure d-lactic acid from raw corn starch, we constructed l-lactate dehydrogenase gene (ldhL1)-deficient Lactobacillus plantarum and introduced a plasmid encoding Streptococcus bovis 148 α-amylase (AmyA). The resulting strain produced only d-lactic acid from glucose and successfully expressed amyA. With the aid of secreting AmyA, direct d-lactic acid fermentation from raw corn starch was accomplished. After 48 h of fermentation, 73.2 g/liter of lactic acid was produced with a high yield (0.85 g per g of consumed sugar) and an optical purity of 99.6%. Moreover, a strain replacing the ldhL1 gene with an amyA-secreting expression cassette was constructed. Using this strain, direct d-lactic acid fermentation from raw corn starch was accomplished in the absence of selective pressure by antibiotics. This is the first report of direct d-lactic acid fermentation from raw starch.Poly-lactic acid (PLA) is an important agro-based plastic that can be produced from inexpensive, renewable, and abundantly available biomass resources, including starchy materials. These resources have advantages over limited oil- and fossil-based sources, as they do not result in any net carbon dioxide release to the atmosphere (7). Recently, stereocomplex PLA, which is composed of both poly-l- and -d-lactic acid, has been attracting much attention due to its high thermostability. Stereocomplex-type polymers show a melting point (ca. 230°C) that is approximately 50°C higher than that of the respective single polymers (8). Therefore, d-lactic acid, in addition to l-lactic acid, which has been the focus of production to date, is of significant importance.Lactic acid bacteria (LAB) are promising microorganisms for the efficient production of lactic acid from various sugars, such as glucose, sucrose, and lactose. However, when starchy materials are used as a carbon source, they must be saccharified by physicochemical and enzymatic treatment because most LAB cannot utilize starchy materials directly (13). This makes the whole process less economically viable. Therefore, many researchers have examined the direct production of lactic acid from starchy materials by using wild amylolytic LAB (ALAB) (6, 24, 25) or genetically modified amylase-producing LAB (15, 16). Although d-lactic acid has been produced by fermentation from pretreated substrates such as rice starch (5) and by simultaneous saccharification and fermentation from cellulose (23), there have been no reports on the direct production of d-lactic acid from starchy materials. This is due to a lack of d-lactic acid-producing ALAB and difficulties in gene manipulation of d-lactic acid-producing LAB, such as Lactobacillus delbrueckii (22).We focused on Lactobacillus plantarum, which is an industrially important strain due to its environmental flexibility and its ability to assimilate a wide range of carbohydrates (9). In recent years, several gene manipulation methods for Lactobacillus plantarum have been established (18, 19). Moreover, the complete genome sequence has been decoded for L. plantarum NCIMB 8826 (9). Based on whole-genome analysis, L. plantarum possesses two types of lactate dehydrogenase (LDH), l-LDH and d-LDH, which convert pyruvate into l- and d-lactic acid, respectively. Ferain et al. (4) reported that chromosomal deletion in the ldhL1 gene of L. plantarum NCIMB 8826 provoked an absence of l-LDH activity and produced d-lactic acid from glucose.In the present study, to produce d-lactic acid directly from starch, we constructed an l-LDH-deficient, α-amylase-secreting L. plantarum strain. The engineered strain expressed α-amylase from Streptococcus bovis 148 (AmyA) (20) and efficiently degraded raw starch with the aid of a C-terminal starch-binding domain (11). Using this strain, we achieved the direct and efficient fermentation of optically pure d-lactic acid from raw corn starch.     

Establishment of Oxidative d-Xylose Metabolism in Pseudomonas putida S12

The oxidative d-xylose catabolic pathway of Caulobacter crescentus, encoded by the xylXABCD operon, was expressed in the gram-negative bacterium Pseudomonas putida S12. This engineered transformant strain was able to grow on d-xylose as a sole carbon source with a biomass yield of 53% (based on g [dry weight] g d-xylose⁻¹) and a maximum growth rate of 0.21 h⁻¹. Remarkably, most of the genes of the xylXABCD operon appeared to be dispensable for growth on d-xylose. Only the xylD gene, encoding d-xylonate dehydratase, proved to be essential for establishing an oxidative d-xylose catabolic pathway in P. putida S12. The growth performance on d-xylose was, however, greatly improved by coexpression of xylXA, encoding 2-keto-3-deoxy-d-xylonate dehydratase and α-ketoglutaric semialdehyde dehydrogenase, respectively. The endogenous periplasmic glucose dehydrogenase (Gcd) of P. putida S12 was found to play a key role in efficient oxidative d-xylose utilization. Gcd activity not only contributes to d-xylose oxidation but also prevents the intracellular accumulation of toxic catabolic intermediates which delays or even eliminates growth on d-xylose.The requirement for renewable alternatives to replace oil-based chemicals and fuels necessitates development of novel technologies. Lignocellulose provides a promising alternative feedstock. However, since the pentose sugar fraction may account for up to 25% of lignocellulosic biomass (12), it is essential that this fraction is utilized efficiently to obtain cost-effective biochemical production. In a previous study, the solvent-tolerant bacterium Pseudomonas putida S12, known for its use as a platform host for the production of aromatic compounds (15, 16, 19, 22), was engineered to use d-xylose as a sole carbon source. This was achieved by introducing genes encoding the phosphorylative d-xylose metabolic pathway of Escherichia coli, followed by laboratory evolution (14). Prior to evolutionary improvement, extensive oxidation of d-xylose to d-xylonate occurred, resulting in a very low biomass-for-substrate yield as d-xylonate is a metabolic dead-end product in P. putida. The evolution approach resulted in elimination of the activity of periplasmic glucose dehydrogenase (Gcd), the enzyme responsible for d-xylose oxidation, which turned out to be a critical step in optimizing phosphorylative d-xylose utilization in P. putida S12.Instead of prevention of endogenous oxidation of d-xylose, this oxidation may be used to our advantage when it is combined with an oxidative d-xylose metabolic pathway, such as the pathways described for several Pseudomonas species, Caulobacter crescentus, and Haloarcula marismortui (7, 11, 18, 20). In these pathways, d-xylonate is dehydrated to 2-keto-3-deoxy-d-xylonate. This intermediate either can be cleaved into pyruvate and glycolaldehyde (7) or is further dehydrated to α-ketoglutaric semialdehyde (α-KGSA). In the final step of the latter pathway, α-KGSA is oxidized to the tricarboxylic acid (TCA) cycle intermediate α-ketoglutarate (18, 20).In addition to Gcd (PP1444), some of the enzymes required for oxidative d-xylose metabolism are expected to be endogenous in P. putida S12. Transport of d-xylonate into the cytoplasm likely occurs through the gluconate transporter (encoded by gntP [PP3417]). The enzyme catalyzing the final step of the pathway, α-KGSA dehydrogenase, is also likely to be present (presumably PP1256 and/or PP3602) because of the requirement for metabolism of 4-hydroxyproline (1), a compound that is efficiently utilized by P. putida S12. In view of these properties, the most obvious approach for constructing d-xylose-utilizing P. putida S12 is reconstruction of a complete oxidative d-xylose metabolic pathway by introducing the parts of such a pathway that complement the endogenous activities. Recently, the genetic information for one such oxidative d-xylose pathway has become available (18), enabling the approach used in the present study, i.e., expression of the oxidative d-xylose metabolic pathway of C. crescentus in P. putida S12 and investigation of the contribution of endogenous enzyme activities.     

Profiling Sex-Specific piRNAs in Zebrafish

Piwi proteins and their partner small RNAs play an essential role in fertility, germ-line stem cell development, and the basic control and evolution of animal genomes. However, little knowledge exists regarding piRNA biogenesis. Utilizing microfluidic chip analysis, we present a quantitative profile of zebrafish piRNAs expressed differentially between testis and ovary. The sex-specific piRNAs are derived from separate loci of repeat elements in the genome. Ovarian piRNAs can be categorized into groups that reach up to 92 members, indicating a sex-specific arrangement of piRNA genes in the genome. Furthermore, precursor piRNAs preferentially form a hairpin structure at the 3′end, which seem to favor the generation of mature sex-specific piRNAs. In addition, the mature piRNAs from both the testis and the ovary are 2′-O-methylated at their 3′ ends.SMALL RNAs, ranging from 19 to 30 nucleotides (nt) in length, constitute a large family of regulatory molecules with diverse functions in invertebrates, vertebrates, plants, and fungi (Bartel 2004; Nakayashiki 2005). Two major classes of small RNAs are microRNAs (miRNAs) and small interfering RNAs (siRNAs). The functions of small RNAs have been conserved through evolution; they have been shown to inhibit gene expression at the levels of mRNA degradation, translational repression, chromatin modification, heterochromatin formation, and DNA elimination (Mochizuki et al. 2002; Bartel 2004; Kim et al. 2005; Brodersen and Voinnet 2006; Lee and Collins 2006; Vaucheret 2006).Over the past few years, focus on the genetics of small RNAs has helped clarify the mechanisms behind the regulation of these molecules. While hundreds of small RNAs have been identified from mammalian somatic tissues, relatively little is known about small RNAs in germ cells. A recent breakthrough has been the identification of small RNAs that associate with Piwi proteins (piRNAs) from Drosophila and mammalian gonads (Aravin et al. 2001, 2006; Girard et al. 2006; Grivna et al. 2006; Vagin et al. 2006; Watanabe et al. 2006). piRNAs and their interacting proteins Ziwi/Zili have also been identified in zebrafish (Houwing et al. 2007, 2008). Increasing evidence indicates that piRNAs play roles mainly in germ cell differentiation and genomic stability (Carthew 2006; Lau et al. 2006; Vagin et al. 2006; Brennecke et al. 2007; Chambeyron et al. 2008; Klattenhoff and Theurkauf 2008; Kuramochi-Miyagawa et al. 2008; Kim et al. 2009; Lim et al. 2009; Unhavaithaya et al. 2009). Moreover, although piRNAs are mostly expressed in germ line cells, recent studies showed piRNA expression in nongerm cells, for example, T-cell lines (Jurkat cells and MT4) (Azuma-Mukai et al. 2008; Yeung et al. 2009), indicating other functions such as in the immune system. piRNAs do not appear to be derived from double-stranded RNA precursors, and their biogenesis mechanisms, although unclear, may be distinct from those of siRNA and miRNA. Recently, two distinct piRNA production pathways were further proposed: the “ping-pong” model (Brennecke et al. 2007; Gunawardane et al. 2007) and the Ago3-independent piRNA pathway centered on Piwi in somatic cells (Li et al. 2009; Malone et al. 2009). However, the mechanistic pathways of piRNA activity and their biogenesis are still largely unknown.Teleost fishes comprise >24,000 species, accounting for more than half of extant vertebrate species, displaying remarkable variation in morphological and physiological adaptations (see review in Zhou et al. 2001). Recently, Houwing et al. (2007, 2008) reported findings on Ziwi/Zili and associated piRNAs, implicating roles in germ cell differentiation, meiosis, and transposon silencing in the germline of the zebrafish. However, some of the identified zebrafish piRNAs are nonrepetitive and nontransposon-related piRNAs, suggesting that piRNAs may have additional unknown roles. In this study, we show that for males and females, piRNAs are specifically derived from separate loci of the repeat elements, and that ovarian piRNAs are far more often associated in groups. Genomic analysis of piRNAs indicates a tendency to folding at the 3′ end of the piRNA precursor, which may favor cleavage of the piRNA precursor to generate mature sex-specific piRNAs. Furthermore, methylation modification occurs at the 2′-O-hydroxyl group on the ribose of the final 3′ nucleotide in both the testis and the ovary.     

A Role for the Class A Penicillin-Binding Protein PonA2 in the Survival of Mycobacterium smegmatis under Conditions of Nonreplication

Class A penicillin-binding proteins (PBPs) are large, bifunctional proteins that are responsible for glycan chain assembly and peptide cross-linking of bacterial peptidoglycan. Bacteria in the genus Mycobacterium have been reported to have only two class A PBPs, PonA1 and PonA2, that are encoded in their genomes. We report here that the genomes of Mycobacterium smegmatis and other soil mycobacteria contain an additional gene encoding a third class A penicillin-binding protein, PonA3, which is a paralog of PonA2. Both the PonA2 and PonA3 proteins contain a penicillin-binding protein and serine/threonine protein kinase-associated (PASTA) domain that we propose may be involved in sensing the cell cycle and a C-terminal proline-rich region (PRR) that may have a role in protein-protein or protein-carbohydrate interactions. We show here that an M. smegmatis ΔponA2 mutant has an unusual antibiotic susceptibility profile, exhibits a spherical morphology and an altered cell surface in stationary phase, and is defective for stationary-phase survival and recovery from anaerobic culture. In contrast, a ΔponA3 mutant has no discernible phenotype under laboratory conditions. We demonstrate that PonA2 and PonA3 can bind penicillin and that PonA3 can partially substitute for PonA2 when ponA3 is expressed from a constitutive promoter on a multicopy plasmid. Our studies suggest that PonA2 is involved in adaptation to periods of nonreplication in response to starvation or anaerobiosis and that PonA3 may have a similar role. However, the regulation of PonA3 is likely different, suggesting that its importance could be related to stresses encountered in the environmental niches occupied by M. smegmatis and other soil-dwelling mycobacteria.The cell envelope of mycobacteria is a complex carbohydrate- and lipid-rich entity and is a major factor contributing to the success of these organisms as saprophytic and pathogenic bacteria (7, 8, 29, 35). The innermost layer of the cell envelope is a peptidoglycan (PG) composed of N-acylmuramic acid and N-acetylglucosamine with l-alanyl (or glycyl in the case of Mycobacterium leprae)-d-isoglutaminyl-meso-diaminopimelyl-d-alanyl-d-alanine pentapeptides attached to the muramic acid residues (13, 16, 54). While some of the muramyl residues are N acetylated, as they are in most other bacteria, a majority of the muramyl residues are N glycolylated (2, 37, 48, 49), a modification that confers lysozyme resistance (53) and also influences the innate immune response to mycobacterial cell walls (10). The pentapeptide chains of the mycobacterial PG can be modified by amidation, glycylation, or methylation, but the functional significance of these modifications is unknown (28, 31, 32, 38, 54).Approximately 80% of the pentapeptides in mycobacterial PG are cross-linked, and a majority of the links are between the carboxyl group of a penultimate d-Ala residue in a pentapeptide precursor and the amino group of the side chain d center of a meso-diaminopimelic acid (DAP) residue from an adjacent peptide (referred to as a 4-3 cross-link), while approximately one-third of the links are between the carboxyl group of the l center of a DAP residue of one peptide and the amino group of the side chain d center of the DAP residue in an adjacent peptide (referred to as a 3-3 cross-link) (17, 65). The 4-3 linkage is considered the “standard” linkage and is catalyzed by classical, penicillin-sensitive dd-transpeptidases, while the novel 3-3 linkage is thought to be catalyzed by the concerted action of dd-carboxypeptidases and novel ld-transpeptidases (31, 34, 39-41). The reasons why bacteria produce both 4-3 and 3-3 linkages are unknown. Some workers have suggested that the 3-3 linkages might reinforce the wall during times of stress and under nonreplicating conditions or stabilize complex cell envelopes (17, 50, 51, 55). In this regard, the high percentage of 3-3 linkages found in the PG of mycobacteria and their predominance in stationary-phase M. tuberculosis cells (31) suggest that these linkages may have an important role in maintaining cell envelope integrity during periods of growth and under nonreplicating conditions.The enzymes involved in peptidoglycan assembly, the penicillin-binding proteins (PBPs), have a triad sequence motif that forms the transpeptidation active site ([SxxK]——[S/YxN/C]——[K/H][T/S]G), which is the target of the β-lactam class of antibiotics (for a review, see reference 18). The PBPs have been grouped into several classes based on this motif, surrounding sequences, and other structural features (18). Of interest here are the class A PBPs, which are high-molecular-weight (HMW) proteins with both a transglycosylase domain (also called a non-penicillin-binding module [n-PB]) and a transpeptidase domain (also called a penicillin-binding module [PB]) (18). These proteins are tethered to the cytoplasmic membrane by a transmembrane helix with the catalytic domains facing the outside of the cell. Mycobacteria have been reported to have only two genes that encode class A PBPs, ponA1 and ponA2, which are annotated Rv0051 and Rv3682 in the sequence genome of M. tuberculosis H37Rv (9, 17). Previous studies that analyzed collections of transposon mutants to obtain clones with various phenotypes identified strains with insertions in these two genes. The phenotypes of these mutants have clearly shown that these PBPs play a complex role in mycobacterial physiology. One group of workers found a ponA1 mutant of M. smegmatis in a search for mutants with an altered dye-binding phenotype (an indicator of changes in the cell envelope) and showed that this slowly growing mutant was hypersusceptible to β-lactam antibiotics and had altered permeability (6). A ponA2 mutant of M. smegmatis was discovered in a screen for mutants defective for survival during long-term culture (25), while other workers isolated an M. tuberculosis ponA2 mutant in a screen for mutants sensitive to low pH (61). The same group of workers also showed that the M. tuberculosis mutant was more sensitive to heat, H₂O₂, and NO and was attenuated for persistence in the mouse model of inhalation tuberculosis (62). We previously identified an M. tuberculosis ponA2 mutant in a screen for mutants hypersusceptible to β-lactam antibiotics (14). All of these studies identified transposon mutants in searches for mutants with specific phenotypes, but there have been no direct genetic studies that have specifically examined the function of these PBPs in peptidoglycan metabolism.In this study we demonstrated that M. smegmatis has three class A PBPs. We show here that a newly recognized protein, which we designated PonA3, is a paralog of the PonA2 protein and is found only in certain environmental species of mycobacteria. We analyzed the phenotypes of M. smegmatis mutants with in-frame deletions of ponA2 and ponA3 singly and in combination to increase our understanding of the role that these PBPs play in mycobacterial peptidoglycan biology.     

Efficient Bioethanol Production by a Recombinant Flocculent Saccharomyces cerevisiae Strain with a Genome-Integrated NADP+-Dependent Xylitol Dehydrogenase Gene

The recombinant industrial Saccharomyces cerevisiae strain MA-R5 was engineered to express NADP⁺-dependent xylitol dehydrogenase using the flocculent yeast strain IR-2, which has high xylulose-fermenting ability, and both xylose consumption and ethanol production remarkably increased. Furthermore, the MA-R5 strain produced the highest ethanol yield (0.48 g/g) from nonsulfuric acid hydrolysate of wood chips.Successful fermentation of lignocellulosic biomass to ethanol is dependent on efficient utilization of d-xylose, which is the second most common fermentable sugar in the hydrolysate. Although the well-known fermentative yeast Saccharomyces cerevisiae is one of the most effective ethanol-producing organisms for hexose sugars, it is not able to ferment d-xylose. However, S. cerevisiae can metabolize an isomerization product of d-xylose, d-xylulose, which is phosphorylated to d-xylulose 5-phosphate, channeled through the pentose phosphate pathway and glycolysis.S. cerevisiae transformed with the XYL1 and XYL2 genes encoding xylose reductase (XR) and xylitol dehydrogenase (XDH) from Pichia stipitis (referred to as PsXR and PsXDH, respectively) acquires the ability to ferment d-xylose to ethanol (2, 5, 6, 9, 10, 12, 22). Furthermore, overexpression of the XKS1 gene encoding xylulokinase (XK) from S. cerevisiae (ScXK) has been shown to aid d-xylose utilization (4, 7, 11, 16, 23), with xylitol still a major by-product. Kuyper et al. (14) also demonstrated the successful fermentation of d-xylose to ethanol using recombinant S. cerevisiae strains expressing fungal xylose isomerase. However, these approaches are insufficient for industrial bioprocesses, mainly due to the low rate of d-xylose fermentation.Xylitol excretion has been ascribed mainly to the difference in coenzyme specificities between PsXR (with NADPH) and PsXDH (with NAD⁺), which creates an intracellular redox imbalance (1). Therefore, modifying the coenzyme specificity of XR and/or XDH by protein engineering is an attractive approach for achieving efficient fermentation of ethanol from d-xylose using recombinant S. cerevisiae. Watanabe et al. (24) previously succeeded in generating several PsXDH mutants (e.g., quadruple ARSdR mutant) with a complete reversal of coenzyme specificity toward NADP⁺ by multiple site-directed mutagenesis on amino acids from the coenzyme-binding domain. The ARSdR mutant (D207A/I208R/F209S/N211R) has more that 4,500-fold-higher catalytic efficiency (k_cat/K_m) with NADP⁺ than the wild-type PsXDH. In addition, we recently found that several laboratory recombinant S. cerevisiae strains, in which the ARSdR mutant, along with PsXR and ScXK, is expressed through a strong constitutive promoter, increased ethanol production from d-xylose at the expense of xylitol excretion (17, 18), probably by maintaining the intracellular redox balance. However, commercialization of lignocellulosic hydrolysate fermentation requires industrial strains that are more robust than laboratory strains (5, 19, 21).A potential host for developing d-xylose-fermenting strains requires an active and efficient pentose phosphate pathway linking the d-xylose-to-d-xylulose pathway to glycolysis. In the case of S. cerevisiae, strains have different d-xylulose fermentation abilities (3, 25), indicating inherent differences in the capacities of these strains to ferment pentose sugars. Furthermore, anaerobic d-xylulose fermentation was investigated to identify genetic backgrounds potentially beneficial to anaerobic d-xylose fermentation in strains not exhibiting product formation related to the redox imbalance generated by the first two steps of the eukaryotic d-xylose metabolism (3), although the physiological purpose of the different d-xylulose fermentation abilities of S. cerevisiae is not yet clear. Therefore, we selected an efficient industrial d-xylulose-fermenting S. cerevisiae strain as a host for constructing a recombinant strain through chromosomal integration of the NADP⁺-dependent XDH gene and the XR and endogenous XK genes. Using this recombinant strain, we characterized the enzyme activity and ability to ferment both d-xylose and lignocellulosic hydrolysate.     

Molecular Basis of Vancomycin Dependence in VanA-Type Staphylococcus aureus VRSA-9

The vancomycin-resistant Staphylococcus aureus VRSA-9 clinical isolate was partially dependent on glycopeptide for growth. The responsible vanA operon had the same organization as that of Tn1546 and was located on a plasmid. The chromosomal d-Ala:d-Ala ligase (ddl) gene had two point mutations that led to Q260K and A283E substitutions, resulting in a 200-fold decrease in enzymatic activity compared to that of the wild-type strain VRSA-6. To gain insight into the mechanism of enzyme impairment, we determined the crystal structure of VRSA-9 Ddl and showed that the A283E mutation induces new ion pair/hydrogen bond interactions, leading to an asymmetric rearrangement of side chains in the dimer interface. The Q260K substitution is located in an exposed external loop and did not induce any significant conformational change. The VRSA-9 strain was susceptible to oxacillin due to synthesis of pentadepsipeptide precursors ending in d-alanyl-d-lactate which are not substrates for the β-lactam-resistant penicillin binding protein PBP2′. Comparison with the partially vancomycin-dependent VRSA-7, whose Ddl is 5-fold less efficient than that of VRSA-9, indicated that the levels of vancomycin dependence and susceptibility to β-lactams correlate with the degree of Ddl impairment. Ddl drug targeting could therefore be an effective strategy against vancomycin-resistant S. aureus.Methicillin-resistant Staphylococcus aureus (MRSA) bacteria that have acquired the vancomycin resistance vanA operon from glycopeptide-resistant enterococci are designated vancomycin-resistant S. aureus (VRSA) (29). Vancomycin acts by binding to the C-terminal acyl-d-Ala-d-Ala of the undecaprenol-diphosphate MurNAc-pentapeptide intermediate and inhibits transglycosylation and transpeptidation reactions in cell wall peptidoglycan polymerization and cross-linking (30). d-Ala-d-Ala is synthesized by the ATP-dependent d-Ala:d-Ala ligase (Ddl) (EC 6.3.2.4) before its incorporation in peptidoglycan precursors (26, 35). VanA-type vancomycin resistance results from the incorporation into peptidoglycan intermediates of a d-alanyl-d-lactate (d-Ala-d-Lac) depsipeptide, synthesized by a d-Ala:d-Lac ligase, which is responsible for diminished binding affinity of glycopeptides for their target. Kinetic analyses of Ddls have established two subsites in the active site for d-Ala binding (24, 27). The reaction mechanism culminates in the transfer of the γ-phosphoryl of ATP to the carboxyl group of d-Ala₁ to produce an acylphosphate and ADP. The acyl carbon atom of the acylphosphate then reacts with the amino group of d-Ala₂ to yield a tetrahedral intermediate. Finally, the intermediate releases phosphate to yield d-Ala-d-Ala.Mutants of Enterococcus faecium (8, 14), Enterococcus faecalis (34), and S. aureus (23) with an impaired Ddl are able to grow because they use the vancomycin resistance pathway for cell wall synthesis. Since resistance is inducible by the drug, these bacteria require the presence of vancomycin in the culture medium for growth. Ddls from vancomycin-dependent enterococci (14) have mutations affecting amino acids highly conserved in the d-Ala:d-Ala ligase superfamily (10). Molecular modeling based on the X-ray structure of Escherichia coli DdlB (11) revealed that all the mutated residues interact directly with one of the substrates of the enzymatic reaction or stabilize the position of critical residues in the active site. However, the degree of enzyme impairment was not evaluated biochemically. Recently, we reported the mechanism of vancomycin dependence in VanA-type S. aureus VRSA-7 and showed that the chromosomal Ddl had the single mutation N308K, which probably affects the binding of the transition-state intermediate, leading to a 1,000-fold decrease in activity relative to that of the wild-type enzyme (23). Glycopeptide-dependent mutants could therefore be considered useful tools to explore structure-activity relationships of the Ddl, which represents an attractive target for designing new drugs. Here we describe the partially vancomycin-dependent VanA-type S. aureus strain VRSA-9 and report the biochemical and structural characterization of its mutated Ddl.     

Escherichia coli Strains Engineered for Homofermentative Production of d-Lactic Acid from Glycerol

Given its availability and low price, glycerol has become an ideal feedstock for the production of fuels and chemicals. We recently reported the pathways mediating the metabolism of glycerol in Escherichia coli under anaerobic and microaerobic conditions. In this work, we engineer E. coli for the efficient conversion of glycerol to d-lactic acid (d-lactate), a negligible product of glycerol metabolism in wild-type strains. A homofermentative route for d-lactate production was engineered by overexpressing pathways involved in the conversion of glycerol to this product and blocking those leading to the synthesis of competing by-products. The former included the overexpression of the enzymes involved in the conversion of glycerol to glycolytic intermediates (GlpK-GlpD and GldA-DHAK pathways) and the synthesis of d-lactate from pyruvate (d-lactate dehydrogenase). On the other hand, the synthesis of succinate, acetate, and ethanol was minimized through two strategies: (i) inactivation of pyruvate-formate lyase (ΔpflB) and fumarate reductase (ΔfrdA) (strain LA01) and (ii) inactivation of fumarate reductase (ΔfrdA), phosphate acetyltransferase (Δpta), and alcohol/acetaldehyde dehydrogenase (ΔadhE) (strain LA02). A mutation that blocked the aerobic d-lactate dehydrogenase (Δdld) also was introduced in both LA01 and LA02 to prevent the utilization of d-lactate. The most efficient strain (LA02Δdld, with GlpK-GlpD overexpressed) produced 32 g/liter of d-lactate from 40 g/liter of glycerol at a yield of 85% of the theoretical maximum and with a chiral purity higher than 99.9%. This strain exhibited maximum volumetric and specific productivities for d-lactate production of 1.5 g/liter/h and 1.25 g/g cell mass/h, respectively. The engineered homolactic route generates 1 to 2 mol of ATP per mol of d-lactate and is redox balanced, thus representing a viable metabolic pathway.Lactic acid (lactate) and its derivatives have many applications in the food, pharmaceutical, and polymer industries (13, 30). An example is polylactic acid, a renewable, biodegradable, and environmentally friendly polymer produced from d- and l-lactate (19). In this context, biological processes have the advantage of being able to produce chirally pure lactate from inexpensive media containing only the carbon source and mineral salts (43). While lactic acid bacteria traditionally have been used in the production of d-lactate from carbohydrate-rich feedstocks, several laboratories recently have reported alternative biocatalysts (13, 30), many of which are engineered Escherichia coli strains that produce d- or l-lactate (4, 8, 50, 51, 52).Unlike the aforementioned reports, which have dealt with the use of carbohydrates, our work focuses on the use of glycerol as a carbon source for the production of d-lactate. Glycerol has become an inexpensive and abundant substrate due to its generation in large amounts as a by-product of biodiesel and bioethanol production (18, 32, 47). The conversion of glycerol to higher-value products has been proposed as a path to economic viability for the biofuels industry (47). One such product is lactate, whose production could be readily integrated into existing biodiesel and bioethanol facilities, thus establishing true biorefineries.Although many microorganisms are able to metabolize glycerol (25), the use of industrial microbes such as E. coli could greatly accelerate the development of platforms to produce fuels and chemicals from this carbon source. We recently reported on the ability of E. coli to metabolize glycerol under either anaerobic or microaerobic conditions and identified the environmental and metabolic determinants of these processes (9, 11, 28). In one of the studies, the pathways involved in the microaerobic utilization of glycerol were elucidated, and they are shown in Fig. ​Fig.11 (9). A common characteristic of glycerol metabolism under either anaerobic or microaerobic conditions is the generation of ethanol as the primary product and the negligible production of lactate (6, 9, 11, 28). In the work reported here, the knowledge base created by the aforementioned studies was used to engineer E. coli for the efficient conversion of glycerol to d-lactate in minimal medium. The engineered strains hold great promise as potential biocatalysts for the conversion of low-value glycerol streams to a higher-value product like d-lactate.Open in a separate windowFIG. 1.Pathways involved in the microaerobic utilization of glycerol in E. coli (9). Genetic modifications supporting the metabolic engineering strategies employed in this work are illustrated by thicker lines (overexpression of gldA-dhaKLM, glpK-glpD, and ldhA) or cross bars (disruption of pflB, pta, adhE, frdA, and dld). Broken lines illustrate multiple steps. Relevant reactions are represented by the names of the gene(s) coding for the enzymes: aceEF-lpdA, pyruvate dehydrogenase complex; adhE, acetaldehyde/alcohol dehydrogenase; ackA, acetate kinase; dhaKLM, dihydroxyacetone kinase; dld, respiratory d-lactate dehydrogenase; fdhF, formate dehydrogenase, part of the formate hydrogenlyase complex; frdABCD, fumarate reductase; gldA, glycerol dehydrogenase; glpD, aerobic glycerol-3-phosphate dehydrogenase; glpK, glycerol kinase; hycB-I, hydrogenase 3, part of the formate hydrogenlyase complex; ldhA, fermentative d-lactate dehydrogenase; pflB, pyruvate formate-lyase; pta, phosphate acetyltransferase; pykF, pyruvate kinase. Abbreviations: DHA, dihydroxyacetone; DHAP, DHA phosphate; G-3-P, glycerol-3-phosphate; PEP, phosphoenolpyruvate; PYR, pyruvate; P/O, amount of ATP produced in the oxidative phosphorylation per pair of electrons transferred through the electron transport system; QH₂, reduced quinones.