Engineering Filamentous Fungi for Conversion of d-Galacturonic Acid to l-Galactonic Acid

d-Galacturonic acid, the main monomer of pectin, is an attractive substrate for bioconversions, since pectin-rich biomass is abundantly available and pectin is easily hydrolyzed. l-Galactonic acid is an intermediate in the eukaryotic pathway for d-galacturonic acid catabolism, but extracellular accumulation of l-galactonic acid has not been reported. By deleting the gene encoding l-galactonic acid dehydratase (lgd1 or gaaB) in two filamentous fungi, strains were obtained that converted d-galacturonic acid to l-galactonic acid. Both Trichoderma reesei Δlgd1 and Aspergillus niger ΔgaaB strains produced l-galactonate at yields of 0.6 to 0.9 g per g of substrate consumed. Although T. reesei Δlgd1 could produce l-galactonate at pH 5.5, a lower pH was necessary for A. niger ΔgaaB. Provision of a cosubstrate improved the production rate and titer in both strains. Intracellular accumulation of l-galactonate (40 to 70 mg g biomass⁻¹) suggested that export may be limiting. Deletion of the l-galactonate dehydratase from A. niger was found to delay induction of d-galacturonate reductase and overexpression of the reductase improved initial production rates. Deletion of the l-galactonate dehydratase from A. niger also delayed or prevented induction of the putative d-galacturonate transporter An14g04280. In addition, A. niger ΔgaaB produced l-galactonate from polygalacturonate as efficiently as from the monomer.     

Amino Acid and vitamin requirements of several bacteroides strains

Nutritional studies were performed on nine Bacteroides strains, by use of the methodology and media of anaerobic rumen microbiology. Ristella perfoetens CCI required l-arginine hydrochloride, l-tryptophan, l-leucine, l-histidine hydrochloride, l-cysteine hydrochloride, dl-valine, dl-tyrosine, and the vitamin calcium-d-pantothenate, since scant turbidity developed in media without these nutrients. R. perfoetens was stimulated by glycine, dl-lysine hydrochloride, dl-isoleucine, l-proline, l-glutamic acid, dl-alanine, dl-phenylalanine, dl-methionine, and the vitamins nicotinamide and p-aminobenzoic acid, since maximal turbidity developed more slowly in media without these nutrients than in complete medium. Medium A-23, which was devised for R. perfoetens, contained salts, 0.0002% nicotinamide and calcium d-pantothenate, 0.00001% p-aminobenzoic acid, 0.044% l-tryptophan, 0.09% l-glutamic acid, and 0.1% of the other 13 amino acids listed above. Zuberella clostridiformis and seven strains of R. pseudoinsolita did not require vitamins, and showed no absolute requirement for any one amino acid. Various strains produced maximal turbidity more slowly in media deficient in l-proline, glycine, l-glutamic acid, dl-serine, l-histidine hydrochloride, dl-alanine, or l-cysteine hydrochloride, than in complete medium. These eight strains grew optimally in medium A-23 plus 0.1% dl-serine but without vitamins.     

Enzyme Characteristics of β-d-Galactosidase- and β-d-Glucuronidase-Positive Bacteria and Their Interference in Rapid Methods for Detection of Waterborne Coliforms and Escherichia coli

Bacteria which were β-d-galactosidase and β-d-glucuronidase positive or expressed only one of these enzymes were isolated from environmental water samples. The enzymatic activity of these bacteria was measured in 25-min assays by using the fluorogenic substrates 4-methylumbelliferyl-β-d-galactoside and 4-methylumbelliferyl-β-d-glucuronide. The enzyme activity, enzyme induction, and enzyme temperature characteristics of target and nontarget bacteria in assays aimed at detecting coliform bacteria and Escherichia coli were investigated. The potential interference of false-positive bacteria was evaluated. Several of the β-d-galactosidase-positive nontarget bacteria but none of the β-d-glucuronidase-positive nontarget bacteria contained unstable enzyme at 44.5°C. The activity of target bacteria was highly inducible. Nontarget bacteria were induced much less or were not induced by the inducers used. The results revealed large variations in the enzyme levels of different β-d-galactosidase- and β-d-glucuronidase-positive bacteria. The induced and noninduced β-d-glucuronidase activities of Bacillus spp. and Aerococcus viridans were approximately the same as the activities of induced E. coli. Except for some isolates identified as Aeromonas spp., all of the induced and noninduced β-d-galactosidase-positive, noncoliform isolates exhibited at least 2 log units less mean β-d-galactosidase activity than induced E. coli. The noncoliform bacteria must be present in correspondingly higher concentrations than those of target bacteria to interfere in the rapid assay for detection of coliform bacteria.Indicators of pollution (e.g., coliforms, fecal coliforms, and Escherichia coli) are traditionally used for monitoring the microbiological safety of water supplies and recreational water. Several techniques for detection of coliforms and E. coli are based on enzymatic hydrolysis of fluorogenic or chromogenic substrates for β-d-galactosidase and β-d-glucuronidase (9, 20). Current methods of recovery are usually culture based, and the analysis time is 18 to 24 h. In addition to enzymatic activity, these techniques use growth at appropriate temperatures in the presence of inhibitors, combined with demonstration of enzymatic activity, to selectively detect target bacteria.Rapid methods which require less than 6 h and are based on chromogenic, fluorogenic, or chemiluminogenic substrates for detection of coliforms, fecal coliforms, or E. coli have been described (1–3, 10, 27, 28). These rapid assays are based on the assumption that β-d-galactosidase and β-d-glucuronidase are markers for coliforms and E. coli, respectively. However, when the incubation time is 1 h or less, growth is not a selective step, and all β-d-galactosidase-positive or β-d-glucuronidase-positive microorganisms in a water sample contribute to the activity measured. At low initial concentrations of target bacteria (i.e., E. coli and total coliforms), increasing the preincubation time to 5 to 6 h did not result in a predominance of target bacteria compared to nontarget bacteria (28).The β-d-galactosidase or β-d-glucuronidase activity calculated per cultivable coliform or fecal coliform bacterium in environmental samples can be 1 to 2 log units higher than the activity per induced E. coli cell in pure culture (11, 26). The presence of active, noncultivable bacteria can be one reason for this. Studies of survival (7, 24, 25) and disinfection (26) of E. coli have shown that loss of cultivability does not necessarily result in a loss of β-d-galactosidase activity. The presence of false-positive bacteria can be another reason.β-d-Galactosidase has been found in numerous microorganisms, including gram-negative bacteria (e.g., strains belonging to the Enterobacteriaceae, Vibrionaceae, Pseudomonadaceae, and Neisseriaceae), several gram-positive bacteria, yeasts, protozoa, and fungi (17, 29). β-d-Glucuronidase is produced by most E. coli strains and also by other members of the Enterobacteriaceae, including some Shigella and Salmonella strains and a few Yersinia, Citrobacter, Edwardia, and Hafnia strains. Production of β-d-glucuronidase by Flavobacterium spp., Bacteroides spp., Staphylococcus spp., Streptococcus spp., anaerobic corynebacteria, and Clostridium has also been reported (12).High numbers of false-positive bacteria in sewage and contaminated water have been revealed by enumeration of β-d-galactosidase- and β-d-glucuronidase-positive CFU on nonselective agar supplemented with fluorogenic or chromogenic substrates (11, 28). Whether the activity from nontarget organisms can be neglected in a rapid assay depends on the number of nontarget organisms compared with the number of target bacteria and also on the level of their enzyme activity. Plant and algal biomass must be present at high concentrations to interfere in rapid bacterial β-d-galactosidase and β-d-glucuronidase assays (8).The main objective of this study was to investigate the enzyme characteristics of β-d-galactosidase- and β-d-glucuronidase-positive bacteria isolated from environmental water samples and to evaluate the potential influence of false-positive bacteria in rapid assays for coliform bacteria or E. coli in water. The effect of temperature on enzyme activity and on the interference of nontarget bacteria in the rapid assays was investigated as an important factor.(Some of the results were presented at the 97th General Meeting of the American Society for Microbiology 1997, Miami Beach, Fla., 4 to 8 May 1997.)     

Biofilm Formation and Quorum-Sensing-Molecule Production by Clinical Isolates of Serratia liquefaciens

Serratia spp. are opportunistic human pathogens responsible for an increasing number of nosocomial infections. However, little is known about the virulence factors and regulatory circuits that may enhance the establishment and long-term survival of Serratia liquefaciens in the hospital environment. In this study, two reporter strains, Chromobacterium violaceum CV026 and VIR24, and high-resolution triple-quadrupole liquid chromatography–mass spectrometry (LC-MS) were used to detect and to quantify N-acyl-homoserine lactone (AHL) quorum-sensing signals in 20 S. liquefaciens strains isolated from clinical samples. Only four of the strains produced sufficient amounts of AHLs to activate the sensors. Investigation of two of the positive strains by high-performance liquid chromatography (HPLC)-MS confirmed the presence of significant amounts of short-acyl-chain AHLs (N-butyryl-l-homoserine lactone [C₄-HSL] and N-hexanoyl-l-homoserine lactone [C₆-HSL]) in both strains, which exhibited a complex and strain-specific signal profile that included minor amounts of other short-acyl-chain AHLs (N-octanoyl-l-homoserine lactone [C₈-HSL] and N-3-oxohexanoyl-l-homoserine lactone [OC₆-HSL]) and long-acyl-chain (C₁₀, C₁₂, and C₁₄) AHLs. No correlation between biofilm formation and the production of large amounts of AHLs could be established. Fimbria-like structures were observed by transmission electron microscopy, and the presence of the type 1 fimbrial adhesin gene fimH in all strains was confirmed by PCR. The ability of S. liquefaciens to adhere to abiotic surfaces and to form biofilms likely contributes to its persistence in the hospital environment, increasing the probability of causing nosocomial infections. Therefore, a better understanding of the adherence properties of this species will provide greater insights into the diseases it causes.     

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.     

Polyamines and root formation in mung bean hypocotyl cuttings : I. Effects of exogenous compounds and changes in endogenous polyamine content

The effect of several polyamines (putrescine, spermidine, and spermine), their precursors (l-arginine and l-ornithine), and some analogs and metabolic inhibitors (l-canavanine, l-canaline, and methylglyoxal-bis [guanylhydrazone]) on root formation have been studied in mung bean (Vigna radiata [L.] Wilczek) hypocotyl cuttings.     

Metabolic Engineering of Fungal Strains for Conversion of d-Galacturonate to meso-Galactarate

d-Galacturonic acid can be obtained by hydrolyzing pectin, which is an abundant and low value raw material. By means of metabolic engineering, we constructed fungal strains for the conversion of d-galacturonate to meso-galactarate (mucate). Galactarate has applications in food, cosmetics, and pharmaceuticals and as a platform chemical. In fungi d-galacturonate is catabolized through a reductive pathway with a d-galacturonate reductase as the first enzyme. Deleting the corresponding gene in the fungi Hypocrea jecorina and Aspergillus niger resulted in strains unable to grow on d-galacturonate. The genes of the pathway for d-galacturonate catabolism were upregulated in the presence of d-galacturonate in A. niger, even when the gene for d-galacturonate reductase was deleted, indicating that d-galacturonate itself is an inducer for the pathway. A bacterial gene coding for a d-galacturonate dehydrogenase catalyzing the NAD-dependent oxidation of d-galacturonate to galactarate was introduced to both strains with disrupted d-galacturonate catabolism. Both strains converted d-galacturonate to galactarate. The resulting H. jecorina strain produced galactarate at high yield. The A. niger strain regained the ability to grow on d-galacturonate when the d-galacturonate dehydrogenase was introduced, suggesting that it has a pathway for galactarate catabolism.d-Galacturonate is the main component of pectin, an abundant and cheap raw material. Sugar beet pulp and citrus peel are both rich in pectin residues. At present, these residues are mainly used as cattle feed. However, since energy-consuming drying and pelletizing of the residues is required to prevent them from rotting, it is not always economical to process the residues, and it is desirable to find alternative uses.Various microbes which live on decaying plant material have the ability to catabolize d-galacturonate using various, completely different pathways (19). Eukaryotic microorganisms use a reductive pathway in which d-galacturonate is first reduced to l-galactonate by an NAD(P)H-dependent reductase (12, 17). In the following steps a dehydratase, aldolase, and reductase convert the l-galactonate to pyruvate and glycerol (9, 11, 14).In Hypocrea jecorina (anamorph Trichoderma reesei) the gar1 gene codes for a strictly NADPH-dependent d-galacturonate reductase. In Aspergillus niger a homologue gene sequence, gar2, exists; however, a different gene, gaaA, is upregulated during growth on d-galacturonate containing medium (16). The gaaA codes for a d-galacturonate reductase with different kinetic properties than the H. jecorina enzyme, having a higher affinity toward d-galacturonate and using either NADH or NADPH as cofactor. It is not known whether gar2 codes for an active protein.Some bacteria, such as Agrobacterium tumefaciens or Pseudomonas syringae, have an oxidative pathway for d-galacturonate catabolism. In this pathway d-galacturonate is first oxidized to meso-galactarate (mucate) by an NAD-utilizing d-galacturonate dehydrogenase. Galactarate is then converted in the following steps to α-ketoglutarate. This route is sometimes called the α-ketoglutarate pathway (20). Galactarate can also be catabolized through the glycerate pathway (20). The products of this pathway are pyruvate and d-glycerate. These pathways have been described in prokaryotes, and it is not certain whether similar pathways also exist in fungi, some of which are able to metabolize galactarate.d-Galacturonate dehydrogenase (EC 1.1.1.203) has been described in Agrobacterium tumefaciens and in Pseudomonas syringae, and the enzymes from these organisms have been purified and characterized (3, 6, 22). Recently, the corresponding genes were also identified (4, 24). Both enzymes are specific for NAD as a cofactor but are not specific for the substrate. They oxidize d-galacturonate and d-glucuronate to meso-galactarate (mucate) and d-glucarate (saccharate), respectively. The reaction product is probably the hexaro-lactone which spontaneously hydrolyzes. The reverse reaction can only be observed at acidic pH where some of the galactarate is in the lactone form (22).We describe here strains of filamentous fungi that have been genetically engineered to produce galactarate by disruption of d-galacturonate reductase and expression of d-galacturonate dehydrogenase (Fig. ​(Fig.1).1). Galactarate is currently commercially produced from d-galactose by oxidation with nitric acid (1) or from d-galacturonate by electrolytic oxidation (8). Oxidation with nitric acid is expensive and produces toxic wastes. Galactarate is used as a chelator and in skin care products. It was formerly used as a leavening agent in self-rising flour (2) and has potential applications in polymer synthesis (10) and as a platform chemical (for a review, see reference 13).Open in a separate windowFIG. 1.Engineering the d-galacturonic acid pathway in fungi. Deletion of the gene encoding d-galacturonate reductase resulted in strains unable to utilize d-galacturonic acid as a carbon source. The expression of a bacterial udh gene, encoding an NAD-dependent d-galacturonate dehydrogenase, resulted in fungal strains, which were able to oxidize d-galacturonic acid to meso-galactaric acid (mucic acid). d-Galacturonate dehydrogenase forms a galactaro-lactone which spontaneously hydrolyzes.     

Utilization of d-Lactate as an Energy Source Supports the Growth of Gluconobacter oxydans

d-Lactate was identified as one of the few available organic acids that supported the growth of Gluconobacter oxydans 621H in this study. Interestingly, the strain used d-lactate as an energy source but not as a carbon source, unlike other lactate-utilizing bacteria. The enzymatic basis for the growth of G. oxydans 621H on d-lactate was therefore investigated. Although two putative NAD-independent d-lactate dehydrogenases, GOX1253 and GOX2071, were capable of oxidizing d-lactate, GOX1253 was the only enzyme able to support the d-lactate-driven growth of the strain. GOX1253 was characterized as a membrane-bound dehydrogenase with high activity toward d-lactate, while GOX2071 was characterized as a soluble oxidase with broad substrate specificity toward d-2-hydroxy acids. The latter used molecular oxygen as a direct electron acceptor, a feature that has not been reported previously in d-lactate-oxidizing enzymes. This study not only clarifies the mechanism for the growth of G. oxydans on d-lactate, but also provides new insights for applications of the important industrial microbe and the novel d-lactate oxidase.     

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.     

Bacterial catabolism of threonine. Threonine degradation initiated by l-threonine acetaldehyde-lyase (aldolase) in species of Pseudomonas

1. The route of l-threonine degradation was studied in four strains of the genus Pseudomonas able to grow on the amino acid and selected because of their high l-threonine aldolase activity. Growth and manometric results were consistent with the cleavage of l-threonine to acetaldehyde+glycine and their metabolism via acetate and serine respectively. 2. l-Threonine aldolases in these bacteria exhibited pH optima in the range 8.0–8.7 and K_m values for the substrate of 5–10mm. Extracts exhibited comparable allo-l-threonine aldolase activities, K_m values for this substrate being 14.5–38.5mm depending on the bacterium. Both activities were essentially constitutive. Similar activity ratios in extracts, independent of growth conditions, suggested a single enzyme. The isolate Pseudomonas D2 (N.C.I.B. 11097) represents the best source of the enzyme known. 3. Extracts of all the l-threonine-grown pseudomonads also possessed a CoA-independent aldehyde dehydrogenase, the synthesis of which was induced, and a reversible alcohol dehydrogenase. The high acetaldehyde reductase activity of most extracts possibly resulted in the underestimation of acetaldehyde dehydrogenase. 4. l-Serine dehydratase formation was induced by growth on l-threonine or acetate+glycine. Constitutively synthesized l-serine hydroxymethyltransferase was detected in extracts of Pseudomonas strains D2 and F10. The enzyme could not be detected in strains A1 and N3, probably because of a highly active `formaldehyde-utilizing' system. 5. Ion-exchange and molecular exclusion chromatography supported other evidence that l-threonine aldolase and allo-l-threonine aldolase activities were catalysed by the same enzyme but that l-serine hydroxymethyltransferase was distinct and different. These results contrast with the specificities of some analogous enzymes of mammalian origin.     

Biosynthesis of l-tyrosine O-sulphate from the methyl and ethyl esters of l-tyrosine

1. Rat-liver supernatant preparations are capable of achieving the biological sulphation of l-tyrosine methyl ester, the reaction proceeding maximally at a substrate concentration of 30 mm and at pH 7·0. 2. Two sulphated products are formed, one of which has been identified as l-tyrosine O-sulphate. On the basis of indirect evidence the other product can be assumed to be l-tyrosine O-sulphate methyl ester. 3. An enzyme present in rat-liver supernatant preparations is capable of converting l-tyrosine O-sulphate methyl ester into l-tyrosine O-sulphate. This enzyme is inhibited by l-tyrosine methyl ester. 4. l-Tyrosine ethyl ester also yields two sulphated products when used as an acceptor in the liver sulphating system. One of these has been identified chromatographically as l-tyrosine O-sulphate and the other may be presumed to be l-tyrosine O-sulphate ethyl ester.     

High-Level Heterologous Production of d-Cycloserine by Escherichia coli

Previously, we successfully cloned a d-cycloserine (d-CS) biosynthetic gene cluster consisting of 10 open reading frames (designated dcsA to dcsJ) from d-CS-producing Streptomyces lavendulae ATCC 11924. In this study, we put four d-CS biosynthetic genes (dcsC, dcsD, dcsE, and dcsG) in tandem under the control of the T7 promoter in an Escherichia coli host. SDS-PAGE analysis demonstrated that the 4 gene products were simultaneously expressed in host cells. When l-serine and hydroxyurea (HU), the precursors of d-CS, were incubated together with the E. coli resting cell suspension, the cells produced significant amounts of d-CS (350 ± 20 μM). To increase the productivity of d-CS, the dcsJ gene, which might be responsible for the d-CS excretion, was connected downstream of the four genes. The E. coli resting cells harboring the five genes produced d-CS at 660 ± 31 μM. The dcsD gene product, DcsD, forms O-ureido-l-serine from O-acetyl-l-serine (OAS) and HU, which are intermediates in d-CS biosynthesis. DcsD also catalyzes the formation of l-cysteine from OAS and H₂S. To repress the side catalytic activity of DcsD, the E. coli chromosomal cysJ and cysK genes, encoding the sulfite reductase α subunit and OAS sulfhydrylase, respectively, were disrupted. When resting cells of the double-knockout mutant harboring the four d-CS biosynthetic genes, together with dcsJ, were incubated with l-serine and HU, the d-CS production was 980 ± 57 μM, which is comparable to that of d-CS-producing S. lavendulae ATCC 11924 (930 ± 36 μM).     

Biosynthesis of l-Sorbose and l-Psicose Based on C—C Bond Formation Catalyzed by Aldolases in an Engineered Corynebacterium glutamicum Strain

The property of loose stereochemical control at aldol products from aldolases helped to synthesize multiple polyhydroxylated compounds with nonnatural stereoconfiguration. In this study, we discovered for the first time that some fructose 1,6-diphosphate aldolases (FruA) and tagatose 1,6-diphosphate (TagA) aldolases lost their strict stereoselectivity when using l-glyceraldehyde and synthesized not only l-sorbose but also a high proportion of l-psicose. Among the aldolases tested, TagA from Bacillus licheniformis (BGatY) showed the highest enzyme activity with l-glyceraldehyde. Subsequently, a “one-pot” reaction based on BGatY and fructose-1-phosphatase (YqaB) generated 378 mg/liter l-psicose and 199 mg/liter l-sorbose from dihydroxyacetone-phosphate (DHAP) and l-glyceraldehyde. Because of the high cost and instability of DHAP, a microbial fermentation strategy was used further to produce l-sorbose/l-psicose from glucose and l-glyceraldehyde, in which DHAP was obtained from glucose through the glycolytic pathway, and some recombination pathways based on FruA or TagA and YqaB were constructed in Escherichia coli and Corynebacterium glutamicum strains. After evaluation of different host cells and combinations of FruA or TagA with YqaB and optimization of gene expression, recombinant C. glutamicum strain WT(pXFTY) was selected and produced 2.53 g/liter total ketoses, with a yield of 0.50 g/g l-glyceraldehyde. Moreover, deletion of gene cgl0331, encoding the Zn-dependent alcohol dehydrogenase in C. glutamicum, was confirmed for the first time to significantly decrease conversion of l-glyceraldehyde to glycerol and to increase yield of target products. Finally, fed-batch culture of strain SY14(pXFTY) produced 3.5 g/liter l-sorbose and 2.3 g/liter l-psicose, with a yield of 0.61 g/g l-glyceraldehyde. This microbial fermentation strategy also could be applied to efficiently synthesize other l-sugars.     

The biological sulphation of l-tyrosyl peptides

1. A rat-liver supernatant preparation can achieve the biological O-sulphation of l-tyrosylglycine and l-tyrosyl-l-alanine at pH7·0. 2. The optimum concentrations of l-tyrosylglycine and l-tyrosyl-l-alanine in this system are 50mm and 60mm respectively. 3. l-Tyrosylglycine yields two sulphated products, whereas l-tyrosyl-l-alanine yields three sulphated products, when used as acceptor for sulphate in the rat-liver system. 4. With both substrates, one of the sulphated products has been identified as the O-sulphate ester of the corresponding parent peptide.     

Structural and Functional Characterization of VanG d-Ala:d-Ser Ligase Associated with Vancomycin Resistance in Enterococcus faecalis

d-Alanyl:d-lactate (d-Ala:d-Lac) and d-alanyl:d-serine ligases are key enzymes in vancomycin resistance of Gram-positive cocci. They catalyze a critical step in the synthesis of modified peptidoglycan precursors that are low binding affinity targets for vancomycin. The structure of the d-Ala:d-Lac ligase VanA led to the understanding of the molecular basis for its specificity, but that of d-Ala:d-Ser ligases had not been determined. We have investigated the enzymatic kinetics of the d-Ala:d-Ser ligase VanG from Enterococcus faecalis and solved its crystal structure in complex with ADP. The overall structure of VanG is similar to that of VanA but has significant differences mainly in the N-terminal and central domains. Based on reported mutagenesis data and comparison of the VanG and VanA structures, we show that residues Asp-243, Phe-252, and Arg-324 are molecular determinants for d-Ser selectivity. These residues are conserved in both enzymes and explain why VanA also displays d-Ala:d-Ser ligase activity, albeit with low catalytic efficiency in comparison with VanG. These observations suggest that d-Ala:d-Lac and d-Ala:d-Ser enzymes have evolved from a common ancestral d-Ala:d-X ligase. The crystal structure of VanG showed an unusual interaction between two dimers involving residues of the omega loop that are deeply anchored in the active site. We constructed an octapeptide mimicking the omega loop and found that it selectively inhibits VanG and VanA but not Staphylococcus aureus d-Ala:d-Ala ligase. This study provides additional insight into the molecular evolution of d-Ala:d-X ligases and could contribute to the development of new structure-based inhibitors of vancomycin resistance enzymes.     

Utilization of d-Ribitol by Lactobacillus casei BL23 Requires a Mannose-Type Phosphotransferase System and Three Catabolic Enzymes

Lactobacillus casei strains 64H and BL23, but not ATCC 334, are able to ferment d-ribitol (also called d-adonitol). However, a BL23-derived ptsI mutant lacking enzyme I of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) was not able to utilize this pentitol, suggesting that strain BL23 transports and phosphorylates d-ribitol via a PTS. We identified an 11-kb region in the genome sequence of L. casei strain BL23 (LCABL_29160 to LCABL_29270) which is absent from strain ATCC 334 and which contains the genes for a GlpR/IolR-like repressor, the four components of a mannose-type PTS, and six metabolic enzymes potentially involved in d-ribitol metabolism. Deletion of the gene encoding the EIIB component of the presumed ribitol PTS indeed prevented d-ribitol fermentation. In addition, we overexpressed the six catabolic genes, purified the encoded enzymes, and determined the activities of four of them. They encode a d-ribitol-5-phosphate (d-ribitol-5-P) 2-dehydrogenase, a d-ribulose-5-P 3-epimerase, a d-ribose-5-P isomerase, and a d-xylulose-5-P phosphoketolase. In the first catabolic step, the protein d-ribitol-5-P 2-dehydrogenase uses NAD⁺ to oxidize d-ribitol-5-P formed during PTS-catalyzed transport to d-ribulose-5-P, which, in turn, is converted to d-xylulose-5-P by the enzyme d-ribulose-5-P 3-epimerase. Finally, the resulting d-xylulose-5-P is split by d-xylulose-5-P phosphoketolase in an inorganic phosphate-requiring reaction into acetylphosphate and the glycolytic intermediate d-glyceraldehyde-3-P. The three remaining enzymes, one of which was identified as d-ribose-5-P-isomerase, probably catalyze an alternative ribitol degradation pathway, which might be functional in L. casei strain 64H but not in BL23, because one of the BL23 genes carries a frameshift mutation.     

A Novel Type of Peptidoglycan-binding Domain Highly Specific for Amidated d-Asp Cross-bridge,Identified in Lactobacillus casei Bacteriophage Endolysins

Peptidoglycan hydrolases (PGHs) are responsible for bacterial cell lysis. Most PGHs have a modular structure comprising a catalytic domain and a cell wall-binding domain (CWBD). PGHs of bacteriophage origin, called endolysins, are involved in bacterial lysis at the end of the infection cycle. We have characterized two endolysins, Lc-Lys and Lc-Lys-2, identified in prophages present in the genome of Lactobacillus casei BL23. These two enzymes have different catalytic domains but similar putative C-terminal CWBDs. By analyzing purified peptidoglycan (PG) degradation products, we showed that Lc-Lys is an N-acetylmuramoyl-l-alanine amidase, whereas Lc-Lys-2 is a γ-d-glutamyl-l-lysyl endopeptidase. Remarkably, both lysins were able to lyse only Gram-positive bacterial strains that possess PG with d-Ala⁴→d-Asx-l-Lys³ in their cross-bridge, such as Lactococcus casei, Lactococcus lactis, and Enterococcus faecium. By testing a panel of L. lactis cell wall mutants, we observed that Lc-Lys and Lc-Lys-2 were not able to lyse mutants with a modified PG cross-bridge, constituting d-Ala⁴→l-Ala-(l-Ala/l-Ser)-l-Lys³; moreover, they do not lyse the L. lactis mutant containing only the nonamidated d-Asp cross-bridge, i.e. d-Ala⁴→d-Asp-l-Lys³. In contrast, Lc-Lys could lyse the ampicillin-resistant E. faecium mutant with 3→3 l-Lys³-d-Asn-l-Lys³ bridges replacing the wild-type 4→3 d-Ala⁴-d-Asn-l-Lys³ bridges. We showed that the C-terminal CWBD of Lc-Lys binds PG containing mainly d-Asn but not PG with only the nonamidated d-Asp-containing cross-bridge, indicating that the CWBD confers to Lc-Lys its narrow specificity. In conclusion, the CWBD characterized in this study is a novel type of PG-binding domain targeting specifically the d-Asn interpeptide bridge of PG.     

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.