Genome mining and physiological analyses uncover adaptation strategies and biotechnological potential of Virgibacillus dokdonensis T4.6 isolated from high-salt shrimp paste.

Virgibacillus spp. stand out as a potent starter culture for accelerating the fermention of fish sauces and shrimp pastes. However, the underlying molecular mechanisms responsible for their adaptation and biotechnological potential remain elusive. Therefore, the present study focuses on phenotypic and genomic analyses of a halophilic bacterium Virgibacillus dokdonensis T4.6, derived from Vietnamese high-salt fermented shrimp paste. The draft genome contained 4,096,868 bp with 3780 predicted coding sequences. Genome mining revealed the presence of 143 genes involved in osmotic adaptation explaining its resistant phenotype to 24% (w/v) NaCl. Among them, 37 genes making up the complete ectoine metabolism pathway, confirmed its ability to produce 4.38 ± 0.29 wt% ectoine under 12.5% NaCl stress. A significant finding was the identification of 39 genes responsible for an entire degradation pathway of the toxic biogenic amine histamine, which was in agreement with its histamine degradation rate of 42.7 ± 2.1% in the HA medium containing 5 mM histamine within 10 days at 37 °C. Furthermore, 114 proteolytic and 19 lipolytic genes were detected which might contribute to its survival as well as the nutrient quality and flavor of shrimp paste. Of note, a putative gene vdo2592 was found as a possible novel lipase/esterase due to its unique Glycine-Aspartate-Serine-Leucine (GDSL) sequence motif. This is the first report to reveal the adaptative strategies and related biotechnological potential of Virgibacillus associated with femented foods. Our findings indicated that V. dokdonensis T4.6 is a promising starter culture for the production of fermented shrimp paste products.

New insight into protective effect against oxidative stress and biosynthesis of exopolysaccharides produced by Lacticaseibacillus paracasei NC4 from fermented eggplant.

Fermented eggplant is a traditional fermented food, however lactic acid bacteria capable of producing exopolysaccharide (EPS) have not yet been exploited. The present study focused on the production and protective effects against oxidative stress of an EPS produced by Lacticaseibacillus paracasei NC4 (NC4-EPS), in addition to deciphering its genomic features and EPS biosynthesis pathway. Among 54 isolates tested, strain NC4 showed the highest EPS yield and antioxidant activity. The maximum EPS production (2.04 ± 0.11 g/L) was achieved by culturing in MRS medium containing 60 g/L sucrose at 37 °C for 48 h. Under 2 mM H2O2 stress, the survival of a yeast model Saccharomyces cerevisiae treated with 0.4 mg/mL NC4-EPS was 2.4-fold better than non-treated cells, which was in agreement with the catalase and superoxide dismutase activities measured from cell lysates. The complete genome of NC4 composed of a circular chromosome of 2,888,896 bp and 3 circular plasmids. The NC4 genome comprises more genes with annotated function in nitrogen metabolism, phosphorus metabolism, cell division and cell cycle, and iron acquisition and metabolism as compared to other reported L. paracasei. Of note, the eps gene cluster is not conserved across L. paracasei. Pathways of sugar metabolism for EPS biosynthesis were proposed for the first time, in which gdp pathway only present in few plant-derived bacteria was identified. These findings shed new light on the cell-protective activity and biosynthesis of EPS produced by L. paracasei, paving the way for future efforts to enhance yield and tailor-made EPS production for food and pharmaceutical industries.

Camellia annamensis (Theaceae): phytochemical analysis, cytotoxic, antioxidative, and antimicrobial activities.

Cytotoxic, antioxidative, and antimicrobial activities of Camellia annamensis, and its chemical compositions were first provided in the current study. Phenolic contents in the methanol extracts of its leaves and flowers were 222.73 ± 0.09 and 64.44 ± 0.08 mg GAE/g extract, whereas flavonoid contents in these parts were 108.80 ± 0.28 and 131.26 ± 0.39 mg rutin/g extract, respectively. By using HPLC-DAD analysis, gallic acid (43.72 ± 0.09 - 81.89 ± 1.83 mg/g) and (-)-epigallocatechin gallate (67.31 ± 1.26 - 70.68 ± 7.82 mg/g) were identified as the major compounds. C. annamensis leaf and flower extracts were moderately cytotoxic against A549, HT-29, SK-Mel-2, MCF-7, HepG2, HeLa, and MKN-7. Particularly, they are better than the standards trolox (IC50 7.57 ± 0.23 µg/mL) in lipid peroxidation inhibitory evaluation, and streptomycin (IC50/MIC = 45.34-50.34/128-256 µg/mL) in antimicrobial assay against the Gram-positive bacteria Enterococcus faecalis ATCC299212, Staphylococcus aureus ATCC25923, and the Gram-negative bacterium Salmonella enterica ATCC13076.

Influence of structure phase transition on the thermoelectric properties of Cu2Se1-x Te x liquid-like compounds.

Copper chalcogenide Cu2(Se,Te) compounds are well known as typical p-type thermoelectric materials with a figure of merit (ZT) that can be optimized by the ratio of Se : Te. Here, by using the mechanical alloying and solid-state reaction methods, Te was substituted into Se sites within Cu2Se as the formula Cu2Se1-x Te x (x = 0.1, 0.2, 0.25, and 0.3). The observed changes in structural phase, grain morphologies, and grain size were recorded by XRD and FE-SEM imaging with the appearance of the secondary phase of Cu2Te, with a Te content of x = 0.25. The layered structure morphology was observed more clearly at the high Te content. The electrical conductivity was greatly increased with enriched Te content while the maximum Seebeck coefficient was obtained in the Cu2Se0.75Te0.25 sample. Accordingly, a power factor value of up to 9.84 µW cm-1 K-2 at 773 K was achieved. The appearance of a Cu2Te phase with a Te content of 0.25 created a structural phase transition which results in a ZT value of 1.35 at 773 K in the Cu2Se0.75Te0.25 sample.

Genome-Guided Investigation Provides New Insights into Secondary Metabolites of Streptomyces parvulus SX6 from Aegiceras corniculatum.

Whole-genome sequencing and genome mining are recently considered an efficient approach to shine more light on the underlying secondary metabolites of Streptomyces. The present study unearths the biosynthetic potential of endophytic SX6 as a promising source of biologically active substances and plant-derived compounds for the first time. Out of 38 isolates associated with Aegiceras corniculatum (L.) Blanco, Streptomyces parvulus SX6 was highly active against Pseudomonas aeruginosa ATCC® 9027™ and methicillin-resistant Staphylococcus epidermidis (MRSE) ATCC® 35984™. Additionally, S. parvulus SX6 culture extract showed strong cytotoxicity against Hep3B, MCF-7, and A549 cell lines at a concentration of 30 µg/ml, but not in non-cancerous HEK-293 cells. The genome contained 7.69 Mb in size with an average G + C content of 72.8% and consisted of 6,779 protein-coding genes. AntiSMASH analysis resulted in the identification of 29 biosynthetic gene clusters (BGCs) for secondary metabolites. Among them, 4 BGCs showed low similarity (28-67% of genes show similarity) to actinomycin, streptovaricin, and polyoxypeptin gene clusters, possibly attributed to antibacterial and anticancer activities observed. In addition, the complete biosynthetic pathways of plant-derived compounds, including daidzein and genistein were identified using genome mining and HPLC-DAD-MS analysis. These findings portray an exciting avenue for future characterization of promising secondary metabolites from mangrove endophytic S. parvulus.

Molecular docking and molecular dynamics approach to identify potential compounds in Huperzia squarrosa for treating Alzheimer's disease.

OBJECTIVES: Alzheimer's disease (AD) is a lingering progressive neurodegenerative disorder that causes patients to lose cognitive function. The enzyme Acetylcholinesterase (AChE), Butyrylcholinesterase (BuChE), Monoamine oxidase A (MAO A), Beta-secretase cleavage enzyme (BACE 1) and N-methyl-D-aspartate (NMDA) receptors play an important role in the pathogenesis of Alzheimer's disease. Therefore, inhibiting enzymes is an effective method to treat Alzheimer disease. In this study, we evaluated in silico inhibitory effects of AChE, BuChE, MAO A, BACE 1 and NMDA enzyme of Huperzia squarrosa's compounds. METHODS: The three-dimensional (3D) of N-methyl-D-aspartate receptor (PDB ID: 1PBQ), enzyme ß-secretase 1 (PDB ID: 4X7I), enzyme monoamine oxidase A (PDB ID: 2Z5X), enzyme butyrylcholinesterase (PDB ID: 4BDS) and enzyme acetylcholinesterase (PDB ID: 1EVE) were retrieved from the Protein Data Bank RCSB. Molecular docking was done by Autodock vina software and molecular dynamics (MD) simulation of the ligand-protein complex with the least binding energy pose was perfomed by MOE. Lipinski Rule of Five is used to compare compounds with drug-like and non-drug-like properties. Pharmacokinetic parameters of potential compounds were evaluated using the pkCSM tool. RESULTS: Based on previous publication of Huperzia squarrosa, we have collected 15 compounds. In these compounds, huperzine B, huperzinine, lycoposerramine U N-oxide, 12-epilycodine N-oxide showed strongly inhibit the five AChE, BuChE, MAO A, BACE 1 and NMDA targets for Alzheimer's treatment. Lipinski rule of five and ADMET predict have shown that four above compounds have drug-likeness properties, good absorption ability and cross the blood-brain barrier, which have the most potential to become drugs for the treatment of Alzheimer's in the future. Furthermore, MD study showed that huperzine B and huperzinine have stability of the docking pose with NMDA target. CONCLUSIONS: In this study, we found two natural compounds in Huperzia squarrosa including Huperzine B and Huperzinine have drug-likeness properties, good absorption ability and cross the blood-brain barrier, which have potential to become drugs for the treatment of Alzheimer's in the future.

Plant-derived bioactive compounds produced by Streptomyces variabilis LCP18 associated with Litsea cubeba (Lour.) Pers as potential target to combat human pathogenic bacteria and human cancer cell lines.

To date, endophytic actinomycetes have been well-documented as great producers of novel antibiotics and important pharmaceutical leads. The present study aimed to evaluate potent bioactivities of metabolites synthesized by the strain LCP18 residing in the Vietnamese medicinal plant Litsea cubeba (Lour.) Pers towards human pathogenic bacteria and human cancer cell lines. Endophytic actinomycete strain LCP18 showed considerable inhibition against seven bacterial pathogens and three human tumor cell lines and was identified as species Streptomyces variabilis. Strain S. variabilis LCP18 was phenotypically resistant to fosfomycin, trimethoprim-sulfamethoxazole, dalacin, cefoxitin, rifampicin, and fusidic acid and harbored the two antibiotic biosynthetic genes such as PKS-II and NRPS. Further purification and structural elucidation of metabolites from the LCP18 extract revealed five plant-derived bioactive compounds including isopcrunetin, genistein, daidzein, syringic acid, and daucosterol. Among those, isoprunetin, genistein, and daidzein exhibited antibacterial activity against Salmonella typhimurium ATCC 14,028 and methicillin-resistant Staphylococcus epidermidis ATCC 35,984 with the MIC values ranging from 16 to 128 µg/ml. These plant-derived compounds also exhibited cytotoxic effects against human lung cancer cell line A549 with IC50 values of less than 46 µM. These findings indicated that endophytic S. variabilis LCP18 can be an alternative producer of plant-derived compounds which significantly show potential applications in combating bacterial infections and inhibition against lung cancer cell lines.

Two new terpenoids from the leaves of callicarpa macrophylla.

Two new terpenoids (1-2) and seven known compounds (3-9) were isolated from methanol extract of Callicarpa macrophylla leaves. Their structures were determined to be ent-7α,16ß,17,18-tetrahydroxykaur-15-one (1), 3ß-acetoxy-urs-12-ene-11-one-12-ol (2), ent-1ß-acetoxy-7α,14ß-dihydroxykaur-16-en-15-one (3), 3ß-acetoxy-11α,13ß-dihydroxyolean-12-one (4), ß-amyrin (5), spinasterol (6), ursolic acid (7), ß-sitosterol (8), and daucosterol (9) by analyses of their MS, NMR spectroscopic data and by comparison with those reported in the literature. Compounds 1 - 4, and 7 displayed potential cytotoxic activity towards HepG-2, LU-1, and MCF-7 human cancer cell lines with IC50 values ranging from 0.46 ± 0.21 to 18.14 ± 0.33 µM. Compound 6 showed IC50 values of 14.17 ± 0.21 and 5.72 ± 0.42 µM against Hep-G2 and MCF-7 cell lines, respectively.

Coxsackieviruses A6 and A16 associated with hand, foot, and mouth disease in Vietnam, 2008-2017: Essential information for rational vaccine design.

Development of multivalent hand, foot, and mouth disease (HFMD) vaccines against enterovirus A71 (EV-A71) and several non-EV-A71 enteroviruses is needed for this life-threatening disease with a huge economic burden in Asia-Pacific countries. Comprehensive studies on the molecular epidemiology and genetic and antigenic characterization of major causative enteroviruses will provide information for rational vaccine design. Compared with molecular studies on EV-A71, that for non-EV-A71 enteroviruses remain few and limited in Vietnam. Therefore, we conducted a 10-year study on the circulation and genetic characterization of coxsackievirus A16 (CV-A16) and CV-A6 isolated from patients with HFMD in Northern Vietnam between 2008 and 2017. Enteroviruses were detected in 2228 of 3212 enrolled patients. Of the 42 serotypes assigned, 28.4% and 22.4% accounted for CV-A6 and CV-A16, being the second and the third dominant serotypes after EV-A71 (31.7%), respectively. The circulation of CV-A16 and CV-A6 showed a wide geographic distribution and distinct periodicity. Phylogenetic analyses revealed that the majority of Vietnamese CV-A6 and CV-A16 strains were located within the largest sub-genotypes or sub-genogroups. These comprised strains isolated from patients with HFMD worldwide during the past decade and the Vietnamese strains have been evolving in a manner similar to the strains circulating worldwide. Amino acid sequences of the putative functional loops on VP1 and other VPs among Vietnamese CV-A6 and CV-A16 isolates were highly conserved. Moreover, the functional loop patterns of VP1 were similar to the dominant patterns found worldwide, except for the T164K substitution on the EF loop in Vietnamese CV-A16. The findings suggest that the development of a universal HFMD vaccine, at least in Vietnam, must target CV-A6 and CV-A16 as two of the three major HFMD-causing serotypes. Vietnamese isolates or their genome sequences can be considered for rational vaccine design.

Improvement of regio-specific production of myricetin-3-O-α-L-rhamnoside in engineered Escherichia coli.

Myricetin is an important flavonol whose medically important properties include activities as an antioxidant, anticarcinogen, and antimutagen. The solubility, stability, and other biological properties of the compounds can be enhanced by conjugating aglycon with sugar moieties. The type of sugar moiety also plays a significant role in the biological and physical properties of the natural product glycosides. Reconstructed Escherichia coli containing thymidine diphosphate-α-L-rhamnose sugar gene cassette and Arabidopsis-derived glycosyltransferase were used for rhamnosylation of myricetin. Myricetin (100 µM) was exogenously supplemented to induced cultures of engineered E. coli. The formation of target product-myricetin-3-O-α-L-rhamnoside-was confirmed by chromatographic and NMR analyses. The yield of product was improved by using various mutants and methylated cyclodextrin as a molecular carrier for myricetin in combination with E. coli M3G3. The maximal yield of product is 55.6 µM (3.31-fold higher than the control E. coli MG3) and shows 55.6 % bioconversion of substrate under optimized conditions.

Hepatitis C virus in Vietnam: high prevalence of infection in dialysis and multi-transfused patients involving diverse and novel virus variants.

Hepatitis C virus (HCV) is a genetically diverse pathogen infecting approximately 2-3% of the world's population. Herein, we describe results of a large, multicentre serological and molecular epidemiological study cataloguing the prevalence and genetic diversity of HCV in five regions of Vietnam; Ha Noi, Hai Phong, Da Nang, Khanh Hoa and Can Tho. Individuals (n=8654) with varying risk factors for infection were analysed for the presence of HCV Ab/Ag and, in a subset of positive specimens, for HCV RNA levels (n=475) and genotype (n=282). In lower risk individuals, including voluntary blood donors, military recruits and pregnant women, the prevalence of infection was 0.5% (n=26/5250). Prevalence rates were significantly higher (p<0.001) in intravenous drug users (IDUs; 55.6%, n=556/1000), dialysis patients (26.6%, n=153/575) commercial sex workers (CSWs; 8.7%, n=87/1000), and recipients of multiple blood transfusions (6.0%, n=32/529). The prevalence of HCV in dialysis patients varied but remained high in all regions (11-43%) and was associated with the receipt of blood transfusions [OR: 2.08 (1.85-2.34), p=0.001], time from first transfusion [OR: 1.07 (1.01-1.13), p=0.023], duration of dialysis [OR: 1.31 (1.19-1.43), p<0.001] and male gender [OR: 1.60 (1.06-2.41), p=0.026]. Phylogenetic analysis revealed high genetic diversity, particularly amongst dialysis and multi-transfused patients, identifying subtypes 1a (33%), 1b (27%), 2a (0.4%), 3a (0.7%), 3b (1.1%), 6a (18.8%), 6e (6.0%), 6h (4.6%), 6l (6.4%) and 2 clusters of novel genotype 6 variants (2.1%). HCV genotype 1 predominated in Vietnam (60%, n=169/282) but the proportion of infections attributable to genotype 1 varied between regions and risk groups and, in the Southern part of Vietnam, genotype 6 viruses dominated in dialysis and multi-transfused patients (73.9%). This study confirms a high prevalence of HCV infection in Vietnamese IDUs and, notably, reveals high levels of HCV infection associated with dialysis and blood transfusion.

A multicentre molecular analysis of hepatitis B and blood-borne virus coinfections in Viet Nam.

Hepatitis B (HBV) infection is endemic in Viet Nam, with up to 8.4 million individuals estimated to be chronically infected. We describe results of a large, multicentre seroepidemiological and molecular study of the prevalence of HBV infection and blood-borne viral coinfections in Viet Nam. Individuals with varying risk factors for infection (n = 8654) were recruited from five centres; Ha Noi, Hai Phong, Da Nang, Khanh Hoa and Can Tho. A mean prevalence rate of 10.7% was observed and levels of HBsAg were significantly higher in injecting drug users (IDUs) (17.4%, n = 174/1000) and dialysis patients (14.3%, n = 82/575) than in lower-risk groups (9.4%; p<0.001). Coinfection with HIV was seen in 28% of HBV-infected IDUs (n = 49/174) and 15.2% of commercial sex workers (CSWs; n = 15/99). HCV infection was present in 89.8% of the HBV-HIV coinfected IDUs (n = 44/49) and 40% of HBV-HIV coinfected CSWs (n = 16/40). Anti-HDV was detected in 10.7% (n = 34/318) of HBsAg positive individuals. Phylogenetic analysis of HBV S gene (n = 187) showed a predominance of genotype B4 (82.6%); genotypes C1 (14.6%), B2 (2.7%) and C5 (0.5%) were also identified. The precore mutation G1896A was identified in 35% of all specimens, and was more frequently observed in genotype B (41%) than genotype C (3%; p<0.0001). In the immunodominant 'a' region of the surface gene, point mutations were identified in 31% (n = 58/187) of sequences, and 2.2% (n = 4/187) and 5.3% (n = 10/187) specimens contained the major vaccine escape mutations G145A/R and P120L/Q/S/T, respectively. 368 HBsAg positive individuals were genotyped for the IL28B SNP rs12979860 and no significant association between the IL28B SNP and clearance of HBsAg, HBV viral load or HBeAg was observed. This study confirms the high prevalence of HBV infection in Viet Nam and also highlights the significant levels of blood-borne virus coinfections, which have important implications for hepatitis-related morbidity and development of effective management strategies.

Prevalence of HIV type 1 antiretroviral drug resistance mutations in Vietnam: a multicenter study.

The prevalence of HIV-1 drug resistance mutations (DRMs) was determined for a cross-section of individuals (n=8654) in five centers across Vietnam (Hanoi, Hai Phong, Da Nang, Khanh Hoa, and Can Tho) between 2008 and 2009. Following serological screening for HIV infection, HIV-1 viral load was determined, using an in-house real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay. Samples with quantifiable viral loads [all either commercial sex workers (CSW) or intravenous drug users (IDU)] underwent DRM analysis. Sequences were obtained for 92 treatment-naive individuals, the majority of whom were infected with HIV-1 CRF01_AE (99%), with one instance of subtype A1 also detected. DRMs were detected in seven treatment-naive individuals (7.6%). The most common DRMs observed were M184V, V75A/M, M41L, and K65R (NRTI) and K103N, G190A, and Y181C (NNRTI). Overall, the data from this first multicenter survey of DRMs in Vietnam indicate that the problem of transmitted drug resistance is of major concern in the highest-risk groups of IDU and CSW.

Identification and characterization of gerPI and gerPII involved in epoxidation and hydroxylation of dihydrochalcolactone in Streptomyces species KCTC 0041BP.

The macrolide antibiotics are biosynthesized by initial assembly of a macrolactone ring, followed by a series of post-polyketide (PKS) modifications. In general, the additional hydroxyl or epoxy groups are installed by cytochrome P450 enzymes, improving the bioactivity profile through structural diversification of natural products. The biosynthetic gene cluster for the 16-membered macrolide antibiotic dihydrochalcomycin (DHC) has been cloned from Streptomyces sp. KCTC 0041BP. Three cytochrome P450 genes are found in the DHC biosynthetic gene (ger) cluster. Two P450 enzymes were characterized from this cluster. Disruption of gerPI accumulated predominantly 12,13-de-epoxydihydrochalcomycin while disruption of gerPII accumulated 8-dehydroxy-12,13-de-epoxydihydrochalcomycin; DHC production was abolished in both cases. The results suggest that GerPII P450 catalyzes hydroxylation at the C(8) position followed by an epoxidation reaction catalyzed by GerPI P450 at the C(12)-C(13) position.

Dihydrochalcomycin Production and Glycosyltransferase from Streptomyces SP. KCTC 0041BP.

The dihydrochalcomycin (GERI) synthetic gene cluster from Streptomyces sp. KCTC 0041BP has been isolated. Two open reading frames (ORFs), designated gerT1 and gerT2 as glycosyltransferase genes, has been identified by sequence analysis. GerT1 encodes for the protein function as dTDP-deoxyallosyltransferase and it is responsible to the attachment of dTDP-allose to the macrolide ring. Similarly, gerT2 encodes for peptide named as dTDP-chacosyltransferase which can transfers the dTDP-4,6-dideoxyglucose to macrolactone core. During process of compound isolation, a new compound has been isolated with molecular weight m/z 755 [M+Na+]. This compound could be the dihydrochalcomycin derivative. The compound has been shown the same antibacterial activity as GERI compound.

Characterization of a chalcosyltransferase (gerGTII) in dihydrochalcomycin biosynthesis.

An open reading frame, designated GerGTII and located downstream of the polyketide synthase genes, has been identified as a chalcosyltransferase by sequence analysis in the dihydrochalcomycin biosynthetic gene cluster of Streptomyces sp. KCTC 0041BP. The deduced product of gerGTII is similar to several glycosyltransferases, authentic and putative, and it displays a consensus sequence motif that appears to be characteristic of a sub-group of these enzymes. Specific disruption of gerGTII within the S. sp. KCTC 0041BP genome by insertional in-frame deletion method, resulted complete abolishment of dihydrochalcomycin and got the 20-O-mycinosyl-dihydrochalconolide as intermediate product in dihydrochalcomycin biosynthesis which was confirmed by electron spray ionization-mass spectrometry and liquid chromatography-mass spectrometry. Dihydrochalcomycin also was recovered after complementation of gerGTII.

Biosynthesis of dTDP-6-deoxy-beta-D-allose, biochemical characterization of dTDP-4-keto-6-deoxyglucose reductase (GerKI) from Streptomyces sp. KCTC 0041BP.

dTDP-6-deoxy-d-allose, an unusual deoxysugar, has been identified as an intermediate in the mycinose biosynthetic pathway of several macrolide antibiotics. In order to characterize the biosynthesis of this deoxysugar, we have cloned and heterologously overexpressed gerK1 in Escherichia coli BL21 (DE3) cells. This gene encodes for a protein with the putative function of a dTDP-4-keto-6-deoxyglucose reductase, which appears to be involved in the dihydrochalcomycin (GERI-155) biosynthesis evidenced by Streptomyces sp KCTC 0041BP. Our results revealed that GerK1 exhibited a specific reductive effect on the 4-keto carbon of dTDP-4-keto-6-deoxy-d-allose, with the hydroxyl group in an axial configuration at the C3 position only. The enzyme catalyzed the conversion of dTDP-4-keto-6-deoxyglucose to dTDP-6-deoxy-beta-D-allose, according to the results of an in vitro coupled enzyme assay, in the presence of GerF (dTDP-4-keto-6-deoxyglucose 3-epimerase). The product was isolated, and its stereochemistry was determined via nuclear magnetic resonance analysis.

Functional characterizations of novWUS involved in novobiocin biosynthesis from Streptomyces spheroides.

NovW, novU, and novS gene products represent dTDP-4-keto-6-deoxy-D-glucose 3,5 epimarase, C-methyltransferase and dTDP-glucose-4-ketoreductase involved in noviose biosynthetic pathway, respectively. We have expressed three genes to elucidate the functions of NovW, NovU, and NovS in Escherichia coli. NovW and NovU catalyze the formation of dTDP-4-keto-6-deoxy-5-C-methyl-L-lyxo-hexose from dTDP-4-keto-6-deoxy-D-glucose. NovS reduces the product formed from the reaction of NovW with dTDP-4-keto-6-deoxy-D-glucose in the presence of NADH to result in dTDP-l-rhamnose. Furthermore, a pathway for the biosynthesis of noviose is proposed.