Substrate binding and catalytic mechanism of the Se-glycosyltransferase SenB in the biosynthesis of selenoneine.

Selenium is an essential multifunctional trace element in diverse organisms. The only Se-glycosyltransferase identified that catalyzes the incorporation of selenium in selenoneine biosynthesis is SenB from Variovorax paradoxus. Although the biochemical function of SenB has been investigated, its substrate specificity, structure, and catalytic mechanism have not been elucidated. Here, we reveal that SenB exhibits sugar donor promiscuity and can utilize six UDP-sugars to generate selenosugars. We report crystal structures of SenB complexed with different UDP-sugars. The key elements N20/T23/E231 contribute to the sugar donor selectivity of SenB. A proposed catalytic mechanism is tested by structure-guided mutagenesis, revealing that SenB yields selenosugars by forming C-Se glycosidic bonds via spontaneous deprotonation and disrupting Se-P bonds by nucleophilic water attack, which is initiated by the critical residue K158. Furthermore, we functionally and structurally characterize two other Se-glycosyltransferases, CbSenB from Comamonadaceae bacterium and RsSenB from Ramlibacter sp., which also exhibit sugar donor promiscuity.

Complete pathway elucidation and heterologous reconstitution of (+)-nootkatone biosynthesis from Alpinia oxyphylla.

(+)-Nootkatone is a natural sesquiterpene ketone widely used in food, cosmetics, pharmaceuticals, and agriculture. It is also regarded as one of the most valuable terpenes used commercially. However, plants contain trace amounts of (+)-nootkatone, and extraction from plants is insufficient to meet market demand. Alpinia oxyphylla is a well-known medicinal plant in China, and (+)-nootkatone is one of the main components within the fruits. By transcriptome mining and functional screening using a precursor-providing yeast chassis, the complete (+)-nootkatone biosynthetic pathway in Alpinia oxyphylla was identified. A (+)-valencene synthase (AoVS) was identified as a novel monocot-derived valencene synthase; three (+)-valencene oxidases AoCYP6 (CYP71BB2), AoCYP9 (CYP71CX8), and AoCYP18 (CYP701A170) were identified by constructing a valencene-providing yeast strain. With further characterisation of a cytochrome P450 reductase (AoCPR1) and three dehydrogenases (AoSDR1/2/3), we successfully reconstructed the (+)-nootkatone biosynthetic pathway in Saccharomyces cerevisiae, representing a basis for its biotechnological production. Identifying the biosynthetic pathway of (+)-nootkatone in A. oxyphylla unravelled the molecular mechanism underlying its formation in planta and also supported the bioengineering production of (+)-nootkatone. The highly efficient yeast chassis screening method could be used to elucidate the complete biosynthetic pathway of other valuable plant natural products in future.

Understanding base and backbone contributions of phosphorothioate DNA for molecular recognition with SBD proteins.

Bacterial DNA phosphorothioate (PT) modification provides a specific anchoring site for sulfur-binding proteins (SBDs). Besides, their recognition patterns include phosphate links and bases neighboring the PT-modified site, thereby bringing about genome sequence-dependent properties in PT-related epigenetics. Here, we analyze the contributions of the DNA backbone (phosphates and deoxyribose) and bases bound with two SBD proteins in Streptomyces pristinaespiralis and coelicolor (SBDSco and SBDSpr). The chalcogen-hydrophobic interactions remained constantly at the anchoring site while the adjacent bases formed conditional and distinctive non-covalent interactions. More importantly, SBD/PT-DNA interactions were not limited within the traditional "4-bp core" range from 5'-I to 3'-III but extended to upstream 5'-II and 5'-III bases and even 5''-I to 5''-III at the non-PT-modified complementary strand. From the epigenetic viewpoint, bases 3'-II, 5''-I, and 5''-III of SBDSpr and 3'-II, 5''-II, and 5''-III of SBDSco present remarkable differentiations in the molecular recognitions. From the protein viewpoint, H102 in SBDSpr and R191 in SBDSco contribute significantly while proline residues at the PT-bound site are strictly conserved for the PT-chalcogen bond. The mutual and make-up mutations are proposed to alter the SBD/PT-DNA recognition pattern, besides additional chiral phosphorothioate modifications on phosphates 5'-II, 5'-II, 3'-I, and 3'-II.

Metabolic Engineering of Saccharomyces cerevisiae for Vitamin B5 Production.

Vitamin B5, also called d-pantothenic acid, is an essential vitamin in the human body and is widely used in pharmaceuticals, nutritional supplements, food, and cosmetics. However, few studies have investigated the microbial production of d-pantothenic acid, especially in Saccharomyces cerevisiae. By employing a systematic optimization strategy, we screened seven key genes in d-pantothenic acid biosynthesis from diverse species, including bacteria, yeast, fungi, algae, plants, animals, etc., and constructed an efficient heterologous d-pantothenic acid pathway in S. cerevisiae. By adjusting the copy number of the pathway modules, knocking out the endogenous bypass gene, balancing NADPH utilization, and regulating the GAL inducible system, a high-yield d-pantothenic acid-producing strain, DPA171, which can regulate gene expression using glucose, was constructed. By optimizing fed-batch fermentation, DPA171 produced 4.1 g/L d-pantothenic acid, which is the highest titer in S. cerevisiae to date. This study provides guidance for the development of vitamin B5 microbial cell factories.

In vitro DPPH Radical Scavenging Activity of α-Pyrones from Odontonema strictum (Acanthaceae).

An ethyl acetate leaf extract from Odontonema strictum has been reported to have potent antihypertensive activity by inhibiting coronary artery contractions in porcine heart. However, the phytochemistry of the active fraction was unknown. Here we report, for the first time, the isolation and characterization of four known α-pyrones from the active fraction. The antioxidant activity of umuravumbolide (IC50 = 55.7±0.027 µg/mL), deacetylumuravumbolide (IC50 = 0.24±0.0002 µg/mL), dideacetylboronolide (IC50 = 149±0 µg/mL) and deacetylboronolide (IC50 = 24±0 µg/mL) was evaluated in vitro against 2,2-diphenyl-1-picrylhydrazyl radicals. Ascorbic acid was used as a positive control (IC50 = 1.73×10-3±0.3 µg/mL). The presence of 6-substituted 5,6-dihydro-α-pyrones and phenylpropanoid glucosides in the active fraction was suggested to be responsible for the antihypertensive activity. This is the first time that the antioxidant potential of these phytochemicals has been evaluated, and the results indicate that O. strictum has potential as an herbal medicine. Thus, further chemotaxonomic studies among the genera Odontonema and Tetradenia, a known source of α-pyrones, are recommended.

Pharmacological Activities of Safflower Yellow and Its Clinical Applications.

Background: Safflower is an annual herb used in traditional Chinese herbal medicine. It consists of the dried flowers of the Compositae plant safflower. It is found in the central inland areas of Asia and is widely cultivated throughout the country. Its resistance to cold weather and droughts and its tolerance and adaptability to salts and alkalis are strong. Safflower has the effect of activating blood circulation, dispersing blood stasis, and relieving pain. A natural pigment named safflower yellow (SY) can be extracted from safflower petals. Chemically, SY is a water-soluble flavonoid and the main active ingredient of safflower. The main chemical constituents, pharmacological properties, and clinical applications of SY are reviewed in this paper, thereby providing a reference for the use of safflower in preventing and treating human diseases. Methods: The literature published in recent years was reviewed, and the main chemical components of SY were identified based on chemical formula and structure. The pharmacological properties of hydroxysafflor yellow A (HSYA), SYA, SYB, and anhydrosafflor yellow B (AHSYB) were reviewed. Results: The main chemical constituents of SY included HSYA, SYA, SYB, and AHSYB. These ingredients have a wide range of pharmacological activities. SY has protective effects on the heart, kidneys, liver, nerves, lungs, and brain. Moreover, its effects include, but are not limited to, improving cardiovascular and cerebrovascular diseases, abirritation, regulating lipids, and treating cancer and diabetic complications. HSYA is widely recognised as an effective ingredient to treat cardiovascular and cerebrovascular diseases. Conclusion: SY has a wide range of pharmacological activities, among which improving cardiovascular and cerebrovascular diseases are the most significant.

Rapid Profiling of Chemical Constituents in Qingfei Paidu Granules Using High Performance Liquid Chromatography Coupled with Q Exactive Mass Spectrometry.

Qingfei Paidu (QFPD) granules have played a critical role during the Coronavirus Disease 2019 (COVID-19) in China. However, worldwide acceptance has been a problem because of the complex ingredients and unique theory of treatment. In this study, high-performance liquid chromatography (HPLC)-Q Exactive Orbitrap-mass spectrometry (MS) and the Orbitrap traditional Chinese medicine library (OTCML) were used to investigate the chemical constituents of QFPD granules. By comparing retention times, masses, isotope ion patterns, and MS2 profiles, 108 compounds were putatively identified using the OTCML combined with manual verification, including 12 alkaloids, 49 flavonoids, 13 terpenoids, 14 phenylpropanoids, 4 phenolic acids, 5 phenols, and 11 other phytochemicals. Of these compounds, 17 were confirmed using reference standards. In addition, representative compounds of these different chemical types were used as examples to analyze the fragmentation pathways and characteristic product ions. Moreover, 20 herbs within the QFPD granules were also identified to establish the sources of these chemical components. This is the first rapid profiling of the chemical constituents of QFPD granules using HPLC-Q Exactive Orbitrap-MS and yields valuable information for further quality control and mechanistic studies of QFPD granules. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10337-021-04085-0.

Qualitative analysis of chemical components in Lianhua Qingwen capsule by HPLC-Q Exactive-Orbitrap-MS coupled with GC-MS.

The Lianhua Qingwen (LHQW) capsule is a popular traditional Chinese medicine for the treatment of viral respiratory diseases. In particular, it has been recently prescribed to treat infections caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, due to its complex composition, little attention has been directed toward the analysis of chemical constituents present in the LHQW capsule. This study presents a reliable and comprehensive approach to characterizing the chemical constituents present in LHQW by high-performance liquid chromatography-Q Exactive-Orbitrap mass spectrometry (HPLC-Q Exactive-Orbitrap-MS) coupled with gas chromatography-mass spectrometry (GC-MS). An automated library alignment method with a high mass accuracy (within 5 ppm) was used for the rapid identification of compounds. A total of 104 compounds, consisting of alkaloids, flavonoids, phenols, phenolic acids, phenylpropanoids, quinones, terpenoids, and other phytochemicals, were successfully characterized. In addition, the fragmentation pathways and characteristic fragments of some representative compounds were elucidated. GC-MS analysis was conducted to characterize the volatile compounds present in LHQW. In total, 17 compounds were putatively characterized by comparing the acquired data with that from the NIST library. The major constituent was menthol, and all the other compounds were terpenoids. This is the first comprehensive report on the identification of the major chemical constituents present in the LHQW capsule by HPLC-Q Exactive-Orbitrap-MS, coupled with GC-MS, and the results of this study can be used for the quality control and standardization of LHQW capsules.

Two putative parallel pathways for naringenin biosynthesis in Epimedium wushanense.

Flavonoids that exhibit various biological activities such as antioxidant, antitumor, antiviral, antibacterial and anti-inflammatory properties are found in a wide range of medicinal plants. Among the flavonoid-producing plants identified so far, the genus Epimedium is recognised as a group of prolific prenyl-flavonoid glycoside producers with high economic value in the global dietary supplement market. To date, the biosynthetic genes for prenyl-flavonoid glycosides still remain elusive in Epimedium. Here, we identified five genes in Epimedium wushanense responsible for the biosynthesis of naringenin, the common precursor for flavonoid natural products. We successfully set up the biosynthetic pathway of naringenin using l-tyrosine as the precursor through enzymatic assays of these genes' encoding products, including phenylalanine ammonia-lyase (EwPAL), 4-coumarate-CoA ligase (Ew4CL1), chalcone synthase (EwCHS1), chalcone isomerase (EwCHI1) and CHI-like protein (EwCHIL3). Intriguingly, in vitro characterisation of the above catalytic enzymes' substrate specificity indicated a route parallel to naringenin biosynthesis, which starts from l-phenylalanine and ends in pinocembrin. The fact that there is no pinocembrin or pinocembrin-derived flavonoid accumulated in E. wushanense prompted us to propose that pinocembrin is likely converted into naringenin in vivo, constituting two parallel biosynthetic pathways for naringenin. Therefore, our study provides a basis for the full elucidation of the biosynthetic logic of prenyl-flavonoid glycoside in Epimedium, paving the way for future metabolite engineering and molecular breeding of E. wushanense to acquire a higher titre of desired, bioactive flavonoid compounds.

Defense Mechanism of Phosphorothioated DNA under Peroxynitrite-Mediated Oxidative Stress.

DNA phosphorothioation (PT) exists in many pathogenic bacteria; however, the mechanism of PT-DNA resistance to the immune response is unclear. In this work, we meticulously investigated the peroxynitrite (PN) tolerance using PT-bioengineered E. coli strains. The in vivo experiment confirms that the S+ strain survives better than the S- strain under moderately oxidative stress. The LCMS, IC, and GCMS experiments demonstrated that phosphorothioate partially converted to phosphate, and the byproduct included sulfate and elemental sulfur. When O,O-diethyl thiophosphate ester (DETP) was used, the reaction rate k1 was determined to be 4.3 ± 0.5 M-1 s-1 in the first-order for both phosphorothioate and peroxynitrite at 35 °C and pH of 8.0. The IC50 values of phosphorothioate dinucleotides are dramatically increased by 400-700-fold compared to DETP. The SH/OH Yin-Yang mechanism rationalizes the in situ DNA self-defense against PN-mediated oxidative stress at the extra bioenergetic cost of DNA modification.

Conjugated linoleic acid attenuates 2,4-dinitrofluorobenzene-induced atopic dermatitis in mice through dual inhibition of COX-2/5-LOX and TLR4/NF-κB signaling.

Conjugated linoleic acid (CLA), commonly found in beef, lamb and dairy products, has been reported to exhibit anti-inflammatory and antipruritus effects and to inhibit the release of chemical mediators such as histamine and eicosanoid in laboratory rodents. The chief objective of the study is to assess the efficacy of CLA on atopic dermatitis (AD) in mice and to explore possible mechanisms with CLA treatments. To develop a new therapy for AD, the anti-AD potential of CLA was investigated by inducing AD-like skin lesions in mice using 2,4-dinitrofluorobenzene. We evaluated dermatitis severity; histopathological changes; serum levels of T helper (Th) cytokines (interferon-γ, interleukin-4); changes in protein expression by western blotting and immunohistochemistry staining for cyclooxygenase-2 (COX-2), 5-lipoxygenase (5-LOX), toll like receptor 4 (TLR-4), myeloid differentiation factor 88 (MyD88), nuclear factor-κB (NF-κB) and tumor necrosis factor α (TNF-α); and production of the proinflammatory lipid mediators, such as prostaglandin E2 and leukotriene B4, in the skin lesions. Treatment with CLA ameliorated the development of AD-like clinical symptoms and effectively inhibited epidermal hyperplasia and infiltration of mast cells and CD4+ T cells in the AD mouse skin. Total serum immunoglobulin E levels and the expression levels of Th1/Th2 cytokines and lipid mediators in dorsal skin were dramatically suppressed by CLA. Furthermore, CLA down-regulated the expressions of COX-2, 5-LOX, TLR4, MyD88, NF-κB and TNF-α. Taken together, our findings demonstrate the potential usefulness of CLA as an anti-inflammatory dietary supplement or drug for the prevention and management of AD skin diseases by modulating the COX-2/5-LOX and TLR4/MyD88/NF-κB signaling pathways.

Characterization of an efficient estrogen-degrading bacterium Stenotrophomonas maltophilia SJTH1 in saline-, alkaline-, heavy metal-contained environments or solid soil and identification of four 17ß-estradiol-oxidizing dehydrogenases.

The efficient bioremediation of estrogen contamination in complex environments is of great concern. Here the strain Stenotrophomonas maltophilia SJTH1 was found with great and stable estrogen-degradation efficiency even under stress environments. The strain could utilize 17ß-estradiol (E2) as a carbon source and degrade 90% of 10 mg/L E2 in a week; estrone (E1) was the first degrading intermediate of E2. Notably, diverse pH conditions (3.0-11.0) and supplements of 4% salinity, 6.25 mg/L of heavy metal (Cd2+ or Cu2+), or 1 CMC of surfactant (Tween 80/ Triton X-100) had little effect on its cell growth and estrogen degradation. The addition of low concentrations of copper and Tween 80 even promoted its E2 degradation. Bioaugmentation of strain SJTH1 into solid clay soil achieved over 80% removal of E2 contamination (10 mg/kg) within two weeks. Further, the whole genome sequence of S. maltophilia SJTH1 was obtained, and a series of potential genes participating in stress-tolerance and estrogen-degradation were predicted. Four dehydrogenases similar to 17ß-hydroxysteroid dehydrogenases (17ß-HSDs) were found to be induced by E2, and the four heterogenous-expressed enzymes could oxidize E2 into E1 efficiently. This work could promote bioremediation appliance potential with microorganisms and biodegradation mechanism study of estrogens in complex real environments.

Hexafluoroisopropanol-based hydrophobic deep eutectic solvents for dispersive liquid-liquid microextraction of pyrethroids in tea beverages and fruit juices.

A series of new hydrophobic deep eutectic solvents (DESs), which are liquid at room temperature and have high density (>1.4 g mL-1), were synthesized using hexafluoroisopropanol (HFIP) as hydrogen-bond donor and l-carnitine/betaine as hydrogen-bond acceptor. Then these hydrophobic DESs were used as extraction solvents to establish dispersive liquid-liquid microextraction (DLLME) method for extraction of pyrethroids. The DES extraction phase was in the bottom after DLLME, being easy to be collected for analysis. After optimization by one-variable-at-a-time and response surface methodology, the enrichment factors of 265-360 were achieved for five pyrethroids. The proposed DLLME method coupled with HPLC has good performance: linear ranges of 0.25/0.5/1-100/200/400 ng/mL (r ≥ 0.9990), limits of detection of 0.06-0.17 ng mL-1, relative recoveries of 85.1-109.4%, intra-day and inter-day RSDs below 7.5%. The novel DLLME method is simple, rapid, highly efficient and eco-friendly for extraction of pyrethroids in real tea beverages and fruit juices.

Near-Infrared II Dye-Protein Complex for Biomedical Imaging and Imaging-Guided Photothermal Therapy.

The development of novel biodegradable and nontoxic fluorophores that integrate diagnosis and therapy for effective cancer treatment has obtained tremendous attention in the past decades. In this report, water-soluble and biocompatible small-molecule near-infrared II (NIR-II) fluorescent dye H2a-4T complexed with fetal bovine serum (FBS) and Cetuximab proteins with excellent optical properties and targeting ability is prepared. High spatial and temporal resolution imaging of hind limb vasculature and the lymphatic system of living mice using H2a-4T@FBS complex is demonstrated in precise NIR-II imaging-guided sentinel lymph node surgery. More importantly, H2a-4T@Cetuximab complex not only exhibits a remarkable cell-killing ability but also achieves highly active tumor targeting efficiency for epidermal growth factor receptor, overexpressing colorectal cancer which is beneficial to in vivo NIR-II fluorescent imaging-guided photothermal therapy of colon tumors. To the best of our knowledge, it is the first time that the concept of light-harvesting complex is exploited for enhancing the NIR-II signals and photothermal energy conversion in molecule-protein complex theranostic agent, making them a promising candidate for future clinical applications in cancer theranostics.

Biosynthesis of plant-derived ginsenoside Rh2 in yeast via repurposing a key promiscuous microbial enzyme.

Ginsenoside Rh2 is a potential anticancer drug isolated from medicinal plant ginseng. Fermentative production of ginsenoside Rh2 in yeast has recently been investigated as an alternative strategy compared to extraction from plants. However, the titer was quite low due to low catalytic capability of the key ginseng glycosyltransferase in microorganisms. Herein, we have demonstrated high-level production of ginsenoside Rh2 in Saccharomyces cerevisiae via repurposing an inherently promiscuous glycosyltransferase, UGT51. The semi-rationally designed UGT51 presented an ~1800-fold enhanced catalytic efficiency (kcat/Km) for converting protopanaxadiol to ginsenoside Rh2 in vitro. Introducing the mutant glycosyltransferase gene into yeast increased Rh2 production from 0.0032 to 0.39mg/g dry cell weight (DCW). Further metabolic engineering, including preventing Rh2 degradation and increasing UDP-glucose precursor supply, increased Rh2 production to 2.90mg/g DCW, which was more than 900-fold higher than the starting strain. Finally, fed-batch fermentation in a 5-L bioreactor led to production of ~300mg/L Rh2, which was the highest titer reported.

Genome mining of astaxanthin biosynthetic genes from Sphingomonas sp. ATCC 55669 for heterologous overproduction in Escherichia coli.

As a highly valued keto-carotenoid, astaxanthin is widely used in nutritional supplements and pharmaceuticals. Therefore, the demand for biosynthetic astaxanthin and improved efficiency of astaxanthin biosynthesis has driven the investigation of metabolic engineering of native astaxanthin producers and heterologous hosts. However, microbial resources for astaxanthin are limited. In this study, we found that the α-Proteobacterium Sphingomonas sp. ATCC 55669 could produce astaxanthin naturally. We used whole-genome sequencing to identify the astaxanthin biosynthetic pathway using a combined PacBio-Illumina approach. The putative astaxanthin biosynthetic pathway in Sphingomonas sp. ATCC 55669 was predicted. For further confirmation, a high-efficiency targeted engineering carotenoid synthesis platform was constructed in E. coli for identifying the functional roles of candidate genes. All genes involved in astaxanthin biosynthesis showed discrete distributions on the chromosome. Moreover, the overexpression of exogenous E. coli idi in Sphingomonas sp. ATCC 55669 increased astaxanthin production by 5.4-fold. This study described a new astaxanthin producer and provided more biosynthesis components for bioengineering of astaxanthin in the future.

Microbial production strategies and applications of lycopene and other terpenoids.

Terpenoids are a large class of compounds that have far-reaching applications and economic value, particularly those most commonly found in plants; however, the extraction and synthesis of these compounds is often expensive and technically challenging. Recent advances in microbial metabolic engineering comprise a breakthrough that may enable the efficient, cost-effective production of these limited natural resources. Via the engineering of safe, industrial microorganisms that encode product-specific enzymes, and even entire metabolic pathways of interest, microbial-derived semisynthetic terpenoids may soon replace plant-derived terpenoids as the primary source of these valuable compounds. Indeed, the recent metabolic engineering of an Escherichia coli strain that produces the precursor to lycopene, a commercially and medically important compound, with higher yields than those in tomato plants serves as a successful example. Here, we review the recent developments in the metabolic engineering of microbes for the production of certain terpenoid compounds, particularly lycopene, which has been increasingly used in pharmaceuticals, nutritional supplements, and cosmetics. Furthermore, we summarize the metabolic engineering strategies used to achieve successful microbial production of some similar compounds. Based on this overview, there is a reason to believe that metabolic engineering comprises an optimal approach for increasing the production of lycopene and other terpenoids.

Genome-scale screening of drug-target associations relevant to Ki using a chemogenomics approach.

The identification of interactions between drugs and target proteins plays a key role in genomic drug discovery. In the present study, the quantitative binding affinities of drug-target pairs are differentiated as a measurement to define whether a drug interacts with a protein or not, and then a chemogenomics framework using an unbiased set of general integrated features and random forest (RF) is employed to construct a predictive model which can accurately classify drug-target pairs. The predictability of the model is further investigated and validated by several independent validation sets. The built model is used to predict drug-target associations, some of which were confirmed by comparing experimental data from public biological resources. A drug-target interaction network with high confidence drug-target pairs was also reconstructed. This network provides further insight for the action of drugs and targets. Finally, a web-based server called PreDPI-Ki was developed to predict drug-target interactions for drug discovery. In addition to providing a high-confidence list of drug-target associations for subsequent experimental investigation guidance, these results also contribute to the understanding of drug-target interactions. We can also see that quantitative information of drug-target associations could greatly promote the development of more accurate models. The PreDPI-Ki server is freely available via: http://sdd.whu.edu.cn/dpiki.

Over-expression of UDP-glucose pyrophosphorylase increases validamycin A but decreases validoxylamine A production in Streptomyces hygroscopicus var. jinggangensis 5008.

During the fermentation of Streptomyces hygroscopicus TL01 to produce validamycin A (18 g/L), a considerable amount of an intermediate validoxylamine A (4.0 g/L) is accumulated. Chemical or enzymatic hydrolysis of validamycin A was not observed during the fermentation process. Over-expression of glucosyltransferase ValG in TL01 did not increase the efficiency of glycosylation. However, increased validamycin A and decreased validoxylamine A production were observed in both the cell-free extract and fermentation broth of TL01 supplemented with a high concentration of UDP-glucose. The enzymatic activity of UDP-glucose pyrophosphorylase (Ugp) in TL01, which catalyzes UDP-glucose formation, was found to be much lower than the activities of other enzymes involved in the biosynthesis of UDP-glucose and the glucosyltransferase ValG. An ugp gene was cloned from S. hygroscopicus 5008 and verified to code for Ugp. In TL01 with an extra copy of ugp, the transcription of ugp was increased for 1.5 times, and Ugp activity was increased by 100%. Moreover, 22 g/L validamycin A and 2.5 g/L validoxylamine A were produced, and the validamycin A/validoxylamine A ratio was increased from 3.15 in TL01 to 5.75. These data prove that validamycin A biosynthesis is limited by the supply of UDP-glucose, which can be relieved by Ugp over-expression.