Validamycin Research Articles

Enhanced Antifungal Efficacy of Validamycin A Co-Administered with Bacillus velezensis TCS001 against Camellia anthracnose

Anthracnose, a fungal disease harming fruit trees and crops, poses a threat to agriculture. Traditional chemical pesticides face issues like environmental pollution and resistance. A strategy combining low-toxicity chemicals with biopesticides is proposed to enhance disease control while reducing chemical use. Our study found that mixing validamycin A (VMA) and Bacillus velezensis TCS001 effectively controlled anthracnose in Camellia oleifera. The combination increased antifungal efficacy by 65.62% over VMA alone and 18.83% over TCS001 alone. It caused pathogen deformities and loss of pathogenicity. Transcriptomic analysis revealed that the mix affected the pathogen’s metabolism and redox processes, particularly impacting cellular membrane functions and inducing apoptosis via glycolysis/gluconeogenesis. In vivo tests showed the treatment activated C. oleifera’s disease resistance, with a 161.72% increase in polyphenol oxidase concentration in treated plants. This research offers insights into VMA and TCS001’s mechanisms against anthracnose, supporting sustainable forestry and national edible oil security.

Exploring the sorption and degradation dynamics of validamycin-A in agricultural soils for environmental management.

Validamycin A (VA) is one of the antibiotics that have been utilized in agriculture in Asia; nevertheless, there haven't been many investigations on what happens to VA in soil. The rate at which pesticides are adsorbed into the soil must be determined, since their usage in agriculture is growing. In order to accomplish this, the current study investigated the sorption and degradation of VA in ten distinct soil samples via batch equilibrium studies while maintaining strict laboratory controls. In thermodynamic analysis with a C-type curve, the negative values of Gibbs free energy (ΔG) are thoroughly evaluated using both linear and Freundlich models. These values vary from - 16.8 to - 22.2kJ/mol. Impact of temperature (18, 23, and 30°C) and pH (5, 7, and 9) on the degradation of this antibiotic in soil was also scrutinized. Our findings demonstrated that, as a result of enhanced microbial activity at higher temperatures, VA deteriorated more quickly at 23°C and 30°C than at 18°C. In comparison to lower pH values, the VA removal efficiencies with sample-4 was significantly greater at pH 7.4 (92.9%) and pH 9 (97.4%). Moreover, first order reaction kinetics were followed in the degradation of VA. The results demonstrated that VA bound to the selected soils, resulting in medium to low persistence as demonstrated by degradation values. In summary, this study provides important information regarding the behavior and fate of VA in different types of soil, information that might be useful in developing workable management strategies and environmental risk assessments.

Validamycin A Inhibited FB1 Biosynthesis by the Target FvNth in Fusarium verticillioides.

Validamycin A (VMA) is an antifungal antibiotic derived from Streptomyces hygroscopicus commonly used in plant disease management. Surprisingly, VMA was discovered to impede the production of fumonisin B1 (FB1) in agricultural settings. However, the specific target of VMA in Fusarium verticillioides remained unclear. To unravel the molecular mechanism of VMA, ultrastructural observations unveiled damage to mitochondrial membranes. Trehalase (FvNth) was pinpointed as the target of VMA by utilizing a 3D-printed surface plasmon resonance sensor. Molecular docking identified Trp285, Arg447, Asp452, and Phe665 as the binding sites between VMA and FvNth. A ΔFvnth mutant lacking amino acids 250-670 was engineered through homologous recombination. Transcriptome analysis indicated that samples treated with VMA and ΔFvnth displayed similar expression patterns, particularly in the suppression of the FUM gene cluster. VMA treatment resulted in reduced trehalase and ATPase activity as well as diminished production of glucose, pyruvic acid, and acetyl-CoA. Conversely, these effects were absent in samples treated with ΔFvnth. This research proposes that VMA hinders acetyl-CoA synthesis by trehalase, thereby suppressing the FB1 biosynthesis. These findings present a novel target for the development of mycotoxin control agents.

Assessment of the Novel Anti-Seizure Potential of Validamycin A Using Zebrafish Epilepsy Model.

Epilepsy is a prevalent neurological disorder characterized by recurrent seizures. Validamycin A (VA) is an antibiotic fungicide that inhibits trehalase activity and is widely used for crop protection in agriculture. In this study, we identified a novel function of VA as a potential anti-seizure medication in a zebrafish epilepsy model. Electroencephalogram (EEG) analysis demonstrated that VA reduced pentylenetetrazol (PTZ)-induced seizures in the brains of larval and adult zebrafish. Moreover, VA reduced PTZ-induced irregular movement in a behavioral assessment of adult zebrafish. The developmental toxicity test showed no observable anatomical alteration when the zebrafish larvae were treated with VA up to 10 µM within the effective range. The median lethal dose of VA in adult zebrafish was > 14,000 mg/kg. These results imply that VA does not demonstrate observable toxicity in zebrafish at concentrations effective for generating anti-seizure activity in the EEG and alleviating abnormal behavior in the PTZ-induced epileptic model. Furthermore, the effectiveness of VA was comparable to that of valproic acid. These results indicate that VA may have a potentially safer anti-seizure profile than valproic acid, thus offering promising prospects for its application in agriculture and medicine.

Effect of pH and temperature on the biodegradation of oxytetracycline, streptomycin, and validamycin A in soil

Residual antibiotics in agricultural soils can be of concern due to the development of antibiotic resistant microorganisms. Among various antibiotics, oxytetracycline (OTC), streptomycin (ST), and validamycin A (VA) have been used for agricultural purposes in South Korea; however, studies on the biodegradation of these antibiotics in soil are limited. Therefore, this study investigated the effects of pH (5.5, 6.8, and 7.4) and temperature (1.8, 23.0, and 31.2 °C) conditions on the biodegradation of these antibiotics in soil. The biodegradation tests were carried out in the field soil (FS) and rice paddy soil (RS) for 30 d with OTC and ST and 10 d with VA, and the residual antibiotics concentrations were monitored over the degradation period. Under various conditions, the degradation rates of ST was lower (11–69%) than that of OTC (60–90%) and VA (15–96%). The degradation half-lives of OTC and VA tend to decrease with increasing pH value, while the degradation half-life of ST tend to increase with increasing pH value. But, the effect of soil pH on the antibiotics degradation was not statistically significant, except for ST in the FS and RS and VA in the FS. The degradation of three antibiotics was greater at higher temperatures (23.0 °C and 31.2 °C) than at lower temperature (1.8 °C), and the degradation half-lives decreased with increasing temperature. The different degradation characteristics of different antibiotics in soil can be explained by the different characteristics of the antibiotics (e.g., sorption affinity, chemical forms) and soil (e.g., organic matter content). The results suggest that the degradation characteristics of antibiotics need to be considered in order to properly manage the residual antibiotics in soil.

Mechanism of validamycin A inhibiting DON biosynthesis and synergizing with DMI fungicides against Fusarium graminearum.

Deoxynivalenol (DON) is a vital virulence factor of Fusarium graminearum, which causes Fusarium head blight (FHB). We recently found that validamycin A (VMA), an aminoglycoside antibiotic, can be used to control FHB and inhibit DON contamination, but its molecular mechanism is still unclear. In this study, we found that both neutral and acid trehalase (FgNTH and FgATH) are the targets of VMA in F. graminearum, and the deficiency of FgNTH and FgATH reduces the sensitivity to VMA by 2.12‐ and 1.79‐fold, respectively, indicating that FgNTH is the main target of VMA. We found FgNTH is responsible for vegetative growth, FgATH is critical to sexual reproduction, and both of them play an important role in conidiation and virulence in F. graminearum. We found that FgNTH resided in the cytoplasm, affected the localization of FgATH, and positively regulated DON biosynthesis; however, FgATH resided in vacuole and negatively regulated DON biosynthesis. FgNTH interacted with FgPK (pyruvate kinase), a key enzyme in glycolysis, and the interaction was reduced by VMA; the deficiency of FgNTH affected the localization of FgPK under DON induction condition. Strains with a deficiency of FgNTH were more sensitive to demethylation inhibitor (DMI) fungicides. FgNTH regulated the expression level of FgCYP51A and FgCYP51B by interacting with FgCYP51B. Taken together, VMA inhibits DON biosynthesis by targeting FgNTH and reducing the interaction between FgNTH and FgPK, and synergizes with DMI fungicides against F. graminearum by decreasing FgCYP51A and FgCYP51B expression.

Field Evaluation of Chemotherapy on HLB-Affected Citrus Trees With Emphasis on Fruit Yield and Quality.

Huanglongbing (HLB) is one of the most devastating diseases of citrus, which is associated with Candidatus Liberibacter asiaticus (Las) in the United States. To date, no effective antimicrobial compound is commercially available to control the disease. In this study, we investigated the effects of different antimicrobial chemicals with suitable surfactants on HLB-affected matured citrus trees with emphasis on the fruit yield and quality. Each treatment was applied three times in a 2-week interval during the spring flush period, one time in summer and three times during the autumn flushing period. We extensively examined different parameters such as pathogenic index, disease index, tree canopy, fruit yield, quality, and nutritional status. The results showed that among the treatments, penicillin (PEN) with surfactant was most effective in suppressing Las titer in infected citrus trees, followed by Fosetyl-Al (ALI), Carvacrol (CARV), and Validamycin (VA). Fruit quality analysis revealed that PEN treatment increased the soluble solids content (SSC), whereas Oxytetracycline (OXY) treatment significantly reduced titratable acidity (TA) level and increased the SSC/TA ratio compared to the control. Nutrient analysis showed increased N and Zn levels in ALI and PEN treatments, and OXY treatment increased leaf P, K, S, and Mg levels compared to untreated control. Furthermore, B, Ca, Cu, Fe, and Mn in leaves were reduced in all chemical treatments than that of the untreated control. These findings revealed that some of the chemical treatments were able to suppress Las pathogen, enhance nutritional status in leaves, and improve tree growth and fruit quality of HLB-affected trees.

Validamycin A Induces Broad-Spectrum Resistance Involving Salicylic Acid and Jasmonic Acid/Ethylene Signaling Pathways

Validamycin A (VMA) is an aminoglycoside antibiotic used to control rice sheath blight. Although it has been reported that VMA can induce the plant defense responses, the mechanism remains poorly understood. Here, we found that reactive oxygen species (ROS) bursts and callose deposition in Arabidopsis thaliana, rice (Oryza sativa L.), and wheat (Triticum aestivum L.) were induced by VMA and were most intense with 10 μg of VMA per milliliter at 24 h. Moreover, we showed that VMA induced resistance against Pseudomonas syringae, Botrytis cinerea, and Fusarium graminearum in Arabidopsis leaves, indicating that VMA induces broad-spectrum disease resistance in both dicots and monocots. In addition, VMA-mediated resistance against P. syringae was not induced in NahG transgenic plants, was partially decreased in npr1 mutants, and VMA-mediated resistance to B. cinerea was not induced in npr1, jar1, and ein2 mutants. These results strongly indicated that VMA triggers plant defense responses to both biotrophic and necrotrophic pathogens involved in salicylic acid (SA) and jasmonic acid/ethylene (JA/ET) signaling pathways and is dependent on NPR1. In addition, transcriptome analysis further revealed that VMA regulated the expression of genes involved in SA, JA/ET, abscisic acid (ABA), and auxin signal pathways. Taken together, VMA induces systemic resistance involving in SA and JA/ET signaling pathways and also exerts a positive influence on ABA and auxin signaling pathways. Our study highlights the creative application of VMA in triggering plant defense responses against plant pathogens, providing a valuable insight into applying VMA to enhance plant resistance and reduce the use of chemical pesticides.[Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Effect of furfural addition on validamycin-A production in fermentation of Streptomyces hygroscopicus 5008

Furfural is one of main inhibitors in hemicellulose hydrolysates such as xylose mother liquor, but its positive effect on the production of validamycin-A (VAL-A), a widely used agricultural antibiotic, was interestingly found in fermentation of Streptomyces hygroscopicus 5008. The furfural level in medium up to 1 g/L was effectively converted to furfuryl alcohol and furoic acid by the microorganism. Both intracellular H2O2 level and ValG enzyme activity of the cells were enhanced by furfural addition. Xylose mother liquor medium with supplementation of about 1 g/L furfural could enhance the VAL-A titer by 39 %. This work is helpful to VAL-A fermentation using the hemicellulose hydrolysate.

Positive and negative regulation of GlnR in validamycin A biosynthesis by binding to different loci in promoter region.

Validamycin A (VAL-A) is a C7N aminocyclitol antibiotic produced by Streptomyces hygroscopicus var. jinggangensis 5008, which has been widely used as antifungal agent against rice sheath blight disease. VAL-A biosynthesis has been proven to be affected by γ-butyrolactone and temperature. Herein, we showed that GlnR, a global regulator in nitrogen metabolism, is specifically associated with valK-valA intergenic promoter region by DNA-affinity chromatography and MS-based protein identification. Subsequent EMSA and DNase I footprinting assays revealed two GlnR binding sites in this promoter region. Targeted disruption of glnR in S. hygroscopicus 5008 led to a significant increase in the transcription of VAL-A structural genes, albeit the VAL-A production was reduced by 80% and the sporulation of the mutant was impaired. Compared with the wild-type 5008, site-specific mutagenesis of GlnR binding site I enhanced VAL-A production by 2.5-fold, whereas the mutation of GlnR binding site II resulted in a 50% reduction of VAL-A yield. Moreover, tandem mutation of site I in the site II mutant led to a 66% increase of VAL-A production. The result suggested that GlnR not only serves as an inhibitor by binding site I but also as an activator by binding site II for VAL-A biosynthesis. Furthermore, overexpression of glnR in the site I mutant JG45 improved VAL-A production for 41% compared with the control strain containing the vector. Therefore, the obtained data illustrate a novel regulatory feature of the global regulator GlnR. GlnR is firstly proved to act simultaneously as an activator and a repressor in validamycin biosynthesis by binding to different loci within a promoter region of the gene cluster.

Production of validamycin A from hemicellulose hydrolysate by Streptomyces hygroscopicus 5008

Validamycin A (VAL-A) is an important agricultural antibiotic produced by Streptomyces hygroscopicus 5008, which uses starch as carbon source occupying about 20% of total production cost. To reduce the medium cost, corncob hydrolysate – a hemicellulose hydrolysate was applied as a low-cost substrate to VAL-A fermentation. It was found that three major sugars in corncob hydrolysate including d-glucose, d-xylose and l-arabinose could all be utilized by S. hygroscopicus 5008 to produce VAL-A while d-xylose was the main contributor. A higher VAL-A production titer from d-xylose was achieved by using a genetically engineered strain TC03 derived from S. hygroscopicus 5008, which resulted in 1.27-fold improvement of VAL-A production from the medium containing 13% (v/v) corncob hydrolysate compared to that by its original strain. A medium cost analysis was done and compared with previous reports. This work indicates a great potential of the hemicellulose hydrolysate as substrate for antibiotic fermentation.

Enhanced production of validamycin A in Streptomyces hygroscopicus 5008 by engineering validamycin biosynthetic gene cluster.

Validamycin A (VAL-A) is a widely used antifungal antibiotic for the treatment of sheath blight disease of rice and other plants. It can be produced from agro-industrial by-products by Streptomyces hygroscopicus 5008. To enhance its production titer, in this work, the entire val gene cluster was amplified in tandem in S. hygroscopicus 5008 by integrating the zouA-mediated DNA amplification system into between the two boundaries of val gene cluster, resulting in multiple copies (mainly three to five) of the val gene cluster. The genetic stability of the amplified copies was confirmed by Southern blot and fermentation experiments. In shake flask fermentation, the recombinant strain (TC03) led to a 34% enhancement of VAL-A production titer compared to that of the wild-type strain, while the accumulation of intermediate validoxylamine A was decreased in TC03. Additionally, both the structural gene transcription levels and the ValG enzyme activity were significantly increased in TC03. This work demonstrated that the amplification of the val gene cluster was an efficient strategy to enhance VAL-A production by S. hygroscopicus 5008, and the information obtained would be helpful for engineering other interesting antibiotic biosynthesis by gene cluster amplification.

Analysis of validamycin as a potential antifungal compound against Candida albicans.

Validamycin A has been successfully applied in the fight against phytopathogenic fungi. Here, the putative antifungal effect of this pseudooligosaccharide against the prevalent human pathogen Candida albicans was examined. Validamycin A acts as a potent competitive inhibitor of the cell-wall-linked acid trehalase (Atc1p). The estimated MIC50 for the C. albicans parental strain CEY.1 was 500 mg/l. The addition of doses below MIC50 to exponentially growing CEY.1 cells caused a slight reduction in cell growth. A concentration of 1 mg/ml was required to achieve a significant degree of cell killing. The compound was stable as evidenced by the increased reduction of cell growth with increasing incubation time. A homozygous atc1delta/atc1delta mutant lacking functional Atc1p activity showed greater resistance to the drug. The antifungal power of validamycin A was limited compared with the drastic lethal action caused by exposure to amphotericin B. The endogenous content of trehalose rose significantly upon validamycin and amphotericin B addition. Neither serum-induced hypha formation nor the level of myceliation recorded in macroscopic colonies were affected by exposure to validamycin A. Our results suggest that, although validamycin A cannot be considered a clinically useful antifungal against C. albicans, its mechanism of action and antifungal properties provide the basis for designing new, clinically interesting, antifungal-related compounds.

Exogenous 1,4‐butyrolactone stimulates A‐factor‐like cascade and validamycin biosynthesis in Streptomyces hygroscopicus 5008

γ-Butyrolactones (GBLs), such as A-factor, are one type of signaling molecules produced by Streptomyces species and have been reported to regulate secondary metabolism. However, they are usually produced in very small amount, which has hindered their structural elucidation and application for antibiotic overproduction. In this work, 1,4-butyrolactone (1,4-BL), as an easily accessible and cheap analogue of GBLs, was applied to the fermentation of validamycin A (VAL-A), an important antifungal antibiotic produced by Streptomyces hygroscopicus 5008. The addition of 1,4-BL enhanced VAL-A production by 30% in both shaking flasks and bioreactors. The transcriptional levels of the adpA homologue (adpA-H) and VAL-A biosynthetic genes were significantly increased. Among the three A-factor receptor homologous genes identified in the genome of S. hygroscopicus 5008, shbR3 was proved to be responsible for the inducing activity of 1,4-BL by gene disruption and circular dichroism analysis, and ShbR3 could bind to the promoter region of adpA-H as indicated by EMSA analysis. Furthermore, the mutation of adpA-H abolished the transcription of VAL-A biosynthetic genes and VAL-A productivity. In EMSA analysis, AdpA-H could directly bind to the promoter regions of VAL-A gene cluster. Moreover, addition of the 1,4-BL also improved the VAL-A production in a high-yielding strain TL01. The results showed that 1,4-BL could stimulate A-factor-like cascade and subsequently enhance VAL-A production in S. hygroscopicus 5008.

Temperature shift-induced reactive oxygen species enhanced validamycin A production in fermentation of Streptomyces hygroscopicus 5008

In order to enhance the production of validamycin A (VAL-A), a widely used agricultural antibiotic, a temperature shift strategy was developed in the fermentation of Streptomyces hygroscopicus 5008. VAL-A production and the transcriptional levels of its structural genes were enhanced in the optimal temperature shift condition. The addition of diphenyleneiodonium [DPI, reactive oxygen species (ROS) inhibitor] inhibited intracellular ROS level and VAL-A production, which indicated that ROS signal might contribute to the enhancement of VAL-A production in the temperature shift process. The transcriptional levels of stress response sigma factors SigmaB and SigmaH as well as global regulator PhoRP were enhanced, which suggested that these regulators might participate in the signal pathway. This study developed a useful strategy for VAL-A production. It will help to further understand the regulation mechanism of ROS on VAL-A synthesis. The involvement of ROS in this process will encourage researchers to develop new ROS induction strategies to enhance VAL-A production.

Impact of nitrogen concentration on validamycin A production and related gene transcription in fermentation of Streptomyces hygroscopicus 5008

Validamycin A (VAL-A) is an important and widely used agricultural antibiotic. In this study, statistical screening designs were applied to identify significant medium variables for VAL-A production and to find their optimal levels. The optimized medium caused 70% enhancement of VAL-A production. The difference between optimized medium and original medium suggested that low nitrogen source level might attribute to the enhancement of VAL-A production. The addition of different nitrogen sources to the optimized medium inhibited VAL-A production, which confirmed the importance of nitrogen concentration for VAL-A production. Furthermore, differences in structural gene transcription and enzyme activity between the two media were assayed. The results showed that lower nitrogen level in the optimized medium could regulate VAL-A production in gene transcriptional level. Our previous study indicated that the transcription of VAL-A structural genes could be enhanced at elevated temperature. In this work, the increased fermentation temperature from 37 to 42 °C with the optimized medium enhanced VAL-A production by 39%, which testified to the importance of structural gene transcription in VAL-A production. The information is useful for further VAL-A production enhancement.

Enhanced production of validamycin A by H2O2-induced reactive oxygen species in fermentation of Streptomyces hygroscopicus 5008

A novel fermentation strategy to enhance antibiotics production was demonstrated by inducing reactive oxygen species (ROS), and validamycin A (VAL-A) production by Streptomyces hygroscopicus 5008 in agro-industrial residues containing medium was taken as an example. By optimizing H2O2 amount and addition time, the intracellular ROS level was increased, and VAL-A production titer was enhanced by 40% on day 4 when 25 μM H2O2 was added at 8th h of fermentation. Addition of diphenyleneiodonium chloride (ROS inhibitor) reduced the H2O2 induction effect. The transcription level of eight VAL-A structure genes was enhanced by ROS, and activities of glucose-6-phosphate dehydrogenase and ValG enzyme were increased while glyceraldehyde 3-phosphate dehydrogenase activity was inhibited. This work demonstrated that ROS induction was a useful strategy for VAL-A fermentation, and the information on gene transcription and enzyme activities may be helpful to further understanding the mechanism of ROS effect on the antibiotic biosynthesis.

Inhibitory effects of validamycin compounds on the termites trehalase

Trehalase, with the target to control insects, nematodes and fungi, is of increasing interest and has been investigated extensively in recent years. Validamycin compounds, as competitive trehalase inhibitors and lead compounds with broad applications have attracted substantial attention as well. In this study, the characterizations of termites trehalase were investigated and the inhibitory effects of validamycin compounds on the termites trehalase were studied as well. Results showed that the termites trehalase is presumably belonging to the acid trehalase with optimal pH of 3.3 and optimal temperature of 37 °C. It was investigated that the concentrations of validoxylamine A (VAA), validoxylamine B (VBB), validamycin A (VA) and validamycin B (VB) required for 50% inhibition IC 50 of termites trehalase were calculated to be 14.73 mg l −1, 20.80 mg l −1, 3.17 × 10 3 mg l −1and 2.24 × 10 3 mg l −1, respectively. The inhibition kinetic constant K i values for the above validamycin compounds were 3.2 × 10 −6 mol l −1, 1.03 × 10 −5 mol l −1 , 4.02 × 10 −4 mol l −1and 2.69 × 10 −4 mol l −1, respectively. Validoxylamine A appeared to be the most potential termites trehalase inhibitor among the four compounds.

Effect of fermentation temperature on validamycin A production by Streptomyces hygroscopicus 5008

Validamycin A (VAL-A), produced by Streptomyces hygroscopicus, is an important anti-fungal agro-antibiotic. In this work, the effect of fermentation temperature on VAL-A biosynthesis by S. hygroscopicus 5008 was investigated between 28 °C and 42 °C, and an interesting threshold of temperature for VAL-A biosynthesis was found between 35 °C and 37 °C. At a relatively higher temperature, a much higher VAL-A productivity was obtained together with faster protein synthesis and sugar consumption. Transcriptional analysis of samples from early, middle and late stages of fermentation at various temperatures demonstrated that three operons, valABC, valKLMN and valG, for all eight necessary structure genes, were dramatically promoted when temperature reached the threshold. Activities of both glucose-6-phosphate dehydrogenase (G6PDH) of pentose-phosphate pathway and ValG of VAL-A biosynthesis were also enhanced at a higher cultivation temperature. The interesting temperature effect with a 2 °C threshold shift from 35 °C to 37 °C on the antibiotic biosynthesis was understood to be related to the gene transcriptional levels and key enzyme activities.

Enhanced validamycin production and gene expression at elevated temperature in Streptomyces hygroscopicus subsp. jingangensis 5008

Cultivation shift from 30° to 37° significantly enhanced validamycin (VAL) production. Analyzed by reverse-transcription PCR, the transcription of three val genes, valA, valK and valG, representing the three operons of the cluster was simultaneously increased at elevated temperature. Furthermore, the transcription of valP and valQ, a pair of two-component regulators in validamycin biosynthetic gene cluster, was also increased at 37°. Inactivation of valP and valQ reduced validamycin production at 37° to the yield level of wild type strain at 30°, and the val genes showed reduced expression in the mutant LL-8 at 37°. These results revealed that the two-component regulator valP and valQ contribute to the elevated validamycin production.