Directed production of aurantizolicin and new members based on a YM-216391 biosynthetic system.

Using a highly effective heterologous expression system, the YM-216391 (1) biosynthetic gene cluster was engineered to yield aurantizolicin (2) and the hybrid compound 3. Both of these compounds were isolated and fully structurally characterised and the bioactivity was evaluated. The results indicate that compound 3 exhibits significantly improved antitumor activity.

A Six-Oxidase Cascade for Tandem C-H Bond Activation Revealed by Reconstitution of Bicyclomycin Biosynthesis.

As a commercial antibiotic, bicyclomycin (BCM) is currently the only known natural product targeting the transcription termination factor rho. It belongs to a family of highly functionalized diketopiperazine (DKP) alkaloids and bears a unique O-bridged bicyclo[4.2.2]piperazinedione ring system, a C1 triol, and terminal exo-methylene groups. We have identified and characterized the BCM biosynthetic pathway by heterologous biotransformations, in vitro biochemical assays, and one-pot enzymatic synthesis. A tRNA-dependent cyclodipeptide synthase guides the heterodimerization of leucine and isoleucine to afford the DKP precursor; subsequently, six redox enzymes, including five α-ketoglutarate/Fe2+ -dependent dioxygenases and one cytochrome P450 monooxygenase, regio- and stereoselectively install four hydroxy groups (primary, secondary, and two tertiary), an exo-methylene moiety, and a medium-sized bridged ring through the functionalization of eight unactivated C-H bonds.

Hijacking a hydroxyethyl unit from a central metabolic ketose into a nonribosomal peptide assembly line.

Nonribosomal peptide synthetases (NRPSs) usually catalyze the biosynthesis of peptide natural products by sequential selection, activation, and condensation of amino acid precursors. It was reported that some fatty acids, α-ketoacids, and α-hydroxyacids originating from amino acid metabolism as well as polyketide-derived units can also be used by NRPS assembly lines as an alternative to amino acids. Ecteinascidin 743 (ET-743), naphthyridinomycin (NDM), and quinocarcin (QNC) are three important antitumor natural products belonging to the tetrahydroisoquinoline family. Although ET-743 has been approved as an anticancer drug, the origin of an identical two-carbon (C(2)) fragment among these three antibiotics has not been elucidated despite much effort in the biosynthetic research in the past 30 y. Here we report that two unexpected two-component transketolases (TKases), NapB/NapD in the NDM biosynthetic pathway and QncN/QncL in QNC biosynthesis, catalyze the transfer of a glycolaldehyde unit from ketose to the lipoyl group to yield the glycolicacyl lipoic acid intermediate and then transfer the C(2) unit to an acyl carrier protein (ACP) to form glycolicacyl-S-ACP as an extender unit for NRPS. Our results demonstrate a unique NRPS extender unit directly derived from ketose phosphates through (α,ß-dihydroxyethyl)-thiamin diphosphate and a lipoyl group-tethered ester intermediate catalyzed by the TKase-ACP platform in the context of NDM and QNC biosynthesis, all of which also highlights the biosynthesis of ET-743. This hybrid system and precursor are distinct from the previously described universal modes involving the NRPS machinery. They exemplify an alternate strategy in hybrid NRPS biochemistry and enrich the diversity of precursors for NRPS combinatorial biosynthesis.

Dietary Immunoglobulin Y by Targeting Both GbpB and GtfB Enhances the Anticaries Effect in Rats.

OBJECTIVE: The purpose of this work was to develop an anti-CAT-SYI immunoglobulin Y (IgY) antibody that targeted both GtfB (glucosyltransferase B) and GbpB (glucan-binding protein B) and test its anticaries properties in rats. METHODS: A new CAT-SYI fusion gene was created utilising functional DNA fragments from the GtfB and GbpB genes. The recombinant antigens, comprising the fused CAT-SYI antigen, GtfB, and GbpB, were expressed and purified using a prokaryotic expression and purification system. The purified recombinant antigens were utilised to immunise laying hens against particular IgY production. The biological activities of these particular IgY antibodies were then assessed both in vitro and in vivo, including their capacity to suppress biofilm formation and tooth caries. RESULTS: Results indicated that these produced IgY antibodies demonstrated a high antibody titer (>0.1 µg/mL) and could precisely recognise and bind to their respective antigens. Furthermore, it was discovered that these specific IgY antibodies successfully bind to Streptococcus mutans and significantly reduce biofilm development. After 8 weeks of ingesting antigen-specific IgY meals, comprising anti-GtfB IgY and anti-GbpB IgY, the severity of dental caries was dramatically reduced in S mutans-infected Sprague-Dawley rats (P < .01). Anti-CAT-SYI IgY therapy significantly reduced tooth cavities by 89.0% in vivo (P < .05) compared to other treatment groups. CONCLUSIONS: The anti-CAT-SYI IgY, a multitarget antibody that targets both GtfB and GbpB, displayed excellent inhibitory effects against S mutans, making it a promising targeted method with improved anticaries efficacy and significant application opportunities.

Economic burden attributable to healthcare-associated infections at western China hospitals: 6 Year, prospective cohort study.

OBJECTIVES: Healthcare-associated infections (HAIs) represent a major threat to patient safety and are associated with significant economic burden. Calculating the costs attributable to HAIs is challenging given the various sources of bias. Although HAIs as a reasonably preventable medical harm should have been closely linked to medical insurance incentives, there was little linkage between HAIs and medicare in western China owing to the lack of economic evaluation data. The present study aimed to generate estimates of the attributable costs associated with HAIs and the magnitude of costs growth. METHODS: In this cohort study designed horizontally and vertically from 2016 to 2022, we compared outcomes of randomly sampling patients with HAIs and individually matched patients without HAIs in two cohorts at a 6-year interval at 34 hospitals in western China. The primary outcome was the direct medical cost for the entire hospital stay, converted to US dollars ($ for the benchmark year), discounted at 3% annually, and estimated separately in the full analysis set (FAS) and the per protocol set (PPS). We used multiple linear regression to adjust the discounted costs and to assess subgroups effects within each cohort. We nested a dynamic vertical comparison of costs attributable to HAIs between the front and rear cohorts. RESULTS: A total of 230 patients with HAIs in 2016 and 204 patients with HAIs in 2022 were enrolled. After a 1:1 match, all 431 pairs were recruited as FAS, of which 332 pairs as PPS met all matching restrictions. Compared to the 2016 cohort in FAS, the patients with HAIs in 2022 had a significantly older age (64.40 ± 16.45 years), higher repeat hospitalization rate (65 [32.02%] of 203), and lower immune function (69 [33.99%] of 203). The discounted costs and adjusted-discounted costs for patients with HAIs in the 2022 cohort were found to be significantly higher than those of patients without HAIs (discounted costs: $5484.60 [IQR 8426.03] vs $2554.04(4530.82), P < 0.001; adjusted-discounted costs: $5235.90 [3772.12] vs $3040.21(1823.36), P < 0.001, respectively), and also higher than those of patients with HAIs in the 2016 cohort (discounted costs: $5484.60 [8426.03] vs $3553.00 [6127.79], P < 0.001; adjusted-discounted costs: $5235.90 [3772.12] vs $3703.82 [3159.14], P < 0.001, respectively). In vertical comparison of PPS, the incremental costs of the 2022 cohort are 1.48 times higher than those of the 2016 cohort ($964.63(4076.15) vs $652.43 [2533.44], P = 0.084). CONCLUSIONS: This meticulously designed study in western China has successfully and accurately examined the economic burden attributable to HAIs. Their rapidly increasing tendency poses a serious challenge to patients, hospitals, and the medical insurance. A closer linkage between HAIs and ongoing motivating system changes is urgently needed in western China.

Recent progress in understanding mitokines as diagnostic and therapeutic targets in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most prevalent tumors worldwide and the leading contributor to cancer-related deaths. The progression and metastasis of HCC are closely associated with altered mitochondrial metabolism, including mitochondrial stress response. Mitokines, soluble proteins produced and secreted in response to mitochondrial stress, play an essential immunomodulatory role. Immunotherapy has emerged as a crucial treatment option for HCC. However, a positive response to therapy is typically dependent on the interaction of tumor cells with immune regulation within the tumor microenvironment. Therefore, exploring the specific immunomodulatory mechanisms of mitokines in HCC is essential for improving the efficacy of immunotherapy. This study provides a comprehensive overview of the association between HCC and the immune microenvironment and highlights recent progress in understanding the involvement of mitochondrial function in preserving liver function. In addition, a systematic review of mitokines-mediated immunomodulation in HCC is presented. Finally, the potential diagnostic and therapeutic roles of mitokines in HCC are prospected and summarized. Recent progress in mitokine research represents a new prospect for mitochondrial therapy. Considering the potential of mitokines to regulate immune function, investigating them as a relevant molecular target holds great promise for the diagnosis and treatment of HCC.

Heterologous characterization of mechercharmycin A biosynthesis reveals alternative insights into post-translational modifications for RiPPs.

Mechercharmycin A (MCM-A) is a marine natural product belonging to a family of polyazole cyclopeptides with remarkable bioactivities and unique structures. Identification, heterologous expression, and genetic characterizations of the MCM biosynthetic gene cluster in Bacillus subtilis revealed that it is a ribosomally synthesized and post-translationally modified peptide (RiPP) possessing complex with distinctive modifications. Based on this heterologous expression system, two MCM analogs with comparable antitumor activity are generated by engineering the biosynthetic pathway. Combinatorial co-production of a precursor peptide with different modifying enzymes in Escherichia coli identifies a different timing of modifications, showing that a tRNAGlu-dependent highly regioselective dehydration is the first modification step, followed by polyazole formation through heterocyclization and dehydrogenation in an N- to C-terminal direction. Therefore, a rational biosynthetic pathway of MCMs is proposed, which unveils a subfamily of azol(in)e-containing RiPPs and sets the stage for further investigations of the enzymatic mechanism and synthetic biology.

Atractylenolide III reduces depressive- and anxiogenic-like behaviors in rat depression models.

Atractylenolide III, a major component of the atractylodes macrocephala Koidz, derived from the rhizoma atractylodes, has been reported to produce various pharmacological effects including anti-aging, anti-inflammation, anti-tumor, and other effects. Growing evidence suggests that proinflammatory cytokines, such as interleukin (IL)-1, IL-6 and tumor necrosis factor (TNF)-α, are increased in depressed patients. The present study was aimed at investigating the antidepressant- and anxiolytic-like effects of atractylenolide III in lipopolysaccharide (LPS) challenge and chronic unpredictable mild stress (CUMS) rat model. We found that 30 mg/kg of atractylenolide III administered by oral gavage for 14 days, significantly reduced the immobility time in a forced swimming test (FST), but did not alter the number of crossings in an open field test (OFT), respectively. The results indicated that atractylenolide III has an antidepressant-like effect without affecting locomotor activity. We then used the LPS-induced depression model to assess the effects of atractylenolide III on behaviors in FST, sucrose preference test (SPT), and novelty-suppressed feeding test (NSFT). Interestingly, in addition to the antidepressant-like effects, 30 mg/kg of atractylenolide III also produced an anxiolytic-like effect. To further identify the antidepressant- and anxiolytic-like effects of atractylenolide III, we used the CUMS model with 28 consecutive days of the atractylenolide III treatment, followed by the SPT, FST, and NSFT. Atractylenolide III prevented CUMS-induced depressive- and anxiety-like behaviors in rats. To illustrate the underlying possible mechanisms of action of atractylenolide III, we measured the proinflammatory cytokines levels. The results showed that atractylenolide III decreased the proinflammatory cytokines levels in the hippocampus of CUMS exposed rats. In summary, our findings demonstrated that atractylenolide III produces antidepressant- and anxiolytic-like effects in rats, and these effects appear to be mediated by inhibition of hippocampal neuronal inflammation.

Disrupting Reconsolidation by Systemic Inhibition of mTOR Kinase via Rapamycin Reduces Cocaine-Seeking Behavior.

Drug addiction is considered maladaptive learning, and drug-related memories aroused by the presence of drug related stimuli (drug context or drug-associated cues) promote recurring craving and reinstatement of drug seeking. The mammalian target of rapamycin signaling pathway is involved in reconsolidation of drug memories in conditioned place preference and alcohol self-administration (SA) paradigms. Here, we explored the effect of mTOR inhibition on reconsolidation of addiction memory using cocaine self-administration paradigm. Rats received intravenous cocaine self-administration training for 10 consecutive days, during which a light/tone conditioned stimulus was paired with each cocaine infusion. After acquisition of the stable cocaine self-administration behaviors, rats were subjected to nosepoke extinction (11 days) to extinguish their behaviors, and then received a 15 min retrieval trial with or without the cocaine-paired tone/light cue delivery or without. Immediately or 6 h after the retrieval trial, rapamycin (10 mg/kg) was administered intraperitoneally. Finally, cue-induced reinstatement, cocaine-priming-induced reinstatement and spontaneous recovery of cocaine-seeking behaviors were assessed in rapamycin previously treated animals, respectively. We found that rapamycin treatment immediately after a retrieval trial decreased subsequent reinstatement of cocaine seeking induced by cues or cocaine itself, and these effects lasted at least for 28 days. In contrast, delayed intraperitoneal injection of rapamycin 6 h after retrieval or rapamycin injection without retrieval had no effects on cocaine-seeking behaviors. These findings indicated that mTOR inhibition within the reconsolidation time-window impairs the reconsolidation of cocaine associated memory, reduces cocaine-seeking behavior and prevents relapse, and these effects are retrieval-dependent and temporal-specific.

A radical S-adenosyl-L-methionine enzyme and a methyltransferase catalyze cyclopropane formation in natural product biosynthesis.

Cyclopropanation of unactivated olefinic bonds via addition of a reactive one-carbon species is well developed in synthetic chemistry, whereas natural cyclopropane biosynthesis employing this strategy is very limited. Here, we identify a two-component cyclopropanase system, composed of a HemN-like radical S-adenosyl-L-methionine (SAM) enzyme C10P and a methyltransferase C10Q, catalyzes chemically challenging cyclopropanation in the antitumor antibiotic CC-1065 biosynthesis. C10P uses its [4Fe-4S] cluster for reductive cleavage of the first SAM to yield a highly reactive 5'-deoxyadenosyl radical, which abstracts a hydrogen from the second SAM to produce a SAM methylene radical that adds to an sp2-hybridized carbon of substrate to form a SAM-substrate adduct. C10Q converts this adduct to CC-1065 via an intramolecular SN2 cyclization mechanism with elimination of S-adenosylhomocysteine. This cyclopropanation strategy not only expands the enzymatic reactions catalyzed by the radical SAM enzymes and methyltransferases, but also sheds light on previously unnoticed aspects of the versatile SAM-based biochemistry.

Unified Biosynthetic Origin of the Benzodipyrrole Subunits in CC-1065.

CC-1065 is the first characterized member of a family of naturally occurring antibiotics including yatakemycin and duocarmycins with exceptionally potent antitumor activity. CC-1065 contains three benzodipyrroles (1a-, 1b-, and 1c-) of which the 1a-subunit is remarkable by being composed of a cyclopropane ring, and the mechanism for the biological formation of benzodipyrrole rings remains elusive. Previously, biosynthetic studies of CC-1065 were limited to radioactively labeled precursor feeding experiments, which showed that tyrosine (Tyr) and serine (Ser) were incorporated into the two benzodipyrrole (1b- and 1c-) subunits via the same mode but that this was different from the key cyclopropabenzodipyrrole (1a-) subunit with N1-C2-C3 derived from Ser. Herein, the biosynthetic gene cluster of CC-1065 has been cloned, analyzed, and characterized by a series of gene inactivations. Significantly, a key intermediate bearing a C7-OH group derived from a Δc10C mutant exhibited improved cytotoxicity. Moreover, this data inspired us to suspect that the 1a-subunit might employ the same precursor incorporation mode as the 1b- and 1c-subunits. Subsequently, 13C-labeled Tyr feeding experiments confirmed that the N1-C2-C3 is originated from Tyr via DOPA as an intermediate. Collectively, a biosynthetic pathway of benzodipyrrole is proposed featuring a revised and unified precursor incorporation mode, which implicates an oxidative cyclization strategy for the assembly of benzodipyrrole. This work sets the stage for further study of enzymatic mechanisms and combinatorial biosynthesis for new DNA alkylating analogues.

Inhibiting histone deacetylase 6 partly protects cultured rat cortical neurons from oxygen­glucose deprivation­induced necroptosis.

Necroptosis has an important role in ischemia-reperfusion damage. The expression of histone deacetylase 6 (HDAC6) is upregulated in neurons following ischemia-reperfusion, however, whether HDAC6 is closely involved in the necroptosis, which occurs during ischemia-reperfusion damage remains to be elucidated. In the present study, the roles of HDAC6 in the necroptosis of cultured rat cortical neurons were investigated in a oxygen-glucose deprivation (OGD) model. The results demonstrated that OGD induced marked necroptosis of cultured rat cortical neurons and upregulated the expression of HDAC6 in the cultured neurons, compared with the control (P<0.05). The necroptosis inhibitor, necrostatin-1 (Nec-1), decreased The expression of HDAC6 in the OGD-treated cultured neurons, accompanied by the inhibition of necroptosis. Further investigation revealed that, compared with OGD treatment alone, inhibiting the activity of HDAC6 with tubacin, a specific HDAC6 inhibitor, reduced the OGD-induced necroptosis of the cultured rat cortical neurons (P<0.05), which was similar to the change following treatment with Nec-1 (P>0.05). In addition, inhibiting the activity of HDAC6 reversed the OGD-induced increase of reactive oxygen species (ROS) and the OGD-induced decrease of acetylated tubulin in the cultured rat cortical neurons (P<0.05), compared with the neurons treated with OGD alone). The levels of acetylated tubulin in the cultured neurons following treatment with OGD and tubacin were significantly higher than those in the control (P<0.05). These results suggested that HDAC6 was involved in the necroptosis of neurons during ischemia-reperfusion by modulating the levels of ROS and acetylated tubulin.

Degenerative alterations in noradrenergic neurons of the locus coeruleus in Alzheimer's disease.

Mice carrying mutant amyloid-ß precursor protein and presenilin-1 genes (APP/PS1 double transgenic mice) have frequently been used in studies of Alzheimer's disease; however, such studies have focused mainly on hippocampal and cortical changes. The severity of Alzheimer's disease is known to correlate with the amount of amyloid-ß protein deposition and the number of dead neurons in the locus coeruleus. In the present study, we assigned APP/PS1 double transgenic mice to two groups according to age: young mice (5-6 months old) and aged mice (16-17 months old). Age-matched wild-type mice were used as controls. Immunohistochemistry for tyrosine hydroxylase (a marker of catecholaminergic neurons in the locus coeruleus) revealed that APP/PS1 mice had 23% fewer cells in the locus coeruleus compared with aged wild-type mice. APP/PS1 mice also had increased numbers of cell bodies of neurons positive for tyrosine hydroxylase, but fewer tyrosine hydroxylase-positive fibers, which were also short, thick and broken. Quantitative analysis using unbiased stereology showed a significant age-related increase in the mean volume of tyrosine droxylase-positive neurons in aged APP/PS1 mice compared with young APP/PS1 mice. Moreover, the mean volume of tyrosine hydroxylase-positive neurons was positively correlated with the total volume of the locus coeruleus. These findings indicate that noradrenergic neurons and fibers in the locus coeruleus are predisposed to degenerative alterations in APP/PS1 double transgenic mice.

Analysis of YM-216391 biosynthetic gene cluster and improvement of the cyclopeptide production in a heterologous host.

YM-216391, an antitumor natural product, represents a new class of cyclic peptides containing a polyoxazole-thiazole moiety. Herein we describe its gene cluster encoding the biosynthetic paradigm featuring a ribosomally synthesizing precursor peptide followed by a series of novel posttranslational modifications, which include (i) cleavage of both N-terminal leader peptide and C-terminal extension peptide and cyclization in a head-to-tail fashion, (ii) conversion of an L-Ile to D-allo-Ile, and (iii) ß-hydroxylation of Phe by a P450 monooxygenase followed by further heterocyclization and oxidation to form a phenyloxazole moiety. The cluster was heterologously expressed in Streptomyces lividans to bypass difficult genetic manipulation. Deletion of the ymR3 gene, encoding a putative transcriptional regulator, increased the YM-216391 yield about 20-fold higher than the original yields for the heterologous expression of wild-type cluster, which set the stage for further combinatorial biosynthesis.

Insulin increases shedding of syndecan-1 in the serum of patients with type 2 diabetes mellitus.

AIMS: To detect the level of serum syndecan-1 of patients with type 2 diabetes. METHODS: Subjects with diabetes were categorized into 4 subgroups, oral-agents, insulin therapy for 12 months. Serum syndecan-1 was detected by ELISA, and potential correlation between syndecan-1 levels and clinical characteristics was analyzed. RESULTS: Sixty-two diabetic patients and 20 healthy subjects (controls) were enrolled. Syndecan-1 in diabetic patients (24.616+/-1.993 ng/ml) was higher than that of the controls (18.907+/-2.638 ng/ml). The average concentration of syndecan-1 in the group of oral-agents, insulin therapy for 12 months was 19.157+/-2.556 ng/ml (n=20), 24.447+/-3.173 ng/ml (n=23), 35.005+/-4.749 ng/ml (n=11), and 27.593+/-8.304 ng/ml (n=8), respectively. An association between serum syndecan-1 and intake of exogenous insulin was found (r=0.266, p=0.035). Serum syndecan-1 of insulin-therapy group (27.811+/-2.669 ng/ml) enhanced significantly compared to that of the controls (p=0.030) and that of the oral-agents group (p=0.035). Syndecan-1 of the insulin therapy for 1-12 months group enhanced predominantly compared to that of the controls (p=0.005) and the oral-agents group (p=0.005). CONCLUSIONS: Chronic inflammation and exogenous insulin usage increases serum syndecan-1 level. Exogenous insulin can promote shedding of syndecan-1 ectodomains to the serum in a time-dependent manner.

Identification of genes necessary for jinggangmycin biosynthesis from Streptomyces hygroscopicus 10-22.

A series of large chromosomal deletions in Streptomyces hygroscopicus 10-22 were aligned on the physical map of the wild-type strain and the mutants were assessed for their ability to produce the aminocyclitol antibiotic 5102-I (jinggangmycin). Twenty-eight mutants were blocked for jinggangmycin production and all of them were found to lack a 300 kb AseI-F fragment of the wild-type chromosome. An ordered cosmid library of the 300 kb AseI-F fragment was made and one of the cosmids conferred jinggangmycin productivity to Streptomyces lividans ZX1. Three of the overlapping cosmids (18G7, 5H3 and 9A2) also hybridized to the valA gene of the validamycin pathway from S. hygroscopicus 5008 as a probe. This gene resembles acbC from Actinoplanes sp. 50/110, which encodes a C7-cyclitol synthase that catalyses the transformation of sedoheptulose 7-phosphate into 2-5-epi-valiolone for acarbose biosynthesis. The valA/acbC-homolog (orf1) of S. hygroscopicus 10-22 was shown to be essential for jinggangmycin biosynthesis as an engineered mutant with a specific in-frame deletion removing a 609 bp sequence internal to orf1 completely abolished jinggangmycin production and the corresponding knock-out mutant (JXH4) could be complemented for jinggangmycin production by the introduction of an orf1-containing construct. Concurrently, the identities of the genes common to S. hygroscopicus strains 10-22 and 5008 prompted a comparison of the chemical structures of jinggangmycin and validamycin, which led to a clear demonstration that they are identical.

Gene cluster responsible for validamycin biosynthesis in Streptomyces hygroscopicus subsp. jinggangensis 5008.

A gene cluster responsible for the biosynthesis of validamycin, an aminocyclitol antibiotic widely used as a control agent for sheath blight disease of rice plants, was identified from Streptomyces hygroscopicus subsp. jinggangensis 5008 using heterologous probe acbC, a gene involved in the cyclization of D-sedoheptulose 7-phosphate to 2-epi-5-epi-valiolone of the acarbose biosynthetic gene cluster originated from Actinoplanes sp. strain SE50/110. Deletion of a 30-kb DNA fragment from this cluster in the chromosome resulted in loss of validamycin production, confirming a direct involvement of the gene cluster in the biosynthesis of this important plant protectant. A sequenced 6-kb fragment contained valA (an acbC homologue encoding a putative cyclase) as well as two additional complete open reading frames (valB and valC, encoding a putative adenyltransferase and a kinase, respectively), which are organized as an operon. The function of ValA was genetically demonstrated to be essential for validamycin production and biochemically shown to be responsible specifically for the cyclization of D-sedoheptulose 7-phosphate to 2-epi-5-epi-valiolone in vitro using the ValA protein heterologously overexpressed in E. coli. The information obtained should pave the way for further detailed analysis of the complete biosynthetic pathway, which would lead to a complete understanding of validamycin biosynthesis.