Ultrasensitive SERS substrate for label-free therapeutic drug monitoring of chlorpromazine hydrochloride and aminophylline in human serum.

Surface-enhanced Raman spectroscopy (SERS) has been widely used in the field of therapeutic drug monitoring (TDM) because of its powerful fingerprinting capability. In this paper, we used an in situ synthesis method to anchor Ag nanoparticles (AgNPs) on the surface of MIL-101(Cr) to obtain MIL-101(Cr)@Ag. Owing to the large specific surface area and ultra-high porosity of MIL-101(Cr)@Ag, we developed a method for the determination of chlorpromazine hydrochloride (CPZ) and aminophylline (AMP) in human serum by using it as a solid-phase extraction sorbent and SERS substrate. The label-free TDM-SERS method was able to evaluate the levels of CPZ and AMP in serum samples with detection limits as low as 8.91 × 10-2 µg/mL and 3.4 × 10-2 µg/mL, respectively. In addition, influencing factors including sample solution pH, AgNO3 concentration, drug adsorption time, and the amount of sample solution were optimized. This protocol provides a new method with good selectivity, stability, reproducibility, homogeneity, and sensitivity for the determination of small-molecule drug content in serum samples. This label-free TDM-SERS method will help to achieve rapid individualized dosing regimens in clinical practice and has potential applications in the field of TDM.

Discovery and characterization of a novel PKD-Fn3 domains containing GH44 endoglucanase from a Tibetan metagenomic library.

AIMS: To explore novel microbial endoglucanases with unique properties derived from extreme environments by using metagenomics approach. METHODS AND RESULTS: A Tibetan soil metagenomic library was applied for screening cellulase-active clones by function-based metagenomics. The candidate genes in the active clones were identified through bioinformatic analyses and heterologously expressed using an Escherichia coli system. The recombinant endoglucanases were purified and characterized using enzyme assays to determine their bioactivities, stabilities, substrate specificities, and other enzymatic properties. A novel endoglucanase gene Zfeg1907 was identified, which consisted of a glycoside hydrolase family 44 (GH44) catalytic domain along with a polycystic kidney disease (PKD) domain and a fibronectin type Ⅲ (Fn3) domain at the C terminal. Recombinant enzyme ZFEG1907 and its truncated mutant ZFEG1907t (ΔPKDΔFn3) were successfully expressed and purified. The two recombinants exhibited catalytic activities toward carboxymethyl cellulose, konjac glucomannan (KGM), and lichenan. Both enzymes had an optimal temperature of 50°C and an optimal pH value of 5.0. The catalytic activities of both recombinant enzymes were promoted by adding Zn2+ and Ca2+ at the final concentration of 10 mM. The Km value of ZFEG1907 was lower, while the kcat/Km value of ZFEG1907 was higher than those of of ZFEG1907t when using carboxymethyl cellulose, KGM, and lichenan as substrates. Structure prediction of two recombinants revealed that PKD-Fn3 domains consisted of a flexible linker and formed a ß-sandwich structure. CONCLUSIONS: A novel endoglucanase ZFEG1907 contained a GH44 catalytic domain and a PKD-Fn3 domain was characterized. The PKD-Fn3 domains were not indispensable for the activity but contributed to the enzyme binding of the polysaccharide substrates as a carbohydrate-binding module (CBM).

Magnetic/Zeolitic Imidazolate Framework-67 Nanocomposite for Magnetic Solid-Phase Extraction of Five Flavonoid Components from Chinese Herb Dicranopteris pedata.

A magnetically functionalized Fe3O4@ZIF-67 metal-organic framework (MOF) was prepared by electrostatic self-assembly using magnetic Fe3O4 nanoparticles as the core and ZIF-67 as the shell. The composite was characterized by electron microscopy, X-ray diffraction, Fourier- transform infrared spectroscopy, and Brunauer-Emmett-Teller measurements. Magnetic solid-phase extraction (MSPE) was performed on five flavonoids from Dicranopteris pedata using Fe3O4@ZIF-67 as an adsorbent. The developed MSPE method was combined with high-performance liquid chromatography-ultraviolet detection to preconcentrate and separate five flavonoids (rutin, quercitrin, kaempferol-3-O-α-L-rhamnoside, quercetin, and kaempferol) from Dicranopteris pedata. The factors affecting the extraction, such as the amount of Fe3O4@ZIF-67 adsorbent, salt ion concentration in the sample solution, vortex time, type and amount of desorbing solvent, concentration of formic acid to acidify the desorbing solvent, and acetonitrile ratio, were optimized. The developed method showed good linearity over the concentration range of 1.09-70.0 µg∙mL-1 for the five flavonoids, with R2 values between 0.9901 and 0.9945. The limits of detection and average recoveries for the five flavonoids were in the ranges of 39.5-56.2 ng∙mL-1 and 92.2-100.7%, respectively. The method presented herein is simple, efficient, and sensitive; it can be used for enrichment analysis of the five flavonoids in Dicranopteris pedata.

Novel amikacin resistance genes identified from human gut microbiota by functional metagenomics.

AIMS: The aim of this study was to evaluate the diversity and potential for horizontal transfer of amikacin resistance genes from the human gut. METHODS AND RESULTS: A library of human faecal microbiota was constructed and subjected to functional screening for amikacin resistance. In total, five amikacin resistance genes that conferred relatively high amikacin resistance, with minimum inhibitory concentrations (MICs) ranging from 64 to >512, were identified from the library, including a novel aminoglycoside acetyltransferase gene and a 16S rRNA methyltransferase (MTase) gene, labelled aac (6')-Iao and rmtI, respectively. AAC(6')-Iao showed the highest identity of 48% to AAC(6')-Ian from a clinical isolate Serratia marcescens, whereas RmtI shared the closest amino acid identity of 32% with ArmA from Klebsiella pneumonia. The MICs of these five subclones to six commonly used aminoglycosides were determined. Susceptibility analysis indicated that RmtI was associated with high resistance phenotype to 4,6-disubstituted 2-DOS aminoglycosides, whereas AAC(6')-Iao conferred resistance to amikacin and kanamycin. In addition, kinetic parameters of AAC(6')-Iao were determined, suggesting a strong catalytic effect on amikacin and kanamycin. CONCLUSIONS: Antibiotic resistance genes with low identity to known sequences can be uncovered by functional metagenomics. In addition, the diversity and prevalence of amikacin resistance genes merit further investigation in extended habitats, especially the 16S rRNA MTase gene that might have been underestimated in previous cognition. SIGNIFICANCE AND IMPACT OF STUDY: Two novel amikacin resistance genes were identified in this study, including a 16S rRNA methyltransferase gene rmtI and an aminoglycoside acetyltransferase gene aac(6')-Iao. This work would contribute to the in-depth study of the diversity and horizontal transfer potential of amikacin resistance genes in the microbiome of the human gut.

Regulated Expression of an Environmental DNA-Derived Type II Polyketide Gene Cluster in Streptomyces Hosts Identified a New Tetracenomycin Derivative TCM Y.

As bacterial natural products have been proved to be the most important source of many therapeutic medicines, the need to discover novel natural products becomes extremely urgent. Despite the fact that the majority of bacterial species are yet to be cultured in a laboratory setting, and that most of the bacterial natural product biosynthetic genes are silent, "metagenomics technology" offers a solution to help clone natural product biosynthetic genes from environmental samples, and genetic engineering enables the silent biosynthetic genes to be activated. In this work, a type II polyketide biosynthetic gene cluster was identified from a soil metagenomic library and was activated by over-expression of a SARP regulator gene in the gene cluster in Streptomyces hosts. A new tetracenomycin type compound tetracenomycin Y was identified from the fermentation broth. This study shows that metagenomics and genetic engineering could be combined to provide access to new natural metabolites.

Identification and heterologous expression of an isoquinolinequinone biosynthetic gene cluster from Streptomyces albus J1074.

Nitrogen heterocycle small molecules display various pharmaceutically important bioactivities and have great potential in drug development and application. Microbes are an important source for discovering nitrogen heterocycle natural products, and the elucidation of their biosynthetic pathways in microbes facilitates genetic manipulation of new nitrogen heterocycle products. In this study, we isolated three isoquinolinequinones from a Streptomyces albus J1074 conjugant and identified their biosynthetic gene cluster in the S. albus J1074 genome. The function of the biosynthetic gene cluster was confirmed by heterologous expression of the gene cluster in S. coelicolor M1146. This study uncovered a new biosynthetic machinery to produce nitrogen heterocycle natural products in microbes.

Identification and characterization of a novel bifunctional cellulase/hemicellulase from a soil metagenomic library.

Microbes, especially the uncultured microbes, have been considered as an important resource for discovery of novel cellulases. In this study, a novel bifunctional cellulase/hemicellulase (ZFYN184) was identified by functional screening of a soil metagenomic library. Sequence analysis indicated that ZFYN184 shared at best 39% identity with glycoside hydrolase family 44 (GH44) proteins and contained a glutamic acid residue at 235 acting as the catalytic proton donor in hydrolysis of polysaccharides. The recombinant ZFYN184 was expressed in Escherichia coli BL21 (DE3), and the biochemical profiles of the enzyme, including optimum pH and temperature, pH and thermal stabilities, tolerance to various additives, and substrate specificity, were determined. ZFYN184 possessed strong endo-ß-1,4-glucanase and endo-1,4-ß-mannanase activities, as well as weak xylanase activity, while all these hydrolytic activities were derived from a single catalytic domain in this GH44 enzyme. KEY POINTS: • Discovery a novel bifunctional glycosyl hydrolase from uncultured microorganism. • ZFYN184 contains a single catalytic domain belonged to GH44.

Salumycin, a New Pyrazolequinone from a Streptomyces albus J1074 Mutant Strain.

Heterocyclic natural products with various bioactivities play significant roles in pharmaceuticals. Here, we isolated a heterocyclic compound salumycin (1) from a Streptomyces albus J1074 mutant strain. The structure of (1) was elucidated via single-crystal X-ray diffraction, mass spectrometry (MS), fourier transform infrared spectrometer (FTIR), and nuclear magnetic resonance (NMR) data analysis. Salumycin (1) contained a novel pyrazolequinone ring, which had never been previously reported in a natural product. Salumycin (1) exhibited moderate 2,2'-diphenyl-1-picrylhydrazyl (DPPH)-radical scavenging activity (EC50 = 46.3 ± 2.2 µM) compared with tert-butylhydroquinone (EC50 = 4.7 ± 0.3 µM). This study provides a new example of discovering novel natural products from bacteria.

Functional analysis of environmental DNA-derived type II polyketide synthases reveals structurally diverse secondary metabolites.

A single gram of soil is predicted to contain thousands of unique bacterial species. The majority of these species remain recalcitrant to standard culture methods, prohibiting their use as sources of unique bioactive small molecules. The cloning and analysis of DNA extracted directly from environmental samples (environmental DNA, eDNA) provides a means of exploring the biosynthetic capacity of natural bacterial populations. Environmental DNA libraries contain large reservoirs of bacterial genetic diversity from which new secondary metabolite gene clusters can be systematically recovered and studied. The identification and heterologous expression of type II polyketide synthase-containing eDNA clones is reported here. Functional analysis of three soil DNA-derived polyketide synthase systems in Streptomyces albus revealed diverse metabolites belonging to well-known, rare, and previously uncharacterized structural families. The first of these systems is predicted to encode the production of the known antibiotic landomycin E. The second was found to encode the production of a metabolite with a previously uncharacterized pentacyclic ring system. The third was found to encode the production of unique KB-3346-5 derivatives, which show activity against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecalis. These results, together with those of other small-molecule-directed metagenomic studies, suggest that culture-independent approaches are capable of accessing biosynthetic diversity that has not yet been extensively explored using culture-based methods. The large-scale functional screening of eDNA clones should be a productive strategy for generating structurally previously uncharacterized chemical entities for use in future drug development efforts.

The "depict" strategy for discovering new compounds in complex matrices: Lycibarbarspermidines as a case.

The comprehensive detection and identification of active ingredients in complex matrices is a crucial challenge. Liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) is the most prominent analytical platform for the exploration of novel active compounds from complex matrices. However, the LC-HRMS-based analysis workflow suffers from several bottleneck issues, such as trace content of target compounds, limited acquisition for fragment information, and uncertainty in interpreting relevant MS2 spectra. Lycibarbarspermidines are vital antioxidant active ingredients in Lycii Fructus, while the reported structures are merely focused on dicaffeoylspermidines due to their low content. To comprehensively detect the new structures of lycibarbarspermidine derivatives, a "depict" strategy was developed in this study. First, potential new lycibarbarspermidine derivatives were designed according to the biosynthetic pathway, and a comprehensive database was established, which enlarged the coverage of lycibarbarspermidine derivatives. Second, the polarity-oriented sample preparation of potential new compounds increased the concentration of the target compounds. Third, the construction of the molecular network based on the fragmentation pathway of lycibarbarspermidine derivatives broadened the comprehensiveness of identification. Finally, the weak response signals were captured by data-dependent scanning (DDA) followed by parallel reaction monitoring (PRM), and the efficiency of acquiring MS2 fragment ions of target compounds was significantly improved. Based on the integrated strategy above, 210 lycibarbarspermidine derivatives were detected and identified from Lycii Fructus, and in particular, 170 potential new compounds were structurally characterized. The integrated strategy improved the sensitivity of detection and the coverage of low-response components, and it is expected to be a promising pipeline for discovering new compounds.

O-methyltransferases selectively modify anthraquinone natural products.

In this issue of Structure, Huber et al. identify five O-methyltransferases, and three of them catalyze the sequential methylation of the Gram-negative bacterium-derived aromatic polyketide anthraquinone AQ-256. They present co-crystal structures with bound AQ-256 and its methylated derivatives, which explains the specificities of these O-methyltransferases.

Discovery of two bifunctional/multifunctional cellulases by functional metagenomics.

Cellulases are widely used in industry, and the usage in bioconversion of biofuels makes cellulases more valuable. In this study, two tandem genes that encoded cellulases ZF994-1 and ZF994-2, respectively, were identified on a cosmid from a soil metagenomic library. Phylogenetic analysis indicated that ZF994-1 and ZF994-2 belonged to glycoside hydrolase family 12 (GH12), and GH3, respectively. Based on the substrate specificity analysis, the recombinant ZF994-1 exhibited weak endoglucanase activity, moderate ß-1,3-glucanase and ß-1,4-mannanase activities, and strong ß-glucosidase activity, while the recombinant ZF994-2 exhibited moderate endoglucanase activity and strong ß-glucosidase activity. More than 45% ß-glucosidase activity of the recombinant ZF994-1 retained in the buffer containing 3 M glucose, indicating the good tolerance against glucose. The recombinant ZF994-2 showed high activity in the presence of metal ions and organic reagents, exhibiting potential industrial applications.

A funnel-type stepwise filtering strategy for identification of potential Q-markers of traditional Chinese medicine formulas.

Quality marker (Q-marker) serves as an important driver for the standardization of quality control in traditional Chinese medicine (TCM) formulas. However, it is still challenging to discover comprehensive and representative Q-markers. This study aimed to identify Q-markers of Hugan tablet (HGT), a famous TCM formula with ideal clinical effects in liver diseases. Here, we proposed a funnel-type stepwise filtering strategy that integrated secondary metabolites characterization, characteristic chromatogram, quantitative analysis, literature mining, biotransformation rules and network analysis. Firstly, the strategy of "secondary metabolites-botanical drugs-TCM formula" was applied to comprehensively identify the secondary metabolites of HGT. Then, the secondary metabolites with specificity and measurability in each botanical drug were identified by HPLC characteristic chromatogram, biosynthesis pathway and quantitative analysis. Based on literature mining, the effectiveness of botanical metabolites that met the above conditions was evaluated. Furthermore, the metabolism of the above metabolites in vivo was studied to reveal their biotransformation forms, which were used for network analysis. At last, according to biotransformation rules of the prototype drugs in vivo, the secondary metabolites were traced and preliminarily chosen as Q-markers. As a result, 128 plant secondary metabolites were identified in HGT, and 11 specific plant secondary metabolites were screened out. Then, the content of specific plant secondary metabolites in 15 batches of HGT was determined, which confirmed their measurability. And the results of literature mining showed that eight secondary metabolites had therapeutic effects in treating liver disease at the in vivo level, and three secondary metabolites inhibited liver disease-related indicators at the in vitro level. After that, 26 compounds absorbed into the blood (11 specific plant metabolites and their 15 metabolites in vivo) were detected in rats. Moreover, 14 compounds, including prototype components and their metabolites, were selected as Q-marker candidates by the "TCM formula-botanical drugs-compounds-targets-pathways" network. Finally, 9 plant secondary metabolites were defined as comprehensive and representative Q-markers. Our study not only provides a scientific basis for the improvement and secondary development of the quality standard of HGT, but also proposes a reference method for discovering and identifying Q-markers of TCM preparations.

Environmental DNA-encoded antibiotics fasamycins A and B inhibit FabF in type II fatty acid biosynthesis.

In a recent study of polyketide biosynthetic gene clusters cloned directly from soil, we isolated two antibiotics, fasamycins A and B, which showed activity against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecalis. To identify the target of the fasamycins, mutants with elevated fasamycin A minimum inhibitory concentrations were selected from a wild-type culture of E. faecalis OG1RF. Next-generation sequencing of these mutants, in conjunction with in vitro biochemical assays, showed that the fasamycins inhibit FabF of type II fatty acid biosynthesis (FASII). Candidate gene overexpression studies also showed that fasamycin resistance is conferred by fabF overexpression. On the basis of comparisons with known FASII inhibitors and in silico docking studies, the chloro-gem-dimethyl-anthracenone substructure seen in the fasamycins is predicted to represent a naturally occurring FabF-specific antibiotic pharmacophore. Optimization of this pharmacophore should yield FabF-specific antibiotics with increased potencies and differing spectra of activity. This study demonstrates that culture-independent antibiotic discovery methods have the potential to provide access to novel metabolites with modes of action that differ from those of antibiotics currently in clinical use.

A designer bleomycin with significantly improved DNA cleavage activity.

The bleomycins (BLMs) are used clinically in combination with a number of other agents for the treatment of several types of tumors, and the BLM, etoposide, and cisplatin treatment regimen cures 90-95% of metastatic testicular cancer patients. BLM-induced pneumonitis is the most feared, dose-limiting side effect of BLM in chemotherapy, which can progress into lung fibrosis and affect up to 46% of the total patient population. There have been continued efforts to develop new BLM analogues in the search for anticancer drugs with better clinical efficacy and lower lung toxicity. We have previously cloned and characterized the biosynthetic gene clusters for BLMs from Streptomyces verticillus ATCC15003, tallysomycins from Streptoalloteichus hindustanus E465-94 ATCC31158, and zorbamycin (ZBM) from Streptomyces flavoviridis SB9001. Comparative analysis of the three biosynthetic machineries provided the molecular basis for the formulation of hypotheses to engineer novel analogues. We now report engineered production of three new analogues, 6'-hydroxy-ZBM, BLM Z, and 6'-deoxy-BLM Z and the evaluation of their DNA cleavage activities as a measurement for their potential anticancer activity. Our findings unveiled: (i) the disaccharide moiety plays an important role in the DNA cleavage activity of BLMs and ZBMs, (ii) the ZBM disaccharide significantly enhances the potency of BLM, and (iii) 6'-deoxy-BLM Z represents the most potent BLM analogue known to date. The fact that 6'-deoxy-BLM Z can be produced in reasonable quantities by microbial fermentation should greatly facilitate follow-up mechanistic and preclinical studies to potentially advance this analogue into a clinical drug.

Natural product biosynthetic gene diversity in geographically distinct soil microbiomes.

The number of bacterial species estimated to exist on Earth has increased dramatically in recent years. This newly recognized species diversity has raised the possibility that bacterial natural product biosynthetic diversity has also been significantly underestimated by previous culture-based studies. Here, we compare 454-pyrosequenced nonribosomal peptide adenylation domain, type I polyketide ketosynthase domain, and type II polyketide ketosynthase alpha gene fragments amplified from cosmid libraries constructed using DNA isolated from three different arid soils. While 16S rRNA gene sequence analysis indicates these cloned metagenomes contain DNA from similar distributions of major bacterial phyla, we found that they contain almost completely distinct collections of secondary metabolite biosynthetic gene sequences. When grouped at 85% identity, only 1.5% of the adenylation domain, 1.2% of the ketosynthase, and 9.3% of the ketosynthase alpha sequence clusters contained sequences from all three metagenomes. Although there is unlikely to be a simple correlation between biosynthetic gene sequence diversity and the diversity of metabolites encoded by the gene clusters in which these genes reside, our analysis further suggests that sequences in one soil metagenome are so distantly related to sequences in another metagenome that they are, in many cases, likely to arise from functionally distinct gene clusters. The marked differences observed among collections of biosynthetic genes found in even ecologically similar environments suggest that prokaryotic natural product biosynthesis diversity is, like bacterial species diversity, potentially much larger than appreciated from culture-based studies.

Biosynthesis of coelulatin for the methylation of anthraquinone featuring HemN-like radical S-adenosyl-L-methionine enzyme.

Bacterial aromatic polyketides are usually biosynthesized by the type II polyketide synthase (PKS-II) system. Advances in deoxyribonucleic acid (DNA) sequencing, informatics, and biotechnologies have broadened opportunities for the discovery of aromatic polyketides. Meanwhile, metagenomics is a biotechnology that has been considered as a promising approach for the discovery of novel natural products from uncultured bacteria. Here, we cloned a type II polyketide biosynthetic gene cluster (BGC) from the soil metagenome, and the heterologous expression of this gene cluster in Streptomyces coelicolor M1146 resulted in the production of three anthraquinones, two of which (coelulatins 2 and 3) had special hydroxymethyl and methyloxymethyl modifications at C2 of the polyketide scaffold. Gene deletion and in vitro biochemical characterization indicated that the HemN-like radical S-adenosyl-L-methionine (SAM) enzyme CoeI exhibits methylation and is involved in C2 modification.

Discovery and Biosynthesis of the Amodesmycins, Aromatic Polyketide-Siderophore Hybrid Conjugates.

A type II polyketide synthase biosynthetic gene cluster (amd) containing three P450 genes was identified from a soil metagenomic library, and novel benz[h]isoquinoline-desferrioxamine B conjugated compound amodesmycins were isolated from Streptomyces albus J1074 harboring the amd gene cluster. Genetic evidence showed that the benz[h]isoquinoline part and desferrioxamine B part in amodesmycins were derived from the amd gene cluster and S. albus J1074, respectively, while P450 enzymes played critical roles in the conjunction of these two parts.

Metabolites from the induced expression of cryptic single operons found in the genome of Burkholderia pseudomallei.

Bacterial genome sequencing projects routinely uncover gene clusters that are predicted to encode the biosynthesis of uncharacterized small molecules. A subset of these cryptic genetic elements appears as individual operons. Here we investigate potential single-operon biosynthetic systems found in the genome of the pathogenic bacterium Burkholderia pseudomallei . Placing these operons under the control of an inducible promoter led to the production of seven new metabolites. Among the molecules we identified are inhibitors of type-4 phosphodiesterases, suggesting that previously cryptic biosynthetic operons may encode metabolites that could contribute to microbial virulence by disrupting host signaling pathways.

Titer improvement of iso-migrastatin in selected heterologous Streptomyces hosts and related analysis of mRNA expression by quantitative RT-PCR.

iso-Migrastatin (iso-MGS) has been actively pursued recently as an outstanding candidate of antimetastasis agents. Having characterized the iso-MGS biosynthetic gene cluster from its native producer Streptomyces platensis NRRL 18993, we have recently succeeded in producing iso-MGS in five selected heterologous Streptomyces hosts, albeit the low titers failed to meet expectations and cast doubt on the utility of this novel technique for large-scale production. To further explore and capitalize on the production capacity of these hosts, a thorough investigation of these five engineered strains with three fermentation media for iso-MGS production was undertaken. Streptomyces albus J1074 and Streptomyces lividans K4-114 were found to be preferred heterologous hosts, and subsequent analysis of carbon and nitrogen sources revealed that sucrose and yeast extract were ideal for iso-MGS production. After the initial optimization, the titers of iso-MGS in all five hosts were considerably improved by 3-18-fold in the optimized R2YE medium. Furthermore, the iso-MGS titer of S. albus J1074 (pBS11001) was significantly improved to 186.7 mg/L by a hybrid medium strategy. Addition of NaHCO(3) to the latter finally afforded an optimized iso-MGS titer of 213.8 mg/L, about 5-fold higher than the originally reported system. With S. albus J1074 (pBS11001) as a model host, the expression of iso-MGS gene cluster in four different media was systematically studied via the quantitative RT-PCR technology. The resultant comparison revealed the correlation of gene expression and iso-MGS production for the first time; synchronous expression of the whole gene cluster was crucial for optimal iso-MGS production. These results reveal new insights into the iso-MGS biosynthetic machinery in heterologous hosts and provide the primary data to realize large-scale production of iso-MGS for further preclinical studies.