In Vitro Investigation of Crosstalk between Fatty Acid and Polyketide Synthases in the Andrimid Biosynthetic Assembly Line

Andrimid (Adm) synthase, which belongs to the type II system of enzymes, produces Adm in Pantoea agglomerans. The adm biosynthetic gene cluster lacks canonical acyltransferases (ATs) to load the malonyl group to acyl carrier proteins (ACPs), thus suggesting that a malonyl‐CoA ACP transacylase (MCAT) from the fatty acid synthase (FAS) complex provides the essential AT activity in Adm biosynthesis. Here we report that an MCAT is essential for catalysis of the transacylation of malonate from malonyl‐CoA to AdmA polyketide synthase (PKS) ACP in vitro. Catalytic self‐malonylation of AdmA (PKS ACP) was not observed in reactions without MCAT, although many type II PKS ACPs are capable of catalyzing self‐acylation. This lack of self‐malonylation was explained by amino acid sequence analysis of the AdmA PKS ACP and the type II PKS ACPs. The results show that MCAT from the organism's FAS complex can provide the missing AT activity in trans, thus suggesting a protein–protein interaction between the fatty acid and polyketide synthases in the Adm assembly line.     

One‐Pot Chemoenzymatic Cascade for Labeling of the Epigenetic Marker 5‐Hydroxymethylcytosine

The epigenetic DNA modification 5‐hydroxymethylcytosine (5‐hmC) is important for the regulation of gene expression during development and in tumorigenesis. 5‐hmC can be selectively glycosylated by T4 β‐glucosyltransferase (β‐GT); introduction of an azide on the attached sugar provides a chemical handle for isolation or fluorescent tagging of 5‐hmC residues by click chemistry. This approach has not been broadly adopted because of the challenging synthesis and limited commercial availability of the glycosylation substrate, 6‐deoxy‐6‐azido‐α‐D ‐glucopyranoside. We report the enzyme‐assisted synthesis of this precursor by the uridylyltransferase from Pasteurella multocida (PmGlmU). We were able to directly label 5‐hmC in genomic DNA by an enzymatic cascade involving successive action of PmGlmU and β‐GT. This is a facile and cost‐effective one‐pot chemoenzymatic methodology for 5‐hmC analysis.     

New Insights into the Glycosylation Steps in the Biosynthesis of Sch47554 and Sch47555

Sch47554 and Sch47555 are antifungal compounds from Streptomyces sp. SCC‐2136. The availability of the biosynthetic gene cluster made it possible to track genes that encode biosynthetic enzymes responsible for the structural features of these two angucyclines. Sugar moieties play important roles in the biological activities of many natural products. An investigation into glycosyltransferases (GTs) might potentially help to diversify pharmaceutically significant drugs through combinatorial biosynthesis. Sequence analysis indicates that SchS7 is a putative C‐GT, whereas SchS9 and SchS10 are proposed to be O‐GTs. In this study, the roles of these three GTs in the biosynthesis of Sch47554 and Sch47555 are characterized. Coexpression of the aglycone and sugar biosynthetic genes with schS7 in Streptomyces lividans K4 resulted in the production of C‐glycosylated rabelomycin, which revealed that SchS7 attached a d ‐amicetose moiety to the aglycone core structure at the C‐9 position. Gene inactivation studies revealed that subsequent glycosylation steps took place in a sequential manner, in which SchS9 first attached either an l ‐aculose or l ‐amicetose moiety to 4′‐OH of the C‐glycosylated aglycone, then SchS10 transferred an l ‐aculose moiety to 3‐OH of the angucycline core.     

Biosynthesis of the Myxobacterial Antibiotic Corallopyronin A

Corallopyronin A is a myxobacterial compound with potent antibacterial activity. Feeding experiments with labelled precursors resulted in the deduction of all biosynthetic building blocks for corallopyronin A and revealed an unusual feature of this metabolite: its biosynthesis from two chains, one solely PKS‐derived and the other NRPS/PKS‐derived. The starter molecule is believed to be carbonic acid or its monomethyl ester. The putative corallopyronin A biosynthetic gene cluster is a trans‐AT‐type mixed PKS/NRPS gene cluster, containing a β‐branching cassette. Striking features of this gene cluster are a NRPS‐like adenylation domain that is part of a PKS‐type module and is believed to be responsible for glycine incorporation, as well as split modules with individual domains occurring on different genes. It is suggested that CorB is a trans‐acting ketosynthase and it is proposed that it catalyses the Claisen condensation responsible for the interconnection of the two chains. Additionally, the stereochemistry of corallopyronin A was deduced by a combination of a modified Mosher's method and ozonolysis with subsequent chiral GC analyses.     

Glycosyltransferases and their Assays

Glycosyltransferases (GTs) are a large family of enzymes that are essential in all domains of life for the biosynthesis of complex carbohydrates and glycoconjugates. GTs catalyse the transfer of a sugar from a glycosyl donor to a variety of acceptor molecules, for example, oligosaccharides, peptides, lipids or small molecules. Such glycosylation reactions are central to many fundamental biological processes, including cellular adhesion, cell signalling and bacterial‐ and plant‐cell‐wall biosynthesis. GTs are therefore of significant interest as molecular targets in chemical biology and drug discovery. In addition, GTs have found wide application as synthetic tools for the preparation of complex carbohydrates and glycoconjugates. In order to exploit the potential of GTs both as molecular targets and synthetic tools, robust and operationally simple bioassays are essential, especially as more and more protein sequences with putative GT activity but unknown biochemical function are being identified. In this minireview, we give a brief introduction to GT biochemistry and biology. We outline the relevance of GTs for medicinal chemistry and chemical biology, and describe selected examples for recently developed GT bioassays, with a particular emphasis on fluorescence‐based formats.     

Gene expression after dietary intervention with trans fatty acids (trans‐11/trans‐12 18:1) in humans

Gene‐by‐diet interactions play an important role in the prevention of several diseases. Conjugated linoleic acids (CLA) are ligands of gene regulators [e.g. peroxisome proliferator‐activated receptors (PPAR)] and have anti‐inflammatory properties. The aim of the study was to investigate the changes in gene expression in monocytes during the intervention with two trans fatty acids (trans‐11 18:1 and trans‐12 18:1) and endogenous CLA from trans‐11 18:1 as precursor in humans. Monocytes were isolated at baseline and after a 6‐week intervention period. The female and male test groups received Σ6.0 g trans‐11 and trans‐12 18:1/day (1 : 1). The control group received control oil. The expression of candidate genes was determined by quantitative RT‐PCR. Gender‐ and treatment‐related gene expression was found. Due to trans fatty acid intake in both gender subgroups, the relative PPARγ expression was up‐regulated. In the female test group, the expression of FAT, SCD, COX2 and BCL2 were induced, while in the male test group E‐FABP, CYP, GLUT4 and PBE were induced. In the male test group compared to controls, a clear increase in gene expression of PPARγ and GLUT4 was shown. The results reveal a gender‐ and treatment‐related gene expression. There is no clear indication as to what extent the supplemented trans fatty acids and the synthesized cis‐9,trans‐11 CLA were involved.     

Modified gas‐translation model for prediction of gas permeation through microporous organosilica membranes

A modified gas‐translation (GT) model was applied for the theoretical analysis of gas permeation through microporous organosilica membranes derived from bis(triethoxysilyl)ethane (BTESE) via a sol–gel method using different water/alkoxide molar ratios. The pore sizes of BTESE‐derived membranes were quantitatively determined by normalized Knudsen‐based permeance analysis, which was based on a modified‐GT model, using experimentally obtained permeances of He, H₂, N₂, C₃H₈, and SF₆. The pore sizes of BTESE‐derived membranes were successfully controlled from 0.65 to 0.46 nm by increasing the H₂O/BTESE ratio from 6 to 240. Furthermore, theoretical correlations of all possible pairs of permeance ratios were calculated based on the modified‐GT model. The experimental data were in good agreement with the theoretical correlation curves, indicating that the modified‐GT model can clearly explain gas permeation mechanisms through microporous membranes, and, thus, can be used to predict the gas permeation properties for these membranes. © 2014 American Institute of Chemical Engineers AIChE J 60: 4199–4210, 2014     

The Phormidolide Biosynthetic Gene Cluster: A trans‐AT PKS Pathway Encoding a Toxic Macrocyclic Polyketide

Phormidolide is a polyketide produced by a cultured filamentous marine cyanobacterium and incorporates a 16‐membered macrolactone. Its complex structure is recognizably derived from a polyketide synthase pathway, but possesses unique and intriguing structural features that prompted interest in investigating its biosynthetic origin. Stable isotope incorporation experiments confirmed the polyketide nature of this compound. We further characterized the phormidolide gene cluster (phm) through genome sequencing followed by bioinformatic analysis. Two discrete trans‐type acyltransferase (trans‐AT) ORFs along with KS‐AT adaptor regions (ATd) within the polyketide synthase (PKS) megasynthases, suggest that the phormidolide gene cluster is a trans‐AT PKS. Insights gained from analysis of the mode of acetate incorporation and ensuing keto reduction prompted our reevaluation of the stereochemistry of phormidolide hydroxy groups located along the linear polyketide chain.     

Dissecting the Molecular Basis of the Role of the O‐Mannosylation Pathway in Disease: α‐Dystroglycan and Forms of Muscular Dystrophy

Dystroglycanopathies form a subgroup of muscular dystrophies that arise from defects in enzymes that are implicated in the recently elucidated O‐mannosylation pathway, thereby resulting in underglycosylation of α‐dystroglycan. The emerging identification of additional brain proteins modified by O‐mannosylation provides a broader context for interpreting the range of neurological consequences associated with dystroglycanopathies. This form of glycosylation is associated with protein mucin‐like domains that present numerous serine and threonine residues as possible sites for modification. Furthermore, the O‐Man glycans coexist in this region with O‐GalNAc glycans (conventionally associated with such protein sequences), thus resulting in a complex glycoconjugate landscape. Sorting out the relationships between the various molecular defects in glycosylation and the modes of disease presentation, as well as the regulatory interplay among the O‐Man glycans and the effects on other modes of glycosylation in the same domain, is challenging. Here we provide a perspective on chemical biology approaches employing synthetic and analytical methods to address these questions.     

The AT2 Domain of KirCI Loads Malonyl Extender Units to the ACPs of the Kirromycin PKS

The antibiotic kirromycin is assembled by a hybrid modular polyketide synthases (PKSs)/nonribosomal peptide synthetases (NRPSs). Five of six PKSs of this complex assembly line do not have acyltransferase (AT) and have to recruit this activity from discrete AT enzymes. Here, we show that KirCI is a discrete AT which is involved in kirromycin production and displays a rarely found three‐domain architecture (AT₁‐AT₂‐ER). We demonstrate that the second AT domain, KirCI‐AT₂, but not KirCI‐AT₁, is the malonyl‐CoA‐specific AT which utilizes this precursor for loading the acyl carrier proteins (ACPs) of the trans‐AT PKS in vitro. In the kirromycin biosynthetic pathway, ACP5 is exclusively loaded with ethylmalonate by the enzyme KirCII and is not recognized as a substrate by KirCI. Interestingly, the excised KirCI‐AT₂ can also transfer malonate to ACP5 and thus has a relaxed ACP‐specificity compared to the entire KirCI protein. The ability of KirCI‐AT₂ to load different ACPs provides opportunities for AT engineering as a potential strategy for polyketide diversification.     

Modulating stability and mechanical properties of silica–gelatin hybrid by incorporating epoxy‐terminated polydimethylsiloxane oligomer

Silica‐gelatin hybrids, particularly GT‐G hybrids prepared by crosslinking gelatin (G) with γ‐glycidoxypropyltrimethoxysilane (GT), have attracted much attention in tissue engineering for diverse applications in hard or soft tissue regeneration; however, scaffolds with tunable properties are needed to meet specific requirements. In this work, a silica‐gelatin hybrid (ES/GT‐G) was synthesized by incorporating epoxy‐terminated polydimethylsiloxane oligomer (ES) to modulate the properties of GT‐G hybrid. The ES/GT‐G hybrid sponge presented a 3D network structure with porosity 86.4% ± 0.9%, determined by the liquid displacement method, and average pore size 340 ± 36 μm, determined by SEM observation. Compared with GT‐G hybrid material, the prepared ES/GT‐G hybrid wet film showed a decrease of tensile strength from 2.79 ± 0.04 MPa to 1.87 ± 0.12 MPa, with an increase of elongation at break from 19.96 ± 0.66% to 29.86 ± 0.87%, and the ES/GT‐G hybrid sponge exhibited a decline of compressive yield strength from 1.21 ± 0.04 MPa to 0.72 ± 0.06 MPa, based on the tensile and compression tests respectively. The introduction of ES enhanced the thermal denaturing temperature of GT‐G by 5°C as determined by a DSC study, and increased in vitro biodegradation slightly, without significantly changing surface wettability and swelling behavior. These findings suggest that silica‐gelatin hybrids with tunable properties are promising for applications from hard to soft tissue regeneration. © 2015 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016 , 133, 43059.     

Active‐Site His85 of Pasteurella dagmatis Sialyltransferase Facilitates Productive Sialyl Transfer and So Prevents Futile Hydrolysis of CMP‐Neu5Ac

Sialyltransferases of the GT‐80 glycosyltransferase family are considered multifunctional because of the array of activities detected. They exhibit glycosyl transfer, trans‐sialylation, and hydrolysis activities. How these enzymes utilize their active‐site residues in balancing the different enzymatic activities is not well understood. In this study of Pasteurella dagmatis α2,3sialyltransferase, we show that the conserved His85 controls efficiency and selectivity of the sialyl transfer. A His85→Asn variant was 200 times less efficient than wild‐type for sialylation of lactose, and exhibited relaxed site selectivity to form not only the α2,3‐ but also the α2,6‐sialylated product (21 %). The H85N variant was virtually inactive in trans‐sialylation but showed almost the same CMP‐Neu5Ac hydrolase activity as wild‐type. The competition between sialyl transfer and hydrolysis in the conversion of CMP‐Neu5Ac was dependent on the lactose concentration; this was characterized by a kinetic partition ratio of 85 m ^?1 for the H85N variant, compared to 17 000 m ^?1 for the wild‐type enzyme. His85 promotes the productive sialyl transfer to lactose and so prevents hydrolysis of CMP‐Neu5Ac in the reaction.     

Stressor-Induced Increases in Circulating,but Not Colonic,Cytokines Are Related to Anxiety-like Behavior and Hippocampal Inflammation in a Murine Colitis Model

Stressor exposure increases colonic inflammation. Because inflammation leads to anxiety-like behavior, we tested whether stressor exposure in mice recovering from dextran-sulfate-sodium (DSS)-induced colitis enhances anxiety-like behavior. Mice received 2% DSS for five consecutive days prior to being exposed to a social-disruption (SDR) stressor (or being left undisturbed). After stressor exposure, their behavior was tested and colitis was assessed via histopathology and via inflammatory-cytokine measurement in the serum and colon. Cytokine and chemokine mRNA levels in the colon, mesenteric lymph nodes (MLNs), hippocampus, and amygdala were measured with RT-PCR. SDR increased anxiety-like behaviors, which correlated with serum and hippocampal IL-17A. The stressor also reduced IL-1β, CCL2, and iNOS in the colonic tissue, but increased iNOS, IFNγ, IL-17A, and TNFα in the MLNs. A network analysis indicated that reductions in colonic iNOS were related to elevated MLN iNOS and IFNγ. These inflammatory markers were related to serum and hippocampal IL-17A and associated with anxiety-like behavior. Our data suggest that iNOS may protect against extra-colonic inflammation, and when suppressed during stress it is associated with elevated MLN IFNγ, which may coordinate gut-to-brain inflammation. Our data point to hippocampal IL-17A as a key correlate of anxiety-like behavior.     

High trans,trans Conjugated Linoleic Acid-Rich Triacylglyceride Production by Photo-Irradiation

Recent studies have shown that a 20 % trans,trans conjugated linoleic acid (CLA)‐rich soy oil significantly reduces heart disease and diabetes risk factors in obese rats. Furthermore, trans,trans‐CLA has been reported to have superior anti‐carcinogenic activity than other CLA isomers. Therefore, a more concentrated source of trans,trans‐CLA oil would be highly desirable. The objectives of this study were to (1) determine the yield of trans,trans‐CLA isomers resulting from photo‐irradiation of Tonalin^® (BASF Global, Florham Park, NJ, USA) and identify trans,trans‐CLA positional isomers; and (2) derive a mathematical model of kinetics of trans,trans‐CLA TAG formation from Tonalin^®. Fifty‐five percent trans,trans‐CLA rich oil was obtained in about 140 min when Tonalin^® was photo‐isomerized with 0.35 % iodine, which is almost three times more than is possible with photo‐isomerized soy oil. Photo‐isomerization of Tonalin^® requires about 2 h, compared to 12 h for photo‐isomerization of soy oil. This reaction is a first‐order reversible reaction with the forward rate constant (k_f) = 13.17 × 10^?3min^?1 and backward rate constant (k_b) = 5.334 × 10^?3min^?1. The major isomers identified were trans‐9,trans‐11‐ and trans‐10,trans‐12‐CLA.     

Iterative One‐Pot α‐Glycosylation Strategy: Application to Oligosaccharide Synthesis

Based on the combined use of dimethylformamide (DMF) modulation and neighboring group participation, three iterative one‐pot α‐glycosylation methods, i.e., one‐pot (α,α)‐, one‐pot (β,α)‐, and one‐pot (α,β)‐glycosylations, were developed. These methods are applicable to a range of thioglycosyl donors, confer stereocontrol in α‐/β‐glycosidic bond formation, and thus provide for rapid access to oligosaccharides with various permutations of anomeric configurations. The utility of these one‐pot glycosylation methods is demonstrated in the synthesis of eight non‐natural and natural oligosaccharide targets, including the core 1 serine conjugate, core 8 serine conjugate, the D ‐Gal‐α(1→3)‐D ‐Glc‐α(1→3)‐L ‐Rha trisaccharide unit of the cell wall component in Streptococcus pneumoniae, and the D ‐Glc‐α(1→2)‐D ‐Glc‐α(1→3)‐D ‐Glc trisaccharide terminus of the N‐linked glycan precursor. Confirmation of the anomeric configurations of these oligosaccharides is evidenced by ¹H, ¹³C, ¹³C‐non‐proton decoupling, and heteronuclear correlation 2D NMR experiments. Global deprotection of selected oligosaccharide targets is illustrated.     

Unlocking the Potential of Leloir Glycosyltransferases for Applied Biocatalysis: Efficient Synthesis of Uridine 5′‐Diphosphate‐Glucose by Sucrose Synthase

Despite the unsurpassed selectivity that enzymes usually offer, biocatalytic transformations repeatedly fall short of the robustness and process efficiency demanded for production‐scale chemical synthesis. Nucleotide sugar‐dependent “Leloir” glycosyltransferases (GTs) are fine catalysts of glycosylation but there is concern as to whether reactions from this enzyme class are fit for industrial process development. We demonstrate in this study of sucrose synthase (SuSy; EC 2.4.1.13) that, in order to unlock the synthetic potential of the GT reaction, it was vital to combine a focused, kinetic characteristics‐based enzyme selection with a reaction design properly aligned to thermodynamic constraints. The equilibrium constant (K_eq) for the conversion of sucrose and uridine 5′‐diphosphate (UDP) into the target product UDP‐α‐d ‐glucose and d ‐fructose decreased with increasing pH due to deprotonation of the β‐phosphate group of UDP above the pK_a of ∼6.0. Proton uptake coupled to the glucosyl transfer made it essential that the pH was carefully controlled throughout the reaction. Comparing two SuSys from Acidithiobacillus caldus and Glycine max (soybean), substrate inhibition by UDP superseded catalytic efficiency as the prior selection criterion, demanding choice of the bacterial GT for use at high UDP concentrations. Reaction at the operational pH optimum, determined as 5.0, gave 255 mM (144 g L⁻¹) of UDP‐glucose in 85% yield from UDP. Using an enzyme concentration of only 0.1 g L⁻¹, a space‐time yield of 25 g L⁻¹ h⁻¹ was obtained. The mass‐based turnover number (g product formed per g enzyme added) reached a value of 1440 from a single batch conversion. Therefore, these parameters of the UDP‐glucose synthesis show that the reaction of a GT can be pushed to a process efficiency typically required for implementation in fine chemicals production.

     

CHoMP: A Chemoenzymatic Histology Method Using Clickable Probes

The characterization of aberrant glycosylation patterns in biopsied patient samples represents a remarkable challenge for scientists and medical doctors due to the lack of specific methods for detection. Here, we report the development of a histological method, dubbed CHoMP—chemoenzymatic histology of membrane polysaccharides—for analyzing glycosylation patterns in mammalian tissues. This method exploits a recombinant glycosyltransferase to transfer a monosaccharide analogue equipped with a chemical handle to a specific cell‐surface glycan target, which can then be derivatized with imaging probes by using bioorthogonal click chemistry for visualization. We applied CHoMP to survey changes in expression of N‐acetyllactosamine (LacNAc) in human samples from patients afflicted with lung adenocarcinoma and observed a sharp decrease in expression levels between normal and early grade tumors, thus suggesting a potential application of this technique in early cancer diagnosis.     

Hydrogen bond interaction in poly(acrylonitrile‐co‐methylacrylate)/attapulgite nanocomposites

In this article, poly(acrylonitrile‐co‐methylacrylate)/attapulgite (AT) nanocomposites have been prepared by mechanical blending. The hydrogen bonding (H‐bonding) behavior between the copolymer and unmodified AT were investigated by Fourier Transform Infrared. The results indicated that the H‐bond index (HBI) decreased with increasing AT content. According to the dynamic mechanical analysis, the storage modulus and heat distortion temperature of the nanocomposites gradually increased with increasing AT content while the glass transition temperature reached the maximum when AT content was 1 wt%. X‐ray diffraction results demonstrated that the sample with the highest HBI value had the lowest crystallinity, suggesting the H‐bond interaction restricted the motion of polymer chains, thus decreased crystallinity of the nanocomposites. POLYM. ENG. SCI., 2009. © 2009 Society of Plastics Engineers     

In Vitro Reconstitution of a PKS Pathway for the Biosynthesis of Galbonolides in Streptomyces sp. LZ35

The galbonolides are 14‐membered macrolide antibiotics with a macrocyclic backbone similar to that of erythromycins. Galbonolides exhibit broad‐spectrum antifungal activities. Retro‐biosynthetic analysis suggests that the backbone of galbonolides is assembled by a type I modular polyketide synthase (PKS). Unexpectedly, the galbonolide biosynthetic gene cluster, gbn, in Streptomyces sp. LZ35 encodes a hybrid fatty acid synthase (FAS)‐PKS pathway. In vitro reconstitution revealed the functions of GbnA (an AT‐ACP didomain protein), GbnC (a FabH‐like enzyme), and GbnB (a novel multidomain PKS module without AT and ACP domains) responsible for assembling the backbone of galbonolides, respectively. To our knowledge, this study is the first biochemical characterization of a hybrid FAS‐PKS pathway for the biosynthesis of 14‐membered macrolides. The identification of this pathway provides insights into the evolution of PKSs and could facilitate the design of modular pools for synthetic biology.     

Cloning and Characterization of the Biosynthetic Gene Cluster of 16‐Membered Macrolide Antibiotic FD‐891: Involvement of a Dual Functional Cytochrome P450 Monooxygenase Catalyzing Epoxidation and Hydroxylation

FD‐891 is a 16‐membered cytotoxic antibiotic macrolide that is especially active against human leukemia such as HL‐60 and Jurkat cells. We identified the FD‐891 biosynthetic (gfs) gene cluster from the producer Streptomyces graminofaciens A‐8890 by using typical modular type I polyketide synthase (PKS) genes as probes. The gfs gene cluster contained five typical modular type I PKS genes (gfsA, B, C, D, and E), a cytochrome P450 gene (gfsF), a methyltransferase gene (gfsG), and a regulator gene (gfsR). The gene organization of PKSs agreed well with the basic polyketide skeleton of FD‐891 including the oxidation states and α‐alkyl substituent determined by the substrate specificities of the acyltransferase (AT) domains. To clarify the involvement of the gfs genes in the FD‐891 biosynthesis, the P450 gfsF gene was inactivated; this resulted in the loss of FD‐891 production. Instead, the gfsF gene‐disrupted mutant accumulated a novel FD‐891 analogue 25‐O‐methyl‐FD‐892, which lacked the epoxide and the hydroxyl group of FD‐891. Furthermore, the recombinant GfsF enzyme coexpressed with putidaredoxin and putidaredoxin reductase converted 25‐O‐methyl‐FD‐892 into FD‐891. In the course of the GfsF reaction, 10‐deoxy‐FD‐891 was isolated as an enzymatic reaction intermediate, which was also converted into FD‐891 by GfsF. Therefore, it was clearly found that the cytochrome P450 GfsF catalyzes epoxidation and hydroxylation in a stepwise manner in the FD‐891 biosynthesis. These results clearly confirmed that the identified gfs genes are responsible for the biosynthesis of FD‐891 in S. graminofaciens.