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Four unusual cyclopeptides, zelkovamycins B-E (1-4), were isolated from an endophytic Kitasatospora sp. Zelkovamycin B was featured by an unprecedented 3-methyl-5-hydroxypyrrolidine-2,4-dione ring system linked to the cyclopeptide skeleton. Their structures and full configurations were established by spectroscopic analysis, Marfey's method, and NMR calculations. A plausible biosynthetic pathway for zelkovamycins was proposed based on gene cluster analysis. Zelkovamycin E displayed potent inhibitory activity against H1N1 influenza A virus.

H.E.L. was supported by an Imperial College President’s Ph.D. Scholarship. We thank UKRI EPSRC (EP/K038648/1, EP/L011573/1 to P.S.F.) and the European Union’s Seventh Framework Programme (FP7/2007–2013/ERC grant agreement no. 614779 GenoChemetics to R.J.M.G.) for funding. A.M.C.O. receives funding from EPSRC CRITICAT, EP/L016419/1. Natural products and their analogues are often challenging to synthesize due to their complex scaffolds and embedded functional groups. Solely relying on engineering the biosynthesis of natural products may lead to limited compound diversity. Integrating synthetic biology with synthetic chemistry allows rapid access to much more diverse portfolios of xenobiotic compounds, which may accelerate the discovery of new therapeutics. As a proof-of-concept, by supplementing an Escherichia coli strain expressing the violacein biosynthesis pathway with 5-bromo-tryptophan in vitro or tryptophan 7-halogenase RebH in vivo, six halogenated analogues of violacein or deoxyviolacein were generated, demonstrating the promiscuity of the violacein biosynthesis pathway. Furthermore, 20 new derivatives were generated from 5-brominated violacein analogues via the Suzuki–Miyaura cross-coupling reaction directly using the crude extract without prior purification. Herein we demonstrate a flexible and rapid approach to access a diverse chemical space that can be applied to a wide range of natural product scaffolds. Publisher PDF

The rare nonproteinogenic amino acid, meta‐l‐tyrosine is biosynthetically intriguing. Whilst the biogenesis of tyrosine from phenylalanine is well characterised, the mechanistic basis for meta‐hydroxylation is unknown. Herein, we report the analysis of 3‐hydroxylase (Phe3H) from Streptomyces coeruleorubidus. Insights from kinetic analyses of the wild‐type enzyme and key mutants as well as of the biocatalytic conversion of synthetic isotopically labelled substrates and fluorinated substrate analogues advance understanding of the process by which meta‐hydroxylation is mediated, revealing T202 to play an important role. In the case of the WT enzyme, a deuterium label at the 3‐position is lost, whereas in in the T202A mutant 75 % retention is observed, with loss of stereospecificity. These data suggest that one of two possible mechanisms is at play; direct, enzyme‐catalysed deprotonation following electrophilic aromatic substitution or stereospecific loss of one proton after a 1,2‐hydride shift. Furthermore, our kinetic parameters for Phe3H show efficient regiospecific generation of meta‐l‐tyrosine from phenylalanine and demonstrate the enzyme's ability to regiospecifically hydroxylate unnatural fluorinated substrates., The final biosynthetic step to meta‐tyrosine, an unnatural amino acid found in biologically active natural products, is mediated by phenylalanine meta‐hydroxylase. An integrated kinetics analysis, isotope labelling and mutational analysis study identified T202 as mediating mechanistic control of this intriguing enzyme. The synthetic utility of this enzyme was expanded to fluorinated phenylalanine analogues.

A nonenzymatic Pictet‐Spengler reaction has been postulated to give rise to a subset of naturally occurring uridyl peptide antibiotics (UPAs). Here, using a combination of strain engineering and synthetic chemistry, we demonstrate that Pictet‐Spengler chemistry may be employed to generate even greater diversity in the UPAs. We use an engineered strain to afford access to meta‐tyrosine containing pacidamycin 4. Pictet‐Spengler diversification of this compound using a small series of aryl‐aldehydes was achieved with some derivatives affording remarkable diastereomeric control., Beyond the natural: We demonstrate the importance of the N terminus to pacidamycin bioactivity. Incorporating meta‐tyrosine at the N‐terminal facilitates diversification of pacidamycins enabling use of Pictet‐Spengler chemistry and may enable the attenuation of compound activity.

Authors gratefully acknowledge the Max Planck Society, ERA-IB project NBCPBH-EIB.13.008 (SO’C and RG) and ERC 788301 (SO’C). Plant secondary metabolites have applications for the food, biofuel, and pharmaceutical industries. Recent advances in pathway elucidation and host expression systems now allow metabolic engineering of plant metabolic pathways to produce “new-to-nature” derivatives with novel biological activities, thereby amplifying the range of industrial uses for plant metabolites. Here we use a transient expression system in the model plant Nicotiana benthamiana to reconstitute the two-step plant-derived biosynthetic pathway for auxin (indole acetic acid) to achieve accumulation up to 500 ng/g fresh mass (FM). By expressing these plant-derived enzymes in combination with either bacterial halogenases and alternative substrates, we can produce both natural and new-to-nature halogenated auxin derivatives up to 990 ng/g FM. Proteins from the auxin synthesis pathway, tryptophan aminotransferases (TARs) and flavin-dependent monooxygenases (YUCs), could be transiently expressed in combination with four separate bacterial halogenases to generate halogenated auxin derivatives. Brominated auxin derivatives could also be observed after infiltration of the transfected N. benthamiana with potassium bromide and the halogenases. Finally, the production of additional auxin derivatives could also be achieved by co-infiltration of TAR and YUC genes with various tryptophan analogs. Given the emerging importance of transient expression in N. benthamiana for industrial scale protein and product expression, this work provides insight into the capacity of N. benthamiana to interface bacterial genes and synthetic substrates to produce novel halogenated metabolites. Publisher PDF

The uridyl peptide antibiotics (UPAs), of which pacidamycin is a member, have a clinically unexploited mode of action and an unusual assembly. Perhaps the most striking feature of these molecules is the biosynthetically unique 3'-deoxyuridine that they share. This moiety is generated by an unusual, small and monomeric dehydratase, Pac13, which catalyses the dehydration of uridine-5'-aldehyde. Here we report the structural characterisation of Pac13 with a series of ligands, and gain insight into the enzyme's mechanism demonstrating that H42 is critical to the enzyme's activity and that the reaction is likely to proceed via an E1cB mechanism. The resemblance of the 3'-deoxy pacidamycin moiety with the synthetic anti-retrovirals, presents a potential opportunity for the utilisation of Pac13 in the biocatalytic generation of antiviral compounds.

We thank the European Research Council under the European Union’s Seventh Framework Programme (FP7/2007–2013/ERC grant agreement no. 614779 GenoChemetics to R.J.M.G.), Syngenta and Wellcome ISSF (grant no. 204821/Z/16/Z to D.S.G.) for generous financial support. Oceanic cyanobacteria are the most abundant oxygen-generating phototrophs on our planet and are therefore important to life. These organisms are infected by viruses called cyanophages, which have recently shown to encode metabolic genes that modulate host photosynthesis, phosphorus cycling and nucleotide metabolism. Herein we report the characterization of a wild-type flavin-dependent viral halogenase (VirX1) from a cyanophage. Notably, halogenases have been previously associated with secondary metabolism, tailoring natural products. Exploration of this viral halogenase reveals it capable of regioselective halogenation of a diverse range of substrates with a preference for forming aryl iodide species; this has potential implications for the metabolism of the infected host. Until recently, a flavin-dependent halogenase that is capable of iodination in vitro had not been reported. VirX1 is interesting from a biocatalytic perspective as it shows strikingly broad substrate flexibility and a clear preference for iodination, as illustrated by kinetic analysis. These factors together render it an attractive tool for synthesis. Postprint

Plants are an excellent source of drug leads. However availability is limited by access to source species, low abundance and recalcitrance to chemical synthesis. Although plant genomics is yielding a wealth of genes for natural product biosynthesis, the translation of this genetic information into small molecules for evaluation as drug leads represents a major bottleneck. For example, the yeast platform for artemisinic acid production is estimated to have taken >150 person years to develop. Here we demonstrate the power of plant transient transfection technology for rapid, scalable biosynthesis and isolation of triterpenes, one of the largest and most structurally diverse families of plant natural products. Using pathway engineering and improved agro-infiltration methodology we are able to generate gram-scale quantities of purified triterpene in just a few weeks. In contrast to heterologous expression in microbes, this system does not depend on re-engineering of the host. We next exploit agro-infection for quick and easy combinatorial biosynthesis without the need for generation of multi-gene constructs, so affording an easy entrée to suites of molecules, some new-to-nature, that are recalcitrant to chemical synthesis. We use this platform to purify a suite of bespoke triterpene analogs and demonstrate differences in anti-proliferative and anti-inflammatory activity in bioassays, providing proof of concept of this system for accessing and evaluating medicinally important bioactives. Together with new genome mining algorithms for plant pathway discovery and advances in plant synthetic biology, this advance provides new routes to synthesize and access previously inaccessible natural products and analogs and has the potential to reinvigorate drug discovery pipelines., Highlights • Transient plant expression technology provides rapid access to diverse triterpenes. • Gram-scale quantities of purified triterpene can be generated. • Agro-infiltration can be exploited for quick and easy combinatorial biosynthesis. • Bespoke known and new-to-nature compounds can be generated. • The platform allows isolation of analogs for structure-activity investigations.

The authors thank the European Research Council under the European Union’s Seventh Framework Programme (FP7/2007-2013/ERC grant agreement no 614779, and the University of St Andrews for a studentship (to D. R. M. Smith). We report, for the first time, the use of tryptophan synthase in the generation of L- dihalotryptophans and L-alkynyltryptophans. These previously unpublished compounds will be useful tools in the generation of probes for chemical biology, in biosynthetic diversification and as convenient building blocks for synthesis. Postprint

This work was supported by MINECO-FEDER (BIO2016–75327-R) and the Generalitat de Catalunya (XRB and 2014-SGR-521). Júlia García was supported by an FPU grant (AP2012–6464). IRB Barcelona is the recipient of a Severo Ochoa Award of Excellence from MINECO. The IWT Flanders and Janssen Pharmaceutica are thanked for providing financial support of T.W. B.U.W.M ad S.B. thank the Research Foundation Flanders (FWO Vlaanderen; Scientific Research Network (WOG)). While revisiting biologically active natural peptides, the importance of the tryptophan residue became clear. In this article, the incorporation of this amino acid, brominated at different positions of the indole ring, into cyclic peptides was successfully achieved. These products demonstrated improved properties in terms of passive diffusion, permeability across membranes, biostability in human serum and cytotoxicity. Moreover, these brominated tryptophans at positions 5, 6, or 7 proved to be compatible as building blocks to prepare bicyclic stapled peptides by performing on-resin Suzuki-Miyaura cross-coupling reactions. Postprint

Patulin (1) is a mycotoxin contaminant in fruit and vegetable products worldwide. Biocontrol agents, such as the yeast Rhodotorula kratochvilovae strain LS11, can reduce patulin (1) contamination in food. R. kratochvilovae LS11 converts patulin (1) into desoxypatulinic acid (DPA) (5), which is less cytotoxic than the mycotoxin (1) to in vitro human lymphocytes. In the present study, we report our investigations into the pathway of degradation of patulin (1) to DPA (5) by R. kratochvilovae. Isotopic labeling experiments revealed that 5 derives from patulin (1) through the hydrolysis of the γ-lactone ring and subsequent enzymatic modifications. The ability of patulin (1) and DPA (5) to cause genetic damage was also investigated by the cytokinesis-block micronucleus cytome assay on in vitro human lymphocytes. Patulin (1) was demonstrated to cause much higher chromosomal damage than DPA (5).

The market sizes for halogenated compounds are huge; in contrast to synthetic chemical alternatives, halogenating enzymes afford the highly regiospecific incorporation of a halogen into an organic molecule, there is therefore great opportunity for the development of industrially relevant halogenases. Here we overview the main classes of halogenases, the mechanisms through which they operate and their structures. We also look at recent applications of halogenases in areas such as metabolite engineering and late stage CH activation. Opportunities and bottlenecks are discussed.

The authors thank EU BlueGenics (Seventh Framework Programme, Collaborative project “BlueGenics”, Grant no. 311848 RJMG and JP), the SNF (Grant no.205321_165695 to JP), the Helmut Horten Foundation (JP), and ERAIB (Grant no. 031A338A KHVP and RJMG) for funding. Uncultured bacteria from sponges have been demonstrated to be responsible for the generation of many potent, bioactive natural products including halogenated metabolites.1 The identification of gene clusters from the metagenomes of such bacterial communities enables the discovery of enzymes that mediate new and useful chemistries and allows insight to be gained into the biogenesis of potentially pharmacologically important natural products. Here we report a new pathway to the keramamides (krm); the first functional evidence for the existence of a distinct producer in the Theonella swinhoei WA chemotype is revealed, and a key enzyme on the pathway, a unique flavin dependent halogenase with a broad substrate specificity, and with potential as a useful new biocatalytic tool is described. Postprint

Access to natural products and their analogues is crucial. Such compounds have, for many years, played a central role in the area of drug discovery as well as in providing tools for chemical biology­. The ability to quickly and inexpensively acquire genome sequences has accelerated the field of natural product research. Access to genomic data coupled with new technologies for the engineering of organisms is resulting in the identification of large numbers of previously undiscovered natural products as well as an increased understanding of how the biosynthetic pathways responsible for the biogenesis of these compounds may be manipulated. This short review summarizes and reflects upon approaches to accessing natural products and has a particular focus on approaches combining molecular biology and synthetic chemistry. 1 Introduction 2 CHEM: Total Synthesis 3 BIO-CHEM: Semi-Synthesis 4 CHEM-BIO: Precursor Directed Biosynthesis 5 BIO-CHEM-BIO: Mutasynthesis 6 BIO-BIO: Combinatorial Biosynthesis 7 BIO-BIO-CHEM: Genochemetics: Gene Expression Enabling Synthetic Diversification 8 Conclusions and Future Directions in Natural Product Analogue Generation

This work was supported by BBSRC Grant BB/I008713/2 and European Research Council under the European Union’s Seventh Framework Programme (FP7/2007–2013/ERC Grant agreement no 614779) to Goss and BB/I006834/1 to Simmons. Background: Engineering of single-species biofilms for enzymatic generation of fine chemicals is attractive. We have recently demonstrated the utility of an engineered Escherichia coli biofilm as a platform for synthesis of 5-halotryptophan. E. coli PHL644, expressing a recombinant tryptophan synthase, was employed to generate a biofilm. Its rapid deposition, and instigation of biofilm formation, was enforced by employing a spin-down method. The biofilm presents a large three-dimensional surface area, excellent for biocatalysis. The catalytic longevity of the engineered biofilm is striking, and we had postulated that this was likely to largely result from protection conferred to recombinant enzymes by biofilm’s extracellular matrix. SILAC (stable isotopic labelled amino acids in cell cultures), and in particular dynamic SILAC, in which pulses of different isotopically labelled amino acids are administered to cells over a time course, has been used to follow the fate of proteins. To explore within our spin coated biofilm, whether the recombinant enzyme’s longevity might be in part due to its regeneration, we introduced pulses of isotopically labelled lysine and phenylalanine into medium overlaying the biofilm and followed their incorporation over the course of biofilm development. Results: Through SILAC analysis, we reveal that constant and complete regeneration of recombinant enzymes occurs within spin coated biofilms. The striking catalytic longevity within the biofilm results from more than just simple protection of active enzyme by the biofilm and its associated extracellular matrix. The replenishment of recombinant enzyme is likely to contribute significantly to the catalytic longevity observed for the engineered biofilm system. Conclusions: Here we provide the first evidence of a recombinant enzyme’s regeneration in an engineered biofilm. The recombinant enzyme was constantly replenished over time as evidenced by dynamic SILAC, which suggests that the engineered E. coli biofilms are highly metabolically active, having a not inconsiderable energetic demand. The constant renewal of recombinant enzyme highlights the attractive possibility of utilising this biofilm system as a dynamic platform into which enzymes of interest can be introduced in a “plug-and-play” fashion and potentially be controlled through promoter switching for production of a series of desired fine chemicals. Publisher PDF

The pacidamycins belong to a class of antimicrobial nucleoside antibiotics that act by inhibiting the clinically unexploited target translocase I, a key enzyme in peptidoglycan assembly. As with other nucleoside antibiotics, the pacidamycin 4',5'-dehydronucleoside portion is an essential pharmacophore. Here we show that the biosynthesis of the pacidamycin nucleoside in Streptomyces coeruleorubidus proceeds through three steps from uridine. The transformations involve oxidation of the 5'-alcohol by Pac11, transamination of the resulting aldehyde by Pac5, and dehydration by the Cupin-domain protein Pac13.

Biofilm, friend not foe: Single species biofilms can be engineered to form robust biocatalysts with greater catalytic activity and significantly improved catalytic longevity than purified and immobilised enzymes. We report the engineering, structural analysis and biocatalytic capability of a biofilm that can mediate the conversion of serine and haloindoles to halotryptophans.

Rapamycin is a drug with several important clinical uses. Its complex structure means that total synthesis of this natural product and its analogues is demanding and lengthy. A more expeditious approach is to utilise biosynthesis to enable the generation of otherwise synthetically intractable analogues. In order to achieve this, rules governing biosynthetic precursor substrate preference must be established. Through determining these rules and synthesising and administering suitable substrate precursors, we demonstrate the first generation of fluorinated rapamycin analogues. Here we report the generation of six new fluororapamycins.

Pacidamycins, mureidomycins and napsamycins are structurally related uridyl peptide antibiotics that inhibit translocase I, an as yet clinically unexploited target. This potentially important bioactivity coupled to the biosynthetically intriguing structure of pacidamycin make this natural product a fascinating subject for study. A precursor-directed biosynthesis approach was employed in order to access new pacidamycin derivatives. Strikingly, the biosynthetic machinery exhibited highly relaxed substrate specificity with the majority of the tryptophan analogues that were administered; this resulted in the production of new pacidamycin derivatives. Remarkably, 2-methyl-, 7-methyl-, 7-chloro- and 7-bromotryptophans produced their corresponding pacidamycin analogues in larger amounts than the natural pacidamycin. Low levels or no incorporation was observed for tryptophans substituted at positions 4, 5 and 6. The ability to generate bromo- and chloropacidamycins opens up the possibility of further functionalising these compounds through chemical cross-coupling in order to access a much larger family of derivatives.

A sensitive method for the configurational analysis of (R)- and (S)-[2H1]-fluoroacetate has been developed using 2H[1H]-NMR in a chiral liquid crystalline solvent. This has enabled biosynthetic experiments to be conducted which reveal stereochemical details on biological fluorination occurring during the biosynthesis of fluoroacetate and 4-fluorothreonine in the bacterium Streptomyces cattleya. In particular, feeding experiments to S. cattleya with isotopically labeled (1R, 2R)- and (1S, 2R)-[1-2H1]-glycerol 3d and 3e and [2,3-2H(4)]-succinate 4a gave rise to samples of enantiomerically enriched [2-2H1]-fluoroacetates 1a. The predominant enantiomer resulting from each experiment suggests that the stereochemical course of biological fluorination takes place with an overall retention of configuration between a glycolytic intermediate and fluoroacetate 1. Consequently, this outcome suggests that the stereochemical course of the recently identified fluorinase enzyme which mediates a reaction between fluoride ion and S-adenosyl-l-methionine (SAM), occurs with an inversion of configuration.

The pacidamycin and muraymycin uridyl peptide antibiotics show some structural resemblance to an Arg-Trp-x-x-Trp sequence motif for protein–protein interaction between bacteriophage ϕX174 protein E and E. coli translocase MraY. Members of the UPA class, and a synthetic uridine–peptide analogue, were found to show reduced levels of inhibition to F288L or E287A mutant MraY enzymes, implying that the UPAs interact at this extracellular site as part of the enzyme inhibition mechanism.

A simple and scalable one-pot biotransformation enables direct access to L-halotryptophans, including L-7-iodotryptophan, from the corresponding haloindoles. The biotransformation utilizes an easy to prepare bacterial cell lysate that may be stored as the lyophilizate for several months and utilized as a catalyst as and when required.

Introduction of prnA, the halogenase gene from pyrrolnitrin biosynthesis, into Streptomyces coeruleorubidus resulted in efficient in situ chlorination of the uridyl peptide antibotic pacidamycin. The installed chlorine provided a selectably functionalizable handle enabling synthetic modification of the natural product using mild cross-coupling conditions in crude aqueous extracts of the culture broth.

Saponins are polar molecules that consist of a triterpene or steroid aglycone with one or more sugar chains. They are one of the most numerous and diverse groups of plant natural products. These molecules have important ecological and agronomic functions, contributing to pest and pathogen resistance and to food quality in crop plants. They also have a wide range of commercial applications in the food, cosmetics and pharmaceutical sectors. Although primarily found in plants, saponins are produced by certain other organisms, including starfish and sea cucumbers. The under explored biodiversity of this class of natural products is likely to prove to be a vital resource for discovery of high-value compounds. This review will focus on the biological activity of some of the best-studied examples of saponins, on the relationship between structure and function, and on prospects for synthesis of ‘‘designer’’ saponins.

The pacidamycins are antimicrobial nucleoside antibiotics produced by Streptomyces coeruleorubidus that inhibit translocase I, an essential bacterial enzyme yet to be clinically targeted. The novel pacidamycin scaffold is composed of a pseudopeptide backbone linked by a unique exocyclic enamide to an atypical 3'-deoxyuridine nucleoside. In addition, the peptidyl chain undergoes a double inversion caused by the incorporation of a diamino acid residue and a rare internal ureido moiety. The pacidamycin gene cluster was identified and sequenced, thereby providing the first example of a biosynthetic cluster for a member of the uridyl peptide family of antibiotics. Analysis of the 22 ORFs provided an insight into the biosynthesis of the unique structural features of the pacidamycins. Heterologous expression in Streptomyces lividans resulted in the production of pacidamycin D and the newly identified pacidamycin S, thus confirming the identity of the pacidamycin biosynthetic gene cluster. Identification of this cluster will enable the generation of new uridyl peptide antibiotics through combinatorial biosynthesis. The concise cluster will provide a useful model system through which to gain a fundamental understanding of the way in which nonribosomal peptide synthetases interact.

Feeding phenylalanine analogues to Streptomyces coeruleorubidus reveals the remarkable steric and electronic flexibility of this biosynthetic pathway and leads to the generation of a series of new halopacidamycins.

The quest for new antibiotics, especially those with activity against Gram-negative bacteria, is urgent; however, very few new antibiotics have been marketed in the last 40 years, with this limited number falling into only four new structural classes. Several nucleoside natural product antibiotics target bacterial translocase MraY, involved in the lipid-linked cycle of peptidoglycan biosynthesis, and fungal chitin synthase. Biosynthetic studies on the nikkomycin, caprazamycin and pacidamycin/mureidomycin families are also reviewed.

Serine carboxypeptidase-like (SCPL) proteins have recently emerged as a new group of plant acyltransferases. These enzymes share homology with peptidases but lack protease activity and instead are able to acylate natural products. Several SCPL acyltransferases have been characterized to date from dicots, including an enzyme required for the synthesis of glucose polyesters that may contribute to insect resistance in wild tomato (Solanum pennellii) and enzymes required for the synthesis of sinapate esters associated with UV protection in Arabidopsis thaliana. In our earlier genetic analysis, we identified the Saponin-deficient 7 (Sad7) locus as being required for the synthesis of antimicrobial triterpene glycosides (avenacins) and for broad-spectrum disease resistance in diploid oat (Avena strigosa). Here, we report on the cloning of Sad7 and show that this gene encodes a functional SCPL acyltransferase, SCPL1, that is able to catalyze the synthesis of both N-methyl anthraniloyl- and benzoyl-derivatized forms of avenacin. Sad7 forms part of an operon-like gene cluster for avenacin synthesis. Oat SCPL1 (SAD7) is the founder member of a subfamily of monocot-specific SCPL proteins that includes predicted proteins from rice (Oryza sativa) and other grasses with potential roles in secondary metabolism and plant defense.

A one-pot biotransformation for the generation of a series of L-aminotryptophans using a readily prepared protein extract containing tryptophan synthase is reported. The extract exhibits remarkable stability upon freeze-drying, and may be stored and used for long periods after its preparation without significant loss of activity.

We report a convenient synthesis of 4-fluorocyclohexanoic acid, and an insight into the rules governing acceptance of starter acid analogues in the precursor-directed biosynthesis of rapamycin.

Plants produce a huge array of specialized metabolites that have important functions in defense against biotic and abiotic stresses. Many of these compounds are glycosylated by family 1 glycosyltransferases (GTs). Oats (Avena spp.) make root-derived antimicrobial triterpenes (avenacins) that provide protection against soil-borne diseases. The ability to synthesize avenacins has evolved since the divergence of oats from other cereals and grasses. The major avenacin, A-1, is acylated with N-methylanthranilic acid. Previously, we have cloned and characterized three genes for avenacin synthesis (for the triterpene synthase SAD1, a triterpene-modifying cytochrome P450 SAD2, and the serine carboxypeptidase-like acyl transferase SAD7), which form part of a biosynthetic gene cluster. Here, we identify a fourth member of this gene cluster encoding a GT belonging to clade L of family 1 (UGT74H5), and show that this enzyme is an N-methylanthranilic acid O-glucosyltransferase implicated in the synthesis of avenacin A-1. Two other closely related family 1 GTs (UGT74H6 and UGT74H7) are also expressed in oat roots. One of these (UGT74H6) is able to glucosylate both N-methylanthranilic acid and benzoic acid, whereas the function of the other (UGT74H7) remains unknown. Our investigations indicate that UGT74H5 is likely to be key for the generation of the activated acyl donor used by SAD7 in the synthesis of the major avenacin, A-1, whereas UGT74H6 may contribute to the synthesis of other forms of avenacin that are acylated with benzoic acid.

Covering: 2005 to 2012 Natural product analogue generation is important, providing tools for chemical biology, enabling structure activity relationship determination and insight into the way in which natural products interact with their target biomolecules. The generation of analogues is also often necessary in order to improve bioavailability and to fine tune compounds' activity. This review provides an overview of the catalogue of approaches available for accessing series of analogues. Over the last few years there have been major advances in genome sequencing and the development of tools for biosynthetic pathway engineering; it is therefore becoming increasingly easy to combine molecular biology and synthetic organic chemistry in order to enable expeditious access to series of natural products. This review outlines the various ways of combining biology and chemistry that have been applied to analogue generation, drawing upon a series of examples to illustrate each approach.

As with many other antibiotics, pacidamycins are produced as a suite of related compounds. Unlike most other secondary metabolites, however, this diversity is not solely the result of the substrate promiscuity of the biosynthetic enzymes but also arises from a gene duplication event (Pac21, Pac21h) and control of the precursor pool (PhhA). We are demonstrating the ability to harness these three levels of control in order to direct the selective production of specific members of this family of metabolites in a “dial-a-molecule” fashion. Furthermore, PhhA is shown to be a phenylalanine 3-hydroxylase, the first of the iron- and tetrahydropterin-dependent aromatic amino acid hydroxylases to be characterised with this regioselectivity.

Longianone, a metabolite of Xylaria longiana, is an isomer of the well known fungal toxin, patulin; it is demonstrated that longianone is biosynthesised from 6-methylsalicylic acid in a pathway closely related to that found in patulin biosynthesis.

A scalable and general biotransformation for the generation of a series of L-halotryptophans using the lysate of a commercially available microorganism containing tryptophan synthase.