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The cycloaspeptide gene cluster includes a pathway-specific N-methyl transferase. Its disruption allowed incorporation of N-methylated amino acids provided in the culture medium, allowing efficient production of cycloaspeptide E and novel related products., The cycloaspeptides are bioactive pentapeptides produced by various filamentous fungi, which have garnered interest from the agricultural industry due to the reported insecticidal activity of the minor metabolite, cycloaspeptide E. Genome sequencing, bioinformatics and heterologous expression confirmed that the cycloaspeptide gene cluster contains a minimal 5-module nonribosomal peptide synthetase (NRPS) and a new type of trans-acting N-methyltransferase (N-MeT). Deletion of the N-MeT encoding gene and subsequent feeding studies determined that two modules of the NRPS preferentially accept and incorporate N-methylated amino acids. This discovery allowed the development of a system with unprecedented control over substrate supply and thus output, both increasing yields of specific metabolites and allowing the production of novel fluorinated analogues. Furthermore, the biosynthetic pathway to ditryptophenaline, another fungal nonribosomal peptide, was shown to be similar, in that methylated phenylalanine is accepted by the ditryptophenaline NRPS. Again, this allowed the directed biosynthesis of a fluorinated analogue, through the feeding of a mutant strain. These discoveries represent a new paradigm for the production of N-methylated cyclic peptides via the selective incorporation of N-methylated free amino acids.

Flavin‐dependent halogenases are useful enzymes for providing halogenated molecules with improved biological activity, or intermediates for synthetic derivatization. We demonstrate how the fungal halogenase RadH can be used to regioselectively halogenate a range of bioactive aromatic scaffolds. Site‐directed mutagenesis of RadH was used to identify catalytic residues and provide insight into the mechanism of fungal halogenases. A high‐throughput fluorescence screen was also developed, which enabled a RadH mutant to be evolved with improved properties. Finally we demonstrate how biosynthetic genes from fungi, bacteria, and plants can be combined to encode a new pathway to generate a novel chlorinated coumarin “non‐natural” product in E. coli.

Polyketides are a class of specialised metabolites synthesised by both eukaryotes and prokaryotes. These chemically and structurally diverse molecules are heavily used in the clinic and include frontline antimicrobial and anticancer drugs such as erythromycin and doxorubicin. To replenish the clinicians’ diminishing arsenal of bioactive molecules, a promising strategy aims at transferring polyketide biosynthetic pathways from their native producers into the biotechnologically desirable host Escherichia coli. This approach has been successful for type I modular polyketide synthases (PKSs); however, despite more than 3 decades of research, the large and important group of type II PKSs has until now been elusive in E. coli. Here, we report on a versatile polyketide biosynthesis pipeline, based on identification of E. coli–compatible type II PKSs. We successfully express 5 ketosynthase (KS) and chain length factor (CLF) pairs—e.g., from Photorhabdus luminescens TT01, Streptomyces resistomycificus, Streptoccocus sp. GMD2S, Pseudoalteromonas luteoviolacea, and Ktedonobacter racemifer—as soluble heterodimeric recombinant proteins in E. coli for the first time. We define the anthraquinone minimal PKS components and utilise this biosynthetic system to synthesise anthraquinones, dianthrones, and benzoisochromanequinones (BIQs). Furthermore, we demonstrate the tolerance and promiscuity of the anthraquinone heterologous biosynthetic pathway in E. coli to act as genetically applicable plug-and-play scaffold, showing it to function successfully when combined with enzymes from phylogenetically distant species, endophytic fungi and plants, which resulted in 2 new-to-nature compounds, neomedicamycin and neochaetomycin. This work enables plug-and-play combinatorial biosynthesis of aromatic polyketides using bacterial type II PKSs in E. coli, providing full access to its many advantages in terms of easy and fast genetic manipulation, accessibility for high-throughput robotics, and convenient biotechnological scale-up. Using the synthetic and systems biology toolbox, this plug-and-play biosynthetic platform can serve as an engine for the production of new and diversified bioactive polyketides in an automated, rapid, and versatile fashion.

Flavin‐dependent halogenases are potentially useful biocatalysts for the regioselective halogenation of aromatic compounds. Haloaromatic compounds can be utilised in the synthesis and biosynthesis of pharmaceuticals and other valuable products. Here we report the first X‐ray crystal structure of a tryptophan 6‐halogenase (SttH), which enabled key residues that contribute to the regioselectivity in tryptophan halogenases to be identified. Structure‐guided mutagenesis resulted in a triple mutant (L460F/P461E/P462T) that exhibited a complete switch in regioselectivity; with the substrate 3‐indolepropionate 75 % 5‐chlorination was observed with the mutant in comparison to 90 % 6‐chlorination for the wild‐type SttH. This is the first clear example of how regiocomplementary halogenases can be created from a single parent enzyme. The biocatalytic repertoire of SttH was also expanded to include a range of indolic and non‐indolic substrates.

Thaxtomins are diketopiperazine phytotoxins produced by Streptomyces scabies and other actinobacterial plant pathogens that inhibit cellulose biosynthesis in plants. Due to their potent bioactivity and novel mode of action there has been considerable interest in developing thaxtomins as herbicides for crop protection. To address the need for more stable derivatives, we have developed a new approach for structural diversification of thaxtomins. Genes encoding the thaxtomin NRPS from S. scabies, along with genes encoding a promiscuous tryptophan synthase (TrpS) from Salmonella typhimurium, were assembled in a heterologous host Streptomyces albus. Upon feeding indole derivatives to the engineered S. albus strain, tryptophan intermediates with alternative substituents are biosynthesized and incorporated by the NRPS to deliver a series of thaxtomins with different functionalities in place of the nitro group. The approach described herein, demonstrates how genes from different pathways and different bacterial origins can be combined in a heterologous host to create a de novo biosynthetic pathway to 'non‐natural' product target compounds.

Flavin-dependent halogenases are useful enzymes providing halogenated molecules with improved biological activity or intermediates for synthetic derivatization. Here we demonstrate how the fungal halogenase RadH can be used to regioselectively halogenate a range of bioactive aromatic scaffolds. Site-directed mutagenesis of RadH was used to identify catalytic residues and provide insights into the mechanism of fungal halogenases. A high throughput fluorescence screen was also developed enabling a RadH mutant to be evolved with improved properties. Finally we demonstrate how biosynthetic genes from fungi, bacteria and plants can be combined, to encode a new pathway generating a novel chlorinated coumarin ‘non-natural’ product in E. coli.

Targeted mutagenesis increases the activity and alters the regioselectivity of flavin-dependent halogenases., Flavin-dependent halogenases are potentially valuable biocatalysts for the regioselective halogenation of aromatic compounds. These enzymes, utilising benign inorganic halides, offer potential advantages over traditional non-enzymatic halogenation chemistry that often lacks regiocontrol and requires deleterious reagents. Here we extend the biocatalytic repertoire of the tryptophan halogenases, demonstrating how these enzymes can halogenate a range of alternative aryl substrates. Using structure guided mutagenesis we also show that it is possible to alter the regioselectivity as well as increase the activity of the halogenases with non-native substrates including anthranilic acid; an important intermediate in the synthesis and biosynthesis of pharmaceuticals and other valuable products.

The lipopeptides ramoplanin from Actinoplanes sp. ATCC 33076 and enduracidin produced by Streptomyces fungicidicus are effective antibiotics against a number of drug-resistant Gram-positive pathogens. While these two antibiotics share a similar cyclic peptide structure, comprising 17 amino acids with an N-terminal fatty acid side chain, ramoplanin has a di-mannose moiety that enduracidin lacks. The mannosyl substituents of ramoplanin enhance aqueous solubility, which was important in the development of ramoplanin as a potential treatment for Clostridium difficile infections. In this study we have determined the function of the putative mannosyltransferase encoded by ram29 from the ramoplanin biosynthetic gene cluster. Bioinformatics revealed that Ram29 is an integral membrane protein with a putative DxD motif that is suggested to bind to, and activate, a polyprenyl phosphomannose donor and an extracytoplasmic C-terminal domain that is predicted to bind the ramoplanin aglycone acceptor. The ram29 gene was cloned into the tetracycline inducible plasmid pMS17 and integrated into the genome of the enduracidin producer S. fungicidicus. Induction of ram29 expression in S. fungicidicus resulted in the production of monomannosylated enduracidin derivatives, which are not present in the WT strain. Tandem MS analysis showed that mannosylation occurs on the Hpg11 residue of enduracidin. In addition to confirming the function of Ram29, these findings demonstrate how the less common, membrane-associated, polyprenyl phosphosugar-dependent glycosyltransferases can be used in natural product glycodiversification. Such a strategy may be valuable in future biosynthetic engineering approaches aimed at improving the physico-chemical and biological properties of bioactive secondary metabolites including antibiotics.

For enzyme-catalysed biotransformations, continuous in situ detection methods minimise the need for sample manipulation, ultimately leading to more accurate real-time kinetic determinations of substrate(s) and product(s). We have established for the first time an on-line, real-time quantitative approach to monitor simultaneously multiple biotransformations based on UV resonance Raman (UVRR) spectroscopy. To exemplify the generality and versatility of this approach, multiple substrates and enzyme systems were used involving nitrile hydratase (NHase) and xanthine oxidase (XO), both of which are of industrial and biological significance, and incorporate multistep enzymatic conversions. Multivariate data analysis of the UVRR spectra, involving multivariate curve resolution-alternating least squares (MCR-ALS), was employed to effect absolute quantification of substrate(s) and product(s); repeated benchmarking of UVRR combined with MCR-ALS by HPLC confirmed excellent reproducibility.

Flavin-dependent halogenase (Fl-Hal) enzymes have been shown to halogenate a range of synthetic as well as natural aromatic compounds. The exquisite regioselectively of Fl-Hal enzymes can provide halogenated building blocks which are inaccessible using standard halogenation chemistries. Consequently, Fl-Hal are potentially useful biocatalysts for the chemoenzymatic synthesis of pharmaceuticals and other valuable products, which are derived from haloaromatic precursors. However, the application of Fl-Hal enzymes, in vitro, has been hampered by their poor catalytic activity and lack of stability. To overcome these issues, we identified a thermophilic tryptophan halogenase (Th-Hal), which has significantly improved catalytic activity and stability, compared with other Fl-Hal characterised to date. When used in combination with a thermostable flavin reductase, Th-Hal can efficiently halogenate a number of aromatic substrates. X-ray crystal structures of Th-Hal, and the reductase partner (Th-Fre), provide insights into the factors that contribute to enzyme stability, which could guide the discovery and engineering of more robust and productive halogenase biocatalysts.

Despite major recent advances in C-H activation, discrimination between two similar, unactivated C-H positions is beyond the scope of current chemocatalytic methods. Here we demonstrate that integration of regioselective halogenase enzymes with Pd-catalysed cross-coupling chemistry, in one-pot reactions, successfully addresses this problem for the indole heterocycle. The resultant 'chemobio-transformation' delivers a range of functionally diverse arylated products that are impossible to access using separate enzymatic or chemocatalytic C-H activation, under mild, aqueous conditions. This use of different biocatalysts to select different C-H positions contrasts with the prevailing substrate-control approach to the area, and presents opportunities for new pathways in C-H activation chemistry. The issues of enzyme and transition metal compatibility are overcome through membrane compartmentalization, with the optimized process requiring no intermediate work-up or purification steps.

Flavin-dependent halogenase enzymes are potentially useful biocatalysts for the regioselectively halogenation of aromatic compounds. Haloaromatic compounds can be utilised in the synthesis and biosynthesis of pharmaceuticals and other valuable products. Here we report the first X-ray crystal structure of a tryptophan 6-halogenase (SttH), which enabled key residues that contribute to the regioselectivity in tryptophan halogenases to be identified. Structure-guided mutagenesis resulted in a triple mutant (L460F/P461E/P462T) that exhibited a complete switch in regioselectivity; with the substrate 3-indolepropionate 75% 5-chlorination was observed with the mutant in comparison with 90% 6-chlorination for the wild-type SttH. This is the first clear example of how regiocomplementary halogenase enzymes can be created from a single parent enzyme. The biocatalytic repertoire of SttH was also expanded to include a range of indolic and non-indolic substrates.

The Manchester Synthetic Biology Research Centre (SYNBIOCHEM) is a foundry for the biosynthesis and sustainable production of fine and speciality chemicals. The Centre's integrated technology platforms provide a unique capability to facilitate predictable engineering of microbial bio-factories for chemicals production. An overview of these capabilities is described.

A wide variety of enzymatic pathways that produce specialized metabolites in bacteria, fungi and plants are known to be encoded in biosynthetic gene clusters. Information about these clusters, pathways and metabolites is currently dispersed throughout the literature, making it difficult to exploit. To facilitate consistent and systematic deposition and retrieval of data on biosynthetic gene clusters, we propose the Minimum Information about a Biosynthetic Gene cluster (MIBiG) data standard.