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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. [ABSTRACT FROM AUTHOR]

Abstract: Benzylisoquinoline alkaloids (BIAs) are a structurally diverse family of plant secondary metabolites, which have been exploited to develop analgesics, antibiotics, antitumor agents, and other therapeutic agents. Biosynthesis of BIAs proceeds via a common pathway from tyrosine to (S)‐reticulene at which point the pathway diverges. Coclaurine N‐methyltransferase (CNMT) is a key enzyme in the pathway to (S)‐reticulene, installing the N‐methyl substituent that is essential for the bioactivity of many BIAs. In this paper, we describe the first crystal structure of CNMT which, along with mutagenesis studies, defines the enzymes active site architecture. The specificity of CNMT was also explored with a range of natural and synthetic substrates as well as co‐factor analogues. Knowledge from this study could be used to generate improved CNMT variants required to produce BIAs or synthetic derivatives. [ABSTRACT FROM AUTHOR]

Abstract: 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Biosynthetic engineering involves the reprogramming of genes that are involved in the biosynthesis of natural products to generate new "non-natural" products, which might otherwise not exist in nature. Potentially this approach can be used to provide large numbers of secondary metabolite variants, with altered biological activities, many of which are too complex for effective total synthesis. Recently we have been investigating the biosynthesis of the calcium-dependent antibiotics (CDAs) which are members of the therapeutically relevant class of acidic lipopeptide antibiotics. CDAs are assembled by nonribosomal peptide synthetase (NRPS) enzymes. These large modular assembly-line enzymes process intermediates that are covalently tethered to peptidyl carrier protein (PCP) domain bonds, which makes them particularly amenable to reprogramming. The CDA producer, Streptomyces coelicolor, is also a genetically tractable model organism which makes CDAs an ideal template for biosynthetic engineering. To this end, we have elucidated many of the key steps in CDA biosynthesis and utilized this information to develop methods that have enabled the engineered biosynthesis of a wide range of CDA-type lipopeptides. [ABSTRACT FROM AUTHOR]

Natural products continue to fulfill an important role in the development of therapeutic agents. In addition, with the advent of chemical genetics and high-throughput screening platforms, these molecules have become increasingly valuable as tools for interrogating fundamental aspects of biological systems. To access the vast portion of natural-product structural diversity that remains unexploited for these and other applications, genome mining and microbial metagenomic approaches are proving particularly powerful. When these are coupled with recombineering and related genetic tools, large biosynthetic gene clusters that remain intractable or cryptic in the native host can be more efficiently cloned and expressed in a suitable heterologous system. For lead optimization and the further structural diversification of natural-product libraries, combinatorial biosynthetic engineering has also become indispensable. However, our ability to rationally redesign biosynthetic pathways is often limited by our lack of understanding of the structure, dynamics and interplay between the many enzymes involved in complex biosynthetic pathways. Despite this, recent structures of fatty acid synthases should allow a more accurate prediction of the likely architecture of related polyketide synthase and nonribosomal peptide synthetase multienzymes. [ABSTRACT FROM AUTHOR]

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., Chemistry and Chemical Biology

The calcium dependent antibiotic (CDA) is a nonribosomal lipopeptide produced by Streptomyces coelicolor. We constructed a metabolic network of more than 400 reactions for the primary and secondary metabolism of S. coelicolor and used computational metabolic flux balancing to investigate some of the factors affecting growth and production of CDA. Computational results indicated that the CDA production was concomitant with growth. Computational specific growth rates were twice as high as the experimental specific growth rates. Metabolic flux distributions and sensitivity analyses computed for various phases of the batch culture indicated that the specific CDA production rate was affected by nitrogen assimilation, pentose phosphate pathway, shikimate biosynthesis, and oxoglutarate fluxes. Consequently, these metabolic targets were tested using genetic deletions in the model which increased the in silico specific CDA production rate. [Copyright &y& Elsevier]

With the advent of next-generation DNA sequencing technologies, the number of microbial genome sequences has increased dramatically, revealing a vast array of new biosynthetic gene clusters. Genomics data provide a tremendous opportunity to discover new natural products, and also to guide the bioengineering of new and existing natural product scaffolds for therapeutic applications. Notably, it is apparent that the vast majority of biosynthetic gene clusters are either silent or produce very low quantities of the corresponding natural products. It is imperative therefore to devise methods for activating unproductive biosynthetic pathways to provide the quantities of natural products needed for further development. Moreover, on the basis of our expanding mechanistic and structural knowledge of biosynthetic assembly-line enzymes, new strategies for re-programming biosynthetic pathways have emerged, resulting in focused libraries of modified products with potentially improved biological properties. In this review we will focus on the latest bioengineering approaches that have been utilised to optimise yields and increase the structural diversity of natural product scaffolds for future clinical applications. [ABSTRACT FROM AUTHOR]

The biological properties of the calcium-dependent antibiotics (CDAs), daptomycin and related nonribosomal lipopeptides, depend to a large extent on the nature of the N-terminal fatty acid moiety. It is suggested that the chain length of the unusually short (C6) 2,3-epoxyhexanoyl fatty acid moiety of CDA is determined by the specificity of the KAS-II enzyme encoded by fabF3 in the CDA biosynthetic gene cluster. Indeed, deletion of the downstream gene hxcO results in three new lipopeptides, all of which possess hexanoyl side chains (hCDAs). This confirms that HxcO functions as a hexanoyl-CoA or -ACP oxidase. The absence of additional CDA products with longer fatty acid groups further suggests that the CDA lipid chain is biosynthesized on a single ACP and is then transferred directly from this ACP to the first CDA peptide synthetase (CdaPS1). Interestingly, the hexanoyl-containing CDAs retain antibiotic activity. To further modulate the biological properties of CDA by introducing alternative fatty acid groups, a mutasynthesis approach was developed. This involved mutating the key active site Ser residue of the CdaPS1, module 1 PCP domain to Ala, which prevents subsequent phosphopantetheinylation. In the absence of the natural module 1 PCP tethered intermediate, it is possible to effect incorporation of different N-acyl-L-serinyl N-acetylcysteamine (NAC) thioester analogues, leading to CDA products with pentanoyl as well as hexanoyl side chains. [ABSTRACT FROM AUTHOR]

The acidic lipopeptides, including the calcium-dependent antibiotics (CDA), daptomycin, and A54145, are important macrocyclic peptide natural products produced by Streptomyces species. All three compounds contain a 3-methyl glutamate (3-MeGlu) as the penultimate C-terminal residue, which is important for bioactivity. Here, biochemical in vitro reconstitution of the 3-MeGlu biosynthetic pathway is presented, using exclusively enzymes from the CDA producer Streptomyces coelicolor. It is shown that the predicted 3-MeGlu methyltransferase GlmT and its homologues Dptl from the daptomycin producer Streptomyces roseosporus and Lptl from the A54145 producer Streptomyces fradiae do not methylate free glutamic acid, PCP-bound glutamate, or Glu-containing CDA in vitro. Instead, GlmT, Dptl, and Lptl are S-adenosyl methionine (SAM)-dependent α-ketoglutarate methyltransferases that catalyze the stereospecific methylation of α-ketoglutarate (αKG) leading to (3R)-3-methyl-2-oxoglutarate. Subsequent enzyme screening identified the branched chain amino acid transaminase lIvE (SCO5523) as an efficient catalyst for the transformation of (3R)-3-methyl-2-oxoglutarate into (2S,3R)-3-MeGlu. Comparison of reversed-phase HPLC retention time of dabsylated 3-MeGlu generated by the coupled enzymatic reaction with dabsylated synthetic standards confirmed complete stereocontrol during enzymatic catalysis. This stereospecific two-step conversion of αKG to (2S,3R)-3-MeGlu completes our understanding of the biosynthesis and incorporation of β-methylated amino acids into the nonribosomal lipopeptides. Finally, understanding this pathway may provide new possibilities for the production of modified peptides in engineered microbes. [ABSTRACT FROM AUTHOR]

The calcium-dependent antibiotics (CDAs) and daptomycin are therapeutically relevant non-ribosomal lipopeptide antibiotics that contain penultimate C-terminal 3-methyl glutamate (3-MeGlu) residues. Comparison with synthetic standards showed that (2S,3R)-configured 3-MeGlu is present in both CDA and daptomycin. Deletion of a putative methyltransferase gene glmT from the cda biosynthetic gene cluster abolished the incorporation of 3-MeGlu and resulted in the production of Glu-containing CDA exclusively. However, the 3-MeGlu chemotype could be re-established through feeding synthetic 3-methyl-2-oxoglutarate and (2S,3R)-3-MeGlu, but not (2S,3S)-3-MeGlu. This indicates that methylation occurs before peptide assembly, and that the module 10 A-domain of the CDA peptide synthetase is specific for the (2S,3R)-stereoisomer. Further mechanistic analyses suggest that GImT catalyzes the SAM-dependent methylation of α-ketoglutarate to give (3R)-methyl-2-oxoglutarate, which is transaminated to (2S,3R)-3-MeGlu. These insights will facilitate future efforts to engineer lipopeptides with modified glutamate residues, which may have improved bioactivity and/or reduced toxicity. [ABSTRACT FROM AUTHOR]