SciCombinator

Discover the most talked about and latest scientific content & concepts.

Concept: Polyketide

27

Micacocidin is a thiazoline-containing natural product from the bacterium Ralstonia solanacearum that shows significant activity against Mycoplasma pneumoniae. The presence of a pentylphenol moiety distinguishes micacocidin from the structurally related siderophore yersiniabactin, and this residue also contributes to the potent antimycoplasma effects. The biosynthesis of the pentylphenol moiety, as deduced from bioinformatic analysis and stable isotope feeding experiments, involves an iterative type I polyketide synthase (iPKS), which generates a linear tetraketide intermediate from acyl carrier protein-tethered hexanoic acid by three consecutive, decarboxylative Claisen condensations with malonyl-coenzyme A. The final conversion into 6-pentylsalicylic acid depends on a ketoreductase domain within the iPKS, as demonstrated by heterologous expression in E. coli and subsequent site-directed mutagenesis experiments. Our results unveil the early steps in micacocidin biosynthesis and illuminate a bacterial enzyme that functionally resembles fungal polyketide synthases.

Concepts: Protein, Bacteria, Enzyme, Mycoplasma, Polyketide, Nonribosomal peptide, Ralstonia solanacearum, Polyketide synthase

27

New assembly line, new compound: SIA7248, a new symmetric macrolide, was isolated from a marine-derived Streptomyces strain. Bioinformatic analyses of the identified biosynthetic gene cluster (sia) for SIA7248 suggested a polyketide biosynthesis utilizing an iteratively trans-acting ketoreductase (KR). We characterized SiaM as a trans-KR to catalyse reductions of various β-ketoacyl-thioesters with D-stereospecificity.

Concepts: Amino acid, Molecular biology, Assembly line, Biosynthesis, Macrolide, Polyketide, Malonyl-CoA

16

Erythromycin, avermectin and rapamycin are clinically useful polyketide natural products produced on modular polyketide synthase multienzymes by an assembly-line process in which each module of enzymes in turn specifies attachment of a particular chemical unit. Although polyketide synthase encoding genes have been successfully engineered to produce novel analogues, the process can be relatively slow, inefficient, and frequently low-yielding. We now describe a method for rapidly recombining polyketide synthase gene clusters to replace, add or remove modules that, with high frequency, generates diverse and highly productive assembly lines. The method is exemplified in the rapamycin biosynthetic gene cluster where, in a single experiment, multiple strains were isolated producing new members of a rapamycin-related family of polyketides. The process mimics, but significantly accelerates, a plausible mechanism of natural evolution for modular polyketide synthases. Detailed sequence analysis of the recombinant genes provides unique insight into the design principles for constructing useful synthetic assembly-line multienzymes.

Concepts: DNA, Evolution, Amino acid, Module, Natural products, Macrolide, Polyketide, Polyketide synthase

4

Acyltransferase (AT) domains of polyketide synthases (PKSs) select extender units for incorporation into polyketides and dictate large portions of the structures of clinically relevant natural products. Accordingly, there is significant interest in engineering the substrate specificity of PKS ATs in order to site-selectively manipulate polyketide structure. However, previous attempts to engineer ATs have yielded mutant PKSs with relaxed extender unit specificity, rather than an inversion of selectivity from one substrate to another. Here, by directly screening the extender unit selectivity of mutants from active site saturation libraries of an AT from the prototypical PKS, 6-deoxyerythronolide B synthase, a set of single amino acid substitutions was discovered that dramatically impact the selectivity of the PKS with only modest reductions of product yields. One particular substitution (Tyr189Arg) inverted the selectivity of the wild-type PKS from its natural substrate toward a non-natural alkynyl-modified extender unit while maintaining more than twice the activity of the wild-type PKS with its natural substrate. The strategy and mutations described herein form a platform for combinatorial biosynthesis of site-selectively modified polyketide analogues that are modified with non-natural and non-native chemical functionality.

Concepts: Amino acid, Polyketide, Discodermolide, Polyketide synthase, Domain engineering

4

Natural product discovery efforts have focused primarily on microbial biosynthetic gene clusters (BGCs) containing large multimodular polyketide synthases and nonribosomal peptide synthetases; however, sequencing of fungal genomes has revealed a vast number of BGCs containing smaller NRPS-like genes of unknown biosynthetic function. Using comparative metabolomics, we show that a BGC in the human pathogen Aspergillus fumigatus named fsq, which contains an NRPS-like gene lacking a condensation domain, produces several new isoquinoline alkaloids known as the fumisoquins. These compounds derive from carbon-carbon bond formation between two amino acid-derived moieties followed by a sequence that is directly analogous to isoquinoline alkaloid biosynthesis in plants. Fumisoquin biosynthesis requires the N-methyltransferase FsqC and the FAD-dependent oxidase FsqB, which represent functional analogs of coclaurine N-methyltransferase and berberine bridge enzyme in plants. Our results show that BGCs containing incomplete NRPS modules may reveal new biosynthetic paradigms and suggest that plant-like isoquinoline biosynthesis occurs in diverse fungi.

Concepts: Bacteria, Amino acid, Molecular biology, Organism, Fungus, Alkaloid, Polyketide, Nonribosomal peptide

4

Polyketide natural products constitute a broad class of compounds with diverse structural features and biological activities. Their biosynthetic machinery, represented by type I polyketide synthases (PKSs), has an architecture in which successive modules catalyse two-carbon linear extensions and keto-group processing reactions on intermediates covalently tethered to carrier domains. Here we used electron cryo-microscopy to determine sub-nanometre-resolution three-dimensional reconstructions of a full-length PKS module from the bacterium Streptomyces venezuelae that revealed an unexpectedly different architecture compared to the homologous dimeric mammalian fatty acid synthase. A single reaction chamber provides access to all catalytic sites for the intramodule carrier domain. In contrast, the carrier from the preceding module uses a separate entrance outside the reaction chamber to deliver the upstream polyketide intermediate for subsequent extension and modification. This study reveals for the first time, to our knowledge, the structural basis for both intramodule and intermodule substrate transfer in polyketide synthases, and establishes a new model for molecular dissection of these multifunctional enzyme systems.

Concepts: Metabolism, Fatty acid, Enzyme, Fatty acid metabolism, Module, Fatty acid synthase, Polyketide, Polyketide synthase

3

In this study, we engineered fatty acid synthases (FAS) for the biosynthesis of short-chain fatty acids and polyketides, guided by a combined in vitro and in silico approach. Along with exploring the synthetic capability of FAS, we aim to build a foundation for efficient protein engineering, with the specific goal of harnessing evolutionarily related megadalton-scale polyketide synthases (PKS) for the tailored production of bioactive natural compounds.

Concepts: Protein, Amino acid, Nutrition, Fatty acid, Hydrolysis, Butyric acid, Biosynthesis, Polyketide

3

Considering that 70% of our planet’s surface is covered by oceans, it is likely that undiscovered biodiversity is still enormous. A large portion of marine biodiversity consists of microbiomes. They are very attractive targets of bioprospecting because they are able to produce a vast repertoire of secondary metabolites in order to adapt in diverse environments. In many cases secondary metabolites of pharmaceutical and biotechnological interest such as nonribosomal peptides (NRPs) and polyketides (PKs) are synthesized by multimodular enzymes named nonribosomal peptide synthetases (NRPSes) and type-I polyketide synthases (PKSes-I), respectively. Novel findings regarding the mechanisms underlying NRPS and PKS evolution demonstrate how microorganisms could leverage their metabolic potential. Moreover, these findings could facilitate synthetic biology approaches leading to novel bioactive compounds. Ongoing advances in bioinformatics and next-generation sequencing (NGS) technologies are driving the discovery of NRPs and PKs derived from marine microbiomes mainly through two strategies: genome-mining and metagenomics. Microbial genomes are now sequenced at an unprecedented rate and this vast quantity of biological information can be analyzed through genome mining in order to identify gene clusters encoding NRPSes and PKSes of interest. On the other hand, metagenomics is a fast-growing research field which directly studies microbial genomes and their products present in marine environments using culture-independent approaches. The aim of this review is to examine recent developments regarding discovery strategies of bioactive compounds synthesized by NRPS and type-I PKS derived from marine microbiomes and to highlight the vast diversity of NRPSes and PKSes present in marine environments by giving examples of recently discovered bioactive compounds.

Concepts: Bioinformatics, Bacteria, Molecular biology, Metabolism, Biotechnology, Peptide, Polyketide, Nonribosomal peptide

3

Leinamycin (LNM) is a sulfur-containing antitumor antibiotic featuring an unusual 1,3-dioxo-1,2-dithiolane moiety that is spiro-fused to a thiazole-containing 18-membered lactam ring. The 1,3-dioxo-1,2-dithiolane moiety is essential for LNM’s antitumor activity, by virtue of its ability to generate an episulfonium ion intermediate capable of alkylating DNA. We have previously cloned and sequenced the lnm gene cluster from Streptomyces atroolivaceus S-140. In vivo and in vitro characterizations of the LNM biosynthetic machinery have since established that: (i) the 18-membered macrolactam backbone is synthesized by LnmP, LnmQ, LnmJ, LnmI, and LnmG, (ii) the alkyl branch at C-3 of LNM is installed by LnmK, LnmL, LnmM, and LnmF, and (iii) leinamycin E1 (LNM E1), bearing a thiol moiety at C-3, is the nascent product of the LNM hybrid nonribosomal peptide synthetase (NRPS)-acyltransferase (AT)-less type I polyketide synthase (PKS). Sulfur incorporation at C-3 of LNM E1, however, has not been addressed. Here we report that: (i) the bioinformatics analysis reveals a pyridoxal phosphate (PLP)-dependent domain, we termed cysteine lyase (SH) domain (LnmJ-SH), within PKS module-8 of LnmJ; (ii) the LnmJ-SH domain catalyzes C-S bond cleavage by using l-cysteine and l-cysteine S-modified analogs as substrates through a PLP-dependent β-elimination reaction, establishing l-cysteine as the origin of sulfur at C-3 of LNM; and (iii) the LnmJ-SH domain, sharing no sequence homology with any other enzymes catalyzing C-S bond cleavage, represents a new family of PKS domains that expands the chemistry and enzymology of PKSs and might be exploited to incorporate sulfur into polyketide natural products by PKS engineering.

Concepts: DNA, Bioinformatics, Bacteria, Amino acid, Enzyme, Disulfide bond, Polyketide, Nonribosomal peptide

2

Macrolides are a large group of natural products that display broad and potent biological activities and are biosynthesized by type I polyketide synthases (PKSs) and associated enzymatic machinery. There is an urgent need to access macrolides and unnatural macrolide derivatives for drug discovery, drug manufacture, and probe development. Typically, efforts to engineer the biosynthesis of macrolides and macrolide analogues in various microbial hosts are hampered by the complexity of macrolide biosynthetic pathways and our limited ability to rationally reprogram type I PKSs and post-PKS machinery. High-throughput approaches based on synthetic biology and directed evolution could overcome this problem by testing the function of large libraries of variants. Yet, methods that can identify mutant enzymes, pathways, and strains that produce the desired macrolide target are not generally available. Here we show that the promiscuous macrolide sensing transcription factor MphR is a powerful platform for engineering variants with tailored properties. We identified variants that displayed improved sensitivity towards erythromycin, tailored the inducer specificity, and significantly improved sensitivity to macrolides that were very poor inducers of the wild-type MphR biosensor. Designer macrolide biosensors should find broad utility and enable applications related to high-throughput synthetic biology and directed evolution of macrolide biosynthesis.

Concepts: DNA, Gene, Metabolism, Adenosine triphosphate, Biology, Biotechnology, Macrolide, Polyketide