Biosynthetic gene clusters with biotechnological applications in novel Antarctic isolates from Actinomycetota.

Actinomycetota have been widely described as valuable sources for the acquisition of secondary metabolites. Most microbial metabolites are produced via metabolic pathways encoded by biosynthetic gene clusters (BGCs). Although many secondary metabolites are not essential for the survival of bacteria, they play an important role in their adaptation and interactions within microbial communities. This is how bacteria isolated from extreme environments such as Antarctica could facilitate the discovery of new BGCs with biotechnological potential. This study aimed to isolate rare Actinomycetota strains from Antarctic soil and sediment samples and identify their metabolic potential based on genome mining and exploration of biosynthetic gene clusters. To this end, the strains were sequenced using Illumina and Oxford Nanopore Technologies platforms. The assemblies were annotated and subjected to phylogenetic analysis. Finally, the BGCs present in each genome were identified using the antiSMASH tool, and the biosynthetic diversity of the Micrococcaceae family was evaluated. Taxonomic annotation revealed that seven strains were new and two were previously reported in the NCBI database. Additionally, BGCs encoding type III polyketide synthases (T3PKS), beta-lactones, siderophores, and non-ribosomal peptide synthetases (NRPS) have been identified, among others. In addition, the sequence similarity network showed a predominant type of BGCs in the family Micrococcaceae, and some genera were distinctly grouped. The BGCs identified in the isolated strains could be associated with applications such as antimicrobials, anticancer agents, and plant growth promoters, among others, positioning them as excellent candidates for future biotechnological applications and innovations. KEY POINTS: • Novel Antarctic rare Actinomycetota strains were isolated from soil and sediments • Genome-based taxonomic affiliation revealed seven potentially novel species • Genome mining showed metabolic potential for novel natural products.

Diverse Combinatorial Biosynthesis Strategies for C-H Functionalization of Anthracyclinones.

Streptomyces spp. are "nature's antibiotic factories" that produce valuable bioactive metabolites, such as the cytotoxic anthracycline polyketides. While the anthracyclines have hundreds of natural and chemically synthesized analogues, much of the chemical diversity stems from enzymatic modifications to the saccharide chains and, to a lesser extent, from alterations to the core scaffold. Previous work has resulted in the generation of a BioBricks synthetic biology toolbox in Streptomyces coelicolor M1152ΔmatAB that could produce aklavinone, 9-epi-aklavinone, auramycinone, and nogalamycinone. In this work, we extended the platform to generate oxidatively modified analogues via two crucial strategies. (i) We swapped the ketoreductase and first-ring cyclase enzymes for the aromatase cyclase from the mithramycin biosynthetic pathway in our polyketide synthase (PKS) cassettes to generate 2-hydroxylated analogues. (ii) Next, we engineered several multioxygenase cassettes to catalyze 11-hydroxylation, 1-hydroxylation, 10-hydroxylation, 10-decarboxylation, and 4-hydroxyl regioisomerization. We also developed improved plasmid vectors and S. coelicolor M1152ΔmatAB expression hosts to produce anthracyclinones. This work sets the stage for the combinatorial biosynthesis of bespoke anthracyclines using recombinant Streptomyces spp. hosts.

Engineered Biosynthesis and Anticancer Studies of Ring-Expanded Antimycin-Type Depsipeptides.

Respirantins are 18-membered antimycin-type depsipeptides produced by Streptomyces sp. and Kitasatospora sp. These compounds have shown extraordinary anticancer activities against a panel of cancer cell lines with nanomolar levels of IC50 values. However, further investigation has been impeded by the low titers of the natural producers and the challenging chemical synthesis due to their structural complexity. The biosynthetic gene cluster (BGC) of respirantin was previously proposed based on a bioinformatic comparison of the four members of antimycin-type depsipeptides. In this study, we report the first successful reconstitution of respirantin in Streptomyces albus using a synthetic BGC. This heterologous system serves as an accessible platform for the production and diversification of respirantins. Through polyketide synthase pathway engineering, biocatalysis, and chemical derivatization, we generated nine respirantin compounds, including six new derivatives. Cytotoxicity screening against human MCF-7 and Hela cancer cell lines revealed a unique biphasic dose-response profile of respirantin. Furthermore, a structure-activity relationship study has elucidated the essential functional groups that contribute to its remarkable cytotoxicity. This work paves the way for respirantin-based anticancer drug discovery and development.

Molecular evolution and characterization of type III polyketide synthase gene family in Aquilaria sinensis.

2-(2-Phenylethyl) chromone (PEC) and its derivatives are markers of agarwood formation and are also related to agarwood quality. However, the biosynthetic and regulatory mechanisms of PECs still remain mysterious. Several studies suggested that type III polyketide synthases (PKSs) contribute to PEC biosynthesis in Aquilaria sinensis. Furthermore, systematic studies on the evolution of PKSs in A. sinensis have rarely been reported. Herein, we comprehensively analyzed PKS genes from 12 plant genomes and characterized the AsPKSs in detail. A unique branch contained only AsPKS members was identified through evolutionary analysis, including AsPKS01 that was previously indicated to participate in PEC biosynthesis. AsPKS07 and AsPKS08, two tandem-duplicated genes of AsPKS01 and lacking orthologous genes in evolutionary models, were selected for their transient expression in the leaves of Nicotiana benthamiana. Subsequently, PECs were detected in the extracts of N. benthamiana leaves, suggesting that AsPKS07 and AsPKS08 promote PEC biosynthesis. The interaction between the promoters of AsPKS07, AsPKS08 and five basic leucine zippers (bZIPs) from the S subfamily indicated that their transcripts could be regulated by these transcription factors (TFs) and might further contribute to PECs biosynthesis in A. sinensis. Our findings provide valuable insights into the molecular evolution of the PKS gene family in A. sinensis and serve as a foundation for advancing PEC production through the bioengineering of gene clusters. Ultimately, this contribution is expected to shed light on the mechanism underlying agarwood formation.

Genome-guided discovery of two undescribed 6,6-spiroketal polyketides and stereochemical correction of bafilomycins P and Q from the marine-derived Streptomyces sp. SCSIO 66814.

Bafilomycins are macrocyclic polyketides with intriguing structures and therapeutic value. Genomic analysis of Streptomyces sp. SCSIO 66814 revealed a type I polyketide synthase biosynthetic gene cluster (BGC), namely blm, which encoded bafilomycins and featured rich post-modification genes. The One strain many compounds (OSMAC) strategy led to the discovery of six compounds related to the blm BGC from the strain, including two previously undescribed 6,6-spiroketal polyketides, streptospirodienoic acids D (1) and E (2), and four known bafilomycins, bafilomycins P (3), Q (4), D (5), and G (6). The structures of 1 and 2 were determined by extensive spectroscopic analysis, quantum calculation, and biosynthetic analysis. Additionally, the absolute configurations of the 6/5/5 tricyclic ring moiety containing six consecutive chiral carbons in the putative structures of 3 and 4 were corrected through NOE analysis, DP4+ calculation, and single-crystal X-ray diffraction data. Bioinformatic analysis uncovered a plausible biosynthetic pathway for compounds 1-6, indicating that both streptospirodienoic acids and bafilomycins were derived from the same blm BGC. Additionally, sequence analysis revealed that the KR domains of module 2 from blm BGC was B1-type, further supporting the configurations of 1-4. Notably, compounds 3 and 4 displayed significant cytotoxic activities against A-549 human non-small cell lung cancer cells and HCT-116 human colon cancer cells.

Polyketide synthase positive Escherichia coli one-time measurement in stool is not informative of colorectal cancer risk in a screening setting.

Environmental factors like the pathogenicity island polyketide synthase positive (pks+) Escherichia coli (E. coli) could have potential for risk stratification in colorectal cancer (CRC) screening. The association between pks+ E. coli measured in fecal immunochemical test (FIT) samples and the detection of advanced neoplasia (AN) at colonoscopy was investigated. Biobanked FIT samples were analyzed for both presence of E. coli and pks+ E. coli and correlated with colonoscopy findings; 5020 CRC screening participants were included. Controls were participants in which no relevant lesion was detected because of FIT-negative results (cut-off ≥15 µg Hb/g feces), a negative colonoscopy, or a colonoscopy during which only a nonadvanced polyp was detected. Cases were participants with AN [CRC, advanced adenoma (AA), or advanced serrated polyp (ASP)]. Existing DNA isolation and quantitative polymerase chain reaction (qPCR) procedures were used for the detection of E. coli and pks+ E. coli in stool. A total of 4542 (90.2%) individuals were E. coli positive, and 1322 (26.2%) were pks+ E. coli positive. The prevalence of E. coli in FIT samples from individuals with AN was 92.9% compared to 89.7% in FIT samples of controls (p = 0.010). The prevalence of pks+ E. coli in FIT samples from individuals with AN (28.6%) and controls (25.9%) was not significantly different (p = 0.13). The prevalences of pks+ E. coli in FIT samples from individuals with CRC, AA, or ASP were 29.6%, 28.3%, and 32.1%, respectively. In conclusion, the prevalence of pks+ E. coli in a screening population was 26.2% and did not differ significantly between individuals with AN and controls. These findings disqualify the straightforward option of using a snapshot measurement of pks+ E. coli in FIT samples as a stratification biomarker for CRC risk. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Comparative genomic analysis of Sanghuangporus sanghuang with other Hymenochaetaceae species.

Sanghuangporus sanghuang is a medicinal macrofungus with antioxidant and antitumor activities, and it is enriched with secondary metabolites such as polysaccharides, terpenes, polyphenols, and styrylpyrone compounds. To explore the putative core genes and gene clusters involved in sanghuang biosynthesis, we sequenced and assembled a 40.5-Mb genome of S. sanghuang (SH1 strain). Using antiSMASH, local BLAST, and NCBI comparison, 12 terpene synthases (TPSs), 1 non-ribosomal peptide synthase, and five polyketide synthases (PKSs) were identified in SH1. Combining the transcriptome analysis with liquid chromatography mass spectrometry-ion trap-time of flight analysis, we determined that ShPKS1, one phenylalanine aminolyase (ShPAL), and one P450 monooxygenase (ShC4H1) were associated with hispidin biosynthesis. Structural domain comparison indicated that ShPKS2 and ShPKS3 are involved in the biosynthesis of orsellinic acid and 2-hydroxy-6-methylbenzoic acid, respectively. Furthermore, comparative genomic analysis of SH1 with 14 other fungi from the Hymenochaetaceae family showed variation in the number of TPSs among different genomes, with Coniferiporia weirii exhibiting only 9 TPSs and Inonotus obliquus having 20. The number of TPSs also differed among the genomes of three strains of S. sanghuang, namely Kangneng (16), MS2 (9), and SH1 (12). The type and number of PKSs also varied among species and even strains, ranging from two PKSs in Pyrrhoderma noxium to five PKSs in S. sanghuang SH1. Among the three strains of S. sanghuang, both the structural domains and the number of PKSs in strains MS2 and SH1 were consistent, whereas strain Kangneng exhibited only four PKSs and lacked the PKS with the structural domain KS-AT-DH-KR-ACP. Additionally, Sanghuangporus species exhibited more similar PKSs to Inonotus, with higher gene similarity around five PKSs, while showing differences from those of other fungi in the same family, including Phellinus lamaoensis. This result supports the independent taxonomic significance of the genus Sanghuangporus to some extent.

Association between physical activity and the prevalence of tumorigenic bacteria in the gut microbiota of Japanese adults: a cross-sectional study.

Escherichia coli harboring polyketide synthase (pks+ E. coli) has been suggested to contribute to colorectal cancer development. Physical activity is strongly associated with lower colorectal cancer risks, but its effects on pks+ E. coli remain unclear. The aim of this study was to investigate the association between pks+ E. coli prevalence and physical activity. A cross-sectional study was conducted on 222 Japanese adults (27-79-years-old, 73.9% female). Triaxial accelerometers were used to measure light-intensity physical activity, moderate-to-vigorous intensity physical activity, the physical activity level, step-count, and time spent inactive. Fecal samples collected from participants were used to determine the prevalence of pks+ E. coli. Multivariate logistic regression analysis and restricted cubic spline curves were used to examine the association between pks+ E. coli prevalence and physical activity. The prevalence of pks+ E. coli was 26.6% (59/222 participants). The adjusted odds ratios (ORs) and 95% confidence intervals (CIs) for the highest tertile with reference to the lowest tertile of physical activity variables were as follows: light-intensity physical activity (OR 0.63; 95% CI 0.26-1.5), moderate-to-vigorous intensity physical activity (OR 0.85; 95% CI 0.39-1.87), physical activity level (OR 0.69; 95% CI 0.32-1.51), step-count (OR 0.92; 95% CI 0.42-2.00) and time spent inactive (OR 1.30; 95% CI 0.58-2.93). No significant dose-response relationship was found between all physical activity variables and pks+ E. coli prevalence. Our findings did not suggest that physical activity has beneficial effects on the prevalence of pks+ E. coli. Longitudinal studies targeting a large population are needed to clarify this association.

Abi Family Protein, DidK, Is Involved in the Maturation of Anticancer Depsipeptide, Didemnin B.

Didemnin B is a marine-derived depsipeptide with potent antiviral and anticancer activities. A prodrug activation mechanism was previously proposed for the biosynthesis of didemnin B by the nonribosomal peptide synthetase/polyketide synthase (NRPS/PKS) assembly line, but the enzyme involved in the maturation process remained unknown. Herein, we demonstrated that DidA, a dimodular NRPS predicted with unrelated activity to didemnin B structure assembly, was indispensable to produce didemnin B, confirming the prodrug mechanism in didemnin B biosynthesis. We further identified an Abi family transmembrane protease, DidK, that functioned as an esterase in the maturation step of didemnin B by in vivo gene knockout and heterologous expression. DidK is structurally distinct from other known hydrolytic enzymes involved in the maturation of bacterial nonribosomal peptides and is the first Abi family protein known to participate in NRPS/PKS-derived natural product production. Further bioinformatic analysis revealed more than 20 DidK homologues encoded in bacterial NRPS/PKS BGCs, suggesting that the involvement of Abi family proteins in natural product biosynthesis might not be rare. These results not only clarify the priming and maturation steps of didemnin B biosynthesis but also expand the function scope of Abi family proteins.

Animal FAS-like polyketide synthases produce diverse polypropionates.

Animal cytoplasmic fatty acid synthase (FAS) represents a unique family of enzymes that are classically thought to be most closely related to fungal polyketide synthase (PKS). Recently, a widespread family of animal lipid metabolic enzymes has been described that bridges the gap between these two ubiquitous and important enzyme classes: the animal FAS-like PKSs (AFPKs). Although very similar in sequence to FAS enzymes that produce saturated lipids widely found in animals, AFPKs instead produce structurally diverse compounds that resemble bioactive polyketides. Little is known about the factors that bridge lipid and polyketide synthesis in the animals. Here, we describe the function of EcPKS2 from Elysia chlorotica, which synthesizes a complex polypropionate natural product found in this mollusc. EcPKS2 starter unit promiscuity potentially explains the high diversity of polyketides found in and among molluscan species. Biochemical comparison of EcPKS2 with the previously described EcPKS1 reveals molecular principles governing substrate selectivity that should apply to related enzymes encoded within the genomes of photosynthetic gastropods. Hybridization experiments combining EcPKS1 and EcPKS2 demonstrate the interactions between the ketoreductase and ketosynthase domains in governing the product outcomes. Overall, these findings enable an understanding of the molecular principles of structural diversity underlying the many molluscan polyketides likely produced by the diverse AFPK enzyme family.

Exploring the Chain Release Mechanism from an Atypical Apicomplexan Polyketide Synthase.

Polyketide synthases (PKSs) are megaenzymes that form chemically diverse polyketides and are found within the genomes of nearly all classes of life. We recently discovered the type I PKS from the apicomplexan parasite Toxoplasma gondii, TgPKS2, which contains a unique putative chain release mechanism that includes ketosynthase (KS) and thioester reductase (TR) domains. Our bioinformatic analysis of the thioester reductase of TgPKS2, TgTR, suggests differences compared to other systems and hints at a possibly conserved release mechanism within the apicomplexan subclass Coccidia. To evaluate this release module, we first isolated TgTR and observed that it is capable of 4 electron (4e-) reduction of octanoyl-CoA to the primary alcohol, octanol, utilizing NADH. TgTR was also capable of generating octanol in the presence of octanal and NADH, but no reactions were observed when NADPH was supplied as a cofactor. To biochemically characterize the protein, we measured the catalytic efficiency of TgTR using a fluorescence assay and determined the TgTR binding affinity for cofactor and substrates using isothermal titration calorimetry (ITC). We additionally show that TgTR is capable of reducing an acyl carrier protein (ACP)-tethered substrate by liquid chromatography mass spectrometry and determine that TgTR binds to holo-TgACP4, its predicted cognate ACP, with a KD of 5.75 ± 0.77 µM. Finally, our transcriptional analysis shows that TgPKS2 is upregulated ∼4-fold in the parasite's cyst-forming bradyzoite stage compared to tachyzoites. Our study identifies features that distinguish TgPKS2 from well-characterized systems in bacteria and fungi and suggests it aids the T. gondii cyst stage.

Docking Domain Engineering in a Modular Polyketide Synthase and Its Impact on Structure and Function.

Modular polyketide synthases (PKSs) are attractive targets for the directed, biosynthetic production of platform chemicals and pharmaceuticals by protein engineering. In this study, we analyze docking domains from the 6-deoxyerythronolide B synthase, SYNZIP domains, and the SpyCatcher:SpyTag complex as engineering tools to couple the polypeptides VemG and VemH to functional venemycin synthases. Our data show that the high-affinity interaction or covalent connection of modules, enabled by SYNZIP domains and the SpyCatcher:SpyTag complex, can be advantageous, e.g., in synthesis at low protein concentrations, but their rigidity and steric demand decrease synthesis rates. However, we also show that efficiency can be recovered when inserting a hinge region distant from the rigid interface. This study demonstrates that engineering approaches should take the conformational properties of modular PKSs into account and establishes a three-polypeptide split venemycin synthase as an exquisite in vitro platform for the analysis and engineering of modular PKSs.

Discovery and Biosynthesis of the Cytotoxic Polyene Terpenomycin in Human Pathogenic Nocardia.

Nocardia are opportunistic human pathogens that can cause a range of debilitating and difficult to treat infections of the lungs, brain, skin, and soft tissues. Despite their close relationship to the well-known secondary metabolite-producing genus, Streptomyces, comparatively few natural products are known from the Nocardia, and even less is known about their involvement in the pathogenesis. Here, we combine chemistry, genomics, and molecular microbiology to reveal the production of terpenomycin, a new cytotoxic and antifungal polyene from a human pathogenic Nocardia terpenica isolate. We unveil the polyketide synthase (PKS) responsible for terpenomycin biosynthesis and show that it combines several unusual features, including "split", skipped, and iteratively used modules, and the use of the unusual extender unit methoxymalonate as a starter unit. To link genes to molecules, we constructed a transposon mutant library in N. terpenica, identifying a terpenomycin-null mutant with an inactivated terpenomycin PKS. Our findings show that the neglected actinomycetes have an unappreciated capacity for the production of bioactive molecules with unique biosynthetic pathways waiting to be uncovered and highlights these organisms as producers of diverse natural products.

Guidelines for Optimizing Type S Nonribosomal Peptide Synthetases.

Bacterial biosynthetic assembly lines, such as nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs), play a crucial role in the synthesis of natural products that have significant therapeutic potential. The ability to engineer these biosynthetic assembly lines offers opportunities to produce artificial nonribosomal peptides, polyketides, and their hybrids with improved properties. In this study, we introduced a synthetic NRPS variant, termed type S NRPS, which simplifies the engineering process and enables biocombinatorial approaches for generating nonribosomal peptide libraries in a parallelized high-throughput manner. However, initial generations of type S NRPSs exhibited a bottleneck that led to significantly reduced production yields. To address this challenge, we employed two optimization strategies. First, we truncated SYNZIPs from the N- and/or C-terminus of the NRPS. SYNZIPs comprise a large set of well-characterized synthetic protein interaction reagents. Second, we incorporated a structurally flexible glycine-serine linker between the NRPS protein and the attached SYNZIP, aiming to improve dynamic domain-domain interactions. Through an iterative optimization process, we achieved remarkable improvements in production yields, with titer increases of up to 55-fold compared to the nonoptimized counterparts. These optimizations successfully restored production levels of type S NRPSs to those observed in wild-type NRPSs and even surpassed them. Overall, our findings demonstrate the potential of engineering bacterial biosynthetic assembly lines for the production of artificial nonribosomal peptides. In addition, optimizing the SYNZIP toolbox can have valuable implications for diverse applications in synthetic biology, such as metabolic engineering, cell signaling studies, or engineering of other multienzyme complexes, such as PKSs.

Structure of a modular polyketide synthase reducing region.

The chemical scaffolds of numerous therapeutics are polyketide natural products, many formed by bacterial modular polyketide synthases (PKS). The large and flexible dimeric PKS modules have distinct extension and reducing regions. Structures are known for all individual enzyme domains and several extension regions. Here, we report the structure of the full reducing region from a modular PKS, the ketoreductase (KR), dehydratase (DH), and enoylreductase (ER) domains of module 5 of the juvenimicin PKS. The modular PKS-reducing region has a different architecture than the homologous fatty acid synthase (FAS) and iterative PKS systems in its arrangement of domains and dimer interface. The structure reveals a critical role for linker peptides in the domain interfaces, leading to discovery of key differences in KR domains dependent on module composition. Finally, our studies provide insight into the mechanism underlying modular PKS intermediate shuttling by carrier protein (ACP) domains.

Iterative-Acting Thioesterase from Polyketide Biosynthesis Accepts Diverse Nucleophilic Alcohols to Yield Oxazole-Containing Esters.

The biosynthesis of antitumor oxazole-containing conglobatin is directed by a multienzyme assembly line of nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS), in which an uncanonical iterative-acting C-terminal thioesterase domain, Cong-TE, ligated two fully elongated chains/conglobatin monomers on the terminal acylcarrier protein and subsequently cyclized the resulting dimer to a C2-symmetric macrodiolide. Screening of the conglobatin producer for secondary metabolites led to the discovery of two new compounds conglactones A (1) and B (2), possessing inhibitory activities to phytopathogenic microorganisms and cancer cells, respectively. The compounds 1 and 2 feature the ester bond-linked hybrid structures consisting of an aromatic polyketide benwamycin I (3) and one (for 1)/two (for 2) molecules of the conglobatin monomer (5). Genetic mutational analysis revealed that the production of 1 and 2 was correlated with the biosynthetic pathways of 3 and 5. Biochemical analysis indicated that 1 and 2 were produced by Cong-TE from 3 and an N-acetylcysteamine thioester form of 5 (7). Furthermore, the substrate compatibility of Cong-TE was demonstrated by enzymatically generating a bunch of ester products from 7 and 43 exotic alcohols. This property of Cong-TE was further validated by producing 36 hybrid esters in the fermentation of conglobatin producer fed with nonindigenous alcohols. This work shows a prospect of developing Cong-TE for green synthesis of valuable oxazole-containing esters, thus complementing the environmentally unfriendly chemosynthesis strategies.

Boosting titers of engineered triketide and tetraketide synthases to record levels through T7 promoter tuning.

Modular polyketide synthases (PKS's) are promising platforms for the rational engineering of designer polyketides and commodity chemicals, yet their low productivities are a barrier to the practical biosynthesis of these compounds. Previously, we engineered triketide lactone synthases such as Pik167 using the recently updated module definition and showed they generate hundreds of milligrams of product per liter of Escherichia coli K207-3 shake flask culture. As the molar ratio between the 2 polypeptides of Pik167 is highly skewed, we sought to attenuate the strength of the T7 promoter controlling the production of the smaller, better-expressing polypeptide and thereby increase production of the first polypeptide under the control of an unoptimized T7 promoter. Through this strategy, a 1.8-fold boost in titer was obtained. After a further 1.5-fold boost obtained by increasing the propionate concentration in the media from 20 to 80 mM, a record titer of 791 mg L-1 (627 mg L-1 isolated) was achieved, a 2.6-fold increase overall. Spurred on by this result, the tetraketide synthase Pik1567 was engineered and the T7 promoter attenuation strategy was applied to its second and third genes. A 5-fold boost, from 20 mg L-1 to 100 mg L-1, in the titer of its tetraketide product was achieved.

Metabolic Engineering and Synthetic Biology Approaches for the Heterologous Production of Aromatic Polyketides.

Polyketides are a diverse set of natural products with versatile applications as pharmaceuticals, nutraceuticals, and cosmetics, to name a few. Of several types of polyketides, aromatic polyketides comprising type II and III polyketides contain many chemicals important for human health such as antibiotics and anticancer agents. Most aromatic polyketides are produced from soil bacteria or plants, which are difficult to engineer and grow slowly in industrial settings. To this end, metabolic engineering and synthetic biology have been employed to efficiently engineer heterologous model microorganisms for enhanced production of important aromatic polyketides. In this review, we discuss the recent advancement in metabolic engineering and synthetic biology strategies for the production of type II and type III polyketides in model microorganisms. Future challenges and prospects of aromatic polyketide biosynthesis by synthetic biology and enzyme engineering approaches are also discussed.

Thioester Capture Strategy for the Identification of Nonribosomal Peptide and Polyketide Intermediates.

Nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) are multi-domainal megasynthases. While they are capable of generating a structurally diverse array of metabolites of therapeutic relevance, their mere size and complex nature of their assembly (intermediates are tethered and enzyme bound) make them inherently difficult to characterize. In order to facilitate structural characterization of these metabolites, a thioester capture strategy that enables direct trapping and characterization of the thioester-bound enzyme intermediates was developed. Specifically, a synthetic Biotin-Cys agent was designed and utilized, enabling direct analysis by LCMS/MS and NMR spectroscopy. In the long term, the approach might facilitate the discovery of novel scaffolds from cryptic biosynthetic pathways, paving the way for the development of drug leads and therapeutic initiatives.

Polyketide Synthase-Mediated O-Methyloxime Formation in the Biosynthesis of the Oximidine Anticancer Agents.

Bacterial trans-acyltransferase polyketide synthases (trans-AT PKSs) are modular megaenzymes that employ unusual catalytic domains to assemble diverse bioactive natural products. One such PKS is responsible for the biosynthesis of the oximidine anticancer agents, oxime-substituted benzolactone enamides that inhibit vacuolar H+ -ATPases. Here, we describe the identification of the oximidine gene cluster in Pseudomonas baetica and the characterization of four novel oximidine variants, including a structurally simpler intermediate that retains potent anticancer activity. Using a combination of in vivo, in vitro and computational approaches, we experimentally elucidate the oximidine biosynthetic pathway and reveal an unprecedented mechanism for O-methyloxime formation. We show that this process involves a specialized monooxygenase and methyltransferase domain and provide insight into their activity, mechanism and specificity. Our findings expand the catalytic capabilities of trans-AT PKSs and identify potential strategies for the production of novel oximidine analogues.