Abstract
One major bottleneck in natural product drug development is derivatization, which is pivotal for fine tuning lead compounds. A promising solution is modifying the biosynthetic machineries of ...middle molecules such as macrolides. Although intense studies have established various methodologies for protein engineering of type I modular polyketide synthase(s) (PKSs), the accurate targeting of desired regions in the PKS gene is still challenging due to the high sequence similarity between its modules. Here, we report an innovative technique that adapts in vitro Cas9 reaction and Gibson assembly to edit a target region of the type I modular PKS gene. Proof-of-concept experiments using rapamycin PKS as a template show that heterologous expression of edited biosynthetic gene clusters produced almost all the desired derivatives. Our results are consistent with the promiscuity of modular PKS and thus, our technique will provide a platform to generate rationally designed natural product derivatives for future drug development.
From our in-house microbial genome database of secondary metabolite producers, we identified a candidate biosynthetic gene cluster for desertomycin from Streptomyces nobilis JCM4274. We report herein ...the cloning of the 127-kb entire gene cluster for desertomycin biosynthesis using bacterial artificial chromosome vector. The entire biosynthetic gene cluster for desertomycin was introduced in the heterologous host, Streptomyces lividans TK23, with an average yield of more than 130 mg l
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Using genome mining approach, we identified a novel biosynthetic gene cluster containing trans-AT type PKS genes from Streptomyces versipellis 4083-SVS6. A bacterial artificial chromosome (BAC) ...clone, pKU503JL68_PN1_P10-C12, accommodating the entire biosynthetic gene cluster was obtained from a BAC library. Heterologous expression of the biosynthetic gene cluster in Streptomyces lividans TK23 led to the production of a novel polyene compound, JBIR-159. We report herein the biosynthetic gene cluster for JBIR-159, and the heterologous expression, isolation, structure determination and a brief biological activity.
Quinolidomicin A1 is the largest macrolide compound from terrestrial sources, consisting of a 60-membered ring, and its biosynthetic gene cluster was revealed to be over 200 kb. The gene cluster for ...quinolidomicin was cloned and heterologously expressed. Confirmation of the product led to a structural revision, in which the hydroxy group in the chromophore moiety of the reported structure was replaced by an amine group.
Versipelostatin (VST) is an unusual 17-membered macrocyclic polyketide product that contains a spirotetronate skeleton. In this study, the entire VST biosynthetic gene cluster (vst) spanning 108 kb ...from Streptomyces versipellis 4083-SVS6 was identified by heterologous expression using a bacterial artificial chromosome vector. Here, we demonstrate that an enzyme, VstJ, catalyzes the stereoselective 4+2-cycloaddition between the conjugated diene and the exocyclic olefin of a newly identified tetronate-containing intermediate to form the spirotetronate skeleton during VST biosynthesis.
Nitrogen–nitrogen bond-containing functional groups are rare, but they are found in a considerably wide class of natural products. Recent clarifications of the biosynthetic routes for such functional ...groups shed light onto overlooked biosynthetic genes distributed across the bacterial kingdom, highlighting the presence of yet-to-be identified natural products with peculiar functional groups. Here, the genome-mining approach targeting a unique hydrazine-forming gene led to the discovery of actinopyridazinones A (1) and B (2), the first natural products with dihydropyridazinone rings. The structure of actinopyridazinone A was unambiguously established by total synthesis. Biosynthetic studies unveiled the structural diversity of natural hydrazines derived from this family of N–N bond-forming enzymes.
Reprogramming of the NRPS/PKS assembly line is an attractive method for the production of new bioactive molecules. However, it is usually hampered by the loss of intimate domain/module interactions ...required for the precise control of chain transfer and elongation reactions. In this study, we first establish heterologous expression systems of the unique antimycin-type cyclic depsipeptides: JBIR-06 (tri-lactone) and neoantimycin (tetra-lactone), and engineer their biosyntheses by taking advantage of bioinformatic analyses and evolutionary insights. As a result, we successfully accomplish three manipulations: (i) ring contraction of neoantimycin (from tetra-lactone to tri-lactone), (ii) ring expansion of JBIR-06 (from tri-lactone to tetra-lactone), and (iii) alkyl chain diversification of JBIR-06 by the incorporation of various alkylmalonyl-CoA extender units, to generate a set of unnatural derivatives in practical yields. This study presents a useful strategy for engineering NRPS-PKS module enzymes, based on nature's diversification of the domain and module organizations.
Engineering polyketide synthases is one of the most promising ways of producing a variety of polyketide derivatives. Exploring the undiscovered chemical space of this medicinally important class of ...middle molecular weight natural products will aid in the development of improved drugs in the future. In previous work, we established methodology designated 'module editing' to precisely manipulate polyketide synthase genes cloned in a bacterial artificial chromosome. Here, in the course of investigating the engineering capacity of the rapamycin PKS, novel rapamycin derivatives 1-4, which lack the hemiacetal moiety, were produced through the heterologous expression of engineered variants of the rapamycin PKS. Three kinds of module deletions in the polyketide synthase RapC were designed, and the genetically engineered vectors were prepared by the in vitro module editing technique. Streptomyces avermitilis SUKA34 transformed with these edited PKSs produced new rapamycin derivatives. The planar structures of 1-4 established based on 1D and 2D NMR, ESI-TOF-MS and UV spectra revealed that 2 and 3 had skeletons well-matched to the designs, but 1 and 4 did not. The observations provide important insights into the mechanisms of the later steps of rapamycin skeletal formation as well as the ketone-forming oxygenase RapJ.
Heterologous expression of natural compound biosynthetic gene clusters (BGCs) is a robust approach for not only revealing the biosynthetic mechanisms leading to the compounds, but also for ...discovering new products from uncharacterized BGCs. We established a heterologous expression technique applicable to huge biosynthetic gene clusters for generating large molecular secondary metabolites such as type-I polyketides. As an example, we targeted concanamycin BGC from Streptomyces neyagawaensis IFO13477 (the cluster size of 99 kbp), and obtained a bacterial artificial chromosome (BAC) clone with an insert size of 211 kbp that contains the entire concanamycin BGC. Interestingly, heterologous expression for this BAC clone resulted in two additional aromatic polyketides, ent-gephyromycin, and a new compound designated as JBIR-157, together with the expected concanamycin. Bioinformatic and biochemical analyses revealed that a cryptic biosynthetic gene cluster in this BAC clone was responsible for the production of these type-II polyketide synthases (PKS) compounds. Here, we describe the production, isolation, and structure elucidation of JBIR-157, determined primarily by a series of NMR spectral analyses.
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