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Miharamycins are peptidyl nucleoside antibiotics with a unique branched C9 pyranosyl amino acid core and a rare 2-aminopurine moiety. Inactivation of 19 genes in the biosynthetic gene cluster and identification of several unexpected intermediates suggest an alternative biosynthetic pathway, which is further supported by feeding experiments and in vitro characterization of an unusual adenylation domain recognizing a complex nucleoside derivative as the substrate. These results thereby provide an unprecedented biosynthetic route of high-carbon sugar catalyzed by atypical hybrid nonribosomal peptide synthetase-polyketide synthase.

The biochemical elucidation of the early biosynthetic pathways of miharamycins and amipurimycin revealed the roles of several enzymes, which include GMP hydrolase, represented by MihD/ApmD, and hypothetical proteins, MihI/ApmI, unexpectedly exhibiting the dual function of the guanylglucuronic acid assembly and GMP cleavage. In addition, MihE, a carbonyl reductase that functions on the C2 branch of high-carbon sugars, and MihF, a rare guanine O-methyltransferase, were also functionally verified.

The structural puzzle of amipurimycin, a peptidyl nucleoside antibiotic, is solved by total synthesis and X-ray diffraction analysis, with the originally proposed configurations at C3' and C8' inverted and those at C6', C2'', and C3'' corrected. A similar structural revision of the relevant miharamycins is proposed via chemical transformations and then validated by X-ray diffraction analysis. The miharamycins bear an unusual trans-fused dioxabicyclo[4.3.0]nonane sugar scaffold, which was previously assigned as being in the cis configuration.

Bacterial type II polyketides, an important class of natural products biosynthesized by type II polyketide synthases, are rich in chemical scaffolds and biological activities. In the past three decades, the relative biosynthesis has resulted in significant progress in elucidating the assembly machinery, enzymatic reactions and catalytic mechanisms, which contributed greatly to the discovery of new natural products and engineering the biosynthetic pathway for novel derivatives. In this review, we mainly focus on the new advances on biosynthetic investigations of type II polyketides during the last nine years. We also summarize new tools developed for exploring potent antibiotics, such as genome mining, environmental DNA, and bioengineering.

Oxygen-containing heterocycles are widely encountered in natural products that display diverse pharmacological properties and have potential benefits to human health. The formation of O-heterocycles catalyzed by different types of enzymes in the biosynthesis of natural products not only contributes to the structural diversity of these compounds, but also enriches our understanding of nature's ability to construct complex molecules. This minireview focuses on the various modes of enzymatic O-heterocyclization identified in natural product biosynthesis and summarizes the possible mechanisms involved in ring closure.

Using a highly effective heterologous expression system, the YM-216391 (1) biosynthetic gene cluster was engineered to yield aurantizolicin (2) and the hybrid compound 3. Both of these compounds were isolated and fully structurally characterised and the bioactivity was evaluated. The results indicate that compound 3 exhibits significantly improved antitumor activity.

One biosynthetic gene cluster (BGC) usually governs the biosynthesis of a series of compounds exhibiting either the same or similar molecular scaffolds. Reported here is a multiplex activation strategy to awaken a cryptic BGC associated with tetracycline polyketides, resulting in the discovery of compounds having different core structures. By constitutively expressing a positive regulator gene in tandem mode, a single BGC directed the biosynthesis of eight aromatic polyketides with two types of frameworks, two pentacyclic isomers and six glycosylated tetracyclines. The proposed biosynthetic pathway, based on systematic gene inactivation and identification of intermediates, employs two sets of tailoring enzymes with a branching point from the same intermediate. These findings not only provide new insights into the role of tailoring enzymes in the diversification of polyketides, but also highlight a reliable strategy for genome mining of natural products.

Feeding studies indicate a possible synthetic pattern for the N-terminal cis-aminocyclopentane carboxylic acid (ACPC) and suggest an unusual source of the high-carbon sugar skeleton of amipurimycin (APM). The biosynthetic gene cluster of APM was identified and confirmed by in vivo experiments. A C9 core intermediate was discovered from null mutants of ACPC pathway, and an ATP-grasp enzyme (ApmA8) was reconstituted in vitro for ACPC loading. Our observations allow a first proposal of the APM biosynthetic pathway.

As a commercial antibiotic, bicyclomycin (BCM) is currently the only known natural product targeting the transcription termination factor rho. It belongs to a family of highly functionalized diketopiperazine (DKP) alkaloids and bears a unique O-bridged bicyclo[4.2.2]piperazinedione ring system, a C1 triol, and terminal exo-methylene groups. We have identified and characterized the BCM biosynthetic pathway by heterologous biotransformations, in vitro biochemical assays, and one-pot enzymatic synthesis. A tRNA-dependent cyclodipeptide synthase guides the heterodimerization of leucine and isoleucine to afford the DKP precursor; subsequently, six redox enzymes, including five α-ketoglutarate/Fe2+ -dependent dioxygenases and one cytochrome P450 monooxygenase, regio- and stereoselectively install four hydroxy groups (primary, secondary, and two tertiary), an exo-methylene moiety, and a medium-sized bridged ring through the functionalization of eight unactivated C-H bonds.

Different starter unit and complex tailoring steps for type II polyketide synthase in trioxacarcin biosynthesis., Trioxacarcins (TXNs) are highly oxygenated, polycyclic aromatic natural products with remarkable biological activity and structural complexity. Evidence from 13C-labelled precursor feeding studies demonstrated that the scaffold was biosynthesized from one unit of l-isoleucine and nine units of malonyl-CoA, which suggested a different starter unit in the biosynthesis. Genetic analysis of the biosynthetic gene cluster revealed 56 genes encoding a type II polyketide synthase (PKS), combined with a large amount of tailoring enzymes. Inactivation of seven post-PKS modification enzymes resulted in the production of a series of new TXN analogues, intermediates, and shunt products, most of which show high anti-cancer activity. Structural elucidation of these new compounds not only helps us to propose the biosynthetic pathway, featuring a type II PKS using a novel starter unit, but also set the stage for further characterization of the enzymatic reactions and combinatorial biosynthesis.

In this work, two enzymes responsible for the biogenesis of possible [4 + 2] reaction precursors in the quartromicin biosynthetic pathway were characterized: acetylation of 1 to yield 2 was catalyzed by QmnD3, and subsequent acetic acid elimination of 2 to form double bond product 3 was catalyzed by QmnD4. Site-directed mutagenesis assay of QmnD3 and QmnD4 was investigated, and a general base-catalyzed mechanism for QmnD4 is proposed.

Ramoplanins are lipopeptides effective against a wide range of Gram-positive pathogens. Ramoplanin A2 is in Phase III clinical trials. The structure-activity relationship of the unique 2Z,4E-fatty acid side-chain of ramoplanins indicates a significant contribution to the antimicrobial activities but ramoplanin derivatives with longer 2Z,4E-fatty acid side-chains are not easy to obtain by semi-synthetic approaches. To construct a strain that produces such analogues, an acyl-CoA ligase gene in a ramoplanin-producing Actinoplanes was inactivated and a heterologous gene from an enduracidin producer (Streptomyces fungicidicus) was introduced into the mutant. The resulting strain produced three ramoplanin analogues with longer alkyl chains, in which X1 was purified. The MIC value of X1 was ~0.12 μg/ml against Entrococcus sp. and was also active against vancomycin-resistant Staphylococcus aureus (MIC = 2 μg/ml).

Ramoplanins produced by Actinoplanes are new structural class of lipopeptide and are currently in phase III clinical trials for the prevention of vancomycin-resistant enterococcal infections. The depsipeptide structures of ramoplanins are synthesized by non-ribosomal peptide synthetases (NRPS). Romo-orf17, a stand-alone NRPS, is responsible for the recruitment of Thr into the linear NRPS pathways for which the corresponding adenylation domain is absent. Here, systematical gene inactivation and complementation have been carried out in a Actinoplanes sp. using homologous recombination and site-specific integration methods. A hybrid gene coding for the N-terminal region of the stand-alone NRPS and the A-PCP domains of a heterologous NRPS restored production of ramoplanins. The results elucidate the unusual N-terminal region which is essential for the biosynthesis of ramoplanins.

Spinosyns A and D are novel macrolide secondary metabolites containing two deoxysugars. Each of the sugars is derived from a common intermediate, which is biosynthesized sequentially by NDP-glucose synthase and glucose dehydratase. Systematical duplication of gtt and gdh was carried out by genetic engineering in Saccharopolyspora spinosa. Introduction of additional gtt and gdh genes under the control of PermE* promoter showed significant increase on their transcript levels. The production of spinosad was increased by over threefold to reach 800 μg/ml in the recombinant strain SIPI-M2093.

Trioxacarcin A is a polyoxygenated, structurally complex antibiotic produced by Streptomyces spp., which possesses high anti-bacterial, anti-malaria, and anti-tumor activities. The trioxacarcin biosynthetic pathway involves type II polyketide synthases (PKSs) with L-isoleucine as a unique starter unit, as well as many complex post-PKS tailoring enzymes and resistance and regulatory proteins. In this work, two regulatory genes, txn9 coding for a Streptomyces antibiotic regulatory protein family regulator and txn11 for a two-component response regulator, were revealed to be absolutely required for trioxacarcin production by individually inactivating all the six annotated regulatory genes in the txn cluster. Complementation assay suggested that these two activators do not have a regulatory cascade relationship. Moreover, transcriptional analysis showed that they activate 15 of the 28 txn operons, indicating that a complicated regulatory network is involved in the trioxacarcin production. Information gained from this study may be useful for improving the production of the highly potent trioxacarcin A.

A size exclusion chromatography (SEC) process, in the presence of denaturant in the refolding buffer was developed to refold recombinant human interferon-γ (rhIFN-γ) at a high concentration. The rhIFN-γ was overexpressed inE. coli, resulting in the formation of inactive inclusion bodies (IBs). The IBs were first solubilized in 8 M urea as the denaturant, and then the refolding process performed by decreasing the urea concentration on the SEC column to suppress protein aggregation. The effects of the urea concentration, protein loading mode and column height during the refolding step were investigated. The combination of the bufferexchange effect of SEC and a moderate urea concentration in the refolding buffer resulted in an efficient route for producing correctly folded rhIFN-γ, with protein recovery of 67.1% and specific activity up to 1.2×107 IU/mg.

Bacterial aromatic polyketides, exemplified by anthracyclines, angucyclines, tetracyclines, and pentangular polyphenols, are a large family of natural products with diverse structures and biological activities and are usually biosynthesized by type II polyketide synthases (PKSs). Since the starting point of biosynthesis and combinatorial biosynthesis in 1984–1985, there has been a continuous effort to investigate the biosynthetic logic of aromatic polyketides owing to the urgent need of developing promising therapeutic candidates from these compounds. Recently, significant advances in the structural and mechanistic identification of enzymes involved in aromatic polyketide biosynthesis have been made on the basis of novel genetic, biochemical, and chemical technologies. This review highlights the progress in bacterial type II PKSs in the past three years (2013–2016). Moreover, novel compounds discovered or created by genome mining and biosynthetic engineering are also included.

Phoslactomycins (PLMs) are inhibitors of protein serine/threonine phosphatase 2A showing diverse and important antifungal, antibacterial and antitumor activity. PLMs are polyketide natural products and produced by several Streptomyces species. The PLMs biosynthetic gene clusters were identified from Streptomyces platensis SAM-0654 and localized in two separate genomic regions, consisting of 27 open reading frames that encode polyketide synthases (PKSs), enzymes for cyclohexanecarboxyl-CoA (CHC-CoA) and ethylmalonyl-CoA (Em-CoA) synthesis, enzymes for post-PKS modifications, proposed regulators, and putative resistance transporters. Bioinformatic analysis and inactivation experiment of regulatory genes suggest that PnR1 and PnR2 are two positive regulators of PLMs biosynthesis. Gene transcription analysis by reverse transcriptase PCR (RT-PCR) of the PLMs gene cluster demonstrated that PnR1 and PnR2 activate the transcription of the structural biosynthetic genes while PnR2 specially governs the transcription of pnR1 in a higher level.

In an attempt to construct a strain that produces doramectin, the loading module of Ave polyketide synthase (PKS) from Streptomyces avermitilis M1 was replaced with a cyclohexanecarboxylic (CHC) unique loading module from phoslactomycin PKS. Additionally, the CHC-CoA biosynthetic gene cassette was introduced into the engineered strain, which provided the precursor for directed biosynthesis of doramectin. The doramectin production ability of the final mutant S. avermitilis TG2002 was increased about six times and the ratio of Dor to Ave was enhanced 300 times more than the original strain.

Nosiheptide (NOS), belonging to the e series of thiopeptide antibiotics that exhibit potent activity against various bacterial pathogens, bears a unique indole side ring system and regiospecific hydroxyl groups on the characteristic macrocyclic core. Here, cloning, sequencing, and characterization of the nos gene cluster from Streptomyces actuosus ATCC 25421 as a model for this series of thiopeptides has unveiled new insights into their biosynthesis. Bioinformatics-based sequence analysis and in vivo investigation into the gene functions show that NOS biosynthesis shares a common strategy with recently characterized b or c series thiopeptides for forming the characteristic macrocyclic core, which features a ribosomally synthesized precursor peptide with conserved posttranslational modifications. However, it apparently proceeds via a different route for tailoring the thiopeptide framework, allowing the final product to exhibit the distinct structural characteristics of e series thiopeptides, such as the indole side ring system. Chemical complementation supports the notion that the S-adenosylmethionine-dependent protein NosL may play a central role in converting tryptophan to the key 3-methylindole moiety by an unusual carbon side chain rearrangement, most likely via a radical-initiated mechanism. Characterization of the indole side ring-opened analogue of NOS from the nosN mutant strain is consistent with the proposed methyltransferase activity of its encoded protein, shedding light into the timing of the individual steps for indole side ring biosynthesis. These results also suggest the feasibility of engineering novel thiopeptides for drug discovery by manipulating the NOS biosynthetic machinery.

Characterization of the biosynthetic gene cluster of glycosylated antibiotic nocathiacin I (NOC-I) here adds new insights to thiopeptide biosynthesis, showing the NOC-specific tailoring and unusual sugar formation. NOC-I biosynthesis shares the paradigm for forming a common thiopeptide core and the generality for converting to an e series member, as that of the parent compound nosiheptide (NOS). This may permit the production of NOC-I in the genetically amenable, NOS-producing strain by building NOC-specific genes for pathway engineering.

SummaryThe antiviral compounds quartromicins represent unique members of a family of spirotetronate natural products. In this study, a biosynthetic gene cluster of quartromicins was identified by degenerate primer PCR amplification of specific genes involved in the biosynthesis of the tetronate moiety. The biochemical results confirmed that 1,3-bisphosphoglycerate was incorporated into the tetronate ring, and the intermediates of this ring were also reconstructed in vitro. The data also suggested a module skipping strategy for the production of two alternative polyketide chains by the same polyketide synthase assembly line. These findings set the stage for further investigations of the stereodivergent intermolecular cyclization mechanism, and highlight how nature has constructed this type of C2 symmetric molecule through intermolecular dimerization.