Identification and characterization of a strong constitutive promoter stnYp for activating biosynthetic genes and producing natural products in streptomyces.

BACKGROUND: Streptomyces are well known for their potential to produce various pharmaceutically active compounds, the commercial development of which is often limited by the low productivity and purity of the desired compounds expressed by natural producers. Well-characterized promoters are crucial for driving the expression of target genes and improving the production of metabolites of interest. RESULTS: A strong constitutive promoter, stnYp, was identified in Streptomyces flocculus CGMCC4.1223 and was characterized by its effective activation of silent biosynthetic genes and high efficiency of heterologous gene expression. The promoter stnYp showed the highest activity in model strains of four Streptomyces species compared with the three frequently used constitutive promoters ermEp*, kasOp*, and SP44. The promoter stnYp could efficiently activate the indigoidine biosynthetic gene cluster in S. albus J1074, which is thought to be silent under routine laboratory conditions. Moreover, stnYp was found suitable for heterologous gene expression in different Streptomyces hosts. Compared with the promoters ermEp*, kasOp*, and SP44, stnYp conferred the highest production level of diverse metabolites in various heterologous hosts, including the agricultural-bactericide aureonuclemycin and the antitumor compound YM-216391, with an approximately 1.4 - 11.6-fold enhancement of the yields. Furthermore, the purity of tylosin A was greatly improved by overexpressing rate-limiting genes through stnYp in the industrial strain. Further, the yield of tylosin A was significantly elevated to 10.30 ± 0.12 g/L, approximately 1.7-fold higher than that of the original strain. CONCLUSIONS: The promoter stnYp is a reliable, well-defined promoter with strong activity and broad suitability. The findings of this study can expand promoter diversity, facilitate genetic manipulation, and promote metabolic engineering in multiple Streptomyces species.

Trioxacarcin A Interactions with G-Quadruplex DNA Reveal Its Potential New Targets as an Anticancer Agent.

Trioxacarcin (TXN) A was reported to be an anticancer agent through alkylation of dsDNA. G-quadruplex DNA (G4-DNA) is frequently formed in the promoter regions of oncogenes and the ends of telomerase genes, considered as promising drug targets for anticancer therapy. There are no reports about TXN A interactions with G4-DNA. Here, we tested TXN A's interactions with several G4-DNA oligos with parallel, antiparallel, or hybrid folding, respectively. We demonstrated that TXN A preferred to alkylate one flexible guanine in the loops of parallel G4-DNA. The position of the alkylated guanine is in favor of interactions of G4-DNA with TXN A. The structure of TXN A covalently bound RET G4-DNA indicated that TXN A alkylation on RET G4-DNA stabilizes the G4-DNA conformation. These studies opened a new window of how TXN A interacted with G4-DNA, which might hint a new mode of its function as an anticancer agent.

Biosynthesis of DNA-Alkylating Antitumor Natural Products.

DNA-alkylating natural products play an important role in drug development due to their significant antitumor activities. They usually show high affinity with DNA through different mechanisms with the aid of their unique scaffold and highly active functional groups. Therefore, the biosynthesis of these natural products has been extensively studied, especially the construction of their pharmacophores. Meanwhile, their producing strains have evolved corresponding self-resistance strategies to protect themselves. To further promote the functional characterization of their biosynthetic pathways and lay the foundation for the discovery and rational design of DNA alkylating agents, we summarize herein the progress of research into DNA-alkylating antitumor natural products, including their biosynthesis, modes of action, and auto-resistance mechanisms.

Heterologous characterization of mechercharmycin A biosynthesis reveals alternative insights into post-translational modifications for RiPPs.

Mechercharmycin A (MCM-A) is a marine natural product belonging to a family of polyazole cyclopeptides with remarkable bioactivities and unique structures. Identification, heterologous expression, and genetic characterizations of the MCM biosynthetic gene cluster in Bacillus subtilis revealed that it is a ribosomally synthesized and post-translationally modified peptide (RiPP) possessing complex with distinctive modifications. Based on this heterologous expression system, two MCM analogs with comparable antitumor activity are generated by engineering the biosynthetic pathway. Combinatorial co-production of a precursor peptide with different modifying enzymes in Escherichia coli identifies a different timing of modifications, showing that a tRNAGlu-dependent highly regioselective dehydration is the first modification step, followed by polyazole formation through heterocyclization and dehydrogenation in an N- to C-terminal direction. Therefore, a rational biosynthetic pathway of MCMs is proposed, which unveils a subfamily of azol(in)e-containing RiPPs and sets the stage for further investigations of the enzymatic mechanism and synthetic biology.

Reductive inactivation of the hemiaminal pharmacophore for resistance against tetrahydroisoquinoline antibiotics.

Antibiotic resistance is becoming one of the major crises, among which hydrolysis reaction is widely employed by bacteria to destroy the reactive pharmacophore. Correspondingly, antibiotic producer has canonically co-evolved this approach with the biosynthetic capability for self-resistance. Here we discover a self-defense strategy featuring with reductive inactivation of hemiaminal pharmacophore by short-chain dehydrogenases/reductases (SDRs) NapW and homW, which are integrated with the naphthyridinomycin biosynthetic pathway. We determine the crystal structure of NapW·NADPH complex and propose a catalytic mechanism by molecular dynamics simulation analysis. Additionally, a similar detoxification strategy is identified in the biosynthesis of saframycin A, another member of tetrahydroisoquinoline (THIQ) antibiotics. Remarkably, similar SDRs are widely spread in bacteria and able to inactive other THIQ members including the clinical anticancer drug, ET-743. These findings not only fill in the missing intracellular events of temporal-spatial shielding mode for cryptic self-resistance during THIQs biosynthesis, but also exhibit a sophisticated damage-control in secondary metabolism and general immunity toward this family of antibiotics.

Characterization of the Rifamycin-Degrading Monooxygenase From Rifamycin Producers Implicating Its Involvement in Saliniketal Biosynthesis.

Rifamycin derivatives, such as rifampicin, have potent antibiotic activity and have long been used in the clinic as mainstay components for the treatment of tuberculosis, leprosy, and AIDS-associated mycobacterial infections. However, the extensive usage of these antibiotics has resulted in the rapid development of bacterial resistance. The resistance mechanisms mainly include mutations of the rifamycin target RNA polymerase of bacteria and enzymatic modifications of rifamycin antibiotics. One modification is the recently characterized rifamycin degradation catalyzed by Rox enzymes, which belong to the widely occurring flavin monooxygenases. Intriguingly, our recent sequence analysis revealed the rifamycin producers also encode Rox homologs that are not yet characterized. In this work, we expanded the study of the Rox-catalyzed rifamycin degradation. We first showed that the Rox proteins from rifamycin producers have the enzymatic rifamycin SV-degrading activity. Then we used the structurally diverse rifamycin compounds rifampicin and 16-demethylrifamycin W to probe the substrate scope and found that they each have a slightly different substrate scope. Finally, we demonstrated that Rox proteins can also catalyze the transformation of 16-demethylsalinisporamycin to 16-demethylsaliniketal A. Since 16-demethylsalinisporamycin and 16-demethylsaliniketal A are the counterpart analogs of salinisporamycin and saliniketal A, our biochemical findings not only uncover a previously uncharacterized self-resistance mechanism in the rifamycin producers, but also bridge the gap between the biosynthesis of the potential antitumor compound saliniketal A.

Recent advances in HemN-like radical S-adenosyl-l-methionine enzyme-catalyzed reactions.

Covering: 2012 to 2019HemN-like radical S-adenosyl-l-methionine (SAM) enzymes have been recently disclosed to catalyze diverse chemically challenging reactions from primary to secondary metabolic pathways. In this highlight, we summarize the reaction examples catalyzed by HemN-like enzymes to date and the enzymatic mechanisms reported. From the recent mechanistic investigations, we reason that there is a shared initiating mechanism wherein a characteristic SAM methylene radical is proposed to abstract a hydrogen atom from an sp3 carbon or add onto an sp2 carbon center although variations occur thereafter from reaction to reaction, as well as providing a brief insight into some future prospects.

Directed Biosynthesis of Iso-aclacinomycins with Improved Anticancer Activity.

A four-enzyme catalyzed hydroxy regioisomerization of anthracycline was integrated into the biosynthetic pathway of aclacinomycin A (ALM-A), to generate a series of iso-ALMs via directed combinatorial biosynthesis combined with precursor-directed mutasynthesis. Most of the newly acquired iso-ALMs exhibit obviously (1-5-fold) improved antitumor activity. Therefore, we not only developed iso-ALMs with potential as clinical drugs but also demonstrated the utility of this tailoring tool for modification of anthracycline antibiotics in drug discovery and development.

Identification of the Amipurimycin Gene Cluster Yields Insight into the Biosynthesis of C9 Sugar Nucleoside Antibiotics.

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.

Discovery of Druggability-Improved Analogues by Investigation of the LL-D49194α1 Biosynthetic Pathway.

The biosynthetic gene cluster of antitumor antibiotic LL-D49194α1 (LLD) was identified and comparatively analyzed with that of trioxacarcins. The tailoring genes encoding glycosyltransferase, methyltransferase and cytochrome P450 were systematically deleted, which led to the discovery of eight compounds from the mutants. Preliminary pharmaceutical evaluation revealed two intermediates exhibiting higher cytotoxicity, stability and solubility. These results highlighted the modification pathway for LLD biosynthesis, and provided highly potent, structurally simplified "unnatural" natural products with improved druggability.

Discovery of 16-Demethylrifamycins by Removing the Predominant Polyketide Biosynthesis Pathway in Micromonospora sp. Strain TP-A0468.

A number of strategies have been developed to mine novel natural products based on biosynthetic gene clusters and there have been dozens of successful cases facilitated by the development of genomic sequencing. During our study on biosynthesis of the antitumor polyketide kosinostatin (KST), we found that the genome of Micromonospora sp. strain TP-A0468, the producer of KST, contains other potential polyketide gene clusters, with no encoded products detected. Deletion of kst cluster led to abolishment of KST and the enrichment of several new compounds, which were isolated and characterized as 16-demethylrifamycins (referred to here as compounds 3 to 6). Transcriptional analysis demonstrated that the expression of the essential genes related to the biosynthesis of compounds 3 to 6 was comparable to the level in the wild-type and in the kst cluster deletion strain. This indicates that the accumulation of these compounds was due to the redirection of metabolic flux rather than transcriptional activation. Genetic disruption, chemical complementation, and bioinformatic analysis revealed that the production of compounds 3 to 6 was accomplished by cross talk between the two distantly placed polyketide gene clusters pks3 and M-rif This finding not only enriches the analogue pool and the biosynthetic diversity of rifamycins but also provides an auxiliary strategy for natural product discovery through genome mining in polyketide-producing microorganisms.IMPORTANCE Natural products are essential in the development of novel clinically used drugs. Discovering new natural products and modifying known compounds are still the two main ways to generate new candidates. Here, we have discovered several rifamycins with varied skeleton structures by redirecting the metabolic flux from the predominant polyketide biosynthetic pathway to the rifamycin pathway in the marine actinomycetes species Micromonospora sp. strain TP-A0468. Rifamycins are indispensable chemotherapeutics in the treatment of various diseases such as tuberculosis, leprosy, and AIDS-related mycobacterial infections. This study exemplifies a useful method for the discovery of cryptic natural products in genome-sequenced microbes. Moreover, the 16-demethylrifamycins and their genetically manipulable producer provide a new opportunity in the construction of novel rifamycin derivates to aid in the defense against the ever-growing drug resistance of Mycobacterium tuberculosis.

Directed production of aurantizolicin and new members based on a YM-216391 biosynthetic system.

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.

Enzymology of Anthraquinone-γ-Pyrone Ring Formation in Complex Aromatic Polyketide Biosynthesis.

Aromatic-fused γ-pyrones are structural features of many bioactive natural products and valid scaffolds for medicinal chemistry. However, the enzymology of their formation has not been completely established. Now it is demonstrated that TxnO9, a CalC-like protein belonging to a START family, functions as an unexpected anthraquinone-γ-pyrone synthase involved in the biosynthesis of antitumor antibiotic trioxacarcin A (TXN-A). Structural analysis by NMR identified a likely substrate/product-binding mode and putative key active sites of TxnO9, which allowed an enzymatic mechanism to be proposed. Moreover, a subset of uncharacterized homologous proteins bearing an unexamined Lys-Thr dyad exhibit the same function. Therefore, the functional assignment and mechanistic investigation of this γ-pyrone synthase elucidated an undescribed step in TXN-A biosynthesis, and the discovery of this new branch of polyketide heterocyclases expands the functions of the START superfamily.

A radical S-adenosyl-L-methionine enzyme and a methyltransferase catalyze cyclopropane formation in natural product biosynthesis.

Cyclopropanation of unactivated olefinic bonds via addition of a reactive one-carbon species is well developed in synthetic chemistry, whereas natural cyclopropane biosynthesis employing this strategy is very limited. Here, we identify a two-component cyclopropanase system, composed of a HemN-like radical S-adenosyl-L-methionine (SAM) enzyme C10P and a methyltransferase C10Q, catalyzes chemically challenging cyclopropanation in the antitumor antibiotic CC-1065 biosynthesis. C10P uses its [4Fe-4S] cluster for reductive cleavage of the first SAM to yield a highly reactive 5'-deoxyadenosyl radical, which abstracts a hydrogen from the second SAM to produce a SAM methylene radical that adds to an sp2-hybridized carbon of substrate to form a SAM-substrate adduct. C10Q converts this adduct to CC-1065 via an intramolecular SN2 cyclization mechanism with elimination of S-adenosylhomocysteine. This cyclopropanation strategy not only expands the enzymatic reactions catalyzed by the radical SAM enzymes and methyltransferases, but also sheds light on previously unnoticed aspects of the versatile SAM-based biochemistry.

GyrI-like proteins catalyze cyclopropanoid hydrolysis to confer cellular protection.

GyrI-like proteins are widely distributed in prokaryotes and eukaryotes, and recognized as small-molecule binding proteins. Here, we identify a subfamily of these proteins as cyclopropanoid cyclopropyl hydrolases (CCHs) that can catalyze the hydrolysis of the potent DNA-alkylating agents yatakemycin (YTM) and CC-1065. Co-crystallography and molecular dynamics simulation analyses reveal that these CCHs share a conserved aromatic cage for the hydrolytic activity. Subsequent cytotoxic assays confirm that CCHs are able to protect cells against YTM. Therefore, our findings suggest that the evolutionarily conserved GyrI-like proteins confer cellular protection against diverse xenobiotics via not only binding, but also catalysis.

Catalysis of Extracellular Deamination by a FAD-Linked Oxidoreductase after Prodrug Maturation in the Biosynthesis of Saframycin†A.

The biosynthesis of antibiotics in bacteria is usually believed to be an intracellular process, at the end of which the matured compounds are exported outside the cells. The biosynthesis of saframycin A (SFM-A), an antitumor antibiotic, requires a cryptic fatty acyl chain to guide the construction of a pentacyclic tetrahydroisoquinoline scaffold; however, the follow-up deacylation and deamination steps remain unknown. Herein we demonstrate that SfmE, a membrane-bound peptidase, hydrolyzes the fatty acyl chain to release the amino group; and SfmCy2, a secreted oxidoreductase covalently associated with FAD, subsequently performs an oxidative deamination extracellularly. These results not only fill in the missing steps of SFM-A biosynthesis, but also reveal that a FAD-binding oxidoreductase catalyzes an unexpected deamination reaction through an unconventional extracellular pathway in Streptmyces bacteria.

Unified Biosynthetic Origin of the Benzodipyrrole Subunits in CC-1065.

CC-1065 is the first characterized member of a family of naturally occurring antibiotics including yatakemycin and duocarmycins with exceptionally potent antitumor activity. CC-1065 contains three benzodipyrroles (1a-, 1b-, and 1c-) of which the 1a-subunit is remarkable by being composed of a cyclopropane ring, and the mechanism for the biological formation of benzodipyrrole rings remains elusive. Previously, biosynthetic studies of CC-1065 were limited to radioactively labeled precursor feeding experiments, which showed that tyrosine (Tyr) and serine (Ser) were incorporated into the two benzodipyrrole (1b- and 1c-) subunits via the same mode but that this was different from the key cyclopropabenzodipyrrole (1a-) subunit with N1-C2-C3 derived from Ser. Herein, the biosynthetic gene cluster of CC-1065 has been cloned, analyzed, and characterized by a series of gene inactivations. Significantly, a key intermediate bearing a C7-OH group derived from a Δc10C mutant exhibited improved cytotoxicity. Moreover, this data inspired us to suspect that the 1a-subunit might employ the same precursor incorporation mode as the 1b- and 1c-subunits. Subsequently, 13C-labeled Tyr feeding experiments confirmed that the N1-C2-C3 is originated from Tyr via DOPA as an intermediate. Collectively, a biosynthetic pathway of benzodipyrrole is proposed featuring a revised and unified precursor incorporation mode, which implicates an oxidative cyclization strategy for the assembly of benzodipyrrole. This work sets the stage for further study of enzymatic mechanisms and combinatorial biosynthesis for new DNA alkylating analogues.

New insights into bacterial type II polyketide biosynthesis.

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.

Hydroxyl regioisomerization of anthracycline catalyzed by a four-enzyme cascade.

Ranking among the most effective anticancer drugs, anthracyclines represent an important family of aromatic polyketides generated by type II polyketide synthases (PKSs). After formation of polyketide cores, the post-PKS tailoring modifications endow the scaffold with various structural diversities and biological activities. Here we demonstrate an unprecedented four-enzyme-participated hydroxyl regioisomerization process involved in the biosynthesis of kosinostatin. First, KstA15 and KstA16 function together to catalyze a cryptic hydroxylation of the 4-hydroxyl-anthraquinone core, yielding a 1,4-dihydroxyl product, which undergoes a chemically challenging asymmetric reduction-dearomatization subsequently acted by KstA11; then, KstA10 catalyzes a region-specific reduction concomitant with dehydration to afford the 1-hydroxyl anthraquinone. Remarkably, the shunt product identifications of both hydroxylation and reduction-dehydration reactions, the crystal structure of KstA11 with bound substrate and cofactor, and isotope incorporation experiments reveal mechanistic insights into the redox dearomatization and rearomatization steps. These findings provide a distinguished tailoring paradigm for type II PKS engineering.

Characterization of LnmO as a pathway-specific Crp/Fnr-type positive regulator for leinamycin biosynthesis in Streptomyces atroolivaceus and its application for titer improvement.

The cyclic adenosine monophosphate (cAMP) receptor protein/fumarate and nitrate reductase regulatory protein (Crp/Fnr) family of transcriptional regulators are pleiotropic transcriptional regulators that control a broad range of cellular functions. Leinamycin (LNM) is a potent antitumor antibiotic produced by Streptomyces atroolivaceus S-140. We previously cloned and characterized the lnm biosynthetic gene cluster from S. atroolivaceus S-140. We here report inactivation of lnmO in S. atroolivaceus S-140 and overexpression of lnmO in the S. atroolivaceus S-140 wild-type and ∆lnmE mutant SB3033 to investigate its role in LNM biosynthesis. Bioinformatics analysis revealed LnmO as the only regulator within the lnm gene cluster, exhibiting high sequence similarity to known Crp/Fnr family regulators. The inactivation of lnmO in S. atroolivaceus S-140 completely abolished LNM production but caused no apparent morphological changes, supporting that LnmO is indispensable and specific to LNM biosynthesis. Overexpression of lnmO in S. atroolivaceus S-140 and SB3033 resulted in three- and fourfold increase in LNM and LNM E1 production, respectively, supporting that LnmO acts as a positive regulator. While all of the Crp/Fnr family regulators studied to date appeared to be pleiotropic, our results support LnmO as the first Crp/Fnr family regulator that is pathway-specific. LnmO joins the growing list of regulators that could be exploited to improve secondary metabolite production in Streptomyces. Engineered strains overproducing LNM and LNM E1 will facilitate further mechanistic studies and clinical evaluation of LNM and LNM E1 as novel anticancer drugs.