A Secreted BBE-Like Enzyme Acting as a Drug-Binding Efflux Carrier Confers Microbial Self-Resistance to Mitomycin C.

The berberine bridge enzyme (BBE)-like flavoproteins have attracted continuous attention for their capability to catalyze various oxidative reactions. Here we demonstrate that MitR, a secreted BBE-like enzyme, functions as a special drug-binding efflux protein evolved from quinone reductase. Moreover, this protein provides self-resistance to its hosts toward the DNA-alkylating agent mitomycin C with a distinctive strategy, featured by independently performing drug binding and efflux.

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.

Newly Discovered Mechanisms of Antibiotic Self-Resistance with Multiple Enzymes Acting at Different Locations and Stages.

Self-resistance determinants are essential for the biosynthesis of bioactive natural products and are closely related to drug resistance in clinical settings. The study of self-resistance mechanisms has long moved forward on the discovery of new resistance genes and the characterization of enzymatic reactions catalyzed by these proteins. However, as more examples of self-resistance have been reported, it has been revealed that the enzymatic reactions contribute to self-protection are not confined to the cellular location where the final toxic compounds are present. In this review, we summarize representative examples of self-resistance mechanisms for bioactive natural products functional at different cell locations to explore the models of resistance strategies involved. Moreover, we also highlight those resistance determinants that are widespread in nature and describe the applications of self-resistance genes in natural product mining to interrogate the landscape of self-resistance genes in drug resistance-related new drug discovery.

Identification and Characterization of Enzymes Catalyzing Early Steps in Miharamycin and Amipurimycin Biosynthesis.

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.

Characterization of Miharamycin Biosynthesis Reveals a Hybrid NRPS-PKS to Synthesize High-Carbon Sugar from a Complex Nucleoside.

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.

Activation and Characterization of Cryptic Gene Cluster: Two Series of Aromatic Polyketides Biosynthesized by Divergent Pathways.

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.

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.

Elucidation of the Herbicidin Tailoring Pathway Offers Insights into Its Structural Diversity.

The biosynthetic gene clusters for herbicidins ( hbc) and aureonuclemycin ( anm) were identified in Streptomyces sp. KIB-027 and Streptomyces aureus, respectively. The roles of genes possibly involved in post-core-assembly steps in herbicidin biosynthesis in these clusters and a related her cluster were studied. Through systematic gene deletions, structural elucidation of the accumulated intermediates in the mutants, and in vitro verification of the encoded enzymes, the peripheral modification pathway for herbicidin biosynthesis is now fully established.

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.

Enzymatic Formation of Oxygen-Containing Heterocycles in Natural Product Biosynthesis.

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.

A Six-Oxidase Cascade for Tandem C-H Bond Activation Revealed by Reconstitution of Bicyclomycin Biosynthesis.

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.

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.

The SARP Family Regulator Txn9 and Two-Component Response Regulator Txn11 are Key Activators for Trioxacarcin Biosynthesis in Streptomyces bottropensis.

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.

Biosynthesis of trioxacarcin revealing a different starter unit and complex tailoring steps for type II polyketide synthase.

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.

Characterization of QmnD3/QmnD4 for double bond formation in quartromicin 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.

Functional identification of the gene encoding the enzyme involved in mannosylation in ramoplanin biosynthesis in Actinoplanes sp.

Ramoplanin is a lipopeptide antibiotic active against multi-drug-resistant, Gram-positive pathogens. Structurally, it contains a di-mannose moiety attached to the peptide core at Hpg(11). The biosynthetic gene cluster of ramoplanin has already been reported and the assembly of the depsipeptide has been elucidated but the mechanism of transferring sugar moiety to the peptide core remains unclear. Sequence analysis of the biosynthetic gene cluster indicated ramo-orf29 was a mannosyltransferase candidate. To investigate the involvement of ramo-orf29 in ramoplanin biosynthesis, gene inactivation and complementation have been conducted in Actinoplanes sp. ATCC 33076 by homologous recombination. Metabolite analysis revealed that the ramo-orf29 inactivated mutant produced no ramoplanin but the ramoplanin aglycone. Thus, ramo-orf29 codes for the mannosyltransferase in the ramoplanin biosynthesis pathway. This lays the foundation for further exploitation of the ramoplanin mannosyltransferase and aglycone in combinatorial biosynthesis.

Production of ramoplanin analogues by genetic engineering of Actinoplanes sp.

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).

Unconventional origin and hybrid system for construction of pyrrolopyrrole moiety in kosinostatin biosynthesis.

Kosinostatin (KST), an antitumor antibiotic, features a pyrrolopyrrole moiety spirally jointed to a five-membered ring of an anthraquinone framework glycosylated with a γ-branched octose. By a combination of in silico analysis, genetic characterization, biochemical assay, and precursor feeding experiments, a biosynthetic pathway for KST was proposed, which revealed (1) the pyrrolopyrrole moiety originates from nicotinic acid and ribose, (2) the bicyclic amidine is constructed by a process similar to the tryptophan biosynthetic pathway, and (3) a discrete adenylation enzyme and a peptidyl carrier protein (PCP) are responsible for producing a PCP-tethered building block parallel to type II polyketide synthase (PKS) rather than for the PKS priming step by providing the starter unit. These findings provide an opportunity to further explore the inexplicable enzymatic logic that governs the formation of pyrrolopyrrole moiety and the spirocyclic skeleton.