Guohui Pan

Angucyclines as signals modulate the behaviors of Streptomyces coelicolor

Significance This work addresses the molecular basis for interspecies signaling effects of antibiotics, which have been a controversial but potentially significant emerging topic over the last few years. The “pseudo” gamma-butyrolactone (GBL) receptor (i.e., those GBL receptor homologues often found in Streptomyces genomes, but apparently not binding or responding to GBLs), ScbR2, was identified as the receptor of JdB. It has an extraordinary ability to bind and respond to exogenous angucyclines, as well as to be able to directly regulate the biosynthesis of different endogenous antibiotics and the morphological development of Streptomyces. Our findings significantly extend understanding of antibiotic-mediated signaling mechanisms and the ecological impact of antibiotics. The angucycline antibiotic jadomycin B (JdB) produced by Streptomyces venezuelae has been found here to induce complex survival responses in Streptomyces coelicolor at subinhibitory concentration. The receptor for JdB was identified as a “pseudo” gamma-butyrolactone receptor, ScbR2, which was shown to bind two previously unidentified target promoters, those of redD (redDp) and adpA (adpAp), thus directly regulating undecylprodigiosin (Red) production and morphological differentiation, respectively. Because AdpA also directly regulates the expression of redD, ScbR2, AdpA, and RedD together form a feed-forward loop controlling both differentiation and Red production phenotypes. Different signal strengths (i.e., JdB concentrations) were shown to induce the two different phenotypes by modulating the relative transcription levels of adpA vs. redD. The induction of morphological differentiation and endogenous antibiotic production by exogenous antibiotic exemplifies an important survival strategy more sophisticated than the induction of antibiotic resistance.

JadR*-mediated feed-forward regulation of cofactor supply in jadomycin biosynthesis

Jadomycin production is under complex regulation in Streptomyces venezuelae. Here, another cluster‐situated regulator, JadR*, was shown to negatively regulate jadomycin biosynthesis by binding to four upstream regions of jadY, jadR1, jadI and jadE in jad gene cluster respectively. The transcriptional levels of four target genes of JadR* increased significantly in ΔjadR*, confirming that these genes were directly repressed by JadR*. Jadomycin B (JdB) and its biosynthetic intermediates 2,3‐dehydro‐UWM6 (DHU), dehydrorabelomycin (DHR) and jadomycin A (JdA) modulated the DNA‐binding activities of JadR* on the jadY promoter, with DHR giving the strongest dissociation effects. Direct interactions between JadR* and these ligands were further demonstrated by surface plasmon resonance, which showed that DHR has the highest affinity for JadR*. However, only DHU and DHR could induce the expression of jadY and jadR* in vivo. JadY is the FMN/FAD reductase supplying cofactors FMNH2/FADH2 for JadG, an oxygenase, that catalyses the conversion of DHR to JdA. Therefore, our results revealed that JadR* and early pathway intermediates, particularly DHR, regulate cofactor supply by a convincing case of a feed‐forward mechanism. Such delicate regulation of expression of jadY could ensure a timely supply of cofactors FMNH2/FADH2 for jadomycin biosynthesis, and avoid unnecessary consumption of NAD(P)H.

Identification of JadG as the B ring opening oxygenase catalyzing the oxidative C-C bond cleavage reaction in jadomycin biosynthesis.

Jadomycin B is a member of atypical angucycline antibiotics whose biosynthesis involves a unique ring opening C-C bond cleavage reaction. Here, we firmly identified JadG as the enzyme responsible for the B ring opening reaction in jadomycin biosynthesis. In vitro analysis of the JadG catalyzed reaction revealed that it requires FMNH(2) or FADH(2) as cofactors in the conversion of dehydrorabelomycin to jadomycin A. The cofactors could be supplied by either a cluster-situated flavin reductase JadY or the Escherichia coli Fre. JadY was characterized as a NAD(P)H-dependent FMN/FAD reductase, with FMN as the preferred substrate. Disruption mutant of jadY still produced jadomycin, indicating that the function of JadY could be substituted by other enzymes in the host. JadG represents the biochemically verified member of an enzyme class catalyzing an unprecedented C-C bond cleavage reaction.

Discovery of the leinamycin family of natural products by mining actinobacterial genomes

Significance Leinamycin (LNM) is a promising anticancer drug lead, yet no analog has been isolated since its discovery nearly 30 y ago. By mining bacterial genomes, we discovered 49 potential producers of LNM-type natural products, the structural diversity of which was predicted based on bioinformatics and confirmed by in vitro characterization of selected enzymes and structural elucidation of the guangnanmycins and weishanmycins. These findings demonstrate the power of the discovery-based approach to combinatorial biosynthesis for natural product discovery and structural diversity. New members of the LNM family of natural products should greatly facilitate drug discovery and development. The LNM-type biosynthetic machineries provide outstanding opportunities to dissect and mimic Nature’s strategies for combinatorial biosynthesis and natural product structural diversity. Nature’s ability to generate diverse natural products from simple building blocks has inspired combinatorial biosynthesis. The knowledge-based approach to combinatorial biosynthesis has allowed the production of designer analogs by rational metabolic pathway engineering. While successful, structural alterations are limited, with designer analogs often produced in compromised titers. The discovery-based approach to combinatorial biosynthesis complements the knowledge-based approach by exploring the vast combinatorial biosynthesis repertoire found in Nature. Here we showcase the discovery-based approach to combinatorial biosynthesis by targeting the domain of unknown function and cysteine lyase domain (DUF–SH) didomain, specific for sulfur incorporation from the leinamycin (LNM) biosynthetic machinery, to discover the LNM family of natural products. By mining bacterial genomes from public databases and the actinomycetes strain collection at The Scripps Research Institute, we discovered 49 potential producers that could be grouped into 18 distinct clades based on phylogenetic analysis of the DUF–SH didomains. Further analysis of the representative genomes from each of the clades identified 28 lnm-type gene clusters. Structural diversities encoded by the LNM-type biosynthetic machineries were predicted based on bioinformatics and confirmed by in vitro characterization of selected adenylation proteins and isolation and structural elucidation of the guangnanmycins and weishanmycins. These findings demonstrate the power of the discovery-based approach to combinatorial biosynthesis for natural product discovery and structural diversity and highlight Nature’s rich biosynthetic repertoire. Comparative analysis of the LNM-type biosynthetic machineries provides outstanding opportunities to dissect Nature’s biosynthetic strategies and apply these findings to combinatorial biosynthesis for natural product discovery and structural diversity.

Kinamycin biosynthesis employs a conserved pair of oxidases for B-ring contraction

The biosynthesis of diazobenzofluorene kinamycins requires a hitherto uncharacterized B-ring contraction. Via detailed genetic and enzymatic analyses we unambiguously characterized the conserved pairs of oxidases, AlpJ and AlpK homologs, as natures machinery for benzofluorenone formation, which paves the way for the investigation of the following diazo assembly.

Characterization of the Ketosynthase and Acyl Carrier Protein Domains at the LnmI Nonribosomal Peptide Synthetase-Polyketide Synthase Interface for Leinamycin Biosynthesis.

Leinamycin (LNM) is biosynthesized by a hybrid nonribosomal peptide synthetase (NRPS)–acyltransferase (AT)-less type I polyketide synthase (PKS). Characterization of LnmI revealed ketosynthase (KS)–acyl carrier protein (ACP)–KS domains at the NRPS–PKS interface. Inactivation of the KS domain or ACP domain in vivo abolished LNM production, and the ACP domain can be phosphopantetheinylated in vitro. The LnmI KS–ACP–KS architecture represents a new mechanism for functional crosstalk between NRPS and AT-less type I PKS in the biosynthesis of hybrid peptide–polyketide natural products.

A long-range acting dehydratase domain as the missing link for C17-dehydration in iso-migrastatin biosynthesis

The dehydratase domains (DHs) of the iso-migrastatin (iso-MGS) polyketide synthase (PKS) were investigated by systematic inactivation of the DHs in module-6, -9, -10 of MgsF (i.e., DH6, DH9, DH10) and module-11 of MgsG (i.e., DH11) in vivo, followed by structural characterization of the metabolites accumulated by the mutants, and biochemical characterization of DH10 in vitro, using polyketide substrate mimics with varying chain lengths. These studies allowed us to assign the functions for all four DHs, identifying DH10 as the dedicated dehydratase that catalyzes the dehydration of the C17 hydroxy group during iso-MGS biosynthesis. In contrast to canonical DHs that catalyze dehydration of the β-hydroxy groups of the nascent polyketide intermediates, DH10 acts in a long-range manner that is unprecedented for type I PKSs, a novel dehydration mechanism that could be exploited for polyketide structural diversity by combinatorial biosynthesis and synthetic biology.

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.

New isofuranonaphthoquinones and isoindolequinones from Streptomyces sp. CB01883.

Isofuranonaphthoquinones (IFQs) and Isoindolequinones (IIQs) comprise a small family of natural products, with the latter ones are especially uncommon in nature. Here we report the discovery of seven new IFQs, IFQ A-G (1–7), and three new IIQs, IIQ A-C (8–10), along with the known anthraquinone desoxyerythrolaccin (11), from Streptomyces sp. CB01883, expanding the chemical diversity of this family of natural products. The structures of these natural products were established on the basis of their HR-ESI-MS and nuclear magnetic resonance (NMR) spectroscopic data. All compounds were assessed for antibacterial activity, with 11 and 1, 5–7 exhibiting moderate and weak activities, respectively, against several Gram-positive bacteria tested. Bioinformatics analysis of the Streptomyces sp. CB01883 genome revealed the ifq gene cluster that showed identical genetic organization, with high-sequence identity, to the ifn gene cluster recently cloned from Streptomyces sp. RI-77 and confirmed to encode the biosynthesis of two IFQs, JBIR-76 and JBIR-77. Co-isolation of IFQs with IIQs from Streptomyces sp. CB01883 and facile chemical transformation of selected IFQs to IIQs, as exemplified by 1 to 9, together with the finding of the ifq cluster that most likely only encodes IFQ biosynthesis, support the proposal that IIQs may be derived nonenzymatically from IFQs in the presence of an amine.