Promoter engineering of natural product biosynthetic gene clusters in actinomycetes: concepts and applications.

Covering 2011 to 2022Low titers of natural products in laboratory culture or fermentation conditions have been one of the challenging issues in natural products research. Many natural product biosynthetic gene clusters (BGCs) are also transcriptionally silent in laboratory culture conditions, making it challenging to characterize the structures and activities of their metabolites. Promoter engineering offers a potential solution to this problem by providing tools for transcriptional activation or optimization of biosynthetic genes. In this review, we summarize the 10 years of progress in promoter engineering approaches in natural products research focusing on the most metabolically talented group of bacteria actinomycetes.

Acidonemycins A-C, Glycosylated Angucyclines with Antivirulence Activity Produced by the Acidic Culture of Streptomyces indonesiensis.

The genome of Streptomyces indonesiensis is highly enriched with cryptic biosynthetic gene clusters (BGCs). The majority of these cryptic BGCs are transcriptionally silent in normal laboratory culture conditions as determined by transcriptome analysis. When cultured in acidic pH (pH 5.4), this strain has been shown to produce a set of new metabolites that were not observed in cultures of neutral pH (pH 7.4). The organic extract of the acidic culture displayed an antivirulence activity against methicillin-resistant Staphylococcus aureus (MRSA). Here, we report the structures of new glycosylated aromatic polyketides, named acidonemycins A-C (1-3), belonging to the family of angucyclines. Type II polyketide synthase BGC responsible for the production of 1-3 was identified by a transcriptome comparison between acidic (pH 5.4) and neutral (pH 7.4) cultures and further confirmed by heterologous expression in Streptomyces albus J1074. Of the three new compounds, acidonemycins A and B (1 and 2) displayed antivirulence activity against MRSA. The simultaneous identification of both antivirulent compounds and their BGC provides a starting point for the future effort of combinatorial biosynthesis.

Discovery of Bioactive Metabolites by Acidic Stress to a Geldanamycin Producer, Streptomyces samsunensis.

In an effort to activate silent biosynthetic gene clusters, Streptomyces samsunensis DSM42010, a producer of geldanamycin, was cultured at four different pHs (4.5, 5.4, 6.6, and 7.4). An acidic culture condition (pH 5.4) was selected for a chemical investigation since S. samsunensis showed a different metabolic profile compared to when it was cultured under other conditions. Seven new (1-7) and four known (8-11) compounds were isolated from these cultures. The structures of the isolated compounds were determined by spectroscopic techniques and chemical derivatization. Relative and absolute configurations of the new compounds (1-5) were established using JBCA, PGME method, advanced Marfey's method, modified Mosher's method, and comparison of observed and calculated ECD data. Interestingly, compounds 1-3 were truncated versions of geldanamycin, and compound 4 was also deduced to originate from geldanamycin. Compound 5 was composed of 3-methyltyrosine and 6-hydroxy-2,4-hexadienoic acid connected through an amide bond. Compounds 6 and 7 were dihydrogenated forms of geldanamycin with a hydroxy substitution. It is possible that culturing this strain under acidic conditions interfered to some degree with the geldanamycin polyketide synthase, leading to production of truncated versions as well as analogues of geldanamycin. Compounds 1, 8, and 9 showed significant antivirulence activity, inhibiting production of α-toxin by methicillin-resistant Staphylococcus aureus without growth attenuation and global regulatory inhibition; compounds 1, 8, and 9 may become promising α-toxin-specific antivirulence leads with less risk of resistance development.

Top-down synthetic biology approach for titer improvement of clinically important antibiotic daptomycin in Streptomyces roseosporus.

Secondary metabolites are produced at low titers by native producers due to tight regulations of their productions in response to environmental conditions. Synthetic biology provides a rational engineering principle for transcriptional optimization of secondary metabolite BGCs (biosynthetic gene clusters). Here, we demonstrate the use of synthetic biology principles for the development of a high-titer strain of the clinically important antibiotic daptomycin. Due to the presence of large NRPS (non-ribosomal peptide synthetase) genes with multiple direct repeats, we employed a top-down approach that allows transcriptional optimization of genes in daptomycin BGC with the minimum inputs of synthetic DNAs. The repeat-free daptomycin BGC was created through partial codon-reprogramming of a NRPS gene and cloned into a shuttle BAC vector, allowing BGC refactoring in a host with a powerful recombination system. Then, transcriptions of functionally divided operons were sequentially optimized through three rounds of DBTL (design-build-test-learn) cycles that resulted in up to ~2300% improvement in total lipopeptide titers compared to the wild-type strain. Upon decanoic acid feeding, daptomycin accounted for ∼ 40% of total lipopeptide production. To the best of our knowledge, this is the highest improvement of daptomycin titer ever achieved through genetic engineering of S. roseosporus. The top-down engineering approach we describe here could be used as a general strategy for the development of high-titer industrial strains of secondary metabolites produced by BGCs containing genes of large multi-modular NRPS and PKS enzymes.

Ribocyclophanes A-E, Glycosylated Cyclophanes with Antiproliferative Activity from Two Cultured Terrestrial Cyanobacteria.

The cell extracts of two cultured freshwater Nostoc spp., UIC 10279 and UIC 10366, both from the suburbs of Chicago, showed antiproliferative activity against MDA-MB-231 and MDA-MB-435 cancer cell lines. Bioassay-guided fractionation led to the isolation of five glycosylated cylindrocyclophanes, named ribocyclophanes A-E (1-5) and cylindrocyclophane D (6). The structure determination was carried out by HRESIMS and 1D and 2D NMR analyses and confirmed by single-crystal X-ray crystallography. The structures of ribocyclophanes A-E (1-5) contain a ß-d-ribopyranose glycone in the rare 1 C4 conformation. Among isolated compounds, ribocyclophane D (4) showed antiproliferative activity against MDA-MB-435 and MDA-MB-231 cancer cells with an IC50 value of less than 1 µM.

Yeast homologous recombination-based promoter engineering for the activation of silent natural product biosynthetic gene clusters.

Large-scale sequencing of prokaryotic (meta)genomic DNA suggests that most bacterial natural product gene clusters are not expressed under common laboratory culture conditions. Silent gene clusters represent a promising resource for natural product discovery and the development of a new generation of therapeutics. Unfortunately, the characterization of molecules encoded by these clusters is hampered owing to our inability to express these gene clusters in the laboratory. To address this bottleneck, we have developed a promoter-engineering platform to transcriptionally activate silent gene clusters in a model heterologous host. Our approach uses yeast homologous recombination, an auxotrophy complementation-based yeast selection system and sequence orthogonal promoter cassettes to exchange all native promoters in silent gene clusters with constitutively active promoters. As part of this platform, we constructed and validated a set of bidirectional promoter cassettes consisting of orthogonal promoter sequences, Streptomyces ribosome binding sites, and yeast selectable marker genes. Using these tools we demonstrate the ability to simultaneously insert multiple promoter cassettes into a gene cluster, thereby expediting the reengineering process. We apply this method to model active and silent gene clusters (rebeccamycin and tetarimycin) and to the silent, cryptic pseudogene-containing, environmental DNA-derived Lzr gene cluster. Complete promoter refactoring and targeted gene exchange in this "dead" cluster led to the discovery of potent indolotryptoline antiproliferative agents, lazarimides A and B. This potentially scalable and cost-effective promoter reengineering platform should streamline the discovery of natural products from silent natural product biosynthetic gene clusters.

Functional metagenomic discovery of bacterial effectors in the human microbiome and isolation of commendamide, a GPCR G2A/132 agonist.

The trillions of bacteria that make up the human microbiome are believed to encode functions that are important to human health; however, little is known about the specific effectors that commensal bacteria use to interact with the human host. Functional metagenomics provides a systematic means of surveying commensal DNA for genes that encode effector functions. Here, we examine 3,000 Mb of metagenomic DNA cloned from three phenotypically distinct patients for effectors that activate NF-κB, a transcription factor known to play a central role in mediating responses to environmental stimuli. This screen led to the identification of 26 unique commensal bacteria effector genes (Cbegs) that are predicted to encode proteins with diverse catabolic, anabolic, and ligand-binding functions and most frequently interact with either glycans or lipids. Detailed analysis of one effector gene family (Cbeg12) recovered from all three patient libraries found that it encodes for the production of N-acyl-3-hydroxypalmitoyl-glycine (commendamide). This metabolite was also found in culture broth from the commensal bacterium Bacteroides vulgatus, which harbors a gene highly similar to Cbeg12. Commendamide resembles long-chain N-acyl-amides that function as mammalian signaling molecules through activation of G-protein-coupled receptors (GPCRs), which led us to the observation that commendamide activates the GPCR G2A/GPR132. G2A has been implicated in disease models of autoimmunity and atherosclerosis. This study shows the utility of functional metagenomics for identifying potential mechanisms used by commensal bacteria for host interactions and outlines a functional metagenomics-based pipeline for the systematic identification of diverse commensal bacteria effectors that impact host cellular functions.

Phylogeny-guided (meta)genome mining approach for the targeted discovery of new microbial natural products.

Genomics-based methods are now commonplace in natural products research. A phylogeny-guided mining approach provides a means to quickly screen a large number of microbial genomes or metagenomes in search of new biosynthetic gene clusters of interest. In this approach, biosynthetic genes serve as molecular markers, and phylogenetic trees built with known and unknown marker gene sequences are used to quickly prioritize biosynthetic gene clusters for their metabolites characterization. An increase in the use of this approach has been observed for the last couple of years along with the emergence of low cost sequencing technologies. The aim of this review is to discuss the basic concept of a phylogeny-guided mining approach, and also to provide examples in which this approach was successfully applied to discover new natural products from microbial genomes and metagenomes. I believe that the phylogeny-guided mining approach will continue to play an important role in genomics-based natural products research.

Ostalactones A-C, ß- and ε-Lactones with Lipase Inhibitory Activity from the Cultured Basidiomycete Stereum ostrea.

Ostalactones A-C (1-3), three new ß- and ε-lactone natural products, were isolated from the culture broth of the basidiomycete Stereum ostrea. The structures were elucidated by interpretation of HRFABMS and 1D and 2D NMR data. The structures of 1 and 2 are characterized by the presence of a ß-lactone containing a fused 4/5 bicyclic core structure. Compound 3 possesses a 2-oxepinone ring system, which is likely to be a biosynthetic precursor of compounds 1 and 2. Ostalactones A (1) and B (2) displayed potent inhibitory activity against human pancreatic lipase.

Trichormamides C and D, antiproliferative cyclic lipopeptides from the cultured freshwater cyanobacterium cf. Oscillatoria sp. UIC 10045.

Extract from the cultured freshwater cf. Oscillatoria sp. UIC 10045 showed antiproliferative activity against HT-29 cell line. Bioassay-guided fractionation led to the isolation of two new cyclic lipopeptides, named trichormamides C (1) and D (2). The planar structures were determined by combined analyses of HRESIMS, Q-TOF ESIMS/MS, and 1D and 2D NMR spectra. The absolute configurations of the amino acid residues were assigned by advanced Marfey's analysis after partial and complete acid hydrolysis. Trichormamides C (1) is a cyclic undecapeptide and D (2) is a cyclic dodecapeptide, both containing a lipophilic ß-aminodecanoic acid residue. Trichormamide C (1) displayed antiproliferative activities against HT-29 and MDA-MB-435 cancer cell lines with IC50 values of 1.7 and 1.0µM, respectively, as well as anti-Mycobacterium tuberculosis activity with MIC value of 23.8µg/mL (17.3µM). Trichormamide D (2) was found to be less potent against both HT-29 and MDA-MB-435 cancer cell lines with IC50 values of 11.5 and 11.7µM, respectively.

Mining soil metagenomes to better understand the evolution of natural product structural diversity: pentangular polyphenols as a case study.

Sequence-guided mining of metagenomic libraries provides a means of recovering specific natural product gene clusters of interest from the environment. In this study, we use ketosynthase gene (KS) PCR amplicon sequences (sequence tags) to explore the structural and biosynthetic diversities of pentangular polyphenols (PP). In phylogenetic analyses, eDNA-derived sequence tags often fall between closely related clades that are associated with gene clusters known to encode distinct chemotypes. We show that these common "intermediate" sequence tags are useful for guiding the discovery of not only novel bioactive metabolites but also collections of closely related gene clusters that can provide new insights into the evolution of natural product structural diversity. Gene clusters corresponding to two eDNA-derived KSß sequence tags that reside between well-defined KSß clades associated with the biosynthesis of (C24)-pradimicin and (C26)-xantholipin type metabolites were recovered from archived soil eDNA libraries. Heterologous expression of these gene clusters in Streptomyces albus led to the isolation of three new PPs (compounds 1-3). Calixanthomycin A (1) shows potent antiproliferative activity against HCT-116 cells, whereas arenimycins C (2) and D (3) display potent antibacterial activity. By comparing genotypes and chemotypes across all known PP gene clusters, we define four PP subfamilies, and also observe that the horizontal transfer of PP tailoring genes has likely been restricted to gene clusters that encode closely related chemical structures, suggesting that only a fraction of the "natural product-like" chemical space that can theoretically be encoded by these secondary metabolite tailoring genes has likely been sampled naturally.

The chemical arsenal of Burkholderia pseudomallei is essential for pathogenicity.

Increasing evidence has shown that small-molecule chemistry in microbes (i.e., secondary metabolism) can modulate the microbe-host response in infection and pathogenicity. The bacterial disease melioidosis is conferred by the highly virulent, antibiotic-resistant pathogen Burkholderia pseudomallei (BP). Whereas some macromolecular structures have been shown to influence BP virulence (e.g., secretion systems, cellular capsule, pili), the role of the large cryptic secondary metabolome encoded within its genome has been largely unexplored for its importance to virulence. Herein we demonstrate that BP-encoded small-molecule biosynthesis is indispensible for in vivo BP pathogenicity. Promoter exchange experiments were used to induce high-level molecule production from two gene clusters (MPN and SYR) found to be essential for in vivo virulence. NMR structural characterization of these metabolites identified a new class of lipopeptide biosurfactants/biofilm modulators (the malleipeptins) and syrbactin-type proteasome inhibitors, both of which represent overlooked small-molecule virulence factors for BP. Disruption of Burkholderia virulence by inhibiting the biosynthesis of these small-molecule biosynthetic pathways may prove to be an effective strategy for developing novel melioidosis-specific therapeutics.

Trichormamides A and B with Antiproliferative Activity from the Cultured Freshwater Cyanobacterium Trichormus sp. UIC 10339.

Two new cyclic lipopeptides, trichormamides A (1) and B (2), were isolated from the cultured freshwater cyanobacterium Trichormus sp. UIC 10339. The strain was obtained from a sample collected in Raven Lake in Northern Wisconsin. The planar structures of trichormamides A (1) and B (2) were determined using a combination of spectroscopic analyses including HRESIMS and 1D and 2D NMR experiments. The absolute configurations of the amino acid residues were assigned by the advanced Marfey's method after acid hydrolysis. Trichormamide A (1) is a cyclic undecapeptide containing two D-amino acid residues (D-Tyr and D-Leu) and one ß-amino acid residue (ß-aminodecanoic acid). Trichormamide B (2) is a cyclic dodecapeptide characterized by the presence of four nonstandard α-amino acid residues (homoserine, N-methylisoleucine, and two 3-hydroxyleucines) and one ß-amino acid residue (ß-aminodecanoic acid). Trichormamide B (2) was cytotoxic against MDA-MB-435 and HT-29 cancer cell lines with IC50 values of 0.8 and 1.5 µM, respectively.

Carbamidocyclophanes F and G with Anti-Mycobacterium tuberculosis Activity from the Cultured Freshwater Cyanobacterium Nostoc sp.

Two new (1 and 2) and three known (3-5) carbamidocyclophanes were isolated from a cultured freshwater cyanobacterium Nostoc sp. (UIC 10274) obtained from a sample collected at Des Plaines, Illinois. Their planar structures and stereoconfigurations were determined by extensive spectroscopic analysis including 1D/2D NMR experiments, HRESIMS as well as CD spectroscopy. Carbamidocyclophane F (1) showed potent anti-Mycobacterium tuberculosis activity in the microplate Alamar blue assay and low-oxygen-recovery assay with MIC values of 0.8 and 5.4 µM, respectively. Carbamidocyclophane F (1) also displayed antimicrobial activities against the gram positive bacteria Staphylococcus aureus and Enterococcus faecalis with MIC values of 0.1 and 0.2 µM, respectively. Carbamidocyclophane F (1) and Carbamidocyclophane G (2) both showed antiproliferative activity against MDA-MB-435 and HT-29 human cancer cell lines with IC50 values in the range from 0.5 to 0.7 µM.

Arimetamycin A: improving clinically relevant families of natural products through sequence-guided screening of soil metagenomes.

Sequence-tag-guided screening of soil environmental DNA libraries can be used to guide the discovery of new compounds with improved properties. In heterologous expression experiments the eDNA-derived arm cluster encodes arimetamycin A, an anthracycline that is more potent than clinically used natural anthracyclines and retains activity against multidrug-resistant (MDR) cancer cells.

CaExTun: Mitigating Cas9-Related Toxicity in Streptomyces through Species-Specific Expression Tuning with Randomized Constitutive Promoters.

The CRISPR/Cas9 system provides an efficient tool for engineering genomes. However, its application to Streptomyces genome engineering has been hampered by excessive toxicity associated with overexpression of Cas9 protein. As the level of Cas9 toxicity varies significantly between Streptomyces species, species-specific optimization of Cas9 expression is a strategy to mitigate its toxicity while maintaining sufficient double-strand break (DSB) activity for genome engineering. Using a pool of randomized constitutive promoters and a blue pigment indigoidine biosynthetic gene (IndC) as a reporter, we developed the CaExTun (Cas9 Expression Tuning) platform, which enables rapid screening of a large pool of promoter-Cas9 constructs to quickly recover the one with high DSB activity and no apparent toxicity. We demonstrate the utility of CaExTun using four model Streptomyces species. We also show that CaExTun can be applied to the CRISPRi system by allowing the construction of a library of promoter-dCas9 constructs that confer a wide range of gene repression levels. As demonstrated here, CaExTun is a versatile tool for the rapid optimization of the CRISPR/Cas9 system in a species-specific manner and thus will facilitate CRISPR/Cas9-based genome engineering efforts in Streptomyces.

New meroterpenoids from a soil-derived fungus Penicillium sp. SSW03M2 GY and their anti-virulence activity.

Two new berkeley meroterpenoids (1 and 2), along with seven known compounds (3‒9) were isolated from a fungus, Penicillium sp. SSW03M2 GY derived from a sediment at Seosan bay, South Korea. Chemical structures of the isolated compounds were elucidated on the basis of 1D, 2D NMR, HRESIMS, and optical rotation. All the isolated compounds, 1 showed anti-virulence activity by significantly inhibiting α-toxin (Hla) secreted by methicillin-resistant Staphylococcus aureus without its growth inhibition.

Daldipyrenones A-C: Caged [6,6,6,6,6] Polyketides Derived from an Endolichenic Fungus Daldinia pyrenaica 047188.

Daldipyrenones A-C (1-3), three unprecedented caged xanthone [6,6,6,6,6] polyketides featuring a spiro-azaphilone unit, were discovered from an endolichenic fungus, Daldinia pyrenaica 047188. The structures of 1-3 were determined by using spectroscopic analysis and chemical derivatization. Daldipyrenones are likely derived by combining a chromane biosynthesis intermediate, 1-(2,6-dihydroxyphenyl)but-2-en-2-one, and a spiro-azaphilone, pestafolide A, via radical coupling or Michael addition to form a bicyclo[2.2.2]octane ring. Genome sequencing of the strain revealed two separate biosynthetic gene clusters responsible for forming two biosynthetic intermediates, suggesting a proposed biosynthetic pathway. Daldipyrenone A (1) exhibited significant antimelanogenic activity with lower EC50's than positive controls and moderate adiponectin-secretion promoting activity.

Tetarimycin A, an MRSA-active antibiotic identified through induced expression of environmental DNA gene clusters.

The propagation of DNA extracted directly from environmental samples in laboratory-grown bacteria provides a means to study natural products encoded in the genomes of uncultured bacteria. However, gene silencing often hampers the functional characterization of gene clusters captured on environmental DNA clones. Here we show that the overexpression of transcription factors found in sequenced environmental DNA-derived biosynthetic gene clusters, in conjunction with traditional culture-broth extract screening, can be used to identify new bioactive secondary metabolites from otherwise-silent gene clusters. Tetarimycin A, a tetracyclic methicillin-resistant Staphylococcus aureus (MRSA)-active antibiotic, was isolated from the culture-broth extract of Streptomyces albus cultures cotransformed with an environmentally derived type-II polyketide biosynthetic gene cluster and its pathway-specific Streptomyces antibiotic regulatory protein (SARP) cloned under the control of the constitutive ermE* promoter.

Minutissamides E-L, antiproliferative cyclic lipodecapeptides from the cultured freshwater cyanobacterium cf. Anabaena sp.

The extract of UIC 10035, a strain obtained from a sample collected near the town of Homestead, South Florida, showed antiproliferative activity against MDA-MB-435 cells. Bioassay-guided fractionation led to the isolation of a series of cyclic lipodecapeptides, named minutissamides E-L (1-8). The planar structures were determined by analysis of HRESIMS, tandem MS, and 1D and 2D NMR data, and the stereoconfigurations were assigned by LC-MS analysis of the Marfey's derivatives after acid hydrolysis. Minutissamides E-L (1-8) exhibited antiproliferative activity against MDA-MB-435 cells with IC(50) values ranging between 1 and 10 µM. The structures of minutissamides E-L (1-8) were closely related with those of the previously reported lipopeptides, puwainaphycins A-E and minutissamides A-D, characterized by the presence of a lipophilic ß-amino acid and three non-standard amino acids NMeAsn, OMeThr and Dhb (α,ß-dehydro-α-aminobutyric acid). The strain UIC 10035 was designated as cf. Anabaena sp. on the basis of morphological and 16S rRNA gene sequence analyses.