Streptomycetes: Surrogate hosts for the genetic manipulation of biosynthetic gene clusters and production of natural products

Due to the worldwide prevalence of multidrug-resistant pathogens and high incidence of diseases such as cancer, there is an urgent need for the discovery and development of new drugs. Nearly half of the FDA-approved drugs are derived from natural products that are produced by living organisms, mainly bacteria, fungi, and plants. Commercial development is often limited by the low yield of the desired compounds expressed by the native producers. In addition, recent advances in whole genome sequencing and bioinformatics have revealed an abundance of cryptic biosynthetic gene clusters within microbial genomes. Genetic manipulation of clusters in the native host is commonly used to awaken poorly expressed or silent gene clusters, however, the lack of feasible genetic manipulation systems in many strains often hinders our ability to engineer the native producers. The transfer of gene clusters into heterologous hosts for expression of partial or entire biosynthetic pathways is an approach that can be used to overcome this limitation. Heterologous expression also facilitates the chimeric fusion of different biosynthetic pathways, leading to the generation of “unnatural” natural products. The genus Streptomyces is especially known to be a prolific source of drugs/antibiotics, its members are often used as heterologous expression hosts. In this review, we summarize recent applications of Streptomyces species, S. coelicolor, S. lividans, S. albus, S. venezuelae and S. avermitilis, as heterologous expression systems.     

Genome mining of Streptomyces ambofaciens

Since the discovery of the streptomycin produced by Streptomyces griseus in the middle of the last century, members of this bacterial genus have been largely exploited for the production of secondary metabolites with wide uses in medicine and in agriculture. They have even been recognized as one of the most prolific producers of natural products among microorganisms. With the onset of the genomic era, it became evident that these microorganisms still represent a major source for the discovery of novel secondary metabolites. This was highlighted with the complete genome sequencing of Streptomyces coelicolor A3(2) which revealed an unexpected potential of this organism to synthesize natural products undetected until then by classical screening methods. Since then, analysis of sequenced genomes from numerous Streptomyces species has shown that a single species can carry more than 30 secondary metabolite gene clusters, reinforcing the idea that the biosynthetic potential of this bacterial genus is far from being fully exploited. This review highlights our knowledge on the potential of Streptomyces ambofaciens ATCC 23877 to synthesize natural products. This industrial strain was known for decades to only produce the drug spiramycin and another antibacterial compound, congocidine. Mining of its genome allowed the identification of 23 clusters potentially involved in the production of other secondary metabolites. Studies of some of these clusters resulted in the characterization of novel compounds and of previously known compounds but never characterized in this Streptomyces species. In addition, genome mining revealed that secondary metabolite gene clusters of phylogenetically closely related Streptomyces are mainly species-specific.     

Systems biology and biotechnology of Streptomyces species for the production of secondary metabolites

Streptomyces species continue to attract attention as a source of novel medicinal compounds. Despite a long history of studies on these microorganisms, they still have many biochemical mysteries to be elucidated. Investigations of novel secondary metabolites and their biosynthetic gene clusters have been more systematized with high-throughput techniques through inspections of correlations among components of the primary and secondary metabolisms at the genome scale. Moreover, up-to-date information on the genome of Streptomyces species with emphasis on their secondary metabolism has been collected in the form of databases and knowledgebases, providing predictive information and enabling one to explore experimentally unrecognized biological spaces of secondary metabolism. Herein, we review recent trends in the systems biology and biotechnology of Streptomyces species.     

Discovery of Gene Cluster for Mycosporine-Like Amino Acid Biosynthesis from Actinomycetales Microorganisms and Production of a Novel Mycosporine-Like Amino Acid by Heterologous Expression

Mycosporines and mycosporine-like amino acids (MAAs), including shinorine (mycosporine-glycine-serine) and porphyra-334 (mycosporine-glycine-threonine), are UV-absorbing compounds produced by cyanobacteria, fungi, and marine micro- and macroalgae. These MAAs have the ability to protect these organisms from damage by environmental UV radiation. Although no reports have described the production of MAAs and the corresponding genes involved in MAA biosynthesis from Gram-positive bacteria to date, genome mining of the Gram-positive bacterial database revealed that two microorganisms belonging to the order Actinomycetales, Actinosynnema mirum DSM 43827 and Pseudonocardia sp. strain P1, possess a gene cluster homologous to the biosynthetic gene clusters identified from cyanobacteria. When the two strains were grown in liquid culture, Pseudonocardia sp. accumulated a very small amount of MAA-like compound in a medium-dependent manner, whereas A. mirum did not produce MAAs under any culture conditions, indicating that the biosynthetic gene cluster of A. mirum was in a cryptic state in this microorganism. In order to characterize these biosynthetic gene clusters, each biosynthetic gene cluster was heterologously expressed in an engineered host, Streptomyces avermitilis SUKA22. Since the resultant transformants carrying the entire biosynthetic gene cluster controlled by an alternative promoter produced mainly shinorine, this is the first confirmation of a biosynthetic gene cluster for MAA from Gram-positive bacteria. Furthermore, S. avermitilis SUKA22 transformants carrying the biosynthetic gene cluster for MAA of A. mirum accumulated not only shinorine and porphyra-334 but also a novel MAA. Structure elucidation revealed that the novel MAA is mycosporine-glycine-alanine, which substitutes l-alanine for the l-serine of shinorine.     

Strain-Level Diversity of Secondary Metabolism in Streptomyces albus

Streptomyces spp. are robust producers of medicinally-, industrially- and agriculturally-important small molecules. Increased resistance to antibacterial agents and the lack of new antibiotics in the pipeline have led to a renaissance in natural product discovery. This endeavor has benefited from inexpensive high quality DNA sequencing technology, which has generated more than 140 genome sequences for taxonomic type strains and environmental Streptomyces spp. isolates. Many of the sequenced streptomycetes belong to the same species. For instance, Streptomyces albus has been isolated from diverse environmental niches and seven strains have been sequenced, consequently this species has been sequenced more than any other streptomycete, allowing valuable analyses of strain-level diversity in secondary metabolism. Bioinformatics analyses identified a total of 48 unique biosynthetic gene clusters harboured by Streptomyces albus strains. Eighteen of these gene clusters specify the core secondary metabolome of the species. Fourteen of the gene clusters are contained by one or more strain and are considered auxiliary, while 16 of the gene clusters encode the production of putative strain-specific secondary metabolites. Analysis of Streptomyces albus strains suggests that each strain of a Streptomyces species likely harbours at least one strain-specific biosynthetic gene cluster. Importantly, this implies that deep sequencing of a species will not exhaust gene cluster diversity and will continue to yield novelty.     

Nature’s combinatorial biosynthesis and recently engineered production of nucleoside antibiotics in <Emphasis Type="Italic">Streptomyces</Emphasis>

Modified nucleosides produced by Streptomyces and related actinomycetes are widely used in agriculture and medicine as antibacterial, antifungal, anticancer and antiviral agents. These specialized small-molecule metabolites are biosynthesized by complex enzymatic machineries encoded within gene clusters in the genome. The past decade has witnessed a burst of reports defining the key metabolic processes involved in the biosynthesis of several distinct families of nucleoside antibiotics. Furthermore, genome sequencing of various Streptomyces species has dramatically increased over recent years. Potential biosynthetic gene clusters for novel nucleoside antibiotics are now apparent by analysis of these genomes. Here we revisit strategies for production improvement of nucleoside antibiotics that have defined mechanisms of action, and are in clinical or agricultural use. We summarize the progress for genetically manipulating biosynthetic pathways for structural diversification of nucleoside antibiotics. Microorganism-based biosynthetic examples are provided and organized under genetic principles and metabolic engineering guidelines. We show perspectives on the future of combinatorial biosynthesis, and present a working model for discovery of novel nucleoside natural products in Streptomyces.     

Identification and activation of novel biosynthetic gene clusters by genome mining in the kirromycin producer <Emphasis Type="Italic">Streptomyces collinus</Emphasis> Tü 365

Streptomycetes are prolific sources of novel biologically active secondary metabolites with pharmaceutical potential. S. collinus Tü 365 is a Streptomyces strain, isolated 1972 from Kouroussa (Guinea). It is best known as producer of the antibiotic kirromycin, an inhibitor of the protein biosynthesis interacting with elongation factor EF-Tu. Genome Mining revealed 32 gene clusters encoding the biosynthesis of diverse secondary metabolites in the genome of Streptomyces collinus Tü 365, indicating an enormous biosynthetic potential of this strain. The structural diversity of secondary metabolisms predicted for S. collinus Tü 365 includes PKS, NRPS, PKS-NRPS hybrids, a lanthipeptide, terpenes and siderophores. While some of these gene clusters were found to contain genes related to known secondary metabolites, which also could be detected in HPLC–MS analyses, most of the uncharacterized gene clusters are not expressed under standard laboratory conditions. With this study we aimed to characterize the genome information of S. collinus Tü 365 to make use of gene clusters, which previously have not been described for this strain. We were able to connect the gene clusters of a lanthipeptide, a carotenoid, five terpenoid compounds, an ectoine, a siderophore and a spore pigment-associated gene cluster to their respective biosynthesis products.     

Multiplexed site-specific genome engineering for overproducing bioactive secondary metabolites in actinomycetes

Actinomycetes produce a large variety of pharmaceutically active compounds, yet production titers often require to be improved for discovery, development and large-scale manufacturing. Here, we describe a new technique, multiplexed site-specific genome engineering (MSGE) via the ‘one integrase-multiple attB sites’ concept, for the stable integration of secondary metabolite biosynthetic gene clusters (BGCs). Using MSGE, we achieved five-copy chromosomal integration of the pristinamycin II (PII) BGC in Streptomyces pristinaespiralis, resulting in the highest reported PII titers in flask and batch fermentations (2.2 and 2 g/L, respectively). Furthermore, MSGE was successfully extended to develop a panel of powerful Streptomyces coelicolor heterologous hosts, in which up to four copies of the BGCs for chloramphenicol or anti-tumour compound YM-216391 were efficiently integrated in a single step, leading to significantly elevated productivity (2–23 times). Our multiplexed approach holds great potential for robust genome engineering of industrial actinomycetes and novel drug discovery by genome mining.     

Natural biocombinatorics in the polyketide synthase genes of the actinobacterium Streptomyces avermitilis

Modular polyketide synthases (PKSs) of bacteria provide an enormous reservoir of natural chemical diversity. Studying natural biocombinatorics may aid in the development of concepts for experimental design of genes for the biosynthesis of new bioactive compounds. Here we address the question of how the modularity of biosynthetic enzymes and the prevalence of multiple gene clusters in Streptomyces drive the evolution of metabolic diversity. The phylogeny of ketosynthase (KS) domains of Streptomyces PKSs revealed that the majority of modules involved in the biosynthesis of a single compound evolved by duplication of a single ancestor module. Using Streptomyces avermitilis as a model organism, we have reconstructed the evolutionary relationships of different domain types. This analysis suggests that 65% of the modules were altered by recombinational replacements that occurred within and between biosynthetic gene clusters. The natural reprogramming of the biosynthetic pathways was unambiguously confined to domains that account for the structural diversity of the polyketide products and never observed for the KS domains. We provide examples for natural acyltransferase (AT), ketoreductase (KR), and dehydratase (DH)–KR domain replacements. Potential sites of homologous recombination could be identified in interdomain regions and within domains. Our results indicate that homologous recombination facilitated by the modularity of PKS architecture is the most important mechanism underlying polyketide diversity in bacteria.     

Salinomycin biosynthesis reversely regulates the β‐oxidation pathway in Streptomyces albus by carrying a 3‐hydroxyacyl‐CoA dehydrogenase gene in its biosynthetic gene cluster

Streptomyces is well known for synthesis of many biologically active secondary metabolites, such as polyketides and non‐ribosomal peptides. Understanding the coupling mechanisms of primary and secondary metabolism can help develop strategies to improve secondary metabolite production in Streptomyces. In this work, Streptomyces albus ZD11, an oil‐preferring industrial Streptomyces strain, was proved to have a remarkable capability to generate abundant acyl‐CoA precursors for salinomycin biosynthesis with the aid of its enhanced β‐oxidation pathway. It was found that the salinomycin biosynthetic gene cluster contains a predicted 3‐hydroxyacyl‐CoA dehydrogenase (FadB3), which is the third enzyme of β‐oxidation cycle. Deletion of fadB3 significantly reduced the production of salinomycin. A variety of experimental evidences showed that FadB3 was mainly involved in the β‐oxidation pathway rather than ethylmalonyl‐CoA biosynthesis and played a very important role in regulating the rate of β‐oxidation in S. albus ZD11. Our findings elucidate an interesting coupling mechanism by which a PKS biosynthetic gene cluster could regulate the β‐oxidation pathway by carrying β‐oxidation genes, enabling Streptomyces to efficiently synthesize target polyketides and economically utilize environmental nutrients.

Comprehensive understanding the relationship between primary metabolism and secondary metabolism is important for high‐efficiency production of antibiotics in Streptomyces species. In contrast to the well‐known pattern that primary metabolic processes regulate secondary metabolism, this study reports that a secondary biosynthetic pathway can reversely regulate primary metabolic process by carrying a key gene of the primary metabolic pathway.     

<Emphasis Type="Italic">Streptomyces albulus</Emphasis> yields ε-poly-<Emphasis Type="SmallCaps">l</Emphasis>-lysine and other products from salt-contaminated glycerol waste

Actinomycetes are the most important microorganisms for the industrial production of secondary metabolites with antimicrobial and anticancer properties. However, they have not been implicated in biorefineries. Here, we study the ability of the ε-poly-l-lysine producing Streptomyces albulus BCRC 11814 to utilize biodiesel-derived crude glycerol. S. albulus was cultured in a mineral medium supplemented with up to 10% w/v sodium chloride or potassium chloride, and with crude glycerol as the sole carbohydrate source. Under these conditions, the strain produced 0.1 g ε-poly-l-lysine per 1 g of biomass. RNA sequencing revealed upregulation of the ectoine biosynthetic pathway of S. albulus, which provides proof of halotolerance. S. albulus has several silent secondary metabolite biosynthetic clusters predicted within the genome. Based on the results, we conclude that S. albulus BCRC 11814 is a halotolerant microorganism capable of utilizing biodiesel-derived crude glycerol better than other actinomycetes included in the present study. S. albulus has the potential to be established as microbial platform production host for a range of high-value biological products.     

Genome-Guided Investigation Provides New Insights into Secondary Metabolites of Streptomyces Parvulus SX6 from Aegiceras Corniculatum

Whole-genome sequencing and genome mining are recently considered an efficient approach to shine more light on the underlying secondary metabolites of Streptomyces. The present study unearths the biosynthetic potential of endophytic SX6 as a promising source of biologically active substances and plant-derived compounds for the first time. Out of 38 isolates associated with Aegiceras corniculatum (L.) Blanco, Streptomyces parvulus SX6 was highly active against Pseudomonas aeruginosa ATCC® 9027™ and methicillin-resistant Staphylococcus epidermidis (MRSE) ATCC® 35984™. Additionally, S. parvulus SX6 culture extract showed strong cytotoxicity against Hep3B, MCF-7, and A549 cell lines at a concentration of 30 μg/ml, but not in non-cancerous HEK-293 cells. The genome contained 7.69 Mb in size with an average G + C content of 72.8% and consisted of 6,779 protein-coding genes. AntiSMASH analysis resulted in the identification of 29 biosynthetic gene clusters (BGCs) for secondary metabolites. Among them, 4 BGCs showed low similarity (28–67% of genes show similarity) to actinomycin, streptovaricin, and polyoxypeptin gene clusters, possibly attributed to antibacterial and anticancer activities observed. In addition, the complete biosynthetic pathways of plant-derived compounds, including daidzein and genistein were identified using genome mining and HPLC-DAD-MS analysis. These findings portray an exciting avenue for future characterization of promising secondary metabolites from mangrove endophytic S. parvulus.     

链霉菌中高效生产聚酮化合物的研究方法及进展北大核心CSCD

聚酮化合物是通过聚酮合成途径产生的一大类结构和生物活性多样的次级代谢产物,是链霉菌产生的主要次级代谢产物,具有重要的经济价值。为了在链霉菌中提高聚酮化合物产量,以满足工业生产需求,近年来,代谢工程的方法被广泛应用,例如,过表达合成途径中限速酶或途径特异性激活蛋白、强化前体供应、去除产物反馈抑制、合成基因簇异源表达等。本文将从代谢工程改造实例入手,全面综述链霉菌中聚酮化合物高效生物合成的研究方法及进展,并对利用合成生物学策略智能动态适配各个相关途径,进而提高该类化合物产量的研究思路进行展望。     

Heterologous Expression of Novobiocin and Clorobiocin Biosynthetic Gene Clusters

A method was developed for the heterologous expression of biosynthetic gene clusters in different Streptomyces strains and for the modification of these clusters by single or multiple gene replacements or gene deletions with unprecedented speed and versatility. λ-Red-mediated homologous recombination was used for genetic modification of the gene clusters, and the attachment site and integrase of phage C31 were employed for the integration of these clusters into the heterologous hosts. This method was used to express the gene clusters of the aminocoumarin antibiotics novobiocin and clorobiocin in the well-studied strains Streptomyces coelicolor and Streptomyces lividans, which, in contrast to the natural producers, can be easily genetically manipulated. S. coelicolor M512 derivatives produced the respective antibiotic in yields comparable to those of natural producer strains, whereas S. lividans TK24 derivatives were at least five times less productive. This method could also be used to carry out functional investigations. Shortening of the cosmids' inserts showed which genes are essential for antibiotic production.     

S-Adenosylmethionine (SAM) and antibiotic biosynthesis: effect of external addition of SAM and of overexpression of SAM biosynthesis genes on novobiocin production in Streptomyces

The production of antibiotics in different Streptomyces strains has been reported to be stimulated by the external addition of S-adenosylmethionine (SAM) and by overexpression of the SAM synthetase gene metK. We investigated the influence of SAM addition, and of the expression of SAM biosynthetic genes, on the production of the aminocoumarin antibiotic novobiocin in the heterologous producer strain Streptomyces coelicolor M512 (nov-BG1). External addition of SAM did not influence novobiocin accumulation. However, overexpression of a SAM synthase gene stimulated novobiocin formation, concomitant with an increase of the intracellular SAM concentration. Streptomyces genomes contain orthologs of all genes required for the SAM cycle known from mammals. In contrast, most other bacteria use a different cycle for SAM regeneration. Three secondary metabolic gene clusters, coding for the biosynthesis of structurally very different antibiotics in different Streptomyces strains, were found to contain an operon comprising all five putative genes of the SAM cycle. We cloned one of these operons into an expression plasmid, under control of a strong constitutive promoter. However, transformation of the heterologous novobiocin producer strain with this plasmid did not stimulate novobiocin production, but rather showed a detrimental effect on cell viability in the stationary phase and strongly reduced novobiocin accumulation.     

Avermectin: biochemical and molecular basis of its biosynthesis and regulation

Avermectin and its analogues, produced by Streptomyces avermitilis, are major commercial antiparasitic agents in the field of animal health, agriculture, and human infections. They are 16-membered pentacyclic lactone compounds derived from polyketide and linked to a disaccharide of the methylated deoxysugar l-oleandrose. Labeling studies, analyses of the biosynthetically blocked mutants, and the identification of the avermectin gene cluster allows characterization of most of the biosynthetic pathway. Recent completion of S. avermitilis genome sequencing is also expected to help in revealing the precise biosynthetic sequence and the complicated regulatory mechanism for avermectin biosynthesis, which has been long-awaited to be elucidated. The well characterized avermectin biosynthetic pathway and availability of S. avermitilis genome information in combination with the recent development of combinatorial biosynthesis should allow us to redesign more potent avermectin analogues and to engineer S. avermitilis as a more efficient host for the production of important commercial analogues.     

A butenolide signaling system synergized with biosynthetic gene modules led to effective activation and enhancement of silent oviedomycin production in Streptomyces

Secondary metabolic gene clusters widely exist in the genomes of Streptomyces but mostly remain silent. To awaken this hidden reservoir of natural products, various strategies concerning secondary metabolic pathways are applied. Here, we describe that butenolide signaling molecule deficiency and glucose addition can interdependently activate the expression of silent oviedomycin biosynthetic gene clusters in Streptomyces ansochromogenes and Streptomyces antibioticus. Since oviedomycin is a promising anti-tumor lead compound, in order to improve its yield, we use the cluster-situated genes (ovmF, ovmG, ovmI and ovmH) encoding the enzymes for acyl carrier protein modification and precursor biosynthesis, and the discrete precursor biosynthetic genes (pyk2, gap1 and accA2) involved in glycolysis to assemble two gene modules (pFGIH and pPGA). Their co-overexpression in ΔsabA (a disruption mutant of sabA encoding SAB synthase) has superimposed effect on the yield of oviedomycin, which can be further increased to 59-fold in the presence of galactose as optimal carbon source. This is the most unambiguous evidence that butenolide signaling system can synergize with the optimization of primary metabolism to regulate the expression of secondary metabolic gene clusters, providing efficient strategies for mining natural products of Streptomyces.     

Pleiotropic Control of Secondary Metabolism and Morphological Development by KsbC,a Butyrolactone Autoregulator Receptor Homologue in Kitasatospora setae

The γ-butyrolactone autoregulator signaling cascades have been shown to control secondary metabolism and/or morphological development among many Streptomyces species. However, the conservation and variation of the regulatory systems among actinomycetes remain to be clarified. The genome sequence of Kitasatospora setae, which also belongs to the family Streptomycetaceae containing the genus Streptomyces, has revealed the presence of three homologues of the autoregulator receptor: KsbA, which has previously been confirmed to be involved only in secondary metabolism; KsbB; and KsbC. We describe here the characterization of ksbC, whose regulatory cluster closely resembles the Streptomyces virginiae barA locus responsible for the autoregulator signaling cascade. Deletion of the gene ksbC resulted in lowered production of bafilomycin and a defect of aerial mycelium formation, together with the early and enhanced production of a novel β-carboline alkaloid named kitasetaline. A putative kitasetaline biosynthetic gene cluster was identified, and its expression in a heterologous host led to the production of kitasetaline together with JBIR-133, the production of which is also detected in the ksbC disruptant, and JBIR-134 as novel β-carboline alkaloids, indicating that these genes were biosynthetic genes for β-carboline alkaloid and thus are the first such genes to be discovered in bacteria.     

Genetic engineering of antibiotic biosynthesis for the generation of new aminocoumarins

The aminocoumarin antibiotics novobiocin, clorobiocin and coumermycin A₁ are inhibitors of gyrase and highly effective antibacterial agents. Their biosynthetic gene clusters have been cloned from the respective Streptomyces producer strains, and the function of nearly all genes contained therein has been elucidated by genetic and biochemical methods. Efficient methods have been developed for the genetic manipulation and the heterologous expression of the clusters, and more than 100 new derivatives of these antibiotics have been generated by metabolic engineering, mutasynthesis and chemoenzymatic synthesis, providing a model for the power of genetic and genomic methods for the generation of new bioactive compounds.