Hybrid peptide-polyketide natural products: biosynthesis and prospects toward engineering novel molecules

The structural and catalytic similarities between modular nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) inspired us to search for hybrid NRPS-PKS systems. By examining the biochemical and genetic data known to date for the biosynthesis of hybrid peptide-polyketide natural products, we show (1) that the same catalytic sites are conserved between the hybrid NRPS-PKS and normal NRPS or PKS systems, although the ketoacyl synthase domain in NRPS/PKS hybrids is unique, and (2) that specific interpolypeptide linkers exist at both the C- and N-termini of the NRPS and PKS proteins, which presumably play a critical role in facilitating the transfer of the growing peptide or polyketide intermediate between NRPS and PKS modules in hybrid NRPS-PKS systems. These findings provide new insights for intermodular communications in hybrid NRPS-PKS systems and should now be taken into consideration in engineering hybrid peptide-polyketide biosynthetic pathways for making novel "unnatural" natural products.     

Metabolic diversity in myxobacteria: identification of the myxalamid and the stigmatellin biosynthetic gene cluster of Stigmatella aurantiaca Sg a15 and a combined polyketide-(poly)peptide gene cluster from the epothilone producing strain Sorangium cellulosum So ce90.

Myxobacterial strains producing polyketides (PKs) assumed to be biosynthesized by a type I polyketide synthase (PKS) were analysed. Myxobacteria also produce a variety of polypeptides (PP) and PKs with incorporated amino acids ('mixed PK-PP'). In order to be able to identify the biosynthetic gene clusters for these metabolites a PCR based approach has been developed to clone ketosynthase (KS) domains of PKS genes from these organisms. Conserved regions of peptide synthetases of the non-ribosomal type (NRPS) were also amplified via PCR. KS fragments from Stigmatella aurantiaca Sg a15 were used for chromosomal gene inactivation experiments resulting in a series of mutants including such that were unable to produce stigmatellins and myxalamids. A NRPS fragment and PKS fragments from Sorangium cellulosum So ce90 were used to identify cosmids hybridizing with both types of probes from a genomic library. Both a NRPS and a PKS fragment were cloned and sequenced from a relatively short restriction fragment of one of these cosmids. The method described here should be very useful to clone and identify PKS, NRPS and mixed PKS-NRPS from myxobacteria in general and thereby open opportunities to use the biochemical diversity of these bacteria for genetic engineering and combinatorial biosynthesis.     

Naturally mosaic operons for secondary metabolite biosynthesis: variability and putative horizontal transfer of discrete catalytic domains of the epothilone polyketide synthase locus

A putative instance of horizontal gene transfer (HGT) involving adjacent, discrete beta-ketoacyl synthase (KS), acyl carrier protein (ACP) and nonribosomal peptide synthase (NRPS) domains of the epothilone Type I polyketide biosynthetic gene cluster from the myxobacterium Sorangium cellulosom was identified using molecular phylogenetics and sequence analyses. The specific KS domain of the module EPO B fails to cluster phylogenetically with other epothilone KS sequences present at this locus, in contrast to what is typically observed in many other Type I polyketide synthase (PKS) biosynthetic loci. Furthermore, the GC content of the epoB KS, epoA ACP and NRPS domains differs significantly from the base composition of other epothilone domain sequences. In addition, the putatively transferred epothilone loci are located near previously identified transposon-like sequences. Lastly, comparison with other KS loci revealed another possible case of horizontal transfer of secondary metabolite genes in the genus Pseudomonas. This study emphasizes the use of several lines of concordant evidence (phylogenetics, base composition, transposon sequences) to infer the evolutionary history of particular gene and enzyme sequences, and the results support the idea that genes coding for adaptive traits, e.g. defensive natural products, may be prone to transposition between divergent prokaryotic taxa and genomes.     

A PKS/NRPS/FAS Hybrid Gene Cluster from Serratia plymuthica RVH1 Encoding the Biosynthesis of Three Broad Spectrum,Zeamine-Related Antibiotics

Serratia plymuthica strain RVH1, initially isolated from an industrial food processing environment, displays potent antimicrobial activity towards a broad spectrum of Gram-positive and Gram-negative bacterial pathogens. Isolation and subsequent structure determination of bioactive molecules led to the identification of two polyamino antibiotics with the same molecular structure as zeamine and zeamine II as well as a third, closely related analogue, designated zeamine I. The gene cluster encoding the biosynthesis of the zeamine antibiotics was cloned and sequenced and shown to encode FAS, PKS as well as NRPS related enzymes in addition to putative tailoring and export enzymes. Interestingly, several genes show strong homology to the pfa cluster of genes involved in the biosynthesis of long chain polyunsaturated fatty acids in marine bacteria. We postulate that a mixed FAS/PKS and a hybrid NRPS/PKS assembly line each synthesize parts of the backbone that are linked together post-assembly in the case of zeamine and zeamine I. This interaction reflects a unique interplay between secondary lipid and secondary metabolite biosynthesis. Most likely, the zeamine antibiotics are produced as prodrugs that undergo activation in which a nonribosomal peptide sequence is cleaved off.     

Bacteria of the Roseobacter Clade Show Potential for Secondary Metabolite Production

Members of the Roseobacter clade are abundant and widespread in marine habitats and have very diverse metabolisms. Production of acylated homoserine lactones (AHL) and secondary metabolites, e.g., antibiotics has been described sporadically. This prompted us to screen 22 strains of this group for production of signaling molecules, antagonistic activity against bacteria of different phylogenetic groups, and the presence of genes encoding for nonribosomal peptide synthetases (NRPS) and polyketide synthases (PKS), representing enzymes involved in the synthesis of various pharmaceutically important natural products. The screening approach for NRPS and PKS genes was based on polymerase chain reaction (PCR) with degenerate primers specific for conserved sequence motifs. Additionally, sequences from whole genome sequencing projects of organisms of the Roseobacter clade were considered. Obtained PCR products were cloned, sequenced, and compared with genes of known function. With the PCR approach genes showing similarity to known NRPS and PKS genes were found in seven and five strains, respectively, and three PKS and NRPS sequences from genome sequencing projects were obtained. Three strains exhibited antagonistic activity and also showed production of AHL. Overall production of AHL was found in 10 isolates. Phylogenetic analysis of the 16S rRNA gene sequences of the tested organisms showed that several of the AHL-positive strains clustered together. Three strains were positive for three or four categories tested, and were found to be closely related within the genus Phaeobacter. The presence of a highly similar hybrid PKS/NRPS gene locus of unknown function in sequenced genomes of the Roseobacter clade plus the significant similarity of gene fragments from the strains studied to these genes argues for the functional requirement of the encoded hybrid PKS/NRPS complex. Our screening results therefore suggest that the Roseobacter clade is indeed employing PKS/NRPS biochemistry and should thus be further studied as a potential and largely untapped source of secondary metabolites.     

百部内生放线菌的分离、分类及次级代谢潜力

【目的】以对叶百部块根为材料分离内生放线菌,并对分离菌株进行分类、抗菌活性和次级代谢产物合成基因研究。【方法】样品经过严格的表面消毒,选用4种培养基分离百部内生放线菌;分离菌株通过形态观察和16S rRNA序列分析进行分类鉴定;采用琼脂移块法测试分离菌株的抗菌活性;通过PCR检测分离菌株的PKS/NPRS和卤化酶基因;使用HPLC-UV/VIS-ESI-MS/MS分析发酵产物。【结果】从6个样品中获得18株内生放线菌,分属链霉菌属(Streptomyces)、小单孢菌属(Micromonospora)、假诺卡氏菌属(Pseudonocardia)和甲基杆菌属(Methylobacterium)。分离菌株绝大部分具有抗菌活性和次级代谢产物合成基因,其中13株对耐药金黄色葡萄球菌和/或绿脓杆菌有拮抗活性,17株具有PKS/NRPS基因,8株菌具有卤化酶基因,且卤化酶阳性代表菌株的发酵产物具有抗细菌活性和卤代化合物特征。【结论】百部作为一种传统中药,其内生放线菌以链霉菌和小单孢菌为主,在次级代谢产物合成方面具有很好的潜力,可作为一类重要微生物资源进行活性产物开发。     

Application of molecular biology for the discovery of biosynthetic genes of polyketide and peptide antibiotics produced by actinomycetes

Actinomycetes are currently the main source of antibiotics. Genome sequencing reveals the presence in these organisms of multiple gene clusters for the synthesis of yet unidentified secondary metabolites. Technological advances in DNA isolation, cloning and sequencing, as well as development of bioinformatics, facilitate large scale search for new gene clusters in organisms with unknown genome sequence and in environmental DNA. Methods used for detection of polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) genes are described in this article. New PKS and NRPS genes give access to new biologically active natural products which can become drugs or substrates for chemical modifications. Even more inspiring is their use in combinatorial biosynthesis to produce a variety of compounds with rationally designed structures.     

Culturable Endophytes of Medicinal Plants and the Genetic Basis for Their Bioactivity

The bioactive compounds of medicinal plants are products of the plant itself or of endophytes living inside the plant. Endophytes isolated from eight different anticancer plants collected in Yunnan, China, were characterized by diverse 16S and 18S rRNA gene phylogenies. A functional gene-based molecular screening strategy was used to target nonribosomal peptide synthetase (NRPS) and type I polyketide synthase (PKS) genes in endophytes. Bioinformatic analysis of these biosynthetic pathways facilitated inference of the potential bioactivity of endophyte natural products, suggesting that the isolated endophytes are capable of producing a plethora of secondary metabolites. All of the endophyte culture broth extracts demonstrated antiproliferative effects in at least one test assay, either cytotoxic, antibacterial or antifungal. From the perspective of natural product discovery, this study confirms the potential for endophytes from medicinal plants to produce anticancer, antibacterial and antifungal compounds. In addition, PKS and NRPS gene screening is a valuable method for screening isolates of biosynthetic potential.     

Distribution and diversity of natural product genes in marine and freshwater cyanobacterial cultures and genomes

Natural products are a functionally diverse class of biochemically synthesized compounds, which include antibiotics, toxins, and siderophores. In this paper, we describe both the detection of natural product activities and the sequence identification of gene fragments from two molecular systems that have previously been implicated in natural product production, i.e., nonribosomal peptide synthetases (NRPSs) and modular polyketide synthases (PKSs), in diverse marine and freshwater cyanobacterial cultures. Using degenerate PCR and the sequencing of cloned products, we show that NRPSs and PKSs are common among the cyanobacteria tested. Our molecular data, when combined with genomic searches of finished and progressing cyanobacterial genomes, demonstrate that not all cyanobacteria contain NRPS and PKS genes and that the filamentous and heterocystous cyanobacteria are the richest sources of these genes and the most likely sources of novel natural products within the phylum. In addition to validating the use of degenerate primers for the identification of PKS and NRPS genes in cyanobacteria, this study also defines numerous gene fragments that will be useful as probes for future studies of the synthesis of natural products in cyanobacteria. Phylogenetic analyses of the cyanobacterial NRPS and PKS fragments sequenced in this study, as well as those from the cyanobacterial genome projects, demonstrate that there is remarkable diversity and likely novelty of these genes within the cyanobacteria. These results underscore the potential variety of novel products being produced by these ubiquitous organisms.     

Genetic analysis of polyketide synthase and peptide synthetase genes in cyanobacteria as a mining tool for secondary metabolites

Molecular screening using degenerate PCR to determine the presence of secondary metabolite genes in cyanobacteria was performed. This revealed 18 NRPS and 19 PKS genes in the 21 new cyanobacterial strains examined, representing three families of cyanobacteria (Nostocales, Chroococales and Oscillatoriales). A BLAST analysis shows that these genes have similarities to known cyanobacterial natural products. Analysis of the NRPS adenylation domain indicates the presence of novel features previously ascribed to both proteobacteria and cyanobacteria. Furthermore, binding-pocket predictions reveal diversity in the amino acids used during the biosynthesis of compounds. A similar analysis of the PKS ketosynthase domain shows significant structural diversity and their presence in both mixed modules with NRPS domains and individually as part of a PKS module. We have been able to classify the NRPS genes on the basis of their binding-pockets. Further, we show how this data can be used to begin to link structure to function by an analysis of the compounds Scyptolin A and Hofmannolin from Scytonema sp. PCC 7110.     

Reconstitution of the FK228 biosynthetic pathway reveals cross talk between modular polyketide synthases and fatty acid synthase

Functional cross talk between fatty acid biosynthesis and secondary metabolism has been discovered in several cases in microorganisms; none of them, however, involves a modular biosynthetic enzyme. Previously, we reported a hybrid modular nonribosomal peptide synthetase (NRPS)-polyketide synthase (PKS) pathway for the biosynthesis of FK228 anticancer depsipeptide in Chromobacterium violaceum strain 968. This pathway contains two PKS modules on the DepBC enzymes that lack a functional acyltransferase (AT) domain, and no apparent AT-encoding gene exists within the gene cluster or its vicinity. We report here that, through reconstitution of the FK228 biosynthetic pathway in Escherichia coli cells, two essential genes, fabD1 and fabD2, both encoding a putative malonyl coenzyme A (CoA) acyltransferase component of the fatty acid synthase complex, are positively identified to be involved in FK228 biosynthesis. Either gene product appears sufficient to complement the AT-less PKS modules on DepBC for polyketide chain elongation. Concurrently, a gene (sfp) encoding a putative Sfp-type phosphopantetheinyltransferase was identified to be necessary for FK228 biosynthesis as well. Most interestingly, engineered E. coli strains carrying variable genetic components produced significant levels of FK228 under both aerobic and anaerobic cultivation conditions. Discovery of the trans complementation of modular PKSs by housekeeping ATs reveals natural product biosynthesis diversity. Moreover, demonstration of anaerobic production of FK228 by an engineered facultative bacterial strain validates our effort toward the engineering of novel tumor-targeting bioagents.     

The biosynthetic gene cluster for the anticancer drug bleomycin from Streptomyces verticillus ATCC15003 as a model for hybrid peptide–polyketide natural product biosynthesis

The hybrid peptide–polyketide backbone of bleomycin (BLM) is assembled by the BLM megasynthetase that consists of both nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) modules. BlmIX/BlmVIII/BlmVII constitute a natural hybrid NRPS/PKS/NRPS system, serving as a model for both hybrid NRPS/PKS and PKS/NRPS systems. Sequence analysis and functional comparison of domains and modules of BlmIX/BlmVIII/BlmVII with those of nonhybrid NRPS and PKS systems suggest that (1) the same catalytic sites appear to be conserved in both hybrid NRPS–PKS and nonhybrid NRPS or PKS systems, with the exception of the KS domains in the hybrid NRPS/PKS systems that are unique; (2) specific interpolypeptide linkers may play a critical role in intermodular communication to facilitate transfer of the growing intermediates between the interacting NRPS and/or PKS modules; and (3) posttranslational modification of the BLM megasynthetase has been accomplished by a single PPTase with a broad substrate specificity toward the apo forms of both acyl carrier proteins (ACPs) and peptidyl carrier proteins (PCPs). Journal of Industrial Microbiology & Biotechnology (2001) 27, 378–385. Received 08 June 2001/ Accepted in revised form 18 July 2001     

芍药内生真菌的鉴定及产生活性次生代谢产物的评估

【目的】研究药用植物芍药(Paeonia lactiflora Pall.)内生真菌的种群多样性,同时对其可能存在的聚酮合酶(Polyketide synthase,PKS)和非核糖体多肽合成酶(Non-ribosomal peptide synthetase,NRPS)基因多样性进行评估,预测芍药内生真菌产生活性次生代谢产物的潜力。【方法】采用组织分离法获得芍药根部内生真菌菌株,结合形态学特征和ITS序列分析,进行鉴定;利用兼并性引物对内生真菌中存在的聚酮合酶(PKS)基因和非核糖体多肽合成酶(NRPS)基因进行PCR扩增及序列测定分析,构建系统发育树,明确芍药内真菌PKS基因序列和NRPS基因序列的系统进化地位。【结果】从芍药组织块中共分离得到105株内生分离物,去重复后获得52株内生真菌,菌株ITS基因序列信息显示,52株芍药内生真菌隶属于7目、13科、15属,其中小球腔菌属(Leptosphaeria)、土赤壳属(Ilyonectria)和镰孢属(Fusarium)为优势种群;从52株内生真菌中筛选获得13株含PKS基因片段的菌株,8株含NRPS基因片段的菌株,部分菌株功能基因的氨基酸序列与Gen Bank中已知化合物的合成序列具有一定的同源性,预示芍药根部内生真菌具有合成丰富多样的次生代谢产物的潜力。【结论】药用植物芍药根部具有丰富的内生真菌资源,且具有产生活性次生代谢产物的潜力,值得进一步开发研究和应用。     

Genome mining for natural product biosynthetic gene clusters in the Subsection V cyanobacteria

Background

Cyanobacteria are well known for the production of a range of secondary metabolites. Whilst recent genome sequencing projects has led to an increase in the number of publically available cyanobacterial genomes, the secondary metabolite potential of many of these organisms remains elusive. Our study focused on the 11 publically available Subsection V cyanobacterial genomes, together with the draft genomes of Westiella intricata UH strain HT-29-1 and Hapalosiphon welwitschii UH strain IC-52-3, for their genetic potential to produce secondary metabolites. The Subsection V cyanobacterial genomes analysed in this study are reported to produce a diverse range of natural products, including the hapalindole-family of compounds, microcystin, hapalosin, mycosporine-like amino acids and hydrocarbons.

Results

A putative gene cluster for the cyclic depsipeptide hapalosin, known to reverse P-glycoprotein multiple drug resistance, was identified within three Subsection V cyanobacterial genomes, including the producing cyanobacterium H. welwitschii UH strain IC-52-3. A number of orphan NRPS/PKS gene clusters and ribosomally-synthesised and post translationally-modified peptide gene clusters (including cyanobactin, microviridin and bacteriocin gene clusters) were identified. Furthermore, gene clusters encoding the biosynthesis of mycosporine-like amino acids, scytonemin, hydrocarbons and terpenes were also identified and compared.

Conclusions

Genome mining has revealed the diversity, abundance and complex nature of the secondary metabolite potential of the Subsection V cyanobacteria. This bioinformatic study has identified novel biosynthetic enzymes which have not been associated with gene clusters of known classes of natural products, suggesting that these cyanobacteria potentially produce structurally novel secondary metabolites.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1855-z) contains supplementary material, which is available to authorized users.     

Investigation of the biosynthetic potential of endophytes in traditional Chinese anticancer herbs

Traditional Chinese medicine encompasses a rich empirical knowledge of the use of plants for the treatment of disease. In addition, the microorganisms associated with medicinal plants are also of interest as the producers of the compounds responsible for the observed plant bioactivity. The present study has pioneered the use of genetic screening to assess the potential of endophytes to synthesize bioactive compounds, as indicated by the presence of non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) genes. The total DNA extracts of 30 traditional Chinese herbs, were screened for functional genes involved in the biosynthesis of bioactive compounds. The four PCR screens were successful in targeting four bacterial PKS, six bacterial NRPS, ten fungal PKS and three fungal NRPS gene fragments. Analysis of the detected endophyte gene fragments afforded consideration of the possible bioactivity of the natural products produced by endophytes in medicinal herbs. This investigation describes a rapid method for the initial screening of medicinal herbs and has highlighted a subset of those plants that host endophytes with biosynthetic potential. These selected plants can be the focus of more comprehensive endophyte isolation and natural product studies.     

A Single Sfp-Type Phosphopantetheinyl Transferase Plays a Major Role in the Biosynthesis of PKS and NRPS Derived Metabolites in Streptomyces ambofaciens ATCC23877

The phosphopantetheinyl transferases (PPTases) are responsible for the activation of the carrier protein domains of the polyketide synthases (PKS), non ribosomal peptide synthases (NRPS) and fatty acid synthases (FAS). The analysis of the Streptomyces ambofaciens ATCC23877 genome has revealed the presence of four putative PPTase encoding genes. One of these genes appears to be essential and is likely involved in fatty acid biosynthesis. Two other PPTase genes, samT0172 (alpN) and samL0372, are located within a type II PKS gene cluster responsible for the kinamycin production and an hybrid NRPS-PKS cluster involved in antimycin production, respectively, and their products were shown to be specifically involved in the biosynthesis of these secondary metabolites. Surprisingly, the fourth PPTase gene, which is not located within a secondary metabolite gene cluster, appears to play a pleiotropic role. Its product is likely involved in the activation of the acyl- and peptidyl-carrier protein domains within all the other PKS and NRPS complexes encoded by S. ambofaciens. Indeed, the deletion of this gene affects the production of the spiramycin and stambomycin macrolide antibiotics and of the grey spore pigment, all three being PKS-derived metabolites, as well as the production of the nonribosomally produced compounds, the hydroxamate siderophore coelichelin and the pyrrolamide antibiotic congocidine. In addition, this PPTase seems to act in concert with the product of samL0372 to activate the ACP and/or PCP domains of the antimycin biosynthesis cluster which is also responsible for the production of volatile lactones.     

An approach for obtaining perfect hybridization probes for unknown polyketide synthase genes: a search for the epothilone gene cluster

An approach is described for obtaining 'perfect probes' for type I modular polyketide synthase (PKS) gene clusters that in turn enables the identification of all such gene clusters in a genome. The approach involves sequencing small fragments of a random genomic DNA library containing one or more modular PKS gene clusters, and identifying which fragments emanate from PKS genes. Knowing the approximate sizes of the genome and the target gene cluster, one can predict the the frequency that a PKS gene fragment will be present in the library sequenced. Computer simulations of the approach were applied to the known PKS and non-ribosomal peptide synthetase (NRPS) gene clusters in the Bacillus subtilus genome. The approach was then used to identify PKS gene fragments in a strain of Sorangium cellulosum that produces epothilone. In addition to identifying fragments of the epothilone gene cluster, we obtained 11 unique fragments from other PKS gene clusters; the results suggest that there may be six to eight PKS gene clusters in this organism. In addition, we identified four unique fragments of NRPS genes, demonstrating that the approach is also applicable for identification of these modular gene clusters.     

抗菌和细胞毒活性海洋细菌的筛选及其次生代谢基因证据

从不同海域的海水、海泥和海洋生物中分离海洋细菌,利用琼脂扩散法和MTT法对细菌培养液的乙酸乙酯提取物进行了抗菌和细胞毒活性筛选,并对具有细胞毒活性的细菌菌株进行了16SrRNA系统发生学分析和多聚酮合酶(PKSⅠ型)、非核糖体肽合成酶(NRPS)的筛选。结果显示,在分离到的346株海洋细菌中,42株细菌具有抗菌活性,12株具有细胞毒活性。对12株具有细胞毒活性的细菌菌株进行了16SrRNA系统发生学分析,它们分别属于Agrobacterium,Pseudoalteromons,Bacillus,Paracoccus,Rheinheimera,Aerococcus,Exiguobacterium和Alteromonas8个属。对这12株具有细胞毒活性的细菌菌株进行进一步的PKS和NRPS筛选,得到了4株含有PKSⅠ型的KS结构域或NPRS的A结构域的海洋细菌,为从聚酮和非核糖体肽等生物合成途径去深入研究其次生代谢产物提供了基因学的证据。