Session
Poster Session 2
Natural Product Discovery and Development in the Genomic Era
Contributed Poster Abstract
A rare ether-catalyzing cytochrome P450 in the biosynthesis of platensimycin
Natural Product Discovery and Development in the Genomic Era
Platensimycin (PTM) and platencin (PTN) are highly functionalized bacterial diterpenoid natural products that target bacterial and mammalian fatty acid synthases. The biosynthetic pathways of PTM and PTN diverge at the cyclization of ent-copalyl diphosphate. Two distinct type I diterpene synthases form the skeletons of PTM and PTN, (16R)-ent-kauran-16-ol and ent-atiserene, respectively. Before ent-kauranol resumes its parallel biosynthesis with ent-atiserene, it is processed by a cytochrome P450 to form the characteristic 11S,16S-ether linkage of PTM. The isolation of 16R-hydroxyl intermediates and congeners from the PTM producing strain suggests that ether formation goes through an 11S,16R-diol intermediate, due to the necessary inversion of stereochemistry at C-16. First, one 18O atom of 18O2 was found to be incorporated in an in vitro labeling experiment supporting that the ether oxygen in PTM originates from molecular oxygen. Next, we tested a series of alternate diterpenoid substrates with PtmO5 in vitro and found PtmO5 is a promiscuous P450. Isolation and structural characterization of the enzymatic products revealed insights into the mechanism of P450 ether formation including the regio- and stereoselectivity of PtmO5.
Contributed Poster Abstract
Genome mining of biosynthetic gene clusters in fungal hyperparasites
Natural Product Discovery and Development in the Genomic Era
Contributed Poster Abstract
Discovery of the tyrobetaine natural products and their biosynthetic gene cluster via metabologenomics
Natural Product Discovery and Development in the Genomic Era
Natural products have long served as rich sources of antibiotics, but traditional discovery methods of screening crude extracts for novel natural products are now often unsuccessful due to high rates of rediscovery. More recently, genetic approaches for natural product discovery have shown some promise, but progress has been slow due to the difficulty of identifying unique clusters and the issue of cryptic gene clusters. To address the challenge of natural product discovery, the Metcalf laboratory (University of Illinois at Urbana-Champaign) in collaboration with the Kelleher laboratory (Northwestern University) have developed a method called metabologenomics that combines genomic and mass spectrometric data to allow untargeted discovery of new natural products. This method allowed for identification, isolation, and structural elucidation of two novel nonribosomal peptide natural products. Both of these compounds contain a highly unusual trimethylated N-terminus along with the unnatural amino acid 3-hydroxyleucine. These new natural products differ in that one is halogenated (hereafter referred to as chlorotyrobetaine) and the other (tyrobetaine) is not. Utilizing the metabologenomics method along with heterologous expression, the biosynthetic gene cluster responsible for production of tyrobetaine was identified. Interestingly, the gene responsible for the halogenation of chlorotyrobetaine appears to be present on a different cluster, suggesting that either the halogenase or the initial adenylation domain of the nonribosomal peptide synthetase is promiscuous. The discovery of tyrobetaine, chlorotyrobetaine, and their associated biosynthetic gene clusters demonstrates the great power of the metabologenomics method for the discovery of new natural products that could help to refill the antibiotic pipeline.
Contributed Poster Abstract
Biosynthetic study on rare sulfonamide natural products SB-203207 and SB-203208
Natural Product Discovery and Development in the Genomic Era
SB-203207 and SB-203208 are two isoleucyl tRNA synthetase inhibitors isolated from Streptomyces sp. NCIMB40513. These compounds are structurally related to altemicidin, an acaricidal and antitumor compound, and possess unusual 6-azaindene monoterpene ring and sulfonamide group. Although total synthesis of SB-203207 was achieved, its biosynthetic pathway remains unknown. To elucidate the biosynthetic mechanism of these compounds, two biosynthetic gene clusters in the producer strain were heterologously expressed in Streptomyces lividans step by step. The results revealed that the first cluster is responsible for the production of altemicidin while the second cluster is responsible for the transfer of isoleucine onto the sulfonamide group and methylphenyalanine onto the hydroxyl group of altemicidin to afford SB-203208. Furthermore, in vitro assay of Orf-1 (tRNA synthetase like protein) with isoleucyl tRNA and altemicidin revealed that Orf-1 cannot accept ATP and isoleucine to give isoleucyl-AMP but acts as an amide synthase in this transfer reaction, indicating that Orf-1 was evolved to be a biosynthetic enzyme from house keeping proteins. We also characterized the transfer of methylphenylalanine by Orf-2 (AMP-ligase), Orf-3 (Acyltransferase) and Orf-11 (Carrier protein), that Orf-3 requires carrier protein-tethered methylphenylalanine as the acyl donor and altemicidin as the acceptor for the ester bond formation. Currently, we are investigating the biosynthetic pathway of sulfonamide through in vitro assay and gene deletion study.
Figure 1 Structures of SB-203207, SB-203208, altemicidin
Contributed Poster Abstract
Genetic and in vivo functional analysis of Ble (Orf12) from the clavulanic acid biosynthetic pathway of Streptomyces clavuligerus
Natural Product Discovery and Development in the Genomic Era
Clavulanic acid is produced by Streptomyces clavuligerus and is a potent inhibitor of β-lactamases, enzymes that hydrolyze and inactivate conventional β-lactam antibiotics such as penicillins and cephalosporins. It differs from all other known clavams due to its unique 5R stereochemistry, which is also responsible for its bioactivity. Clavulanic acid and the 5S clavams share a common biosynthetic pathway in S. clavuligerus and precursors leading up to the penultimate step during clavulanic acid biosynthesis also have 5S stereochemistry. As to how and when the stereochemical change from 5S to 5R takes place during production has been a long standing question, with many different hypotheses being proposed. One such hypothesis involves the product of orf12 (or Ble) from the clavulanic acid gene cluster, which resembles class A β-lactamases and has an additional N-terminal domain resembling steroid isomerases/cyclases. Previous reports from other groups have shown that Ble exhibits some in vitro β-lactam esterase activity (Acta Crystallogr D Biol Crystallogr. 2013.69:1567-79), the implications of which on clavulanic acid biosynthesis is not clear. We conducted extensive in vivo mutagenesis, expression and complementation studies to show that Ble is most likely a bonafide enzyme from the clavulanic acid biosynthetic pathway of S. clavuligerus. It is intriguing that a protein related to β-lactamases would be involved in the biosynthesis of a β-lactamase inhibitor, the implications of which will also be discussed.
Contributed Poster Abstract
Genomics driven discovery of natural products from rare actinomycetesNocardia spp. CS682
Natural Product Discovery and Development in the Genomic Era
Nocardia sp. CS682, was isolated from Korea and characterized as prominent producer of nargenicin A1. Nargenicin A1 exhibits significant antimicrobial potential against various Gram positive bacteria including methicillin resistant Staphylococcus aureus (MRSA). By whole genome sequencing and annotation, different gene clusters corresponding to various secondary metabolites were identified from Nocardia sp. CS682. The comprehensive analysis of metabolites led to elucidation of structure of such secondary metabolites. In the meantime the functional characterization of genes in biosynthetic gene cluster and characterization of metabolite profile provided insight on mechanism of biogenesis of nargenicin A1 in Nocardia sp. CS682. Concurrently on the basis of information of biosynthetic mechanism, the gene mutation and gene combination strategies were employed for generating different novel derivatives of nargenicin A1. All the derivatives were structurally elucidated by mass spectrometric and NMR spectroscopic analysis as par need. The biological properties of such novel derivatives were accessed by evaluation of antimicrobial and anticancer activities.
References
Dhakal, D. et al. (2016) Genetic Manipulation of Nocardia species, Current protocols in microbiology, 40:10F.2.1-10F.2.18
Dhakal, D. et al. (2015). Enhanced Production of Nargenicin A1 and generation of novel glycosylated derivatives. Applied biochemistry and biotechnology, 175(6), 2934-2949
Dhakal, D et al. (2016). Enhanced production of nargenicin A1 and creation of a novel derivative using a synthetic biology platform. Applied microbiology biotechnology, 100(23):9917-9931
Acknowledgement: This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (NRF-2017R1A2A2A05000939) and (NRF-2017R1D1A1B03036273).
Contributed Poster Abstract
Biosynthesis of natural and non-natural glycosides of genistein.
Natural Product Discovery and Development in the Genomic Era
Contributed Poster Abstract
Production of coronafacoyl phytotoxins involves a novel biosynthetic pathway in the plant pathogen Streptomyces scabiei
Natural Product Discovery and Development in the Genomic Era
Phytotoxic natural products have been shown to play an important role in the ability of phytopathogenic microorganisms to cause disease. They can allow the producing organism to evade pant defense responses during host colonization and infection. The coronafacoyl phytotoxins are a family of natural products that are biosynthesised by phylogenetically distinct plant pathogenic bacteria and which function as virulence factors for these organisms. One of the most studied coronafacoyl phytotoxins is coronatine (COR), which is produced by the phytopathogenic bacterium Pseudomonas syringae. COR functions as a phytohormone mimic and allows manipulation of host defence signalling pathways during infection. COR and other coronafacoyl phytotoxins consist of the polyketide compound coronafacic acid (CFA) linked via an amide bond to different amino acid or amino acid analogs. Recent studies from our lab have demonstrated that the potato common scab pathogen Streptomyces scabiei produces the coronafacoyl phytotoxin N-coronafacoyl-L-isoleucine, and gene deletion analyses have shown that production of the CFA moiety in this organism involves a novel biosynthetic pathway as compared to P. syringae. In addition, we provide evidence that this novel pathway is conserved in other Actinobacteria that are predicted to produce coronafacoyl phytotoxins but not in other phytotoxin – producing organisms. We additionally reveal that production of coronafacoyl phytotoxins may occur in both plant pathogenetic and non-pathogenetic bacteria based on genomic analyses. This suggests that production of these natural products may be more prevalent than previously realized and that their role for the producing organism may not be limited to host-pathogen interactions.
Contributed Poster Abstract
Discovery of novel antibiotics from rare-actinomycetes using combined-culture strategy
Natural Product Discovery and Development in the Genomic Era
Actinomycetes have a large potential to produce antibiotics, and much of efforts for natural product discovery were focused on Streptomyces species. On the other hand, rare-actinomycetes (= non-Streptomyces) are less-exploited resource for natural product discovery. However, recent bacterial genome sequencing projects revealed that some rare actinomycetes possess comparable levels of natural product biosynthetic gene clusters (NPGCs) in their genomes, and the most parts of their biosynthetic genes were not expressed under the standard culture conditions. We previously showed that mycolic acid-containing bacterium (MACB) efficiently induces secondary metabolite production in some Streptomyces strains by co-culture, and we described this method as “combined-culture”. However, the application of “combined-culture” method to rare actinomycetes was quite limited. Therefore, we applied combined-culture method to our collection of rare actinomycetes to obtain novel antibiotics from them.
As the strains for combined-culture screening, we chose three orders of rare-actinomycetes (Pseudonocardiales, Streptosporangiales, and Micromonosporales), which were reported to possess large amount of NPGCs. As a result, we found that MACB (Tsukamurella pulmonis TP-B0596) activated the production of secondary metabolites in approximately 30% of tested strains by co-culture. Furthermore, we could identify five novel compounds from combined-culture of three rare-actinomycete strains. In conclusion, the “combined-culture” method is effective tool for activating silent biosynthetic genes present in rare-actinomycetes, and we could access novel bioactive compounds from them.
Contributed Poster Abstract
Expanding chemical diversity in the Sirenas Biochemometrics Platform
Natural Product Discovery and Development in the Genomic Era
An increasing challenge in the field of natural products research is the discovery of novel chemistry possessing biologically relevant activity. One solution is to diversify the chemical source by collecting taxonomically distinct organisms or isolating and culturing new bacterial strains. However, these options tend to be difficult, costly, and often result in redundancies. An alternative strategy is to expand or maximize the chemical extraction process and increase chemical diversity by optimizing workflows and the discovery process itself. For example, polar organic molecules, broadly defined as those that are not retained under standard reverse-phase HPLC conditions, are frequently overlooked in most natural product drug discovery workflows due to the difficulty in their isolation, as well as the consistent lack of activity in their associated fractions during bioassay guided pipelines and cellular-based assays. Therefore, the therapeutic potential of this area of chemical space is relatively unexplored. Sirenas recently implemented discovery methods to process and fractionate polar organic fractions originating from the extracted aqueous layer. These methods, after rigorous development, include SPE using a hydrophilic/lipophilic balanced sorbent for sample preparation and subsequent HPLC under conditions suitable for the separation of these types of molecules. Complimented with proprietary software and informatics that integrate chemical and biological data for each molecule, these methods have shown promise in the identification of new chemistry with activity against Mycobacterium tuberculosis.
Contributed Poster Abstract
Nature as the ultimate combinatorial biosynthetic chemist: Discovery of the Leinamycin family of natural products
Natural Product Discovery and Development in the Genomic Era
Nature’s ability to generate diverse natural products from simple building blocks has inspired combinatorial biosynthesis. Knowledge-based approach to combinatorial biosynthesis has allowed the production of designer analogues by rational metabolic pathway engineering. While successful, structural alterations are limited, with the designer analogues often produced in significantly compromised titers. 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 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 distinct 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 as the ultimate combinatorial biosynthetic chemist. Comparative analysis of the newly discovered LNM-type biosynthetic machineries provides outstanding opportunities to dissect Nature’s biosynthetic strategies and apply these findings to the practices of combinatorial biosynthesis for natural product structural diversity.
Contributed Poster Abstract
Deciphering the role of genes involved in ethylmalonyl-CoA supply and tailoring reactions inKirromycin biosynthesis in Streptomyces collinus T 365
Natural Product Discovery and Development in the Genomic Era
Kirromycin is a potent inhibitor of protein biosynthesis in prokaryotes as it binds to the elongation factor Tu, leading to conformational changes and ultimately stalling of the bacterial ribosome. The linear molecule is synthesized by a hybrid PKS-I/NRPS and the biosynthetic gene cluster comprises 26 genes spanning an 82 kb DNA region. A combination of in silico bioinformatic predictions and in vitro mutational studies have revealed the role of core genes involved in biosynthesis of kirromycin, however, genes involved in precursor supply and tailoring reactions remained to be fully characterized in order to fill the gaps in the pathway1,2.
In this study, the functions of the putative crotonyl-CoA reductase/carboxylase (CCR) KirN and the tailoring enzymes KirM, KirHVI, KirOI and KirOII were investigated by genetic manipulation. Derivatives were detected in each mutant involved in tailoring reactions and in the case of the KirN mutant a lowered kirromycin production was observed.
Our genetic studies have allowed us to clarify the putative roles of all enzymes studied here, ultimately allowing us to fill many of the missing gaps in the biosynthetic pathway of kirromycin. Furthermore, this collection of mutants serves as a toolbox for production of new derivatives of the original molecule kirromycin and with this a better understanding of the potential of complex polyketides in production of antibiotics with new mode of actions.
Contributed Poster Abstract
Arsinothricin, a novel organoarsenical antibiotic and its resistance mechanism
Natural Product Discovery and Development in the Genomic Era
Due to the dramatic increase in bacterial antimicrobial resistance (AMR), discovery and development of new antibiotics is an urgent need. Organoarsenicals have been used as antimicrobials more than a century since Paul Ehrlich’s magic bullet Salvarsan. Recently a rice rhizosphere bacterium was shown to produce a novel natural product containing arsenic, arsinothricin (AST), a mimetic of the herbicidal antibiotic phosphinothricin (PT). Here we show that AST is a broad-spectrum antibiotic effective against both Gram-negative and Gram-positive bacteria and its antimicrobial activity is 30-time more effective than PT and retained even in the presence of glutamate that reverses PT. These results suggest that AST is a considerably more potent antibiotic than PT, thus we propose that this novel As-containing antibiotic has the potential to provide a countermeasure to combat AMR. The occurrence of antibiotic resistance is inevitable. Here we demonstrate that, ArsN, N-acetyltransferase encoded by arsN gene widely distributed in bacterial ars (arsenic resistance) operons, confers high-level resistance to AST and low resistance to PT, indicating that ArsN is an AST-selective N-acetyltransferase. We crystallized ArsN and solved the structures for both the apo and AST-bound forms, which provided insights into the substrate selectivity of the enzyme. These results will lead to schemes to reverse bacterial resistance to AST, making it a more effective antibiotic against the growing health treat of AMR.
Contributed Poster Abstract
sequestration as a new mechanism for self-resistance to enediyne antitumor antibiotics
Natural Product Discovery and Development in the Genomic Era
The enediyne antibiotics show great structural variety and astonishing bioactivity. The 9- and 10-membered enediyne-producing strains, respectively, employ the apoproteins and the self-sacrifice proteins against the enediyne toxins. However, the mechanism for resistance to the anthraquinone-fused enediynes remained unclear. In this study, the self-resistance mechanism utilized in this class of microbes is revealed. Bioinformatics analysis shows that the genes encoding TnmS1/TnmS2/TnmS3, UcmS1/UcmS2/UcmS3, and DynE14/DynE15, respectively, in the biosynthetic gene clusters of anthraquinone-fused enediynes, tiancimycin (TNM), uncialamycin (UCM), and dynemicin (DYN), belong to the glyoxalase/bleomycin resistance protein/dioxygenase superfamily that may be involved in antibiotic resistance. In vivo resistance assays, together with in vitro protein-ligand binding assays, provide evidence of antibiotic sequestration as a major mechanism for self-resistance to anthraquinone-fused enediynes. Finally, the binding site of TNM was mapped by the crystal structures of TnmS1/TnmS2/TnmS3. Taken together, these finding provide a new mechanism and a missing piece for self-resistance to a set of enediyne antitumor antibiotics.
Contributed Poster Abstract
Phenazine antibiotics via divergent biosynthesis pathways
Natural Product Discovery and Development in the Genomic Era
Entomopathogenic bacteria of the genus Xenorhabdus living in symbiosis with nematodes of the genus Steinernema are able to produce a huge diversity of toxic proteins as well as natural products as signaling molecules and virulence factors to maintain nematode development and protect the insect cadaver from food competitors. Two phenazine biosynthetic gene clusters (BGCs) including a silent one have been identified in Xenorhabdus szentirmaii, and are subdivided into four modules based on their involvement in four different classes of phenazine derivatives. The biosynthesis of simple hydroxyl- and carboxyl-substituted phenazines (occurring mainly in Pseudomonas) were generated by pairing with two monooxygenases, which were further characterized by in vitro assay, resulting in the production of the cytotoxic pigment iodinin. More complex phenazines like griseoluteic acid (mostly distributed in Streptomyces) were produced by enzymes encoded in the core set of genes of the second BGC activated via promoter exchange to reveal their function. Serendipitously, in vivo characterization of the free-standing NRPS enzymes catalyzing ester-bond formation revealed the production of phenazine-amino acid derivatives active against E. coli. Preliminary structure-activity relationship study indicated that the activity was significantly promoted by the 9-methoxyl group. The unusual modular character of these BGCs might be a good example of a stepwise chemical diversification as a response to diverse environments with different other (micro)organisms.
Contributed Poster Abstract
Scope and utility of trans-acyltransferases for natural product diversification
Natural Product Discovery and Development in the Genomic Era
Polyketides are a diverse class of pharmaceutically relevant natural products, whose biological activities include antibiotic, antitumor, and immunosuppressant properties, among others. These compounds are structurally complex, yet they are biosynthesized from a modest pool of small, simple building blocks. Large enzyme complexes known as polyketide synthases (PKSs) are responsible for intricately stitching together these building blocks in a predictable fashion to make the biologically active natural product. PKSs are organized into modules, where each module incorporates a single building block into the final product. This modular templated biosynthesis, enables engineering to program these pathways to produce molecules with improved or modified biological activity. Each module contains an acyltransferase (AT) domain, which is responsible for selecting the appropriate building block and is therefore often the target of engineering through either mutagenesis or entire domain replacement. An alternative approach is to abolish the activity of the natural in-line AT and utilize a discrete AT, known as a trans-AT, to introduce a different building block at a programmed position within the pathway. This approach has had limited success but has been hindered by faulty protein-protein interactions and the narrow substrate scope of known trans-ATs. Herein, we describe the previously unknown substrate promiscuity of a trans-AT from a PKS/NRPS hybrid pathway and probe the molecular basis of its broad specificity. In addition, we describe efforts towards engineering a highly specific trans-AT to broaden its substrate scope. Cumulatively, our results can be leveraged for the production of novel polyketide analogues.
Contributed Poster Abstract
Chemical perturbation of fruit fly behaviour and development byStreptomyces.
Natural Product Discovery and Development in the Genomic Era
The genus Streptomyces is a valuable source of antibiotics and selective modulators of pathway targets. Environmental isolates of 56 strains were cultured and screened using novel assays against multicellular model eukaryotic organisms. 12% of extracts had a wide range of bioactivities that perturbed the behaviour and development of D. melanogaster. WAC-288, a strain of interest produced a molecule with starvation-induced larvicidal activity. In addition, the biosynthetic cluster of a volatile organic compound 2-methylisoborneol (2-MIB) was identified which induced a concentration dependent attraction-aversion response. These results describe an antagonistic relationship between Streptomyces and insects. Furthermore, this study expands the conventional target diversity of natural products produced by Streptomyces and provides novel tools to identify unique bioactive compounds against a wide range of multicellular eukaryotic organisms.
Contributed Poster Abstract
Application of bioelectrochemical system for enhancement of 3-hydroxypropionic acid production by regulation of intracellular redox balance
Natural Product Discovery and Development in the Genomic Era
In spite of the recent advances of genetic and metabolic engineering, the regulation of intracellular redox state for moderating the thermodynamic limitation remains a challenge to develop a bioprocess for platform chemical productions. The aeration and addition of the oxidant/reductant are common strategies for controlling the cellular redox state (NAD+/NADH). Various chemical oxidants and reductants have been applied in anaerobic condition; however increase of operational cost and inhibition by toxic byproducts makes application less feasible. Bioelectrochemical system (BES) is a novel technology for the regulation of intracellular redox balance via electrochemical reactions between bacteria and electrode. 3-hydroxypropionic acid (3-HP) is commercially and industrially valuable platform chemicals which can be produced by glycerol fermentation; however the conversion of 3-HP is required for NAD+ regeneration from NADH anaerobically. In this report, we applied BES for enhancement of 3-HP production through anaerobic NAD+ regeneration. The recombinant Klebsiella pneumoniae L17 overexpressing aldehyde dehydrogenase (AldH) presented 1.8-fold increased 3-HP production yield in BES compared to that of non-BES, also central metabolic shift was found by activation of anaerobic respiration. This is the first report of 3-HP productivity enhancement by the application of BES. The results can provide a strategy of overcoming thermodynamic barrier of fermentation and various biorefinery process using BES by the regulation of bacterial redox balance.
Contributed Poster Abstract
Acetate production from carbon monoxide by syntrophic interaction between Citrobacter amalonaticus Y19 and Sporomusa ovata
Natural Product Discovery and Development in the Genomic Era
Biological volatile acids production from inorganic carbon such as CO2 and CO through chemosynthesis has been of great interest. However, biological CO conversion is difficult because of its high toxicity and low reducing equivalent. According to previous studies, mixed bacterial community consisted of homoacetogen (CO2 + H2 → acetate) and water-gas shift reaction catalyzing bacteria (CO → CO2 + H2) presented higher conversion efficiency compared to single strains which have activity of both reactions. We hypothesized that syntrophic interaction (or ‘division of labor’) between different species is more important in biological CO conversion to produce volatile fatty acids, rather than single bacteria. To demonstrate the hypothesis, defined co-culture fermentation of Citrobacter amalonaticus Y19 (water-gas shift reaction catalyzing bacteria) and Sporomusa ovata (homoacetogen) was examined for CO conversion. Our results showed that significantly higher CO conversion and acetate production were identified in co-culture fermentation with C. amalonaticus Y19 and S. ovata compared to that of mono-culture fermentation. CO consumption and acetate production rate increased with electron shuttle, and redox equivalent transfer between two species was identified. The results suggested the model of putative carbon monoxide conversion of mixed culture of homoacetogen and water-gas shift reaction catalyzing bacteria.
Contributed Poster Abstract
Examination of clavulanic acid biosynthesis using a comparative genomics approach
Natural Product Discovery and Development in the Genomic Era
Clavulanic acid is a clinically used inhibitor of certain β-lactamases, enzymes which are responsible for causing resistance to the penicillin and cephalosporin class of antibiotics. Clavulanic acid is industrially produced by fermenting Streptomyces clavuligerus and the biosynthetic pathway leading to its production has been partially elucidated, but details regarding the exact boundaries of the gene cluster(s) involved is still lacking. In addition to S. clavuligerus, a few other Streptomyces species also have the capacity to produce clavulanic acid and the genome sequences of certain microorganisms have been found to contain homologues of the clavulanic acid gene cluster; however the latter are unable to produce the metabolite. Therefore, we sequenced the genomes of isolates capable of producing clavulanic acid and using a comparative genomics/bioinformatics approach we explored the possibility of identifying the core set of genes that are involved in the production of this important secondary metabolite. Our results provide insights into possible reasons as to why some clavulanic acid like gene clusters are silent in certain Streptomyces species. In addition, we also examined the detailed composition and organization of the respective gene clusters, which can provide avenues to identify metabolites related to clavulanic acid.
Contributed Poster Abstract
Unlocking Hidden Reservoirs of Microbial Natural Products
Natural Product Discovery and Development in the Genomic Era
Microbial natural products have been the source of the majority of modern antibiotic and chemotherapeutic drugs and remain an important reservoir of therapeutic molecules. It is estimated that we have accessed less than 1 % of these natural resources largely from either our inability to detect and identify these within complex biological extracts or from the producing strain’s lack of production when grown under laboratory conditions. We hypothesize that producing organisms use their natural products to respond to environmental cues from competing organisms, to nutrients and other physical factors and that natural products induced from the application of these cues can be prioritized through comparative metabolomic methods. Here we quantify the global metabolomic and secondary metabolite response to stimuli from 21 diverse strains of actinomycetes exposed to six stimuli conditions. This work identified a number of known natural products that responded strongly to stimuli as well as a novel aminopolyol induced through mixed culture in a rare streptosporangeum strain. The comparative metabolomics methods used in this study are well suited to prioritize secondary metabolites for isolation in a discovery pipeline and may additionally enable the connection between activation stimuli and genetic regulatory elements, resulting in more targeted methods of natural product activation.
Contributed Poster Abstract
Engineered production of novel bleomycin analogues by combinatorial biosynthesis
Natural Product Discovery and Development in the Genomic Era
The bleomycins (BLMs), a family of glycopeptide antibiotics produced by several Streptomyces species, are currently used clinically in combination with a number of other agents for the treatment of several types of tumors. Other members of the BLM family include tallysomycins (TLMs), phleomycins and zorbamycin (ZBM). We have previously cloned and characterized the biosynthetic gene clusters for BLMs from Streptomyces verticillus ATCC15003, TLMs from Streptoalloteichus hindustanus E465-94 ATCC31158, and ZBM from Streptomyces flavoviridis SB9001. Applications of combinatorial biosynthesis strategies to the three biosynthetic machineries enabled the engineered production of several BLM analogues with unique structural characteristics and varying DNA cleavage activities, thereby providing an outstanding opportunity to study the structure–activity relationship (SAR) for the BLM family of anticancer drugs. We now report the engineered production of a new BLM–TLM–ZBM hybrid metabolite, named 6′-deoxy-TLM H-1, which consists of the 22-desmethyl-BLM aglycone, the TLM A C-terminal amine and the ZBM disaccharide, by heterologous expression of the zbmGL genes from the ZBM biosynthetic gene cluster in the Streptoalloteichus hindustanus ΔtlmH mutant strain SB8005. Evaluation of the DNA cleavage activities of 6′-deoxy-TLM H-1 as a measurement for its potential anticancer activity, in comparison with TLM H-1 and BLM A2, reveals new insight into the SAR of BLM family of anticancer drugs.
Contributed Poster Abstract
Comparative studies of the biosynthetic pathways for anthraquinone-fused enediynes
Natural Product Discovery and Development in the Genomic Era
Using the genome mining approach, we recently discovered two novel 10-membered enediyne natural products, tiancimycin (TNM) and yangpumicin (YPM). Like dynemicin (DYN) and uncialamycin (UCM), TNM and YPM also feature an enediyne core fused to an anthraquinone moiety. These enediynes exhibit extraordinary cytotoxicity against a broad panel of cancer cell lines and UCM is currently undergoing preclinical investigation. However, little is known about their biosynthetic pathways. Comparative studies of these gene clusters enabled us to prioritize genes for genetic manipulation. Two tailoring genes, tnmH (encoding a SAM-dependent methyltransferase) and tnmL (encoding a cytochrome P450 monooxygenase), which are present in the tnm gene cluster but absent in the ucm gene cluster, were selected for gene inactivation. Five new enediyne intermediates (TNM B, TNM C, TNM C2, TNM D, and TNM E), together with many cycloaromatized congeners, were isolated and characterized from the fermentation broth of Streptomyces sp. CB03234 wild-type strain and its ΔtnmH and ΔtnmL mutants. Interestingly, all the congeners from the ΔtnmH and ΔtnmL mutants possess an unexpected side chain attached to the enediyne core, indicating that the biosynthesis of anthraquinone-fused enediynes is more complicated than we have anticipated. Based on the structures of the isolated congeners, we can propose a unified biosynthetic pathway for the anthraquinone-fused enediynes.
Contributed Poster Abstract
Development of a general heterologous expression host for cyanobacterial natural product production.
Natural Product Discovery and Development in the Genomic Era
Cyanobacteria, particularly marine strains, are prolific producers of bioactive metabolites that utilize interesting biosynthetic logic. Despite the number of potent biologically active compounds, to date only one cyanobacterial compound (dolastatin 10) has been successfully developed as a drug lead (monomethyl auristatin E). Despite isolating significant quantities of bioactive compounds for structural determination and initial biological screening from cyanobacteria, obtaining the larger quantities necessary for in vitro mechanism of action studies, and biological studies (animal and human) is hampered by the slow growth rate, inability to genetically manipulate, difficulty in large scale cultivation, and low compound yields of many cyanobacterial strains. These problems could be overcome with the development of a general heterologous expression host for cyanobacterial natural products. Here we describe our progress toward developing the model cyanobacterium Anabaena sp. PCC 7120 toward this goal. We have used Anabaena 7120 to produce lyngbyatoxin A as a proof of concept molecule showing that Anabaena 7120 can recognize promoters from marine cyanobacteria, translate and properly post-translationally modify non-ribosomal peptide synthetases, and produce the desired natural product. We find that simple genetic manipulations and growth condition modification can greatly increase the yield of lyngbyatoxin A in this heterologous host. We also show that Anabaena 7120 can recognize promoters from a variety of cyanobacterial natural product gene clusters. We will also present our progress toward genome mining and identification of the cyanobacterial natural products coibamide A and fischerellin A.
Contributed Poster Abstract
Natural products discovery from un- and under-explored Actinomycetes strain collection at The Scripps Research Institution (TSRI)
Natural Product Discovery and Development in the Genomic Era
Natural products possess enormous structural and chemical diversity that is unsurpassed by any synthetic libraries, remain the best sources of drugs and drug leads, but are significantly underrepresented in current small molecule libraries. The Natural Products Library Initiative (NPLI) at the Scripps Research Institute (TRSI) aims at constructing a natural products library with unique chemical and structural diversity that complements the small-molecule collection at TSRI. The current library at TSRI consists of Actinomycetales that are isolated from unexplored or underexplored ecological niches and unavailable in public strain collections (strain collection), their corresponding genomic DNA (gDNA collection), crude microbial extracts, medium-pressure liquid chromatography fractions, and purified natural products with fully assigned structures. Traditional methods, including bioactivity-guided and chemical profiling-guided approaches, as well as genome mining methods, are used for discovery of novel natural products.
Contributed Poster Abstract
Searching for a natural product inhibitor of the tetracycline efflux pump TetA
Natural Product Discovery and Development in the Genomic Era
Over the past decade antibiotic resistance has been increasing at an alarming rate. The continuous use of antibiotics has resulted in the emergence of resistant bacteria, thereby rendering many of the available treatments for infectious diseases ineffective. However, while there is an acute need for the discovery of new antimicrobial compounds, there is a major lack of new antibiotics coming to market. A solution to this problem is the development of compounds capable of inhibiting the resistance mechanisms of bacteria. When used in conjunction with an antibiotic, the inhibitor blocks the resistance element to allow for the drug to target the bacteria. This approach provides opportunity for the rescue of antibiotics that are no longer used in clinical settings, including tetracyclines. Tetracyclines once exhibited activity against a wide range of bacteria, and had been extensively used in the past to treat a variety of infections. Today, tetracyclines have limited use due to the development of resistance mechanisms, including bacterial efflux pumps capable of exporting drug from the cell. The identification of an efflux pump inhibitor would aid in restoring the efficacy of this “broad-spectrum” antibiotic class. Using high throughput screening, a novel assay has been devised to screen the Wright Lab’s in-house collection of over 10,000 natural product extracts derived from environmental organisms. With this platform we ultimately hope to find a natural molecule capable of targeting the TetA efflux pump in order to rescue the legacy of tetracycline antibiotics.
Contributed Poster Abstract
An unknown regulatory system controls intrinsic rifamycin resistance in Actinobacteria
Natural Product Discovery and Development in the Genomic Era
Rifamycins are ansamycin natural products synthesized by a variety of soil actinobacteria which possess potent antibacterial activity. Rifampicin and rifabutin (semi-synthetic rifamycins) are both World Health Organization (WHO) essential medicines which are crucial to the successful treatment of Mycobacterium tuberculosis. Rifamycin antibiotics bind the β-subunit (RpoB) of prokaryotic RNA polymerase and inhibit transcription. Despite the ubiquity of their target among bacteria, rapid development of resistance has prevented widespread clinical use of these compounds. High level resistance to these drugs can be acquired by a single amino acid substitution in RpoB. Despite the availability of this quick and easy route to resistance, a wealth of rifamycin inactivating enzymes are present in environmental Actinobacteria. Rifamycin phosphotransferases, ADP-ribosyltransferases, glycosyltransferases, and monooxygenases have all been characterized. Intriguingly, the genes encoding these inactivating enzymes seem to be induced specifically by rifamycins. This process is known to require a cis-regulatory DNA motif termed the rifamycin associated element (RAE), which is present in the promoter region of rifamycin inactivating enzymes across the Actinobacteria. We posit that the RAE is a binding site for a regulatory protein. Preliminary data suggests this system functions via a de-repressible mechanism. We aim to exploit this fact by designing of a two-step genetic selection and screen based on de-repressible phenotypes to isolate mutants in this regulatory pathway. This strategy will allow for the identification of regulatory protein(s) associated with this pathway. Elucidating the mechanism by which environmental Actinobacteria sense and degrade rifamycin antibiotics.
Contributed Poster Abstract
Milbemycins production from Streptomyces avermitilis through engineering of avermectin biosynthetic pathway
Natural Product Discovery and Development in the Genomic Era
Milbemycins are 16-membered macrolides, produced from Streptomyces hygroscopicus subsp. aureoaureolacrim and Streptomyces bingchenggensis. They are structurally related with avermectin produced from Streptomyces avermitilis. Milbemycins have been known to be very potent acaricidal, insecticidal, and anthelmintic agent with low toxicity. Since milbemycins are commercially important and resistance to the avermectins and their derivatives is increasing, it is crucial to develop an efficient combinatorial biosynthesis system to produce the milbemycins and novel analogs in large quantities. In an attempt to construct a S. avermiltilis strain that produces milbemycins, AveA1 and module 7 in AveA3 of avermectin polyketide synthase (PKS) from S. avermitilis SA-01 were replaced with MilA1 and module 7 in MilA3 from milbemycin PKS, respectively. The titers of total milbemycins produced by engineered S. avermitilis was 291.5mg/l and the major products were C5-O-methylated milbemycins B2, B3, and G with milbemycin A3, A4 and D in small amounts. Subsequent inactivation of the C5-O-methyltransferase AveD resulted in the production of milbemycin A3/A4 approximately 225mg/l in the flask and 377mg/l in the 5l fermenter culture with trace amounts of milbemycin D. In this study, it was demonstrated that the production of milbemycin was only slightly decreased by engineering avermectin biosynthetic pathway. Application of the same strategy in the higher producing industrial strain will further increase the desired product titer and also allow generation of novel milbemycin analogs with improved properties in sufficient amounts for further development.
Contributed Poster Abstract
Regulatory and mutational analysis of the biosynthetic gene cluster for lugdunomycin, a novel antibiotic with unprecedented chemical architecture
Natural Product Discovery and Development in the Genomic Era
The introduction of major new classes of natural product-based antibacterial agents has come to a near standstill while antimicrobial resistance is rising sharply. The approval of daptomycin in 2003 marked the launch of the first new natural product-based antibiotic in decades. We have recently discovered a novel compound, called lugdunomycin, produced by Streptomyces sp. QL37. The compound is derived from the well-known angucyclines. The structure of lugdunomycin has been confirmed by NMR, high-resolution mass spectrometry and X-ray crystallography. Our work is focused on understanding the complex biosynthetic pathway for lugdunomycin biosynthesis, and to unravel the transcriptional control of the biosynthetic gene cluster. Lugdunomycin is derived from the angucycline backbone by complex enzymatic reactions. A Baeyer-Villiger oxidative cleavage in the quinone ring of the angucycline backbone is the likely first step in the rearrangement of angucyclines to generate the lugdunomycins. Besides lugdunomycin, 27 other rearranged and/or unrearranged angucyclines were identified in Streptomyces sp. QL37. This study starts from the perspective of the type II polyketide biosynthetic gene cluster that is responsible for angucycline and lugdunomycin biosynthesis. Gene disruption strategies using homologous recombination have been employed to elucidate the function of the biosynthetic and regulatory genes. Streptomyces sp. QL37 produces very low amounts of lugdunomycin and the compound is not produced in submerged cultures, despite testing a wide range of different culturing conditions. Strategies to improve the production of the compound by engineering of the regulatory network will also be discussed.
Contributed Poster Abstract
High-yield production of herbicidal thaxtomins and analogs in a nonpathogenic Streptomyces host
Natural Product Discovery and Development in the Genomic Era
Thaxtomins are virulent factors of several plant pathogenic Streptomyces strains. Because of their potent herbicidal activity, environmental compatibility and biodegradability, thaxtomins are EPA-approved green herbicides. However, the low yield of thaxtomins in native Streptomyces producers limits their wide applications in agriculture. Synthetic biology is an emerging principle and has demonstrated its successes in producing a wide variety of natural and unnatural chemicals. Here, we describe the high-yield production of thaxtomins and the synthesis of one unnatural analog through mutasynthesis in a heterologous host S. albus J1074. The thaxtomin gene cluster from S. scabiei 87.22 was cloned and expressed in S. albus J1074 on a self-replicative plasmid and on its chromosome. The production of thaxtomins and nitro-tryptophan analogs was detected. When cultured in the minimal medium TMDc, the yield of thaxtomin A from S. albus J1074 was 10 times higher than S. scabiei 87.22, and optimization of the medium resulted in the highest yield of over 150 mg/L. Engineering the thaxtomin cluster led to the production of multiple biosynthetic intermediates important to the chemical synthesis of new analogs. Further, the versatility of the biosynthetic system was revealed in the mutasynthesis as the production of 5-F-thaxtomin A whose structure was determined by a combination of MS and NMR analysis. Both natural and unnatural thaxtomins demonstrated weak anticancer activity toward Jurkat and PC-3 cancer cell lines and potent herbicidal activity in radish seedling assays. These results indicated that S. albus J1074 has the potential to overproduce thaxtomins and thereof, fostering their agricultural applications.
Contributed Poster Abstract
Uncovering the biosynthetic origins of cyclic phenolic natural products in pelagophyte algae.
Natural Product Discovery and Development in the Genomic Era
The chrysophaentins are cyclic phenolic natural products isolated from the colonial marine pelagophyte alga Chrysophaeum taylori, collected in the US Virgin Islands. These compounds inhibit the essential bacterial cell division protein FtsZ, a promising but unexploited target for antibiotic drug development, and show excellent therapeutic potential. However, chemical synthesis has so far been unable to provide a route for production of the drugs. We aim to use a synthetic biology approach to the preparation of the chrysophaentins, or advanced intermediates, by identifying the genes responsible for their production in a laboratory-cultivated strain of C. taylori, which we have shown to produce novel chrysophaentin analogues. In our biosynthetic hypothesis, these molecules derive from the action of a type III polyketide synthase on a phenylalanine derivative, followed by phenol coupling catalyzed by a cytochrome P450. We will sequence the genome of C. taylori, and characterize the complement of these gene families identified within.
Contributed Poster Abstract
Enediyne diversification by a cofactor-promiscuous methyltransferase
Natural Product Discovery and Development in the Genomic Era
SAM-dependent methyltransferases are the most prevalent catalysts in biology to perform chemo- and regio-selective alkylations, which play diverse roles ranging from modulating gene expression to tailoring secondary metabolite structures. Previous reports have demonstrated that certain methyltransferases have cofactor promiscuity and can accept SAM analogs. In this study, we present a structural and functional characterization of TnmH, a phenolic O-methyltransferase involved in tailoring the anthraquinone ring of the tiancimycins. We demonstrate its broad substrate scope and elucidate the timing of methylation in the biosynthesis of the tiancimycins. TnmH demonstrates cofactor promiscuity towards SAM analogs, as well as the ability to completely turnover non-native substrates and cofactors in preparative scale enzymatic reactions. We facilitate turnover by including S-adenosylhomocysteine nucleosidase from E. coli to alleviate co-product inhibition. This study sets the stage to use TnmH to generate tiancimycin analogs and bioconjugates.
Contributed Poster Abstract
Identification and heterologous expression of the whole valinomycin gene cluster from the endophytic strain Streptomyces sp. CBMAI 2042
Natural Product Discovery and Development in the Genomic Era
Actinobacteria are a phylum of gram-positive terrestrial or aquatic bacteria of significant importance for pharmaceutical industry and academic research due to their great potential to biosynthesize different types of natural products, including antibiotics and anticancer agents. Streptomyces is the largest bacterial genera from this phylum and several sequenced genomes have revealed an abundance of gene clusters codifying enzymes from secondary metabolism demonstrating an underestimated potential to produce important bioactive metabolites. Among the most useful natural products, those biosynthesized by nonribosomal peptides synthetases (NRPS) and polyketide synthases (PKS) share both high molecular complexity and therapeutic activity.
The NRPS gene cluster encoding the biosynthesis of a cyclic dodecadepsipeptide ionophore, valinomycin, was annotated from the whole genome sequencing of the endophytic strain Streptomyces sp. CBMAI 2042 isolated from Citrus ssp. The association of fermentation process, molecular networking and mass spectrometry imaging revealed the production of the ionophore by the endophytic strain. Additionally, the heterologous expression of the entire valinomycin gene cluster in S. coelicolor host was successfully promoted. Although the valinomycin titer resulting from these experiments (2 mg L-1) is still modest compared to the one achieved after optimization of enzyme-based high cell fed-batch cultivation in heterologous E. coli (10 mg L-1), this represents the first example of whole VLM gene cluster heterologous expression in Streptomyces. The heterologous host Streptomyces carrying the whole enzymatic arsenal required to promote the biosynthesis of VLM provides a versatile and unique platform for production of new valinomycin derivatives in future studies.
Contributed Poster Abstract
Enzymatic substitution of amides with thioamides on peptidic substrates
Natural Product Discovery and Development in the Genomic Era
Ribosomally synthesized post-translationally modified peptides (RiPPs) are a class of natural products that feature various degrees of enzymatic tailoring to a precursor peptide. The resulting chemical structures impart a diversity of bioactivities within this natural product class. Sulfur substitution of peptide backbone amide oxygens is a form of modification present in several RiPPs and methyl-coenzyme M reductase (MCR), the hallmark enzyme for anaerobic methanogenesis. Despite the significant impact thioamides are expected to exert on the conformational dynamics of a peptide or protein, their biosynthetic installation remains enigmatic. A recent study has shown that ycaO and tfuA genes are responsible for this modification on MCR. Analogous to azoline formation catalyzed by some YcaO homologs, we hypothesized that the YcaO protein installs the thioamide onto its peptidic substrate by the ATP-dependent activation of the amide backbone. In vitro reconstitution studies have revealed that YcaO proteins from methanogens indeed catalyze thioamide substitution on MCR-derived peptides in the presence of ATP and inorganic sulfide and that the TfuA partner protein appears to facilitate the reaction. The YcaO from Methanopyrus kandleri was shown to bind ATP in a manner characteristic to the YcaO family by crystallography study, which supported the ATP-dependent activation mechanism. Substrate tolerance and engagement of the YcaO was investigated by mass spectrometry-based enzymatic assays and fluorescent polarization-based binding studies. With the biochemical capability established, we subsequently surveyed the available bacterial and archaeal genomes for TfuA-associated YcaO-encoding biosynthetic gene clusters, which has revealed thioamidated peptides are likely severely underrepresented as RiPP class.
Contributed Poster Abstract
Analysis of dichloroisoeverninic acid biosynthesis in pursuit of novel everninomicin analogs
Natural Product Discovery and Development in the Genomic Era
The everninomicins are complex oligosaccharides with broad antimicrobial activity produced by Micromonospora carbonacea. However, toxicity issues and an inability to easily access analogs stifled their previous development. Recent crystal and cryo-EM structures of everninomicin A bound to the bacterial ribosome provide a detailed map of the vital interactions responsible for the everninomicins’ activity. The structures reveal crucial interactions between the 50S subunit and the aromatic dichloroisoeverninic acid (DCE) moiety of everninomicin. All natural everninomicins contain at least one iterative type I polyketide synthase (iPKS)-derived DCE moiety. We propose to access everninomicins with derivatized aromatic moieties via the biosynthetic machinery. In order to gain a better understanding of DCE biosynthesis, we deleted the four putatively associated genes: an iPKS, an O-methyltransferase (O-MT), a flavin-dependent halogenase (FDH), and a trans acyltransferase (AT). Functional analysis of these genes confirmed their assignment and provided seven novel everninomicin metabolites. These results demonstrate that the iPKS EvdD3 is responsible for the biosynthesis of the DCE core scaffold, orsellinic acid. Orsellinic acid is transferred to the terminal D-olivose sugar residue by AT EvdD1 to be subsequently tailored by O-MT EvdM5 and FDH EvdD2 to yield DCE. This work also revealed that transfer of the evernitrose sugar to D-olivose requires the presence of the nearly complete DCE ring. Current work is focused on in vitro analysis of the DCE-associated enzymes to evaluate incorporation of non-natural substrates. This work will further elucidate the biosynthesis of the everninomicins and provide novel analogs to revitalize this potent class of antibiotics.
Contributed Poster Abstract
Investigations of the biosynthesis and signaling function of salinipostin, an unusual phosphotriester gamma-butyrolactone compound from Salinispora
Natural Product Discovery and Development in the Genomic Era
Salinipostins are anti-malarial compounds from the marine actinomycete, Salinispora pacifica. These molecules possess a unique structure consisting of a rare phosphotriester and a gamma-butyrolactone ring that resembles signaling molecules such as A-factor from Streptomyces griseus. In S. griseus, A-factor regulates morphological development and secondary metabolism via the A-factor signaling cascade. The structural similarities between salinipostin and A-factor suggested that salinipostins play a similar regulatory role in the genus Salinispora, evoking the hypothesis that the salinipostin is the first signaling molecule identified in Salinispora. We identified a key A-factor-like biosynthesis gene (A-factor synthase, spt9) within biosynthetic operon that we suspect is the biosynthetic gene cluster of salinipostin. To confirm the assignment of the salinipostin BGC and investigate its function, we deleted spt9 in three species of Salinispora, which abolished salinipostin production. LC-MS chemical profile analyses using wild-type and salinipostin-deficient mutants indicated the function of the salinispostin as a regulator of the secondary metabolism. Our investigations of salinipostin biosynthesis have validated the BGC and lead to a proposed biosynthetic pathway. In further investigations, we expressed soluble enzymes in heterologous hosts and chemically synthesized substrates. With these resources, the early stage of biosynthesis constructing the gamma-butyrolactone structure was recapitulated in vitro starting from Spt9 reaction with SNAC mimic of beta-ketoacyl-ACP and dihydroxyacetone phosphate. Interrogation of salinipostins may represent a key step in unlocking the biosynthetic potential in Salinispora, and also study of biosynthetic pathway will enhance our basic understanding of the biochemistry responsible for the construction of a rare phosphotriester motif.
Contributed Poster Abstract
Production of novel glycosylated macrolactam analogues of the macrolide antibiotic YC-17 by chemoenzymatic synthesis
Natural Product Discovery and Development in the Genomic Era
YC-17, a 12-membered ring macrolide antibiotic produced from Streptomyces venezuelae ATCC 15439, consists of polyketide macrolactone 10-deoxymethynolide attached with D-desosamine. A combined approach including chemical synthesis and engineered cell-based generation of unnatural deoxyhexose sugar and aglycone biotransformation was used to generate a range of biologically active macrolactam analogues of YC-17. First, a macrolactam analogue of the YC-17 aglycone 10-dml, aza-(10-dml) (AZDM), was chemically synthesized and an engineered strain of S. venezuelae with its substrate-flexible glycosyltransferase was used to assemble the new glycosylated compounds. The S. venezuelae YJ028 mutant strain in which the entire biosynthetic gene cluster encoding the pikromycin PKS and desosamine biosynthetic enzymes were deleted was chosen as the biotransformation host, and different deoxy sugar biosynthetic gene cassettes and the genes encoding DesVII/DesVIII were expressed. Due to inherent flexibility, DesVII/DesVIII can transfer a range of structurally diverse TDP-sugars, demonstrating their potential for the structural diversification of macrolide antibiotics both in vitro and in vivo. As a result, engineered strains of S. venezuelae capable of generating desosamine and varied alternative sugar moieties produced eight novel macrolactam analogues of YC-17. Some of the novel AZDM glycosides present improved in vitro antibacterial activities against both erythromycin-susceptible and erythromycin-resistant pathogens and metabolic stabilities compared to erythromycin. These results demonstrate the successful application combining chemical synthesis with flexible glycosylation enzymes to generate a variety of novel glycosylated macrolactam analogues of YC-17 with potent antibiotic activity for drug discovery and development.
Contributed Poster Abstract
Antifungal activity of FK506 derivatives produced by engineering post-PKS modification steps
Natural Product Discovery and Development in the Genomic Era
FK506 is a 23-membered macrolide polyketide and used as a immunosuppressant drug for preventing the rejection of organ transplants. Furthermore, FK506 possesses various pharmaceutical potentials, including antifungal, neuroprotective, and neuroregenerative activities. A hybrid polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) system is responsible for the biosynthesis of the macrolide ring. This ring is further modified via remarkable two parallel pathways of post-PKS modification steps including C-9 oxidation by FkbD and 31-O-methylation by FkbM. The novel FK506 analogues, 31-O-demethyl-FK506, 9-deoxo-FK506, 9-deoxo-31-O-demethyl-FK506 and 9-deoxo-prolyl-FK506, were produced in fkbD or fkbM deletion mutant strain of Streptomyces sp. KCTC11604BP. FK506 derivatives were tested for fungal infections caused by Cryptococcus neoformans. Antifungal activities of all derivatives were maintained for C. neoformans. Especially, 31-O-demethyl-FK506 displayed significantly increased activity compared to FK506. Furthermore, these analogues did not have any neurotoxicity in vitro, but have remarkably lower immunosuppressive activity than FK506. These results would contribute to the development of new antifungal agents from FK506 with reduced immunosuppressive activity and toxicity. Overall, this study provides the potential of biosynthetic engineering approaches to generate improved bioactive molecules.
Contributed Poster Abstract
GC-MS metabolite profiling of siderophore-producing Bacillus subtilis rhizobacteria isolated from maize (Zea mays L.) rhizosphere
Natural Product Discovery and Development in the Genomic Era
Plant growth promoting rhizobacteria (PGPR) strain A1 identified as Bacillus subtilis through 16S rDNA gene sequencing was assayed for its metabolite production using GC-MS technique. This siderophore producing bacteria was tested against fungal pathogen Fusarium graminearum and bacterial pathogens Enterococcus faecalis and Bacillus cereus. It showed considerable resistance to these pathogens. The strain has also been previously tested for its plant growth promoting ability on maize plants. The metabolites in the form of volatile organic compounds (VOCs) produced were isolated and analyzed with Gas chromatography-mass spectrometry (GC-MS) using eight solvents for extraction. The solvents used are chloroform, ethyl acetate, diethyl ether, n-hexane, methanol, butanol, petroleum ether, and benzene. Two metabolites were identified from butanol extract while benzene, ethyl acetate and hexane all show one notable bioactive metabolite. Petroleum ether and methanol have the largest return of metabolites with five and fifteen respectively. Chloroform and diethyl ether did not detect any hit in their GC-MS chromatograms. The detected volatile compounds could be chemically grouped into ketones, alcohols, aldehydes, pyrazines, acids, esters, pyridines and benzene compounds. This result identifies the metabolite produced by Bacillus subtilis after successful trial in the field.
Contributed Poster Abstract
Improvement of crude extract yield of Streptomyces sp 796.1 with different carbon sources
Natural Product Discovery and Development in the Genomic Era
Production of secondary metabolites used in drugs is a characteristic of the genus Streptomyces. From a starfish an isolate identified as Streptomyces sp 796.1 with antiproliferative activity against nine tumor cell lines. However, the yield of the crude extract is 100 mg/L being about 50% for the bioactive fraction. The objective of this study was to evaluate the effect of the carbon source on the extract yield. A triplicate fermentation test was performed using R2A supplemented with artificial sea water as the base broth, the carbon source (dextrose 0.05%) being modified by L-arabinose 0.05% w/v, glycerol 1% v/v and starch 1% w/v. The chemical profiles obtained by mass spectrometry (UHPLC-ESI-MS) of the crude extracts with modified carbon source, the control and the bioactive fraction were compared. The masses of the control crude extract were 100% detected in the starch broth, 93% in L-arabinose and 78% in glycerol, whereas 91% of the masses of the bioactive fraction were detected in all extracts. However, the yield of the Starch broth was 33 mg/L, but the broth with glycerol increased the yield respect to the control (430 mg/L aprox). This indicates that the synthesis of the secondary metabolites with antiproliferative activity in this microorganism is not affected by the carbon source, with glycerol being the best alternative carbon source to improve yield.
Contributed Poster Abstract
Effect of temperature and pH on the metabolism of Escherichia coli for bioactive molecules.
Natural Product Discovery and Development in the Genomic Era
Biological systems are known to be robust and adaptable to the culture environment and such robustness is inherent in the biochemical and genetic networks. The overall regulation mechanism was clarified under both pH down-shift and temperature up-shift based on fermentation characteristics and gene transcript levels. Upon temperature up-shift down regulation of glucose uptake rate corresponds to the down regulation of ptsG caused by the up-regulation of mlc gene expression. More acetate was formed with lower biomass yield and less glucose consumption rate at pH 5.5 as compared to the case at pH 7.0 under both aerobic and anaerobic conditions. The gene expressions indicate that the down- regulation of the glucose uptake rate corresponds to the down-regulation of ptsG gene expression caused by the up-regulation of mlc gene which is under positive control of Crp. In accordance with up-regulation of arcA gene expression at acidic condition, the expressions of the TCA cycle-related genes such as icdA and gltA, and the respiratory chain gene cyoA were down-regulated, whereas cydB gene expression was up-regulated. The decreased activity of TCA cycle caused more acetate formation at lower pH. The research on stress response of a microorganism contributes to the variety of practical applications such as temperature-induced heterologous protein production simultaneous saccharification and fermentation (SSF) etc
Contributed Poster Abstract
Cell-free synthetic biology for natural product bio-discovery
Natural Product Discovery and Development in the Genomic Era
Cell-free synthetic biology is an emerging field that utilizes clarified lysates, or cell-free systems, for conducting expression from DNA to protein. These systems can be thought of as open factories that conserve the cell’s ability to conduct complex biochemistry but allow for addition and subtraction of necessary cofactors to drive reaction completion. We present Synvitrobio’s cell-free synthetic biology platform for natural product bio-discovery. In particular, we discuss Synvitrobio’s efforts to express functional ribosomal synthesized and post-translationally modified peptides (RiPPs) in its cell-free systems and corresponding antibiotic activity, and work characterizing the technical feasibility of expression of different natural product clusters in its cell-free platform.
Funding:
DARPA W911NF-17-C-0008
NIH 1 R43 AT009522-01
Contributed Poster Abstract
Toward an enzyme-coupled, bioorthogonal platform for methyltransferases
Natural Product Discovery and Development in the Genomic Era
Methyl group transfer from S-adenosyl-l-methionine (AdoMet) to various substrates including DNA, proteins, and natural products (NPs), is accomplished by methyltransferases (MTs). Analogs of AdoMet, bearing an alternative S-alkyl group can be exploited, in the context of an array of wild-type MT-catalyzed reactions, to differentially alkylate DNA, proteins, and NPs. This technology provides a means to elucidate MT targets by the MT-mediated installation of chemoselective handles from AdoMet analogs to biologically relevant molecules and affords researchers a fresh route to diversify NP scaffolds by permitting the differential alkylation of chemical sites vulnerable to NP MTs that are unreactive to traditional, synthetic organic chemistry alkylation protocols. The full potential of this technology is stifled by several impediments largely deriving from the AdoMet-based reagents, including the instability, membrane impermeability, poor synthetic yield and resulting diastereomeric mixtures. To circumvent these main liabilities, we present novel chemoenzymatic strategies that employ methionine adenosyltransferases (MATs) and methionine (Met) analogs to synthesize AdoMet analogs in vitro. Unstable AdoMet analogs are simultaneously utilized in a one-pot reaction by MTs for the alkylrandomization of NP scaffolds. As cell membranes are permeable to Met analogs, this also sets the stage for cell-based and potentially even in vivo applications. We will also present the use of Met and ATP isosteres in the context of MAT-catalyzed reactions toward the generation of highly stable AdoMet analogs and their downstream utilization by MTs. Finally, we will present the development, use, and results of a high-throughput screen to identify mutant-MAT/Met-analog pairs suitable for postliminary bioorthogonal applications.
Contributed Poster Abstract
Discovery, Pathway Engineering and Characterization of a Novel Rapamycin/FK506 Family Member by an Integrated Genome Mining Platform
Natural Product Discovery and Development in the Genomic Era
Natural products derived from polyketide synthases (PKS) represent an important class of commercially and medicinally relevant natural products, e.g., the antibiotic erythromycin, the immunosuppressants rapamycin and FK506, the anthelminthic agent avermectin, and the insecticide spinosad. To discover new natural products including PKS-derived compounds, Warp Drive Bio has assembled an enormous proprietary and searchable database of microbial genomes, many of which contain both known and novel biosynthetic gene clusters. To date, our database is comprised of sequence of ~135,000 genomes, estimated to encode the biosynthesis of ~3.5 million compounds. In our genome mining campaign, we identified WDB-002, a cyclic polyketide/NRPS natural product, by searching for biosynthetic clusters related to FK506 and Rapamycin. We describe methods to engineer production of WDB-002 in native and heterologous host systems by the overexpression of a non-cognate positive regulator. The structure of WDB-002 was determined by 1D- and 2D-NMR combined with mass spectrometry, and FKBP12-binding was confirmed by multiple assays. We describe the unexpected biosynthetic features of WDB-002 as compared to Rapamycin and FK506
Contributed Poster Abstract
Elucidating the biosynthesis of novel nucleoside antibiotic structural features
Natural Product Discovery and Development in the Genomic Era
Drug-resistant bacterial pathogens are rapidly becoming a widespread problem in the United States and across the globe. Meanwhile, new antibiotics entering the clinic are alarmingly scarce. Highly-modified nucleoside antibiotics, a class of natural products, target MraY bacterial translocase I. MraY is a clinically unexploited enzyme target, essential in peptidoglycan cell wall biosynthesis. This class of secondary metabolites have strong antibacterial activity against Gram-positive pathogens, yet are greatly underexplored. A subset of nucleoside antibiotics contains a characteristic 5'-C-glycyluridine (GlyU) core. The newly discovered nucleoside antibiotic, sphaerimicin, has a novel scaffold that makes it distinct from the GlyU-containing nucleosides known to date. In sphaerimicin, the GlyU core is ultimately appended to a dihydroxylated piperidine that is bridged to the 5'' amine of a 5-amino-5-deoxyribosyl moiety. Additionally, sphaerimicin features a unique 3'-sulfate. Our study will investigate these modifications, which we propose are partially achieved via incorporation of the 3'-sulfate using a novel aryl sulfatase, as well as significant scaffold extension with an aminotransferase and transketolase. Importantly, elucidating how this scaffold is biosynthesized may lead to discovery of new enzymatic chemistries that will power innovative chemoenzymatic synthesis and genome mining to uncover new natural products.
Contributed Poster Abstract
The biosynthetic mechanism of the antibiotic Capuramycin
Natural Product Discovery and Development in the Genomic Era
Natural products have played an important role in the discovery of antibacterial agents since the introduction of penicillin in the 1940s. Until 2012, natural products or derivatives of natural products contributed about 75% of the total FDA-approved antibacterial agents. However, the discovery of novel antibiotics has dramatically decreased over the last few decades while infectious disease, and notably tuberculosis (TB), remains a major threat to global health. Thus, the discovery and development of new antibiotics are urgently needed. Capuramycin, was a kind of nucleoside antibiotics discovered in screening programs for new antibiotics in 1980s. Capuramycin-type antibiotics include A-500359s, A-503083s, and A-102395. The biosynthetic gene cluster and pathway for A-500359 and A-503083 have been identified and characterized. But the biosynthesis mechanism for A-102395 has not been fully resolved. The function of several of the gene products is difficult to predict based solely on sequence analysis, which is perhaps not unexpected since the structure of the capuramycin family of antibiotics consists of several novel chemical features. We hope to characterize the function of gene cluster of A-102395 and provide new insight into the resistance and biosynthetic strategies for the capuramycin-type antibiotics.
Contributed Poster Abstract
Conversion of L-arabinose to L-ribulose using genetically engineered Candida tropicalis
Natural Product Discovery and Development in the Genomic Era
For the biological production of L-ribulose, conversion by enzymes or resting cells has been investigated. However, concentrated substrates, an additional purification step to remove borate, and the requirement for a cell cultivation and harvest steps before utilization of resting cells make the production process expensive and complex. Microbial fermentation may help overcome these limitations. In this study, we constructed a genetically engineered Candida tropicalis strain to produce L-ribulose by fermentation with a glucose/L-arabinose mixture. For the uptake of L-arabinose as a substrate and conversion of L-arabinose to L-ribulose, two heterologous genes, L-arabinose transporter and L-arabinose isomerase genes, were constitutively expressed in C. tropicalis under the GAPDH promoter. The Arabidopsis thaliana-originated L-arabinose transporter gene (STP2)-expressing strain exhibited a high L-arabinose uptake rate of 0.65 g/L/h and the expression of L-arabinose isomerase from Lactobacillus sakei 23K showed 30% of conversion (9 g/L) from 30 g/L of L-arabinose. Furthermore, glucose catabolite repression was overcome by the constitutive expression of L-arabinose isomerase under the GAPDH promoter. This genetically engineered strain can be used for L-ribulose production by fermentation using mixed sugars of glucose and L-arabinose.
Contributed Poster Abstract
Delineating the biosynthesis of capuramycin-type antibiotics
Natural Product Discovery and Development in the Genomic Era
Mycobacterium tuberculosis (TB) has been significant global concern for centuries despite the success of modern medicine. The standard therapeutic regimen against TB remains a long process involving an extensive drug cocktail, and the prominence of various drug resistant strains further complicates the treatment of TB. Thus, it is imperative to discover and develop new antibiotic compounds that will improve upon the therapeutic regimen and circumvent resistance mechanisms. An underexplored class of nucleoside natural products—the capuramycins—have excellent anti-TB properties and have been shown to potently inhibit MraY, a ubiquitous and essential enzyme that functions in bacterial cell wall biosynthesis. However, much is still unknown about the capuramycins’ mechanism of action against MraY and which functionalities are key for improving that activity. The biosynthetic pathway of capuramycin has been proposed, but many steps remain uncharacterized. We have chosen to investigate the enzymatic activities of the putative methyltransferase, CapK, and the putative glycosyltransferase, CapG, found in the gene cluster of capuramycin-type compound A-503083 with the intent of improving capuramycins’ bioactivity. From there, we hope to exploit the activities of CapK and CapG in order to diversify the capuramycin scaffold and assemble a library of novel capuramycins with favorable anti-TB properties: moderate potency, maintained target specificity, and no resistance.