A pharmaceutical model for the molecular evolution of microbial natural products

Microbes are talented chemists with the ability to generate tremendously complex and diverse natural products which harbor potent biological activities. Natural products are produced using sets of specialized biosynthetic enzymes encoded by secondary metabolism pathways. Here, we present a two‐step evolutionary model to explain the diversification of biosynthetic pathways that account for the proliferation of these molecules. We argue that the appearance of natural product families has been a slow and infrequent process. The first step led to the original emergence of bioactive molecules and different classes of natural products. However, much of the chemical diversity observed today has resulted from the endless modification of the ancestral biosynthetic pathways. The second step rapidly modulates the pre‐existing biological activities to increase their potency and to adapt to changing environmental conditions. We highlight the importance of enzyme promiscuity in this process, as it facilitates both the incorporation of horizontally transferred genes into secondary metabolic pathways and the functional differentiation of proteins to catalyze novel chemistry. We provide examples where single point mutations or recombination events have been sufficient for new enzymatic activities to emerge. A unique feature in the evolution of microbial secondary metabolism is that gene duplication is not essential but offers opportunities to synthesize more complex metabolites. Microbial natural products are highly important for the pharmaceutical industry due to their unique bioactivities. Therefore, understanding the natural mechanisms leading to the formation of diverse metabolic pathways is vital for future attempts to utilize synthetic biology for the generation of novel molecules.</p

Crystal structure of the glycosyltransferase SnogD from the biosynthetic pathway of nogalamycin in Streptomyces nogalater

The glycosyltransferase SnogD from Streptomyces nogalater transfers a nogalamine moiety to the metabolic intermediate 3′,4′-demethoxynogalose-1-hydroxynogalamycinone during the final steps of biosynthesis of the aromatic polyketide nogalamycin. The crystal structure of recombinant SnogD, as an apo-enzyme and with a bound nucleotide, 2-deoxyuridine-5′-diphosphate, was determined to 2.6 Å resolution. Reductive methylation of SnogD was crucial for reproducible preparation of diffraction quality crystals due to creation of an additional intermolecular salt bridge between methylated lysine residue Lys384 and Glu374* from an adjacent molecule in the crystal lattice. SnogD is a dimer both in solution and in the crystal, and the enzyme subunit displays a fold characteristic of the GT-B family of glycosyltransferases. Binding of the nucleotide is associated with rearrangement of two active-site loops. Site-directed mutagenesis shows that two active-site histidine residues, His25 and His301, are critical for the glycosyltransferase activities of SnogD both in vivo and in vitro. The crystal structures and the functional data are consistent with a role for His301 in binding of the diphosphate group of the sugar donor substrate, and a function of His25 as a catalytic base in the glycosyl transfer reaction.VRPublishe

The Rieske Oxygenase SnoT Catalyzes 2''-Hydroxylation of L-Rhodosamine in Nogalamycin Biosynthesis

Nogalamycin is an anthracycline anti-cancer agent that intercalates into the DNA double helix. The binding is facilitated by two carbohydrate units, L-nogalose and L-nogalamine, that interact with the minor and major grooves of DNA, respectively. However, recent investigations have shown that nogalamycin biosynthesis proceeds through the attachment of l-rhodosamine (2′′-deoxy-4′′-epi-L-nogalamine) to the aglycone. Herein, we demonstrate that the Rieske enzyme SnoT catalyzes 2′′-hydroxylation of L-rhodosamine as an initial post-glycosylation step. Furthermore, we establish that the reaction order continues with 2–5′′ carbocyclization and 4′′ epimerization by the non-heme iron and 2-oxoglutarate-dependent enzymes SnoK and SnoN, respectively. These late-stage tailoring steps are important for the bioactivity of nogalamycin due to involvement of the 2′′- and 4′′-hydroxy groups of ᴸ-nogalamine in hydrogen bonding interactions with DNA.</p

Enzymatic Synthesis of the C-glycosidic Moiety of Nogalamycin R

Carbohydrate moieties are essential for the biological activity of anthracycline anticancer agents such as nogalamycin, which contains l-nogalose and l-nogalamine units. The former of these is attached through a canonical O-glycosidic linkage, but the latter is connected via an unusual dual linkage composed of C–C and O-glycosidic bonds. In this work, we have utilized enzyme immobilization techniques and synthesized l-rhodosamine-thymidine diphosphate (TDP) from α-d-glucose-1-TDP using seven enzymes. In a second step, we assembled the dual linkage system by attaching the aminosugar to an anthracycline aglycone acceptor using the glycosyl transferase SnogD and the α-ketoglutarate dependent oxygenase SnoK. Furthermore, our work indicates that the auxiliary P450-type protein SnogN facilitating glycosylation is surprisingly associated with attachment of the neutral sugar l-nogalose rather than the aminosugar l-nogalamine in nogalamycin biosynthesis.</p

Genotyping-Guided Discovery of Persiamycin A From Sponge-Associated Halophilic Streptomonospora sp. PA3

Microbial natural products have been a cornerstone of the pharmaceutical industry, but the supply of novel bioactive secondary metabolites has diminished due to extensive exploration of the most easily accessible sources, namely terrestrialStreptomycesspecies. The Persian Gulf is a unique habitat for marine sponges, which contain diverse communities of microorganisms including marine Actinobacteria. These exotic ecosystems may cradle rare actinomycetes with high potential to produce novel secondary metabolites. In this study, we harvested 12 different species of sponges from two locations in the Persian Gulf and isolated 45 symbiotic actinomycetes to assess their biodiversity and sponge-microbe relationships. The isolates were classified intoNocardiopsis(24 isolates),Streptomyces(17 isolates) and rare genera (4 isolates) by 16S rRNA sequencing. Antibiotic activity tests revealed that culture extracts from half of the isolates displayed growth inhibitory effects against seven pathogenic bacteria. Next, we identified five strains with the genetic potential to produce aromatic polyketides by genotyping ketosynthase genes responsible for synthesis of carbon scaffolds. The combined data led us to focus onStreptomonosporasp. PA3, since the genus has rarely been examined for its capacity to produce secondary metabolites. Analysis of culture extracts led to the discovery of a new bioactive aromatic polyketide denoted persiamycin A and 1-hydroxy-4-methoxy-2-naphthoic acid. The genome harbored seven gene clusters involved in secondary metabolism, including a tetracenomycin-type polyketide synthase pathway likely involved in persiamycin formation. The work demonstrates the use of multivariate data and underexplored ecological niches to guide the drug discovery process for antibiotics and anticancer agents

Evolution‐Guided Engineering of Non‐Heme Iron Enzymes Involved in Nogalamycin Biosynthesis

Microbes are competent chemists that are able to generate thousands of chemically complex natural products with potent biological activities. Key to the formation of this chemical diversity has been the rapid evolution of secondary metabolism. Many enzymes residing on these metabolic pathways have acquired atypical catalytic properties in comparison to their counterparts found in primary metabolism. The biosynthetic pathway of the anthracycline nogalamycin contains two such proteins, SnoK and SnoN, belonging to non‐heme iron and 2‐oxoglutarate‐dependent mono‐oxygenases. In spite of structural similarity, the two proteins catalyse distinct chemical reactions; SnoK is a C2–C5′′ carbocyclase, whereas SnoN catalyses stereoinversion at the adjacent C4′′ position. Here we have identified four structural regions involved in the functional differentiation and generated 30 chimeric enzymes to probe catalysis. Our analyses indicate that the carbocyclase SnoK is the ancestral form of the enzyme from which SnoN has evolved to catalyse stereoinversion at the neighboring carbon. The critical step in the appearance of epimerization activity has likely been the insertion of three residues near the C‐terminus, which allow repositioning of the substrate in front of the iron center. The loss of the original carbocyclization activity has then occurred with changes in four amino acids near the iron center that prohibit alignment of the substrate for formation of the C2–C5′′ bond. Our study provides detailed insights into the evolutionary processes that have enabled Streptomyces soil bacteria to become the major source of antibiotics and antiproliferative agents.</div

Differential regulation of undecylprodigiosin biosynthesis in the yeast-scavenging Streptomyces strain MBK6

Streptomyces are efficient chemists with a capacity to generate diverse and potent chemical scaffolds. The secondary metabolism of these soil-dwelling prokaryotes is stimulated upon interaction with other microbes in their complex ecosystem. We observed such an interaction when a Streptomyces isolate was cultivated in a media supplemented with dead yeast cells. Whole-genome analysis revealed that Streptomyces sp. MBK6 harbors the red cluster that is cryptic under normal environmental conditions. An interactive culture of MBK6 with dead yeast triggered the production of the red pigments metacycloprodigiosin and undecylprodigiosin. Streptomyces sp. MBK6 scavenges dead-yeast cells and preferentially grows in aggregates of sequestered yeasts within its mycelial network. We identified that the activation depends on the cluster-situated regulator, mbkZ, which may act as a cross-regulator. Cloning of this master regulator mbkZ in S. coelicolor with a constitutive promoter and promoter-deprived conditions generated different production levels of the red pigments. These surprising results were further validated by DNA-protein binding assays. The presence of the red cluster in Streptomyces sp. MBK6 provides a vivid example of horizontal gene transfer of an entire metabolic pathway followed by differential adaptation to a new environment through mutations in the receiver domain of the key regulatory protein MbkZ

Single cell mutant selection for metabolic engineering of actinomycetes

Actinomycetes are important producers of pharmaceuticals and industrial enzymes. However, wild type strains require laborious development prior to industrial usage. Here we present a generally applicable reporter-guided metabolic engineering tool based on random mutagenesis, selective pressure, and single-cell sorting. We developed fluorescence-activated cell sorting (FACS) methodology capable of reproducibly identifying high-performing individual cells from a mutant population directly from liquid cultures. Actinomycetes are an important source of catabolic enzymes, where product yields determine industrial viability. We demonstrate 5-fold yield improvement with an industrial cholesterol oxidase ChoD producer Streptomyces lavendulae to 20.4 U g−1 in three rounds. Strain development is traditionally followed by production medium optimization, which is a time-consuming multi-parameter problem that may require hard to source ingredients. Ultra-high throughput screening allowed us to circumvent medium optimization and we identified high ChoD yield production strains directly from mutant libraries grown under preset culture conditions. Genome-mining based drug discovery is a promising source of bioactive compounds, which is complicated by the observation that target metabolic pathways may be silent under laboratory conditions. We demonstrate our technology for drug discovery by activating a silent mutaxanthene metabolic pathway in Amycolatopsis. We apply the method for industrial strain development and increase mutaxanthene yields 9-fold to 99 mg l−1 in a second round of mutant selection. In summary, the ability to screen tens of millions of mutants in a single cell format offers broad applicability for metabolic engineering of actinomycetes for activation of silent metabolic pathways and to increase yields of proteins and natural products.</p

The mechanism of the nucleo-sugar selection by multi-subunit RNA polymerases

RNA polymerases (RNAPs) synthesize RNA from NTPs, whereas DNA polymerases synthesize DNA from 2dNTPs. DNA polymerases select against NTPs by using steric gates to exclude the 2 ' OH, but RNAPs have to employ alternative selection strategies. In single-subunit RNAPs, a conserved Tyr residue discriminates against 2 ' dNTPs, whereas selectivity mechanisms of multi-subunit RNAPs remain hitherto unknown. Here, we show that a conserved Arg residue uses a two-pronged strategy to select against 2 ' dNTPs in multi-subunit RNAPs. The conserved Arg interacts with the 2 ' OH group to promote NTP binding, but selectively inhibits incorporation of 2 ' dNTPs by interacting with their 3 ' OH group to favor the catalytically-inert 2 ' -endo conformation of the deoxyribose moiety. This deformative action is an elegant example of an active selection against a substrate that is a substructure of the correct substrate. Our findings provide important insights into the evolutionary origins of biopolymers and the design of selective inhibitors of viral RNAPs. RNA and DNA polymerases need to discriminate efficiently against closely related nucleotide triphosphate substrates. Here, the authors show that a conserved Arg residue is the major determinant of selectivity against deoxyribonucleoside substrates by multisubunit RNA polymerases

Characterization and overproduction of cell-associated cholesterol oxidase ChoD from Streptomyces lavendulae YAKB-15

Cholesterol oxidases are important enzymes with a wide range of applications from basic research to industry. In this study, we have discovered and described the first cell-associated cholesterol oxidase, ChoD, from Streptomyces lavendulaeYAKB-15. This strain is a naturally high producer of ChoD, but only produces ChoD in a complex medium containing whole yeast cells. For characterization of ChoD, we acquired a draft genome sequence of S. lavendulaeYAKB-15 and identified a gene product containing a flavin adenine dinucleotide binding motif, which could be responsible for the ChoD activity. The enzymatic activity was confirmed in vitro with histidine tagged ChoD produced in Escherichia coli TOP10, which lead to the determination of basic kinetic parameters with K-m 15.9 mu M and k(cat) 10.4/s. The optimum temperature and pH was 65 degrees C and 5, respectively. In order to increase the efficiency of production, we then expressed the cholesterol oxidase, choD, gene heterologously in Streptomyces lividans TK24 and Streptomyces albus J1074 using two different expression systems. In S. albus J1074, the ChoD activity was comparable to the wild type S. lavendulaeYAKB-15, but importantly allowed production of ChoD without the presence of yeast cells