Glycosylation and Glycodiversification in Polyketide Antibiotics: Unraveling Biosynthetic Steps in Nogalamycin Formation

Soil-dwelling Streptomyces bacteria are known for their ability to produce biologically active compounds such as antimicrobial, immunosuppressant, antifungal and anticancer drugs. S. nogalater is the producer of nogalamycin, a potential anticancer drug exhibiting high cytotoxicity and activity against human topoisomerases I and II. Nogalamycin is an anthracycline polyketide comprising a four-ring aromatic backbone,a neutral deoxy sugar at C7, and an amino sugar attached via an O–C bond at C1 and a C–C bond between C2 and C5´´. This kind of attachment of the amino sugar is unusual thus making the structure of the compound highly interesting. The sugar is also associated with the biological activity of nogalamycin, as it facilitates binding to DNA. Furthermore, the sugar moieties of anthracyclines are often crucial for their biological activity. Together the interesting attachment of the amino sugar and the general reliance of polyketides on the sugar moieties for bioactivity have made the study of the biosynthesis of nogalamycin attractive. The sugar moieties are typically attached by glycosyltransferases, which use two substrates: the donor and the acceptor. The literature review of the thesis is focused on the glycosylation of polyketides and the possibilities to alter their glycosylation patterns. My own thesis work revolves around the biosynthesis of nogalamycin. We have elucidated the individual steps that lead to its rather unique structure. We reconstructed the whole biosynthetic pathway in the heterologous host S. albus using a cosmid and a plasmid. In the process, we were able to isolate new compounds when the cosmid, which contains the majority of the nogalamycin gene cluster, was expressed alone in the heterologous host. The new compounds included true intermediates of the pathway as well as metabolites, which were most likely altered by the endogenous enzymes of the host. The biological activity of the most interesting new products was tested against human topoisomerases I and II, and they were found to exhibit such activities. The heterologous expression system facilitated the generation of mutants with inactivated biosynthetic genes. In that process, we were able to identify the functions of the glycosyltransferases SnogE and SnogD, solve the structure of SnogD, discover a novel C1-hydroxylase system comprising SnoaW and SnoaL2, and establish that the two homologous non-heme α-ketoglutarate and Fe2+ dependent enzymes SnoK and SnoN catalyze atypical reactions on the pathway. We demonstrated that SnoK was responsible for the formation of the additional C–C bond, whereas SnoN is an epimerase. A combination of in vivo and in vitro techniques was utilized to unravel the details of these enzymes. Protein crystallography gave us an important means to understand the mechanisms. Furthermore, the solved structures serve as platforms for future rational design of the enzymes.Maaperässä esiintyvät Streptomyces-suvun bakteerit tunnetaan niiden kyvystä tuottaa biologisesti aktiivisia yhdisteitä. Nogalamysiini on potentiaalinen syöpälääke, joka on solumyrkyllinen ja aktiivinen ihmisen topoisomeraasi I:tä ja II:ta vastaan. S. nogalater -kanta tuottaa sitä. Nogalamysiini on antrasykliinipolyketidi, joka koostuu nelirenkaisesta aromaattisesta rungosta ja siihen C7-asemaan liittyneestä neutraalista sokerista sekä aminosokerista, joka on liittynyt sekä O–C-sidoksella asemaan C1 että C–C-sidoksella C2- ja C5´´-aseman välillä. Yhdisteen rakenne on hyvin mielenkiintoinen, sillä aminosokeri on kiinnittynyt hyvin epätyypillisellä tavalla ja on omalta osaltaan vastuussa nogalamysiinin biologisesta aktiivisuudesta, sillä se auttaa molekyylin sitoutumisessa DNA:han. Myös yleisellä tasolla sokeriosat ovat usein vastuussa polyketidien biologisesta aktiivisuudesta. Yhdessä rakenteen epätavallisuus sekä sokeriosien tärkeys ovat tehneet nogalamysiinin tutkimisesta olennaista. Sokeriosien kiinnittämisestä vastaa tyypillisesti sokerinsiirtäjäentsyymi glykosyylitransferaasi. Tämä entsyymi käyttää substraatteinaan kahta molekyyliä, aktivoitua sokeria ja vastaanottajamolekyyliä, johon sokeri liitetään. Työni kirjallisuuskatsaus keskittyy sokerien liittämiseen polyketideihin sekä mahdollisuuksiin muuttaa näitä sokerointimalleja. Omat tutkimustulokseni liittyvät nogalamysiinin biosynteesiin, jonka yksittäisiä askeleita olemme tutkineet selvittääksemme, mistä yhdisteen lähes ainutlaatuinen rakenne johtuu. Siirsimme nogalamysiinin biosynteesistä vastaavan geeniklusterin heterologiseen isäntään, S. albukseen, kosmidin ja plasmidin avulla. Kun siirsimme isäntäkantaan ainoastaan kosmidin, joka sisälsi suurimman osan klusterista, mutta ei kaikkia geenejä, havaitsimme kannan tuottavan uusia yhdisteitä. Työssä mittasimme uusien yhdisteiden bioaktiivisuuden ihmisen topoisomeraaseja I:tä ja II:ta vastaan, ja niissä huomattiin olevan aktiivisuutta. Biosynteesiin liittyvien geenien ilmentäminen heterologisessa isännässä mahdollisti inaktivointimutanttien teon. Työn aikana pystyimme selvittämään kahden sokerinsiirtäjäentsyymin, SnogE:n ja SnogD:n, toimintaa sekä SnogD:n rakenteen. Lisäksi löysimme uudenlaisen C1-aseman hydroksylaatiosysteemin, joka koostuu SnoaW:stä ja SnoaL2:sta, sekä kaksi homologista hemitöntä α-ketoglutaraatti- ja rauta2+ -riippuvaista entsyymiä: SnoK:n ja SnoN:n. Osoitimme, että SnoK on vastuussa epätyypillisen hiili–hiilisidoksen muodostumisesta, kun taas SnoN on epimeraasi. Entsyymien toiminnan yksityiskohtien selvittämiseksi käytimme sekä in vivo- että in vitro -tekniikoita. Proteiinikristallografian avulla selvitimme kolmen entsyymin kolmiulotteiset rakenteet, joiden avulla pystyimme ymmärtämään entsyymien reaktiomekanismeja paremmin. Lisäksi rakenteet helpottavat proteiinien muokkausta.Siirretty Doriast

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

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 highperforming 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 5fold 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.Peer reviewe

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

Mapping Nanocellulose- and Alginate-Based Photosynthetic Cell Factory Scaffolds:Interlinking Porosity, Wet Strength, and Gas Exchange

To develop efficient solid-state photosynthetic cell factories for sustainable chemical production, we present an interdisciplinary experimental toolbox to investigate and interlink the structure, operative stability, and gas transfer properties of alginate- and nanocellulose-based hydrogel matrices with entrapped wild-type Synechocystis PCC 6803 cyanobacteria. We created a rheological map based on the mechanical performance of the hydrogel matrices. The results highlighted the importance of Ca2+-cross-linking and showed that nanocellulose matrices possess higher yield properties, and alginate matrices possess higher rest properties. We observed higher porosity for nanocellulose-based matrices in a water-swollen state via calorimetric thermoporosimetry and scanning electron microscopy imaging. Finally, by pioneering a gas flux analysis via membrane-inlet mass spectrometry for entrapped cells, we observed that the porosity and rigidity of the matrices are connected to their gas exchange rates over time. Overall, these findings link the dynamic properties of the life-sustaining matrix to the performance of the immobilized cells in tailored solid-state photosynthetic cell factories.</p

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

Photosynthetically produced sucrose by immobilized Synechocystis sp. PCC 6803 drives biotransformation in E. coli

Background: Whole-cell biotransformation is a promising emerging technology for the production of chemicals. When using heterotrophic organisms such as E. coli and yeast as biocatalysts, the dependence on organic carbon source impairs the sustainability and economic viability of the process. As a promising alternative, photosynthetic cyanobacteria with low nutrient requirements and versatile metabolism, could ofer a sustainable platform for the heterologous production of organic compounds directly from sunlight and CO2. This strategy has been applied for the photoautotrophic production of sucrose by a genetically engineered cyanobacterium, Synechocystis sp. PCC 6803 strain S02. As the key concept in the current work, this can be further used to generate organic carbon compounds for diferent heterotrophic applications, including for the whole-cell biotransformation by yeast and bacteria. Results: Entrapment of Synechocystis S02 cells in Ca2+-cross-linked alginate hydrogel beads improves the specifc sucrose productivity by 86% compared to suspension cultures during 7 days of cultivation under salt stress. The process was further prolonged by periodically changing the medium in the vials for up to 17 days of efcient production, giving the fnal sucrose yield slightly above 3000 mg l −1 . We successfully demonstrated that the medium enriched with photosynthetically produced sucrose by immobilized Synechocystis S02 cells supports the biotransformation of cyclohexanone to ε-caprolactone by the E. coli WΔcscR Inv:Parvi strain engineered to (i) utilize low concentrations of sucrose and (ii) perform biotransformation of cyclohexanone to ε-caprolactone. Conclusion: We conclude that cell entrapment in Ca2+-alginate beads is an efective method to prolong sucrose production by the engineered cyanobacteria, while allowing efcient separation of the cells from the medium. This advantage opens up novel possibilities to create advanced autotroph–heterotroph coupled cultivation systems for solar-driven production of chemicals via biotransformation, as demonstrated in this work by utilizing the photosynthetically produced sucrose to drive the conversion of cyclohexanone to ε-caprolactone by engineered E. coli.</p