Presentation on theme: "Metabolic engineering by interspecies protoplast fusion of angucycline producing streptomycetes A.Kobylyanskyy 1, A.Luzhetskyy 2, A.Bechthold 2 and V.Fedorenko."— Presentation transcript:
Metabolic engineering by interspecies protoplast fusion of angucycline producing streptomycetes A.Kobylyanskyy 1, A.Luzhetskyy 2, A.Bechthold 2 and V.Fedorenko 1 Ivan Franko National University, Department of Genetics and Biotechnology, Hrushevskyy str. 4, 79005 Lviv, Ukraine 1, Albert-Ludwigs-Universität, Institut für Pharmazeutische Wissenschaften, Stefan-Meier-Straße 19, 79104 Freiburg, Germany 2 Protoplast fusion proved itself as an important molecular genetic method widely used for the improvement of secondary metabolite production. It allows recombination events to occur throughout the genome and at a long range thereby modifying bunches of genes in order to improve it. This signifies reorientation of attention from directed evolution of single enzymes to optimization of pathways. The approach used in this work involves protoplast fusion of two or more actinomycete strains, belonging to different species. The particularity is however that the parental strains carry resistance marker insertions in angucycline biosynthetic genes clusters. The presence of resistance cassettes within the clusters stipulates for the recombination between them in course of protoplast fusion. The recombination is directed in the way that double resistant fused mutants carry the hymeric cluster, consistent of the fragments of both parental ones. By applying this technology we aimed to provide the production of novel biologically active secondary metabolites and extend our knowledge about recombination events and enzyme substrate specificity. Fig.1. Fused protoplasts of Streptomyces cells under an electron microscope Strain name Gene inactivated Gene function Major II metabolites Resistance marker S.antibioticus ∆simA8simA8 mono- oxygenase 12-dehydro- rabelomycin spectinomycin S.cyanogenus ∆lanGT1lanGT1 glycosyl- transferase tetrangulolspectinomycin S.cyanogenus ∆lanGT2 +urdGT2 lanGT1 glycosyl- transferase 9-C-D-olivosyl- tetrangulol spectinomycin, apramycin S. fradiae ΔurdQ/RurdQ, urdR sugar dehydra- tase, reductase urdamycin Xapramycin S. globisporus Е7lndE mono- oxygenase prejadomycinhygromycin S. globisporus М12lndM2 oxygenase- dehydrogenase tetrangulol, tetrangomycin hygromycin Micromonospora sp.Tü6368 ΔsaqGT3 saqGT3 glycosyl- transferase SaqAE3spectinomycin Tab.1. Strains used in the experiments of protoplast fusion. Angucyclic antibiotics are quinone natural polyketide products bearing a characteristic four-ring frame of the aglycon moiety, which is assembled in an angular manner. There are few hundreds of them characterised so far, all of which to various measure possess antitumor activity. For three decades protoplast fusion has been successfully used to modify the phenotypic traits of streptomycetes (Fig.1). In contrast to other established techniques, a high frequency of gene transfer and recombination can be achieved by fusing complete protoplast genomes. Recently this process was optimized towards a recursive multiparental fusion and combined with high-throughput screening formats and that greatly accelerated the overall process of strain development. 1 2 3 4 5 6 7 1,1 kb Rec5 S. fradiae ∆urdQ/R S. globisporus M12 CONCLUSION Metabolic engineering by means of interspecies protoplast fusion with the consequent screening of antibiotic resistant recombinants turned out to be enormously fruitful in the case of angucycline producing mutants with damadged post-PKS steps. The recombination within antibiotic biosynthetic gene clusters is thus being provoked by the selective antibiotic pressure. As a result of one fusion of properly selected parental strains we obtained a recombinant producing a whole collection of compounds. Generating it by means of sequential unit step targeted mutagenesis took perennial effort. Protoplast fusion is a promising method to proceed in strain modification when inductive mutagenesis reaches its deadlock. It allows breeding and improvement of subgenomic DNA fragments thereby modifying metabolic pathways. Beside pure combinatorial profit simultaneously a great deal of data on gene functions and nature of enzymes interaction can be won. In order to ascertain that Rec5 recombinant includes genes from both parental biosynthetic clusters we managed to amplify urdGT2 and lndGT1 native glycosyltransferase genes (Fig.2) from its genomic DNA tetrangomycin 11-hydroxy tetrangomycin 4-hydroxy tetrangomycin tetrangulol S.globisporus М12 prx I E F A B C D M2 O P G H Q R S T U V W GT2 X GT1 J Z1 lnd- cluster S. fradiae Δ urdQ/R urdamycin X urd -cluster EFA B C D LMO G H QRS T GT2 GT1a KJZ1Z3J2 GT1b Int GT1c Ret.t [min]Mass [M-H] - CompoundUV/Vis spectrum 3,7565100-1tetrangomycin 3,8485Aquayamycinaquayamycin 459912b-DUG, 100-2aquayamycin 4,8581Diolyvosyltetrangomycintetrangomycin 6,1581Urdamycin Aaquayamycin 8,5469Urdamycin Maquayamycin 8,8337Hydroxytetrangomycintetrangomycin 9,2695Urdamycin Baquayamycin 11,2469Urdamycin Kaquayamycin 11,9827Urdamycin Oaquayamycin 14,2321Tetrangomycintetrangomycin 17,3697Urdamycin Raquayamycin 22433Olivosyltetranguloltetrangulol 22,4563Diolivosyltetranguloltetrangulol 22,7693Triolivosyltetranguloltetrangulol Olivosyltetrangulol: R=H Diolyvosyltetrangomycin R= Triolivosyl- tetrangulol: R= Diolivosyl- tetrangulol: Fig.2. The morfology of recombinant strain Rec5 Fig.4. PCR of Rec5 genomic DNA with primers complementing urdGT2 and lndGT1 Fig 3. The mutations and major secondary metabo- lites of the strains applied for protplast fusion Fig 5. Novel metabolites produced by Rec5 The mutant strains recently generated in our laboratory (table1) have been used in our experiments of protoplast fusion. All of them carry risistance marker insertions within different post-polyketidesynthetic (PKS) tailoring genes. Since PKS genes themselves are homologous in genomes of mentioned strains, we provide recombination directly within the biosynthetic clusters of corresponding antibiotics. Such fused mutants should be able to restore the missing glycosylation or oxygenation steps owing to complementation of metabolic pathways by the enzymes of both parental metabolic pools. Moreover, the flexibility of post-PKS modifying enzymes allows the introduction of new functional groups or attachment of additional sugars in unusual for both parents positions. The morphology of the majority of obtained fusants resembled one of the parental strains. Nevertheless, several of them displayed completely different shape and color of colonies. The most remarkable were the recombinants of S. fradiae ∆urdQ/R x S. globisporus M12 called Rec5 (Fig.2). To our astonishment, generated Rec5 fused mutant turned out to produce about 40! different compounds of angucyclic nature. We succeeded to ascertain the structure only for some of them (tab.2). Namely we detected putative aquayamycin, tetrangomycin, tetrangulol and urdamycinone derivatives. The parental strains of Rec5 are the insertional oxygenase gene mutant of S. globisporus 1912 (producer of landomycin E) and olivose-biosynthesis deficient mutant of S. fradiae Tü2717 (Fig.3). Both parents secrete unglycosylated and not fully oxygenated angucyclic intermediates. Lanes: 1.1kb DNA ladder 2. Rec5 with lndGT2 primers 3. Rec5 with urdGT2 primers Positive controls 4. S. fradiae Δ urdQ/R with urdGT2 5. S. globisporus M12 with lndGT2 Negative controls 6. S. fradiae Δ urdQ/R with lndGT2 7. S. globisporus M12 with urdGT2 Tab.2. Some angucyclic products of the recombinant Rec5 strain Among the others there are glycosylated urdamycin derivatives and even fully oxygenated and glycosylated end product – urdamycin A, wich is an evidence of mutual complementarity of missing enzymes in both clusters. So far we isolated four novel compounds, secreted by Rec5 recombinant shown on figure 5.