1. 1. K S Hwang et al., 2014 Biotechnology advances 32(2014)255-269 Presented by NEERAJ CHAUHAN M.Sc 2nd year DMBT www.Fmicrobewiki.kenyon.edu Systems biology and biotechnology of Streptomyces species for the production of secondary metabolites
2. 2. http://imgkid.com/cute-question-mark-wallpaper.shtml
3. 3. System biology Holistic approach To understand biological complexity The study of biological systems, viewed as integrated and interrelated networks of genes, proteins, and biochemical reactions http://genomics.energy.gov/
4. 4. System Biology https://www.systemsbiology.org/about-systems-biology
5. 5. Applications of system biology Investigate complex processes involved in the development of diseases Identify therapeutic targets and drugs. Overcome pathway redundancy causing resistance to treatment in cancer and other diseases. Determine the relevance of specific molecule or pathway for the overall behaviour of the system or in the pathogenesis of a disease. Assess the suitability of new chemical or biological entities as drugs De-risk scientific decisions and reduce research costs
6. 6. Systems Biology is used to understand the development stages from a single Streptomyces cell to a large antibiotic producing bacterial colony.
7. 7. Streptomyces Scientific classification Kingdom: Bacteria Phylum: Actinobacteria Class: Actinobacteria Order: Actinomycetales Family: Streptomycetaceae Genus: Streptomyces Aerobic and Gram-positive soil bacteria . Filamentous growth. (Global Biodiversity Information Facility) http://mml.sjtu.edu.cn/
8. 8. Linear chromosome (8 Mb-10Mb) with high GC content (about 72 % ). Biosynthetic gene cluster Secondary metabolites Polyketide , Nonribosomal peptides , Lactams , Terpenes (K.S Hwang et al.,2014)Tylosin Clavulanate
9. 9. Secondary metabolites http://imgkid.com/cute-question-mark-wallpaper.shtml
10. 10. Secondary metabolites produced during shifting phase from substrate mycelium to sporulation (Dyson, 2011; Flardh and Buttner, 2009). Important source of medicines Antibiotics( e.g., daptomycin) Immunosuppressants (e.g., rapamycin) Antifungals (e.g., amphotericin B) Antiparasitics (e.g., ivermectin ) Anticancers (e.g., doxorubicin) (Newman and Cragg, 2007)
11. 11. Secondary metabolism of Streptomyces species Secondary Metabolites Polyketides Nonribosomal peptides
12. 12. Polyketides Diverse group of natural products Synthesized through decarboxylative condensation of carboxylic acids by polyketide synthases (PKSs). Polyketide synthases Type I Type II Type III (Yu et al., 2012)
13. 13. Type I PKSs- Multifunctional and multimodular proteins Example macrolides ( tylosin ). Consist several modules. Each module carries three core domains: acyltransferase (AT) acyl carrier protein (ACP) ketosynthase. Type II PKSs - Complex of monofunctional proteins producing aromatic polyketides (e.g., tetracyclines and actinorhodin) Type III PKS- Homodimer of keto-reductase enzyme. Five groups are characterized to date . Example-Flaviolin RppA from the Gram-positive, filamentous bacterium Streptomyces griseus. It catalyzes the synthesis of 1,3,6,8-tetrahydroxynaphthalene (THN) (Yu et al., 2012)
14. 14. Nonribosomal peptide Short (2 to about 50 amino acids) Synthesized by nonribosomal peptide synthetases (NRPSs), Multimodular and multifunctional enzymes Each module consists of adenylation peptidyl carrier protein (PCP or thiolation), condensation domains Epimerization domains Examples Actinomycin B , Coelichelin
15. 15. Regulation and signal transduction of secondary metabolism When the cell senses a stressful condition of nutrient depletion, or specific small signaling molecules (butyrolactones ) In regulation global transcriptional regulators ( AdpA ) and two-component systems (PhoP/R ) play role
16. 16. AdpA - Transcriptional Activator (T. Beppu et al.,1995)
17. 17. PhoR-PhoP- for phosphate control in S. coelicolor Secondary metabolites repressed by high inorganic phosphate concentrations. Thus production has to be performed under phosphate-limiting conditions Actinorhodin and undecylprodigiosin increased in phoP or phoR-phoP deletion mutants PhoR -sensor protein of two-component systems PhoP - related to ROII subfamily of response regulators Mutants unable to synthesize alkaline phosphatase Formation of secondary metabolites occur under phosphate-limiting conditions Nothing is known about the molecular mechanism of phosphate control of expression of the corresponding biosynthetic genes
18. 18. Databases to knowledgebases for understanding and engineering secondary metabolism of Streptomyces species Databases provide genomic information of Streptomyces species Emphasize on gene clusters involved in secondary metabolism. Classification of Databases and Knowledgebases Genome annotation/mining database Secondary metabolites Database
19. 19. Classification Database name URL Refs Genome annotation/ mining database DoBISCUIT http://www.bio.nite.go.j p/pks/ Ichikawa et al.(2013) antiSMASH http://antismash.second arymetabolites.org Blin et al.(2013) Secondary metabolites database NORINE http://bioinfo.lifl.fr/nori ne/ Caboche et al.(2008) Novel Antibiotics Database http://www.antibiotics.o r.jp/journal/database/da tabase-top.htm Caboche et al.(2008) (K.S. Hwang et al.,2014)
20. 20. DoBISCUIT
21. 21. antiMASH
22. 22. Norine
23. 23. Novel antibiotics Database
24. 24. Constraint-based Flux analysis of Metabolism Flux balance analysis mathematical approach analyze the flow of metabolites through a metabolic network. Constraint-based reconstruction and analysis (COBRA) Constraint-based flux analyses Simulate metabolic network models, Used to design strategies of systems metabolic engineering for different Streptomyces species Identification of genes Analysis of intracellular flux distributions
25. 25. System metabolic engineering of Streptomyces Engineering biosynthetic pathways for low cost production of useful products . Rational metabolic engineering play role in strain improvement for the overproduction of secondary metabolites using Streptomyces species.
26. 26. Representative engineering approaches include: Improving precursor and cofactor pools Removing competing pathways Over expressing transcriptional regulators Improving enzyme conversion rates Over expressing genes conferring tolerance for the toxicity of produced antibiotics Systems biology provide targets for gene manipulations and bioprocess engineering for the maximal production of secondary metabolites of interest
27. 27. Approaches of system metabolic engineering Approaches of system metabolic engineering Comparative trans- criptome analysis Constraint based flux analysis Bioprocess engineering
28. 28. Comparative transcriptome analysis Transcriptome profiling allow Parallel analysis of dynamic expression of all genes Enhance the production of actinorhodin in S. coelicolor. Gene downregulator (pfkA2) of actinorhodin biosynthesis is deleted. pfkA2 gene- encode 6-phosphofructokinase in S. coelicolor Inactivate pfkA2 gene Redirect fluxes towards the pentose phosphate pathway Increased the NADPH production Enriched acetyl-CoA pool Both necessary for the production of actinorhodin. Hwang et al.,2014
29. 29. Constraint based flux analysis Boost production of Tacrolimus immunosuppressant from Streptomyces tsukubaensis Genome scale metabolic model constructed Subjected to minimization of metabolic adjustment Maximal specific growth rate fixed Non-zero fluxes increased Overexpression targets predicted Selected as targets if contribute to tacrolimus production 2 gene knockout targets (gdhA, ppc) and 4 gene amplification targets (dahp, gdhA, accA2, and zwf2) selected Mutant in which gdhA deleted and dahp, accA2, and zwf2 overexpressed Results in 2.8-fold increase profduction Hwang et al.,2014
30. 30. Constraint-based reconstruction and analysis approaches to the study of Streptomyces species Host organism Metabolic model Simulation method Refs Streptomyces coelicolor Metabolic model having over 200 reactions Maximizing biomass formation rate at limited consumption rates of carbon, nitrogen, sulfur, phosphate, and potassium Naeimpoor and Mavituna (2000) Streptomyces coelicolor Genome-scale metabolic model having over 400 reactions Maximizing CDA production rate under limited glucose consumption rate and constrained biomass formation rate Single gene knockout simulations to improve CDA production rate Kim et al.,2004 Streptomyces roseosprorus A metabolic model having 138 reactions and 109 metabolites Comparison of flux distributions under two optimizations: maximization of daptomycin biosynthesis rate and specific Hung et al.,2012
31. 31. Bioprocess engineering Systems metabolic engineering Considers the effects of variables As effect microbial metabolism Recent examples include effects of varying Initial seeding volumes Components in the medium for cell cultivation and secondary metabolite production. Initial seeding volume represented by pitching ratio, Affect the production titer of streptolydigin from Streptomyces lydicus. Streptolydigin production was greater at 30% pitching ratio (v/v), compared to 1% or 10% ratio. Hwang et al.,2014
32. 32. Production of novel secondary metabolites Homology based approach Search for homologues of secondary metabolite producing genes in the target microorganism. Identification of novel gene clusters Lead to systems metabolic engineering for the enhanced production of secondary metabolites Disadvantage Not suitable for screening of novel compounds as Certain secondary metabolites produce under special circumstances Relationships between the compound of interest and their biosynthetic genes. (Gottelt et al., 2010)
33. 33. Approaches of system biology Approaches of system biology MS + Genome mining Microbial adaptive evolution Genome mining of silent gene clusters
34. 34. Mass spectrometry combined with genome mining (Chen et al., 2012) Identifies novel secondary metabolites . Able to detect expressed genes and their products For discovery of novel gene clusters and their products DNA sequence for novel NRPSs and PKSs and LCMS analysis of the cell supernatant used to deduce the structure of unknown secondary metabolites. Hwang et al.,2014
35. 35. Microbial adaptive evolution (Charusanti et al., 2012) Streptomyces species to produce novel metabolites. Demonstrated with a competition-based adaptive laboratory evolution platform When multiple Streptomyces clavuligerus replicates were adaptively evolved against methicillin resistant Staphylococcus aureus N315 in this manner, a strain emerged that acquired the ability to constitutively produce holomycin. ContinueCharusanti et al., 2012
36. 36. genome resequencing revealed that the evolved strain had lost pSCL4, a large 1.8 Mbp plasmid acquired several single nucleotide polymorphisms in genes that have been shown to affect secondary metabolite biosynthesis. Hwang et al.,2014
37. 37. Genome mining of silent gene clusters (Baltz, 2011) To identify and produce novel secondary metabolites from silent gene clusters that are not expressed under the typical lab settings The key is to select an ideal heterologous host and a set of strong promoters for the proper expression of the silent gene cluster. Bioinformatics analysis of the completely sequenced genome of Streptomyces chattanoogensis L10 revealed a silent angucycline biosynthetic gene cluster. The overexpression of a pathway-specific activator gene under the constitutive ermE* promoter successfully triggered the expression of theangucycline biosynthetic genes. Hwang et al.,2014
38. 38. Conclusion Streptomyces species -source of medically useful compounds Many biochemical mysteries need to be elucidated Search for novel secondary metabolites and their biosynthetic gene clusters Relevant genomic databases on Streptomyces species constructed. Systems biological approaches useful in the discovery and engineering studies of Streptomyces species