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
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. System Biology
https://www.systemsbiology.org/about-systems-biology
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. Systems Biology is used to understand the development stages
from a single Streptomyces cell to a large antibiotic producing
bacterial colony.
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. Secondary metabolism of Streptomyces species Secondary
Metabolites Polyketides Nonribosomal peptides
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. 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. 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. 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. AdpA - Transcriptional Activator (T. Beppu et
al.,1995)
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. 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. 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. DoBISCUIT
21. antiMASH
22. Norine
23. Novel antibiotics Database
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. 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. 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. Approaches of system metabolic engineering Approaches of
system metabolic engineering Comparative trans- criptome analysis
Constraint based flux analysis Bioprocess engineering
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. 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. 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. 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. 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. Approaches of system biology Approaches of system biology
MS + Genome mining Microbial adaptive evolution Genome mining of
silent gene clusters
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. 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. 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. 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. 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