1. Do symbiotic bacteria synthesize anticancer drugs isolated from Annona plants?
Casey Harless

2. Acetogenins Cherimoya Fruit: Annona cherimola
Metabolite group unique to Annona plants
Highly bio-active
Potent antitumor
Enzymes responsible for synthesis?
Up until 2016
Annona shorthand

3. Acetogenins from symbiotic bacteria
Streptomyces
large, well studied genus
Known biosynthetic potential
Examples:
Streptomycin antibiotic
Adriamycin chemotherapy drug
(Taechowisan et al, 2016)

4. Acetogenin Biosynthesis
Unstudied
Identify biosynthetic genes
Biosynthetic Gene Cluster (BGC)
BGC?

5. Why not culture Streptomyces?
Many strains per plant
2. Compound Extraction
Acetogenin Present?
1. Culture
Silent BGC
Un-culturable

6. Biosynthetic Parallels
Acetogenin
Streptomyces hormone
Monensin
Terminal
Lactone

7. Terminal lactone biosynthesis
A-factor like synthases (AfsA)
AfsA is only necessary gene (Kato et. al., 2007)
Acyl ACP Pi
ACP
Adapted from Morin et. al., 2012
Niu et. al., 2016

8. Polyketide Synthesis Similar to fatty acid synthesis Acetogenin
Polyketide Portion

9. Polyketide Synthase Modular megasynthase Module 1 2 3 4 5 6 7
Terminal Domain
ACP
H2O

10. Proposed Recombination Site
Hypothesis
AfsA fused with PKS gene in symbiotic Streptomyces -> acetogenins
Proposed Recombination Site
Terminal Domain
H2O
ACP
Acetogenin Backbone
AfsA
H2O
ACP
Acetogenin Backbone
BGC high recombination
Proximity of PKS and AfsA genes

11. Amplify predicted PKS-AfsA hybrid
Degenerate PCR primers
Forward primer: conserved KS domain of PKS module
Reverse primer: Streptomyces specific AfsA homologue
PKS/AfsA hybrid (partial)
AfsA homologue
KS domain

12. Workflow Transcripts Annona plant tissue PKS/AfsA hybrid transcript
Total RNA extraction
(bead beating)
Annona
cDNA synthesis (reverse strand)
RNA
Streptomyces
cDNA
AfsA primer (r’)
Band Visualization
(2-6kb)
dsDNA PKS/AfsA hybrid
PKS/AfsA hybrid
native AfsA
Gel Electrophoresis
PCR (KS f and AfsA r primers
RNAse treatment
ssDNA

13. Band Visualization (Results)
Positive control: atpD
Multiple reverse primers used to target AfsA
Forward KS primer
ladder
+-control
AfsA 1
AfsA 2
AfsA 3
AfsA 4
AfsA 5
3kb
466bp

14. Questions?
Thank you

15. References
Feher M, Schmidt JM. Property Distributions: Differences Between Drugs, Natural Products, and Molecules from Combinatorial Chemistry. ChemInform. 2003;34. Xue Y, Zhao L, Liu H-W, Sherman DH. A gene cluster for macrolide antibiotic biosynthesis in Streptomyces venezuelae: Architecture of metabolic diversity. Proceedings of the National Academy of Sciences. 1998;95:12111–6. Newman DJ, Cragg GM. Natural Products as Sources of New Drugs from 1981 to Journal of Natural Products. 2016;79:629–61. Taechowisan T, Singtotong C, Phutdhawong W. Antibacterial and Antioxidant Activities of Acetogenins from Streptomyces sp. VE2; An Endophyte in Vernonia cinerea (L.) Less. Journal of Applied Pharmaceutical Science. 2016;:067–72. Niu G, Chater KF, Tian Y, Zhang J, Tan H. Specialised metabolites regulating antibiotic biosynthesis in Streptomyces spp. FEMS Microbiology Reviews. 2016;40:554–73. Morin JB, Adams KL, Sello JK. Replication of biosynthetic reactions enables efficient synthesis of A-factor, a γ-butyrolactone autoinducer from Streptomyces griseus. Organic & Biomolecular Chemistry. 2012;10:1517. Kroiss J, Kaltenpoth M, Schneider B, Schwinger M-G, Hertweck C, Maddula RK, et al. Symbiotic streptomycetes provide antibiotic combination prophylaxis for wasp offspring. Nature Chemical Biology. 2010;6:261–3. Sun Y, Hahn F, Demydchuk Y, Chettle J, Tosin M, Osada H, et al. In vitro reconstruction of tetronate RK-682 biosynthesis. Nature Chemical Biology [Internet]. 2010;6:99–101 Han B, Cao Y-X, Li Z-M, Wu Z-X, Mao Y-Q, Chen H-L, et al. Annonaceous acetogenin mimic AA005 suppresses human colon cancer cell growth in vivo through downregulation of Mcl-1. Acta Pharmacologica Sinica. 2018

16. Acetogenin Biosynthesis: Why should we care?
Annona Tissue
Extraction

17. Control Positive True Positive False Positive
Metabolically active Streptomyces in Annona tissue
False Positive
Unlikely

18. Control Negative True Negative False Negative:
Acetogenins not extracted from S. VE2
Different biosynthetic route
False Negative:
PCR failed

19. Experimental Positive (one band)
True Positive
Hypothesis supported
False Positive
Non-target amplification
PKS/AfsA present but function not related to acetogenin biosynthesis

20. Experimental Negative (no bands)
True Negative
Acetogenins are synthesized by different route
PKS/AfsA trans-acting
False Negative
PKS/AfsA transcript concentration too low
PCR issues
Tm, dimer, hairpins, etc.
AfsA not targeted by any of the 5 r’ primers

21. Native Acetogenin
Synthetic Acetogenin mimic
Han et. al., 2018

22. Likely Alternative Hypothesis
Tetronate Biosythesis in Streptomyces
Target of PCR: “Glycerate utilization operon”
Adapted from Sun et. al., 2010

23. PKS/AfsA hybrids PKS-AfsA hybrids exist in other bacterial phyla
Function unknown
Module 1
Module 3
Module 2
AfsA homologue

24. Poorly Aligning Proteins