Investigations of Pactamycin Biosynthesis Andrew Osborn

Transcription

1 Investigations of Pactamycin Biosynthesis Andrew Osborn Research Mentors: Dr. Taifo Mahmud, Dr. Kerry McPhail A Bioresource Research Thesis Seminar Presentation

2 Streptomyces pactum produces the antitumor antibiotic pactamycin Streptomyces pactum Pactamycin

3 Despite early discovery, pactamycin has never been clinically used Dimethyl Urea Moiety 6-Methylsalicylic Acid Moiety 3-Aminoacetophenone Moiety

4 What are secondary metabolites? Secondary metabolites, also called natural products, are molecules produced by organisms that are not directly involved in their normal development, growth, and reproduction. Secondary metabolites have many functions: Cell-to-cell signaling Protection from stress (ph, salt, etc.) Pigmentation Caffeine Tetracycline Lovastatin wikipedia.org

5 Secondary metabolites can help cure disease

6 Actinobacteria have been a major source of pharmaceutically relevant compounds Streptomycin Erythromycin Vancomycin wikipedia.org

7 Streptomyces are the source of most bioactive secondary metabolites Awad et al. (2013). J. Teknol

8 Fewer antibacterial agents are approved for pharmaceutical use Silver L L Clin. Microbiol. Rev. 2011;24:71-109

9 Drug-resistant microbes are emerging faster than new drugs are developed Clatworthy. NatChemBiol. 2007

10 Antimalarial resistance is increasing worldwide

11 Analogs TM-025 and TM-026 have superior antimalarial activity compared to pactamycin Lu W. Chem&Bio. 2011

12 Pactamycin analogs are created through genetic engineering Lu W. Chem&Bio. 2011

13 Small structural changes can cause differences in biological activity Pactamycin TM-026 TM-025

14 Pactamycin inhibiting protein synthesis different mode of action proposed Pactamycin Brodersen, D. E. et al. Cell Tourigny DS. J Mol Bio

15 Aims 1. Identify the 6-MSA transferase gene 2. To understand the regulation of pactamycin biosynthesis to improve pactamycin production 6-Methylsalicylic Acid Moiety TM-026

16 The gene ptmr was identified as a potential 6- MSA transferase in the gene cluster Ito T. Chembiochem. 2009

17 Ketoacyl-(ACP) synthases (KAS)-III are invovled in the early steps of fatty acid synthesis Lai CY. J Biol Chem. 2003

18 PtmR, a putative KAS-III, has homologs in other biosynthetic gene clusters

19 The pactamycin resistance gene has not been found in the gene cluster No resistance gene?

20 Pactamycin resistance is conferred through methylation of the 16S rrna Pactamycin s mode of action: Bind rrna, prevent protein synthesis Brodersen, D. E. et al. Cell Pactamycin resistance: Methylate rrna, prevent pactamycin from binding Ballesta, J. J Bacteriol

21 A putative acyltransferase gene is clustered with the pactamycin resistance gene SAM-methyltransferase gene Putative Acyltransferase gene 1. Resistance gene sequence was given by Dr. Michael Calcutt (personal communication) 2. Aligned overlapping contigs from the genome sequence of S. pactum 3. Used Frameplot 4.0 to predict genes in the new sequence 4. Searched for similar genes using Blast 5. Assign putative function to any genes present SAM-methyltransferase gene Putative acyltransferase gene

22 To identify the 6MSA transferase, we used genetic engineering techniques Aim 1: Find the 6-MSA transferase gene through the following studies: 1. Confirm pactamycin resistance Express the pactamycin resistance gene in S. lividans 2. Disrupt the putative acyltransferase gene Create a S. pactum strain with a non-functional putative acyltransferase gene Identify pactamycin metabolites 3. Characterize a S. pactum mutant lacking the ptmr gene Identify pactamycin metabolites

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24 The resistance gene was successfully transferred to S. lividans for heterologous expression PCR of the resistance gene 2. Cloning into expression vector 3. Insertion into S. lividans genome

25 Pactamycin resistance was conferred by the SAMdependent methyltransferase gene S. lividans +met S. lividans 1236 WT µl of 27 mm pactamycin 2. 5 µl of 92 mm apramycin 3. 5 µl of 27 mm pactamycin.

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27 The putative acyltransferase gene was successfully disrupted PCR of acyltransferase gene fragment 2. Acyltransferase gene fragment in ptmn PCR of apramycin gene and acyltransferase gene in S. pactum genome

28 Isolation scheme of pactamycin metabolites Seed culture (BTT media) 3 days, 30 o C, 200 rpm Production culture (Modified BTT media) Supernatant Cell pellet Crude Extract (dissolve in MeOH) Ethyl acetate Extraction n-butanol Extraction Mass Spectrometry

29 Intensity Intensity The putative acyltransferase gene does not appear to affect pactamycin biosynthesis S. pactum ΔAT S. pactum WT m/z m/z Pactamycin has an m/z of 559, observed in both cultures

30 The resistance gene and putative acyltransferase gene have homologs in other gene clusters Homology between: Pactamycin resistance gene region Lactonamycin biosynthetic gene cluster In total, the regions of homology are 78% identical

31 The gene ptmr was disrupted in S. pactum ΔptmH, the TM-026 producing strain PtmR? TM-025 TM-026

32 Isolation of TM-025 from the S. pactum ΔptmRH Isolated peak

33 Relative Abundance The isolated peak was identified as TM-025 by Mass Spectroscopy m/z

34 1 H NMR confirmed that the S. pactum ΔptmRH produced the pactamycin analog TM-025 S. pactum ΔptmRH 1 H NMR of TM-025

35 Disruption of ptmr indicates that KAS-III enzymes can transfer aromatic carboxylic acids

36 Aims 1. Identify the 6-MSA transferase gene 2. To understand the regulation of pactamycin biosynthesis to improve pactamycin production TM-026

37 Three known global regulatory genes were identified in the S. pactum chromosome afsa: Enzyme in A-factor biosynthesis arpa: Transcriptional Repressor phop: Transcriptional Regulator

38 ArpA and AfsA are in the A-Factor Regulatory Cascade, which regulates secondary metabolism AfsA ArpA Transcriptional Repression A-Factor Morphological development AdpA Transcriptional Activator Antibiotic Production Ohnishi Y. Biosci Biotech Biochem. 2005

39 PhoP links phosphate uptake with other nutrients and secondary metabolism Martín J. J Bacteriol :

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41 Each gene fragment was successfully cloned in the plasmid ptmn002 arpa fragment PCR In Vector afsa fragment PCR PCR of Vector phop fragment PCR In vector

42 The three regulatory genes were successfully disrupted in S. pactum ΔarpA ΔphoP ΔafsA 1 2 MR 1 2 MR 1 MR 2 1. Apramycin resistance gene primers 2. Reverse primers from homology region and Apramycin resistance

43 Production of TM-026 was quantified using HPLC

44 TM-026 was produced by the ΔarpA mutant at a slightly higher rate than the control

45 The ΔafsA mutant did not consistently produce TM-026 more than the control

46 The ΔphoP mutant produced a similar amount of TM-026 as the control

47 Summary 1. The 6-MSA transferase was identified in the pactamycin biosynthetic gene cluster 2. The pactamycin resistance gene was confirmed, but no pactamycin biosynthetic gene was located near it 3. We identified global regulatory elements that are involved in the regulation of pactamycin biosynthesis, and increased pactamycin production by 50%

48 Acknowledgements American Society of Pharmacognosy OSU College of Pharmacy Dr. Taifo Mahmud Dr. Kerry McPhail Dr. Kate Field Mostafa Abugreen & TM Lab Members Wanda Crannell