Investigations of Pactamycin Biosynthesis
Andrew Osborn
Research Mentors: Dr. Taifo Mahmud, Dr. Kerry McPhail
A Bioresource Research Thesis Seminar Presentation
Streptomyces pactum produces the antitumor
antibiotic pactamycin
Streptomyces pactum
Pactamycin
Despite early discovery, pactamycin has never
been clinically used
Dimethyl
Urea
Moiety
3-Aminoacetophenone
Moiety
6-Methylsalicylic
Acid Moiety
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
Tetracycline
Caffeine
Lovastatin
wikipedia.org
Secondary metabolites can help cure disease
http://digital.nls.uk/scientists/biographies/alexander-fleming/
Actinobacteria have been a major source of
pharmaceutically relevant compounds
Streptomycin
Erythromycin
Vancomycin
wikipedia.org
Streptomyces are the source of most bioactive
secondary metabolites
Awad et al. (2013). J. Teknol
Fewer antibacterial agents are approved for
pharmaceutical use
Silver L L Clin. Microbiol. Rev. 2011;24:71-109
Drug-resistant microbes are emerging faster than
new drugs are developed
Clatworthy. NatChemBiol. 2007
Antimalarial resistance is increasing worldwide
Analogs TM-025 and TM-026 have superior
antimalarial activity compared to pactamycin
Lu W. Chem&Bio. 2011
Pactamycin analogs are created through genetic
engineering
Lu W. Chem&Bio. 2011
Small structural changes can cause differences in
biological activity
Pactamycin
TM-026
TM-025
Pactamycin inhibiting protein synthesis…different
mode of action proposed
Pactamycin
Brodersen, D. E. et al. Cell. 2000.
Tourigny DS. J Mol Bio. 2013.
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
The gene ptmR was identified as a potential 6MSA transferase in the gene cluster
Ito T. Chembiochem. 2009
Ketoacyl-(ACP) synthases (KAS)-III are invovled
in the early steps of fatty acid synthesis
Lai CY. J Biol Chem. 2003
PtmR, a putative KAS-III, has homologs in other
biosynthetic gene clusters
The pactamycin resistance gene has not been
found in the gene cluster
No resistance gene?
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. 2000.
Pactamycin resistance:
Methylate rRNA, prevent
pactamycin from binding
Ballesta, J. J Bacteriol. 1991.
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
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
1.
2.
3.
The resistance gene was successfully transferred
to S. lividans for heterologous expression
1
2
3
1. PCR of the resistance gene
2. Cloning into expression vector
3. Insertion into S. lividans genome
Pactamycin resistance was conferred by the SAMdependent methyltransferase gene
S. lividans +met
S. lividans 1236 WT
1. 10 µL of 27 mM pactamycin
2. 5 µL of 92 mM apramycin
3. 5 µL of 27 mM pactamycin.
1.
2.
3.
The putative acyltransferase gene was
successfully disrupted
1
2
3
1. PCR of acyltransferase gene fragment
2. Acyltransferase gene fragment in pTMN002
3. PCR of apramycin gene and acyltransferase gene
in S. pactum genome
Isolation scheme of pactamycin metabolites
Seed culture (BTT media)
3 days, 30oC, 200 rpm
Production culture (Modified BTT media)
Supernatant
Cell pellet
Ethyl acetate Extraction
n-Butanol Extraction
Crude Extract
(dissolve in MeOH)
Mass Spectrometry
The putative acyltransferase gene does not
appear to affect pactamycin biosynthesis
S. pactum ΔAT
Intensity
Intensity
S. pactum WT
m/z
m/z
Pactamycin has an m/z of 559, observed
in both cultures
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
The gene ptmR was disrupted in S. pactum
ΔptmH, the TM-026 producing strain
PtmR?
TM-025
TM-026
Isolation of TM-025 from the S. pactum ΔptmRH
Isolated peak
Relative Abundance
The isolated peak was identified as TM-025 by
Mass Spectroscopy
m/z
1H
NMR confirmed that the S. pactum ΔptmRH
produced the pactamycin analog TM-025
S. pactum ΔptmRH
1H
NMR of TM-025
Disruption of ptmR indicates that KAS-III enzymes
can transfer aromatic carboxylic acids
Aims
1. Identify the 6-MSA transferase gene

2. To understand the regulation of pactamycin
biosynthesis – to improve pactamycin production
TM-026
Three known global regulatory genes were
identified in the S. pactum chromosome
• afsA: Enzyme in A-factor biosynthesis
• arpA: Transcriptional Repressor
• phoP: Transcriptional Regulator
ArpA and AfsA are in the A-Factor Regulatory
Cascade, which regulates secondary metabolism
AfsA
ArpA
Transcriptional
Repression
A-Factor
AdpA
Morphological
development
Transcriptional
Activator
Antibiotic
Production
Ohnishi Y. Biosci Biotech Biochem. 2005
PhoP links phosphate uptake with other nutrients
and secondary metabolism
Martín J. J Bacteriol. 2004. 186: 5197–5201.
Each gene fragment was successfully cloned in the
plasmid pTMN002
arpA fragment
PCR
In Vector
afsA fragment
phoP fragment
PCR
PCR of Vector PCR
In vector
The three regulatory genes were successfully
disrupted in S. pactum
ΔarpA
1
2 MR
ΔphoP
1
2 MR
ΔafsA
1 MR 2
1. Apramycin resistance gene primers
2. Reverse primers from homology region and Apramycin resistance
Production of TM-026 was quantified using HPLC
TM-026 was produced by the ΔarpA mutant at a
slightly higher rate than the control
The ΔafsA mutant did not consistently produce
TM-026 more than the control
The ΔphoP mutant produced a similar amount of
TM-026 as the control
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%
Acknowledgements
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American Society of Pharmacognosy
OSU College of Pharmacy
Dr. Taifo Mahmud
Dr. Kerry McPhail
Dr. Kate Field
Mostafa Abugreen & TM Lab Members
Wanda Crannell