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

Pactamycin Streptomyces pactum

Despite early discovery, pactamycin has never been clinically used

Dimethyl Urea Moiety

6-Methylsalicylic Acid Moiety

3-Aminoacetophenone 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

Caffeine

Lovastatin

Tetracycline

wikipedia.org

Secondary metabolites can help cure disease

http://digital.nls.uk/scientists/biographies/alexander-fleming/

Erythromycin

Streptomycin

Vancomycin

Actinobacteria have been a major source of pharmaceutically relevant compounds

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 6-MSA 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

Pactamycin resistance: Methylate rRNA, prevent pactamycin from binding

Brodersen, D. E. et al. Cell. 2000.

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

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

To identify the 6MSA transferase, we used genetic engineering techniques

Aim 1: Find the 6-MSA transferase gene through the following studies:

1.

2.

3.

1.PCR of the resistance gene

2. Cloning into expression vector

3.Insertion into S. lividans genome

The resistance gene was successfully transferred to S. lividans for heterologous expression

1 2 3

Pactamycin resistance was conferred by the SAM-dependent methyltransferase gene

1. 10 µL of 27 mM pactamycin 2. 5 µL of 92 mM apramycin 3. 5 µL of 27 mM pactamycin.

S. lividans +met S. lividans 1236 WT

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

Seed culture (BTT media)

Production culture (Modified BTT media)

Supernatant

Crude Extract

(dissolve in MeOH)

Mass Spectrometry

Cell pellet

Ethyl acetate Extraction

n-Butanol Extraction

3 days, 30oC, 200 rpm

Isolation scheme of pactamycin metabolites

The putative acyltransferase gene does not appear to affect pactamycin biosynthesis

Inte

nsity

Inte

nsity

m/z m/z

Pactamycin has an m/z of 559, observed in both cultures

S. pactum ΔAT

S. pactum WT

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

TM-025 TM-026

The gene ptmR was disrupted in S. pactum ΔptmH, the TM-026 producing strain

PtmR?

Isolation of TM-025 from the S. pactum ΔptmRH

Isolated peak

The isolated peak was identified as TM-025 by Mass Spectroscopy

m/z

Rela

tive A

bundance

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

AfsA

A-Factor

ArpA

Transcriptional Repression

AdpA

Transcriptional Activator

Morphological development

Antibiotic Production

Ohnishi Y. Biosci Biotech Biochem. 2005

ArpA and AfsA are in the A-Factor Regulatory Cascade, which regulates secondary metabolism

Martín J. J Bacteriol. 2004. 186: 5197–5201.

PhoP links phosphate uptake with other nutrients and secondary metabolism

Each gene fragment was successfully cloned in the plasmid pTMN002

arpA fragment PCR In Vector

phoP fragment PCR In vector

afsA fragment PCR PCR of Vector

ΔarpA

1 2 MR

ΔphoP

1 2 MR

ΔafsA

1 MR 2

The three regulatory genes were successfully disrupted in S. pactum

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

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%

Summary

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

Acknowledgements