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Novel sources of actinomycete diversity for detection of antimicrobial agents with pharmaceutical applications

   
Project

QLK3-2001-01783

Cell factory area

3.3.4

EU Contribution

2 093 578 Euro

Duration

36 months

Type

Research project

Starting date

01-10-2001

Keywords
actinomycete diversity
Streptomyces lividans
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ABSTRACT
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Novel anti-microbial agents are needed to overcome problems of resistance and provide protection against infection. The control of microbial growth is also an essential part of product preservation in the cosmeceutical industry. Despite high through put screening the discovery rate of new antimicrobial compounds has declined. We aim to reverse this trend by exploiting biodiversity within the uncultured microbial populations in the environment and clone habitat metagenomes using bacterial artificial chromosomes.

To evaluate the cloning procedure, habitat diversity will be analysed at the functional and taxonomic level. Targeted assays will identify antimicrobial activities of clones for use in control of infections, as transdermal applications and as preservatives for pharmaceuticals. We aim to combine chemical novelty with novelty in application.

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OBJECTIVES
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The problems of selective culture will be solved whilst simultaneously evaluating alternative approaches to exploiting bacterial diversity in natural environments, thus obtaining novel antimicrobial agents. This will be achieved by using a systematic approach to produce genetic diversity from the soil both by cultivation and other means. The availability of the streptomycete artificial chromosome provides the opportunity to clone high molecular weight DNA from total community extracts. The biosynthetic potential of uncultured actinomycete groups will be evaluated. Cloned pathways provide considerable advantages because of their ease of detection and the project will also use hosts such as S.lividans. New molecules will be identified and tested for diverse uses in systemic, dermal and transdermal applications and as biocides for preservation of pharmaceutical products.

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DESCRIPTION OF THE WORK
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Total community DNA will be extracted from environmental samples, which have been chosen because of their taxonomic and functional diversity within the actinomycete populations. High molecular weight DNA will be cloned into specialised vectors for expression in a Streptomyces lividans host. Environmental gene libraries will be evaluated for diversity in target genes such as 16S rRNA and recA, biological activity and metabolite profiles. In parallel, new methods will be applied to isolate a diverse range of actinomycetes from the same samples. The culturable groups will be screened in the same way, as both will be subjected to metabolite analysis using HPLC-DAD and MALD-MS for rapid detection of new activities. The ultimate objective is to detect new antimicrobial agents using targeted screens.

WP1 aims to produce a molecular toolkit for screening actinomycete diversity in soils, isolates and clone libraries. WP2 will involve the screening of soil samples using the toolkit and select 4 samples for detailed analysis. WP3 involves the selective isolation of actinomycetes from the selected environments WP4 involves the provision of four environmental clone libraries which together will provide some 3000 isolates and 60,000 clones. WPs 5 and 6 will determine metabolite profiles. WP7 will determine biological activity. Highly specific targeted assays will include in vitro and in vivo activity with a focus on the control of targeted microbial species. The precise chemical structure of new molecules will be established in WP7 using Atmospheric Pressure Chemical Ionisation mass spectra and NMR. Further analysis of stability and toxicology will enable new applications for drug delivery and the use as preservatives to be established in WP8.

The main goal of the project is the provision and testing of an alternative strategy to selective culture.

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DELIVERABLES
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  • Development of a molecular toolkit for evaluating diversity within actinomycete populations.
  • A strategy for cloning high molecular weight DNA from soil and stable integration into the chromosome of S.lividans host.
  • Rapid methods for detection of altered fermentation profiles of clone libraries using MALD-MS.
  • Provision of molecular structure and activity profiles and development of novel applications for use of antimicrobial agents as anti-infectives, dermal patch treatments and biocidal agents.

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CONSORTIUM
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COORDINATOR
  Prof. E M H Wellington
Department of Biological Sciences, University of Warwick
CV4 7AL Coventry, United Kingdom
Tel: +44 02476523184
Fax: +44 02476523701
ewellington@bio.warwick.ac.uk

PARTNERS
  Flavia Marinelli
Microbial Fermentation, Biosearch Italia SpA
Via R. Lepetit, 34
21040 Gerenzano (Varese), Italy
Tel: +39 296474239
Fax: +39 296474400
fmarinelli@biosearch.it

Prof. Dr. Hans-Peter Fiedler
Mikrobiologisches Institut, Universität Tübingen
Auf der Morgenstelle 28
72076 Tübingen, Germany
Tel: +49 70712972079
Fax: +49 7071295999
hans-peter.fiedler@uni-tuebingen.de

Prof. Dr. L. Dijkhuizen
Microbiology
PO Box 14
University of Groningen
9750 AA Aaren, The Netherlands
Tel: +31 503632150
Fax: +31 503632154
l.dijkhuizen@biol.rug.nl

Joachim Vater
Max-Volmer-Institut für Biophysikalische Chemie und Biochemie
Technische Universität Berlin
Franklinstr. 29
10587 Berlin, Germany
Tel: +49 3031425609
Fax: +49 3031424783
Vater@chem.TU-berlin.de

Prof. M Goodfellow & Dr. A.Ward
Department of Agricultural and Environmental Science
University of Newcastle upon Tyne
NE1 7RU Newcastle upon Tyne, United Kingdom
Tel: +44 1912227709
Fax: +44 1912225228
Alan.Ward@ncl.ac.uk

Dr. Spiros Potinos
Institution Lavipharm S A
P O Box 59
19002, Agias Marinas
Peania-Attica, Greece
Tel: +30 16691201
Fax: +30 16691209
sfotinos@lavipharm.gr

Prof. A Karagouni
Department of Biology
Institution University of Athens
157 81 Athens, Greece
Tel: +30 17243345
Fax: +30 17234136
akarag@cc.uoa.gr
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