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Volume 2
     

Demonstration of increased yield and productivity in selected commercial organisms by strategic transformation of key genes from Aspergillus niger (ANTICO)

   
Project

QLK3-2002-02038

Cell factory area

3.3.2

EU Contribution

1,122,358 Euro

Duration

36 months

Type

DM

Starting date

01 November 2002

Keywords
anaplerotic pathways
glycolitic flux
itaconic acid
oxidative phosphorylation
ABSTRACT

This programme will demonstrate that the transfer of specific genes from an Aspergillus niger strain, which produces citric acid with close to theoretical efficiency, into other commercial micro-organisms, will enhance the pool of precursors in those organisms and lead to increased yields and production rates for their end products. The two key enzymes, pfk and aox1, lead to increased glycolytic flux and the uncoupling of respiration from ATP production respectively, which, together with high glucose levels, will lead to increased metabolic fluxes and steady state levels of intermediates. Such a demonstration of high yields and productivity will then be transferred directly to the production strains of European manufacturers.

OBJECTIVES

The main objective of the proposed project is to demonstrate that the transfer of some strategic genes from Aspergillus niger, one of the most efficient industrial micro-organisms, into other commercially important micro-organisms will increase the productivity and/or yield of their bio-products. It will be achieved by transferring specific A. niger genes that enhance anaplerotic pathways, the reactions that replenish citric acid cycle intermediates. Increased productivity will be demonstrated in Aspergillus terreus, producing itaconic acid from primary metabolites and secondary metabolite lovastatin, Pichia pastoris, used as a host organisms for heterologous protein expression, Streptomyces rimosus, excreting antibiotic oxytetracyclins, Streptomyces clavuligerus, making clavulanic acid and Lactococcus lactis, producing the lantibiotic nisin A.

DESCRIPTION OF THE WORK

Anaplerotic pathways and the productivity of recipient organisms will be enhanced by increasing the metabolic flux through glycolysis and by uncoupling oxidative phosphorylation from ATP synthesis. The first task will be achieved by introducing the gene coding for 6-phosphofructo-1-kinase from a A. niger strain which was found to be up-regulated by phosphorylation, while alternative respiration will be enabled by adding the A. niger aox1 gene that encodes for the alternative oxidase. The first step is for genes for both enzymes to be isolated, cloned and sequenced. After appropriate modifications the genes will be introduced into other microbes of commercial value: A. terreus, P pastoris, L. lactis, S. rimosus and S. clavuligerus. After transformation, all the recipient as well as parent strains will be analysed on four levels; genomic, proteomic, metabolomic and finally they will be evaluated for increased overproduction of specific end products. Estimation of increased yields or productivity will be conducted both on laboratory as well as pilot plant level. All the analytical data will be used for further modification of the transferred genes.

An important part of the project will be the studies of glucose uptake by specific strains since high glucose concentrations in the media are known to be toxic for cells, unless it is rapidly metabolised via glycolysis. By introducing specific A. niger genes recipient strains are expected to sustain growth in higher glucose concentrations media which will additionally effect their productivity.

DELIVERABLES

Demonstrating that the transfer of specific genes from an excellent industrial producer into other commercial micro-organisms will increase their productivity, and will be a major deliverable providing European industry with new options. It will help to increase the production of generics and increase their added value. The major milestones correspond to introduction of a gene or genes, and the assessment of the metabolic consequences.

CONSORTIUM
COORDINATOR
 
Dr Matic Legisa
Department for Biotechnology and Industrial Mycology
National Institute of Chemistry
Hajdrihova 19
1001, Ljubljana, Slovenia
Tel: +386-1-4760 200
Fax: +386-1-4760 300
matic.legisa@ki.si
 
PARTNERS
 
Dr Anton Glieder
Enzyme technology and Cell Engineering
Institute of Biotechnology
Petersgasse 12
8010, Graz, Austria
Tel: +43-316-873 8420
Fax: +43-316-873 8434
a.glieder@tugraz.at

Dr Gregor Kopitar
Department of Operative Development
Lek, d.d.
Kolodvorska 27
1234, Menges, Slovenia
Tel: +386-1-721 7603
Fax: +386-1-723 7244
grega.kopitar@lek.si

Dr Michael Mattey
Department of Bioscience
University of Strathclyde
Royal College Building,
204 George Street
G1 1XW, Glasgow, Scotland, United Kingdom
Tel: +44-141-548 3922
Fax: +44-141-553 4124
m.mattey@strath.ac.uk

Prof. Iain Hunter
Department of Pharmaceutical Sciences
Strathclyde University
Royal College Building,
204 George Street
G1 1XW, Glasgow, Scotland, United Kingdom
Tel: +44-141-548 4111
Fax: +44-141-553 4124
i.s.hunter@strath.ac.uk

Dr Maria Papagianni
Department of Hygiene and Technology
of Food of Animal Origin
School of Veterinary Medicine
University of Thessaloniki
54006, Thessaloniki, Greece
Tel: +30-310-999 804
Fax: +30-310-999 812
mp2000@auth.gr

Dr Karen Deans
Beocarta Ltd.
Colvile Building
48 North Portland Street
G1 1XN, Glasgow, Scotland, United Kingdom
Tel: +44-141-548 4804
Fax: +44-141-548 4809
kdeans@beocarta.com