QLK3-2002-02038 Demonstration of increased yield and productivity in selected commercial organisms by strategic transformation of key genes from Aspergillus niger (ANTICO)

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. This should 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 anapleurotic 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.

Activities

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. nigergenes 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.

Contacts

Coordinator

• Matic LEGISA / National Institute of Chemistry Department for Biotechnology and Industrial Mycology / SLOVENIA

Participant

• Beocarta Ltd. UNITED KINGDOM
• Anton GLIEDER / Institute of Biotechnology Enzyme technology and Cell Engineering / AUSTRIA
• Gregor KOPITAR / Lek, d.d. Department of Operative Development / SLOVENIA
• Iain HUNTER / Strathclyde University Department of Pharmaceutical Sciences / UNITED KINGDOM
• Michael MATTEY / University of Strathclyde Department of Bioscience / UNITED KINGDOM
• Maria PAPAGIANNI / University of Thessaloniki School of Veterinary Medicine / GREECE