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PRODUCTION OF A BAKERS YEAST SECRETING LEGUME LIPOXYGENASE DURING PRODUCTION OF BREAD DOUGH

Objective

EXPRESSION OF LIPOXYGENASE GENE IN BAKER YEAST IS EXPECTED TO BLEACH THE YELLOW PIGMENTS OF THE WHEAT FLOUR TO GIVE A BREAD WITH VERY WHITE CRUMB, SIMULTANEOUSLY IMPROVING THE MIXING PROPERTIES OF THE DOUGH INGREDIENTS AND THE STRENGH OF THE DOUGH.

THE CONSTRUCTED YEAST STRAINS WILL ALLOW A TEST OF THE CORRECTNESS OF THE CURRENT HYPOTHESES AS TO HOW LIPOXYGENASES FROM ADDED SOYA FLOUR INFLUENCE THE CHARACTERISTICS OF THE DOUGH.

BY USING STRAINS SECRETING DIFFERENT AMOUNTS OF LIPOXYGENSASE, AN ASSESSMENT WILL BE MADE OF THE FLAVOUR CONTRIBUTIONS CAUSED BY THE LIPOXYGENASE CATALYZED PROCESSES AGAINST THOSE ORIGINATING FROM OTHER INGREDIENTS OF ADDED SOYA FLOUR.
A genetically engineered baker's yeast that produces and secretes legume lipoxygenase could prove valuable in breadmaking. Spore clones of an outstanding industrial strain of baker's yeast have been characterized by tetrad analysis, molecular hybridization with cloned genes and by pulse field gel electrophoresis of chromosomes. Full length complementary deoxyribonucleic acid (cDNA) corresponding to the 2 major pea seed lipoxygenase polypeptides have been isolated, sequenced and shown to direct the transcription of functional beta (mRNA). Vectors for the expression and secretion of the pea lipoxygenase in yeast have been constructed and a plate assay to recognize lipoxygenase producing yeast colonies has been established.

An outstanding industrial strain has been characterized; all genes tested to date seem to be organized in the same linkage relationships as in standard academic yeast strains.
Near full length cDNA clones of the mRNAs corresponding to the 2 major pea seed lipoxygenases were isolated, sequenced and used in an in vitro transcription translation reaction to demonstrate their potential to produce lipoxygenase in a heterologous situation.
Transformation systems were developed to introduce and integrate into the chromosomes of the production strain the cDNAs encoding the pea seed lipoxygenases. Using reporter genes encoding mouse alpha amylase and bacterial beta galactosidase, stable transformants with good expression and secretion were obtained. No enzyme export could be achieved. However with the lipoxygenase clones (so another system based on a successful yeast) an Escherichia coli shuttle vector is being evaluated. A plate assay designed to recognize lipoxygenase producing yeast clones has also been established.
THE AIM IS TO CONSTRUCT BY GENETIC ENGINEERING YEAST STRAINS WHICH DURING THE PRODUCTION OF DOUGH SECRETE LIPOXYGENASE FROM AN HETEROLOGOUS CLONED PLANT GENE.

STEPS :

1. ISOLATION OF FULL-LENGTH CDNA CLONES FOR LIPOXYGENASE FROM PEA SEED MRNAS
2. ISOLATION OF SEVERAL GENOMIC LIPOXYGENASE CLONES FROM PEA
3. SEQUENCE OF THE PLANT LIPOXYGENASE DNAS
4. SELECTION OF AN APPROPRIATE INDUSTRIAL YEAST STRAIN AS A HOST FOR CLONING
5. TRANSFORMATION BY INTEGRATION USING THE EXPRESSION VECTOR PMS12 CONTAINING THE GENE FOR A-AMYLASE WHICH IS SECRETED BY YEAST
6. SUBSTITUTION OF THE A-AMYLASE GENE BY PLANT LIPOXYGENASE SEQUENCE(S).

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

JOHN INNES CENTRE
Address
Norwich Research Park, Colney
Norwich
United Kingdom

Participants (2)

CARLSBERG LABORATORY
Denmark
Address

Copenhagen
DANISH DISTILLERIES LTD.
Denmark
Address

Copenhagen