Agricultural Research Service

February 6, 2001

The invention relates to a novel system and method for the sequential, directional cloning of multiple DNA sequences into a single vector. It is a facile process by which multiple genes, sufficient for the expression and/or regulation of a metabolic pathway, can be simultaneously introduced into the target species in one transformation event. The process uses designed primers (added to DNA sequences via PCR or ligation) which contain sequences that generate compatible cohesive ends upon digestion by the appropriate restriction enzymes. The method ensures the directionality of the cloned sequences, and permits the insertion of additional DNA sequences into multi-gene arrays previously constructed by this process. To demonstrate its utility, the invention method was used to construct three- and four-gene arrays that were introduced into Fusarium sporotrichioides. These arrays enabled the biosynthesis of the carotenoids lycopene and beta-carotene, which are utilized as colorants and nutritional supplements. By constructing multi-gene arrays containing different metabolic enzymes, F. sporotrichioides strains capable of synthesizing other products such as rubber or taxol could be developed. The invention could potentially be used to facilitate the introduction of novel metabolic pathways into any number of fungal or plant species, thereby expanding the productivity and /or economic value of the species.

James D. Jones Biopolymer Research, NCAUR Peoria, IL 61604 (309) 681-6376 / Fax: (309) 681-6689	Thomas M. Hohn Mycotoxin Research, NCAUR Peoria, IL 61604 (309) 681-6380 / Fax: (309) 681-6686
Timothy D. Leathers (Same address as first inventor) (309) 681-6377 / Fax: (309) 681-6689