Technology Description: Agricultural Research Service scientists have developed 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 multigene arrays previously constructed by this process. To demonstrate its utility, the invention method was used to construct three and fourgene arrays that were introduced into Fusarium sporotrichioides. These arrays enabled the biosynthesis of the carotenoids lycopene and betacarotene, which are utilized as colorants and nutritional supplements. By constructing multigene 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. The technology applies to biotechnology companies that produce DNA cloning kits.  Pharmaceutical companies that produce vitamins and medicines can also use this technology.  Agricultural companies interested in developing new crop varieties would also benefit. Reference: Please refer to USPN 6,372,479 (Docket #0214.00), "Fusarium Sporotrichioides for Production of B-Carotene," which issued April 16, 2002, and is a divisional of USPN 6,184,000 (Docket #0259.97), which issued February 6, 2001. Inventors: James D. Jones  2542 W. Fitch, Second Floor  Chicago, IL 60645 (773) 3810619 (Formerly with Fermentation   Biotechnology Research Peoria) Thomas M. Hohn  102 Blakely Drive  Chapel Hill, NC  27514  (919) 9684225 Timothy D. Leathers  Fermentation Biotechnology Resarch Peoria, IL  616043999  (309) 6816377 / Fax: (309) 68166427