Novel strain development strategies were used to produce secreted recombinant human gelatin in Pichia pastoris. Strains were first constructed using antibiotic resistance markers to select for multicopy strains. To determine if use of an additional expression cassette with another antibiotic marker could further increase copy number, the initial set of strains were either re-electroporated or mated with a plasmid or strain containing a different marker. Crossing strains expressing 25 and 50 kDa gelatin resulted in progeny that expressed both gelatins, demonstrating that a genetic strategy may successfully combine expression cassettes. Quantitative evaluation of strains produced by either single or double marker strategies demonstrated that increased expression cassette copy number and gelatin expression levels were obtained by both methods using two antibiotic resistance markers. (C) 2008 Elsevier Inc. All rights reserved.