Transposon Tn5 mutagenesis was used to generate random mutations in Shewanella baltica MAC1, a polyunsaturated fatty acid (PUFA)-producing bacterium. Three mutants produced 3-5 times more eicosapentaenoic acid (EPA 20:5 n-3) compared to the wild type at 10 degrees C. One of the mutants produced 0.3 mg EPA g(-1) stop when grown at high temperature (30 degrees C). Moreover, 2 mg docosahexaenoic acid (DHA 22:6 n-3) g(-1) was produced by S. baltica mutants at 4 degrees C. Sequencing of insertion mutation(s) showed 96% homology to trimethylamine N-oxide (TMAO) reductase gene and 85% homology to rRNA operons of E. coli. Tn5 transposon mutagenesis therefore is a suitable technique to increase PUFA formation in bacteria.