Fig 3. The evolution of auxotrophy.

(A) The evolution of auxotrophs. Ancestral lys⁻ cells (WY950; WY1335) were grown for tens of generations in minimal medium, either in lysine-limited chemostats [35] or via coculturing with a lysine releaser in a cross-feeding yeast community [30,36]. Out of 20 independent lines, we randomly isolated approximately 50 clones for whole-genome sequencing. Chemostat evolution and coculture evolution both yielded met⁻ mutants: 1 (WY1604) out of 9 clones in chemostat evolution; 3 (WY1569, WY2376, WY1591) out of 42 clones in coculture evolution). These mutants, all isolated from independent lines, required an externally supplied organosulfur such as methionine to grow. A glutamine auxotrophic gln1 clone was also identified. In the experiment shown here, clones were grown to exponential phase in SD supplemented with amino acids, washed with SD, starved for 3 hours to deplete cellular storage, and spotted on indicated agar plates at 30°C. (B) The organosulfur synthesis pathway in S. cerevisiae. S. cerevisiae utilizes sulfate supplied in the medium to synthesize the organosulfur homocysteine, which is then used to make a variety of other organosulfurs, including methionine, cysteine, and GSH [37]. All orgS⁻ mutants we have identified (orange) fail to synthesize homocysteine, and thus can be supported by any of the organosulfurs depicted here (blue; note the interconvertibility between organosulfurs). Anc, ancestor; met⁻, methionine-requiring mutant; gln1, glutamine metabolism gene 1 mutant; GSH, reduced glutathione; SD, synthetic minimal glucose medium.