Auxotrophic marker genes have widely used for genetic engineering in yeast. However, the effects of auxotrophic strains that are deficient in synthesis of amino acids or nucleotides on the growth and production are rarely reported. In this study, a total of eight auxotrophic strains with single knockout of selective markers were obtained to evaluate cell growth and free fatty acid (FFA) production in Saccharomyces cerevisiae with supplementing different concentrations of amino acids or nucleotides. Generally, except for gene ADE2, most auxotrophic strains possessed decreased cell growth and FFA production, which could be remedied by the higher concentrations of supplements. leu2Δ damaged both growth and production even with supplementation of 1000 mg/L leucine. Therefore, this study shows that auxotrophs compromise the metabolic engineering endeavor and provides a guidance in supplementing amino acids or nucleotides during fermentations for maximizing bio-productions.