4. DISCUSSION
Auxotrophic genes are commonly applied as screening markers in genetic manipulation of S. cerevisiae , but their negative roles in bio-productions in metabolic engineering are often overlooked. The deficient synthesis of amino acids or nucleotides may interfere with the homeostasis of microbial cell and hinder cell growth[18]. In this study, eight commonly used auxotrophic makers were selected to investigate their effects on cell growth and FFA production in S. cerevisiae .
The six amino acids are divided into three groups based on their synthesis and degradation metabolism. Generally, except geneADE2 , all these markers decreased both cell growth and FFA production of the corresponding auxotrophs under the commonly used concentrations. Interestingly, these observed repressions could be remedied by the much higher concentrations of supplements than expected (≥500 mg/L), which guided us to increase the supplemented concentrations while adopting auxotrophs. Among them, the minimal required amounts of amino acids from acetyl-CoA and succinyl-CoA modules are supposed to be much higher than those from α-ketoglutarate module and nucleotides (500 mg/L vs 60 mg/L), which may be attributed to the competition with cell growth. As reported, the strain ade2 Δ will turn red under limited concentrations, which is suitable for high-throughput screening[25].
In particular, deficiency in leucine synthesis greatly decreased both cell growth and FFA production, even in medium supplementing 1,000 mg/L of leucine, as observed in other species [26-29]. There is a close relationship of gene LEU2 with cell growth and lipogenesis, which has previously been reported that leucine metabolism, but not leucine itself played a key role as a signal to affect TOR activity. Here, leucine metabolism was inferred as an alternative way to offer cytoplasmic acetyl-CoA to regulate the cell growth and FFA production. Therefore, the auxotrophic strains may be not suitable for bio-productions, especially in the long-run fermentations.