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.