## 4 Discussion
The analysis of intracellular fatty acids indicated that _S. cerevisiae_ cells promptly uptake added UFAs and incorporated them into cells, which consequently down-regulate functional genes expressions (_ACC1_, _FAST1_, _FAS2_, _FAT3_, _FAA1_, _OLE1_, and _ELO1_) to inhibit the biosynthesis of saturated fatty acids (SFAs), because cells don't need de novo synthesis of UFAs from SFAs [31]. This can explain the decreased levels of MCFAs in the medium observed in this study. The disturbance of fatty acids syntheses probably leads to the accumulation of acetyl-CoA in cells because acetyl-CoA is the precursor of fatty acid synthesis. The research of [PERSON] et al. [25] indicated that _S. cerevisiae_ might have an alternative anaerobic pathway to \(\beta\)-oxidation, and lead to UFAs (LA) broken down into acetyl-CoA molecules under anaerobic conditions, which leads to acetyl-CoA accumulation in cells. The metabolism of amino acids is of particular interest from a winemaking perspective because they are not only cell building blocks, but also the precursors of aroma compounds [32]. We found that the contents of most amino acids in cells were less compared to the control cells in the early stage, except for glutamate, which was strengthened by UFAs addition (Figure 3). As yeast-preferred amino acids, glutamate can be synthesized from other amino acids through transamination reaction [33]. So, at the beginning of fermentation, most amino acids are consumed to form preferred amino acids for cell construction, and leads to the global reduction of amino acids (Figure 3). The increased expressions of _BAT1_, _PDC1_, _PDC5_, and _ADHI_ in Ehrlich pathway confirmed that the pathway is activated by UFAs addition, which led to high productions of higher alcohols (Table 2). Another explanation for increase of higher alcohols might be that the formations of higher alcohols can re-oxidize excess NADH to NAD\({}^{+}\), because the _ADH_ gene used NADH as the cofactor to catalyze the last step in the Ehrlich pathway [34]. Acetate esters are synthesized from acetyl-CoA with ethanol or a higher alcohol catalyzed by alcohol acetyltransferases (encoded by _ATF1_ and _ATF2_). The inhibition of _ATF1_ expression by single or mixed UFAs addition has been confirmed by different authors [8; 35]. Our results showed that although the both precursors (acetyl-CoA and higher alcohol) are excessive in cells, the production of acetate esters are still suppressed in UFAs added fermentation. This statement confirmed that _ATF1_ is the key factor determining the synthesis of acetate esters, not precursor availability. It is worth noting that _IAH1_ was up-regulatedin the early stage of BDX fermentation and in the late stage of EC1118 fermentation, which might partially explain the decrement of acetate esters in UFAs added fermentation because _IAH1_ encodes an esterase to hydrolyse acetate esters [36]. MCFA ethyl esters are formed from the reactions of medium-chain fatty acid with ethanol catalyzed by two acyl-CoA: ethanol \(O\)-acyltransferases (encoded by _EHT1_ and _EEB1_), in which _EEB1_ has the main function, while _EHT1_ plays a minor role [1,22]. Unlike acetate esters, the formation of MCFA ethyl esters is the precursor's (MCFAs) dependent, because increased production of precursors (MCFA) resulted in a high amount of MCFA ethyl esters, while overexpressing _EHT1_ and _EEB1_ did not significantly increase their formation in wine [37]. While the data presented here indicated that the availability on MCFAs is not limiting for ethyl ester production as their total concentration is above 22 fold higher than that of ethyl esters. More important, we found that the response expression of _EEB1_ to UFAs is strain-specificity. In contrast to the strain of EC1118, UFAs additionally favored the expression of _EEB1_ in BDX strain, which is corresponding to the high production of MCFAs ethyl esters compared to the control. This also confirmed the prior results that _EEB1_ expression is a key factor determining the synthesis of MCFA ethyl esters [8,38].