## 3 Results and Discussion
### Effect of pH on \(p\)-Coumaric Acid Uptake
Interestingly, an inverse tendency was observed between glucose consumption and resveratrolol accumulation. This fact suggests that resveratrolol synthesis begins after glucose reduction as previously reported [14]. Once glucose is almost or totally consumed, the cells uses \(p\)-coumaric acid to produce resveratrolol (Figure 4). Remarkably, ethanol produced by glucose oxidation and exogenous \(p\)-coumaric acid were simultaneously used by recombinant yeasts, suggesting that ethanol is probably channeled as an energetic substrate for the conversion of \(p\)-coumaric acid into resveratrolol. Despite a similar growth pattern being observed for all the strains, resveratrolol yields had evident differences. Among the six lines studied, pESC-Pa4 CL1-PcPKS5 and pESC-Pa4 CL1-MaSTS3 were the most effective for accumulating resveratrolol at the end of the batch period (Figure 4). In this context, thresholds of resveratrolol production varied in time for each strain assayed. For pESC-Pa4 CL1-PhStS and pESC-Pa4 CL1-RtSTS, maximum levels were detected between 45 and 50 h, whereas an asymptotic tendency was observed for pESC-Pa4 CL1-PcPKS5 (from 35 h), pESC-Pa4 CL1-MaSTS3 (from 35 h), \begin{table}
\begin{tabular}{c c c} \hline \hline
**Strain** & **50 mg L\({}^{-1}\)** & **70 mg L\({}^{-1}\)** \\ \hline pESC-Pa4 CL1-PhStS & 23.7 \(\pm\) 2.6 b  & 27.1 \(\pm\) 3.1 c  \\ pESC-Pa4 CL1-PcPKS5 & 34.5 \(\pm\) 4.1 a  & 39.9 \(\pm\) 2.5 a  \\ pESC-Pa4 CL1-MaSTS3 & 31.3 \(\pm\) 4.7 a  & 34.4 \(\pm\) 3.8 b  \\ pESC-Pa4 CL1-RtSTS & 24.2 \(\pm\) 3.1 b  & 28.5 \(\pm\) 2.4 c  \\ pESC-Pa4 CL1-VvVST1 & 25.4 \(\pm\) 1.8 b  & 27.6 \(\pm\) 3.7 c  \\ pESC-Pa4 CL1-AhSTS & 24.7 \(\pm\) 2.9 b  & 30.9 \(\pm\) 4.3 b  \\ \hline \hline \end{tabular}  
Means with diverse letters indicate statistically significant differences (\(p<\) 0.01).  
\end{table} 
Table 2: Production of resveratrolol at two concentrations of \(p\)-coumaric acid (50 and 70 mg L\({}^{-1}\)) after 60 h batch fermentation. Figure 6: Conversion of \(p\)-coumaric acid into resveratrolol by recombinant strains of _S. cerevisiae_ containing six stilbene synthases growing at pH 6. Different letters indicate statistically significant differences (\(p<\) 0.01). Error bars represent standard deviation of ten independent experiments (\(n=10\)). pESC-Pa4 CL1-VvVST1 (from 30 h), pESC-Pa4 CL1-AhSTS (from 30 h). These results suggest that lines containing the STS from _Vitis vinifera_ and _Arachis hypogaea_ generate resveratrol faster than other strains, but they accumulate less amounts at the end of the batch period. This finding should be related with substrate affinity (K\({}_{\text{m}}\)) and saturation points (V\({}_{\text{max}}\)) reported by the STS expressed in these recombinant lines [22; 25]. These qualities should be of the interest to produce resveratrol at industrial level. Currently, resveratrol production in heterologous systems has achieved significant advances [4; 5; 6; 7; 8; 9; 10]. Due to the low number of reactions involved in resveratrol biosynthesis, several orthologs of PAL, TAL, C4H, 4 CL and STS have been used to engineer a wide spectrum of microorganisms that produce substantial amounts of resevatrol. To the best of our knowledge, this is the first work reporting the use of a 4 CL from a liverwort (_Plagiochasma appendiculatum_) to produce resveratrol in a yeast system [26]. According to our results, the use of codon optimized sequences and the experimental conditions described for batch fermentation led to quadruplicate the levels of resveratrol when compared with previous works [14; 20]. In addition, specific pH and light protection had a clear influence on resveratrol yields.