## 3 Results and Discussion
### Effect of pH on \(p\)-Coumaric Acid Uptake  
According to our results, pH produced evident changes in the transformation of \(p\)-coumaric acid into resveratrol (Figure 5). Resveratrol production was evidently different in the strains evaluated. pH 7-8 caused a considerable decrease in the levels of resveratrol, whereas acidic pH values (4-5) produced a favorable effect. In all the studied cases, pH 6 was the optimal value for the biotransformation process. The effect of alkaline pH was more evident in pESC-Pa4 CL1-VvVST1 and pESC-Pa4 CL1-AhSTS than in other recombinant lines. This effect should be associated to the beneficial effect of slightly acidic media on the growth of \(S\). _cerevisiae_[23]. Also, H\({}^{+}\) gradient may increase the permeability of carboxylic acids like \(p\)-coumaric acid [23]. Figure 3: Synthesis of resveratrol (RE) from \(p\)-coumaric acid (CA) by recombinant strains of \(S\). _cerevisiae._ (**A**) Mixture of authentic standards of \(p\)-coumaric acid and _trans_-resveratrol (1), biotransformation products of pESC-Pa4 CL1-PhSt5 (2), biotransformation products of pESC-Pa4 CL1-PcPKS5 (3), biotransformation products of pESC-Pa4 CL1-MaST3 (4), biotransformation products of pESC-Pa4 CL1-RvVST1 (6), biotransformation products of pESC-Pa4 CL1-AhSTS (7). (**B**) Authentic standard of \(p\)-coumaric acid (CA) and biotransformation of \(p\)-coumaric by wild type W303 (2). Samples collected after 30 h of batch fermentation. Retention times are indicated for each peak. Dose-response curves performed under batch fermentation suggested that 70 mg L\({}^{-1}\)\(p\)-coumaric acid were efficiently converted into resveratrol (Figure 6). Despite MIC values for recombinant strains were over 150 mg L\({}^{-1}\), resveratrol yields were affected from 80 mg L\({}^{-1}\)\(p\)-coumaric acid in batch fermentation and no levels of resveratrol were detected in reactions containing over 100 mg L\({}^{-1}\)\(p\)-coumaric acid. These strains containing codon optimized STS were able to metabolize 4.3 more amount of substrate and quadruplicate the production of resveratrol in comparison with other recombinant microorganisms already reported [14, 20]. Between 20% and 27% of \(p\)-coumaric acid was transformed into resveratrol. Figure 4: Changes in the levels of glucose, ethanol, \(p\)-coumaric acid, resveratrol and dry cell mass under fermented batch conditions. Kinetics for the yeast lines pESC-Pa4 CL1-PhStS (**1**), pESC-Pa4 CL1-PcPKS5 (**2**), pESC-Pa4 CL1-MaSTS3 (**3**), pESC-Pa4 CL1-RtSTS (**4**), pESC-Pa4 CL1-VvVST1 (**5**), pESC-Pa4 CL1-AhSTS (**6**). Figure 5: Effect of pH on the conversion of \(p\)-coumaric acid into resveratrol. Diverse letters indicate statistically significant differences (\(p<0.01\)). Error bars represent standard deviation of ten independent experiments (\(n=10\)). Interestingly, a different capacity to produce resveratrolol was observed in each strain evaluated. Under the assayed conditions, pESC-Pa4 CL1-PhStS had a similar activity to pESC-Pa4 CL1-VvVST1, whereas a comparable activity was observed between pESC-Pa4 CL1-MaSTS3 and pESC-Pa4 CL1-AhSTS to produce resveratrolol (Table 2). Among the six strains assayed, pESC-Pa4 CL1-PcPKS5, pESC-Pa4 CL1-MaSTS3 generated more resveratrolol levels after the addition of 70 mg L\({}^{-1}\)\(p\)-coumaric acid. Despite the enzymatic activity of individual STS should be strongly involved in this results, other intrinsic factors that influence the synthesis and regulation of heterologous enzymes in _S. cerevisiae_ should be considered [24]. Thus, at least an additional heterologous system should be used to corroborate the results obtained in this work. Under batch culture, the recombinant strains exhibited similar parameters (Figure 4). The depletion of glucose levels was inverse to the development of cell growth (3.6 \(\pm\) 0.2 g L\({}^{-1}\)) and ethanol production (6.7 \(\pm\) 0.9 g L\({}^{-1}\)), which demonstrated the efficient replication of recombinant cells (Figure 4).