Selection of 4CL and STS enzymes and characterization of resveratrol biosynthesis in minimal medium with supplementation of p-coumaric acid
As an initial step to generate a bacterial platform for the biosynthesis of resveratrol, we first built a plasmid for expressing the genes coding for the last two enzymes required by the biosynthetic pathway: 4CL and STS. We chose to express the 4CL from Streptomyces coelicolor A2 given its microbial origin and its high catalytic efficiency compared to other homologous enzymes . In the case of STS, we decided to test the performance of enzymes from two distinct origins: A. hypogaea (AhSTS) and V. vinifera (VvSTS), since previous studies have shown that these proteins display better catalytic properties compared to others . The transformed resultant E. coli W3110 strains were named W-Ah and W-Vv, respectively. These strains were cultured in M9 minimal medium supplemented with glucose or glycerol, and we added p-coumaric acid as a precursor for resveratrol, given that these strains do not express a TAL enzyme. In this experiment, we tested the expression and functionality of the plasmid-encoded proteins in an environment non-limited for one of the two resveratrol precursors (p-coumaric acid).
We set up shake flask cultures and induced the expression of the plasmid-encoded enzymes by adding 0.5 mM of IPTG when cells reached an O.D.600 of 0.8. The growth kinetic parameters are shown in Table 2. Our results showed that the use of glycerol as a carbon source causes a higher resveratrol production when compared to glucose (Fig. 3; Table 3). When we compared the final resveratrol titers as a function of the carbon source, strain W-Vv showed an almost 16 % increase, while the strain W-Ah showed an even larger increase of about threefold when grown in glycerol compared to glucose (Table 3). The higher difference observed in the case of W-Ah compared to W-Vv could be a consequence of the distinct catalytic properties of both enzymes (AhSTS and VvSTS) and their expression levels. Enzyme VvSTS is highly expressed and has around twice the turnover number when compared to the enzyme from A. hypogaea (Fig. 4) . These two features could explain the observed results.
Clear differences in resveratrol production were also observed concerning the origin of the STS enzyme encoded in each plasmid vector. In shake-flask cultures grown in glycerol, strain W-Vv reached 2.8-fold higher resveratrol titers than strain W-Ah (Table 3). In glucose supplemented medium, the difference was even more evident, since resveratrol titers in strain W-Vv were about sevenfold higher than the strain W-Ah (Table 3).
We performed an SDS-PAGE stained with Coomassie blue to determine the relative protein levels of the enzymes 4CL and STS at the end of the production cultures (Fig. 4). Lower levels of all the heterologous enzymes were observed in cultures grown on glucose compared to glycerol. The levels of 4CL displayed a slightly lower expression in glucose compared to glycerol. These results suggest that a physiological component of the expression apparatus might be sensitive to the nature of the carbon source, affecting the synthesis of these proteins. However, the most evident difference regarding the protein levels of the heterologous enzymes was related to the origin of the expressed genes. We found that the levels of VsSTS were significantly higher than the levels of AhSTS (Fig. 4). Further studies are needed to establish whether the differences in the expression of these two enzymes is a consequence of dissimilarities in the stability of the RNA transcript, the translation efficiency or the protein stability. These results agree well with the production levels of resveratrol as described above (Table 3) and suggests that the higher expression of the heterologous proteins in cultures grown in glycerol and the higher expression of the STS from V. vinifera accounts for the higher resveratrol titers observed when using this carbon source. A possible explanation for this observation is that a higher enzyme activity due to the higher abundance of the stilbene synthase could help in driving the carbon flux from malonyl-CoA towards the synthesis of resveratrol. An alternative explanation arises from the analysis of acetate metabolism and its relation with acetyl-CoA. The inhibition of STS by acetyl-CoA has been studied by the group of Koffas et al. . They reported an apparent competitive inhibition constant of about 460 μM, triggered by acetyl-CoA against STS activity from V. vinifera . However, they assumed that the intracellular levels of acetyl-CoA in E. coli cells were not as high to produce a significant inhibition of STS. Nevertheless, according to Takamura et al., under aerobic and glucose growth conditions, the acetyl-CoA concentration in E. coli cells range from 20–600 μM . Therefore, it is possible that the intracellular concentration of acetyl-CoA exerts a substantial inhibitory effect on the STS. In support for this hypothesis, we found an inverse relation between the titers of resveratrol and the amount of acetate accumulated in the culture medium (Fig. 3, Tables 3 and 4). The higher content of acetate in glucose-grown cultures agrees with the previous observation that glucose-grown cells have higher intracellular acetyl-CoA levels than cells grown in glycerol, given that acetyl-CoA is a direct precursor of acetate . However, other factors such as a different carbon flux distribution, the loss of carbon in acetate formation and the potential toxic effect of this organic acid, could also contribute to the observed differences in resveratrol production when comparing cultures in glucose with respect to glycerol.
Additionally, as was suggested by Watts et al.  the transport of phenylpropanoids into the cytoplasm of E. coli could be inhibited in cells growing in glucose due to the catabolic repression of the operons hca and mhp involved in the transport and catabolism of these compounds. Therefore, cell cultures supplemented with glycerol instead of glucose could help in the maintenance of a constant flux of p-coumaric acid into the cells, driving forward resveratrol biosynthesis.
Characterization of monocultures for the production of p-coumaric acid and resveratrol
The following experiments were performed with a strain expressing the STS from V. vinifera since higher resveratrol titers were produced by the E. coli strain expressing the gene encoding this enzyme. Previously in our group, we reported an engineered strain for the production of p-coumaric acid . This strain harbors a plasmid encoding the TAL enzyme from the yeast Rhodothorula glutinis and another plasmid with the E. coli genes aroG
fbr and tktA encoding a feedback-inhibition-resistant 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase and a transketolase, respectively. Furthermore, to improve l-tyrosine accumulation, in this strain the pheA gene which encodes for a chorismate mutase/prephenate dehydratase, was inactivated. The enzyme TAL from R. glutinis was selected since it showed a higher affinity towards l-tyrosine rather than l-phenylalanine . This E. coli strain was named W(pheA-)Rg.
To characterize strains W(pheA-)Rg and W-Vv, we performed shake-flask cultures individually with each strain in M9 mineral salts medium supplemented with glycerol as the sole carbon source. The results showed that both strains display similar specific growth rates (Fig. 5a, b) (Table 5). When compared to W(pheA-)Rg, strain W-Vv reached around a 20 % higher biomass, but the consumption rate of glycerol appeared lower. The production of p-coumaric acid by strain [W(pheA−)Rg] reached 133 uM (~22 mg/L) and resveratrol from W-Vv, upon addition of 3 mM of p-coumaric acid, reached ~78.1 mg/L (Fig. 5b). These results are the highest resveratrol titers reported with an E. coli recombinant strain cultured in mineral medium without yeast extract supplementation.
Coculture in M9 mineral medium for the production of resveratrol from glycerol
To determine the feasibility of reconstituting a fully functional biosynthetic pathway to produce resveratrol, the strain with the capacity of transforming glycerol to p-coumaric acid (W(pheA-)Rg) and the strain able to convert it to resveratrol (W-Vv), were both grown in a coculture in M9 mineral medium supplemented with glycerol as the sole carbon source. It is important to point out that neither p-coumaric acid nor yeast extract were supplemented to the culture medium.
Under coculture conditions, the growth rate was similar to that observed for single strain cultures (Fig. 6; Tables 5 and 6). Both strains were inoculated at a ratio of 1:1 (initial O.D.600 of 0.1), and the final biomass was 1.78 g/L. Compared to monoculture results, p-coumaric acid accumulation was lower, which can be partially explained considering that this compound is being consumed for resveratrol synthesis. The resveratrol synthesis profile coincided with that of p-coumaric acid production, starting both at 15 h and continuing until the end of the experiment. During the production period that lasted 15 h, glycerol provided carbon and energy for biomass production, p-coumaric acid and resveratrol synthesis. A final resveratrol titer of 22.58 mg/L and 9.16 mg/L of remaining p-coumaric acid were observed at 30 h of total culture time.
These results suggest that both the production process and production strains can be improved. By modifying the inoculum ratio of both strains, it would be possible to couple the rate of p-coumaric acid production to its consumption by the resveratrol-producing strain, which has the potential to increase the yield of resveratrol and avoid the toxic effect of p-coumaric acid accumulation . The success of a coculture production system depends on the efficient exchange of molecules between the strains. Transport functions can be modified with the aim of improving the system. The aaeXAB operon in E. coli is involved in the excretion of various compounds, including p-coumaric acid . It has been shown that overexpression of these genes results in increased tolerance to the toxicity of this aromatic acid . Therefore, it can be expected that overexpression of genes aaeXAB in strain W(pheA-)Rg would lead to a higher secretion rate for p-coumaric acid, while the inactivation of this operon in strain W-Vv could result in increased intracellular availability of this precursor for resveratrol synthesis.
As shown in Table 1, diverse strategies have been employed for the generation and cultivation of E. coli recombinant strains for resveratrol production. Concerning strain engineering, these approaches have in common the heterologous expression of 4CL and STS enzymes. Further strain improvement modifications involve expressing activities that increase the supply of endogenous metabolic precursors for resveratrol. Cultivation conditions employed for resveratrol production differ concerning media composition. However, with the exception of the present report, p-coumaric acid or l-tyrosine have been supplemented as precursors and a complex medium was used. In the work reporting the highest level of resveratrol (2340 mg/L), in addition to supplementation with p-coumaric acid in a complex medium, the inhibitor cerulenin was employed. In contrast to these reports, it should be noted that the coculture system reported here is based on the use of a minimal medium where glycerol provides all the carbon atoms for resveratrol synthesis. As mentioned above, various strain and culture optimization strategies should enable to further increase resveratrol titer and productivity. In a recent report, a Saccharomyces cerevisiae strain was engineered for the production of resveratrol from glucose or ethanol. In fed-batch cultures, a maximum resveratrol titer of 531 mg/L was produced . It remains to be determined if improvement of the E. coli coculture system can achieve similar results.