Engineering success
| Step | Precursor(s) | Products | Enzyme | Enzyme origin |
|---|---|---|---|---|
| 1 | Tyrosine | P-coumaric acid | TAL | From plants,bacteria or yeast |
| 2 | P-coumaric acid | P-Comaril-CoA | 4CL | From plants |
| 3 |
P-Comaril-CoA Malonyl-CoA |
Resveratrol | STS | From plants |
As shown in Figure 1, the metabolic pathway of resveratrol synthesis from glucose involves many enzymes and steps. However, E.coli is known to have the ability to produce tyrosine from glucose. Therefore, by cloning the relevant genes from grapevine, it could be enabled to produce resveratrol from tyrosine. For this reason, cloning of the TAL, 4CL and STS genes, described in Table 1, will be performed in E.coli. Consequently, our E.coli will reproduce the metabolic pathway and will be able to synthesize resveratrol from p-coumaric acid, a precursor found in wine by-products.
However, in order to maximize the production of resveratrol from wine industry waste by-products, all possible precursors have to be considered. As also shown in Figure 1, there are two main precursors of resveratrol: p-Coumaroyl-CoA (from p-coumaric acid) and malonyl-CoA. Malonyl-CoA is a key molecule in the fatty acid biosynthesis pathway and is rapidly consumed in the cell, so it is found in reduced amounts, limiting resveratrol production. Therefore, another plasmid encoding for the enzyme ScACC (this enzyme converts acetyl-CoA to malonyl-CoA) could be constructed to increase malonyl-CoA concentrations in the cell.
Overall, our main goal is to achieve the production of synthetic bacteria with two plasmids of interest: one vector with the grapevine genes and another one for enhanced malonil-CoA production. The resulting synthetic bacteria will be able to increase resveratrol production from p-coumaric acid and malonil-CoA, both precursors obtained from by-products of the wine industry waste.
The Golden Gate Cloning Method
The Golden Gate technique is a cloning method that allows the assembly of multiple DNA fragments in a user-defined arrangement. This technique utilizes type IIS enzymes. Firstly, the recognition site of the enzyme is non-palindromic (between 4 and 7 nucleotides). Additionally, its cutting sequence is outside the enzyme's recognition sequence, making the assembly functionally scarless.
Various type IIS enzymes are used to cut DNA and produce different ends. For example, the enzyme we use, BsaI, leaves a 4-nucleotide overhang on the strand. By making the cut outside the enzyme's recognition site, the ends, also known as fusion sites, can be designed for compatibility between parts. BsaI can generate 256 possible combinations of nucleotides for fusion sites.
Blue: restriction enzyme recognition site.
The orientation of the recognition sites of IIS enzymes and fusion sites is critical for the success of the Golden Gate technique. In the target vector, the sites must be facing away from each other. As a result of cutting after the recognition sequence, the remaining ends are not complementary to each other, preventing the vector from closing once the IIS enzyme is added.
The engineering cycle
In our project, one engineering cycle has been designed, divided into levels L0, L1 and L2. The design - build - test - learn cycle was developed in the laboratory, based on the set of constructs shown below.
At the L0 level, the four genes of interest (STS, 4CL, TAL and PPOR) and the other three fragments (T7-LacO promoter, ribosome binding region or RBS, and T7 terminator) must be synthesized individually. The PPOR gene encodes a red fluorescent protein that will allow us to confirm the correct expression of the other three genes, due PPOR has the same elements as the other constructions. Thus, each fragment is introduced into a pUC19 plasmid with ampicillin resistance using the restriction enzyme SmaI (blunt ends), resulting in seven L0 plasmids.
At the L1 level, four Golden Gate constructs are made, one for each gene of interest (STS, 4CL, TAL and PPOR). Each kanamycin-resistant backbone (A, B, C and D), obtained from the iGEM kit, contains a full construct with a T7-LacO promoter, a RBS and a T7 terminator for each gene, to ensure correct gene expression. To obtain L1 constructs, we employed pJUMP29 as backbone. Both the backbone and each L0 vector are digested with BsaI enzyme and ligated, generating the correct construction.
At the L2 level, a single spectinomycin-resistant construct must be elaborated using the pJUMP49 plasmid, containing all four genes. Each L1 backbone construct (T7-LacO promoter, RBS, gene and T7 terminator) is extracted and then assembled in tandem, resulting in an L2 plasmid with all four constructs. This Golden Gate construct is assembled using the BsmBI enzyme.
Thus, this final 9217 pb construct would enable the bacteria to express the genes and synthesize resveratrol from by-products of the wine industry (Figure 7). However, since the T7-LacO promoter is inducible by IPTG, it would be necessary to remove glucose and provide lactose (or IPTG) to the bacterial culture, in order to achieve a better gene expression.
Fine-tuning of engineered bacteria
Once the engineered bacteria have been successfully obtained, an analysis of bacterial growth can proceed. Bacterial growth can be recorded according to the concentration of sieved by-products supplied. For this, different known sieve concentrations can be determined and fed to our engineered E.coli, which must be monitored periodically (e.g. every 1h) for a specific period of time. This will allow the respective growth curves to be produced. From this data it will be possible to determine the sieving concentration at which the bacteria have an optimal growth, and therefore a better resveratrol synthesis.