Parts
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Introduction
The goal of Symemco is the production of cutting edge drugs in the treatment of Alzheimer’s disease (AD). To this effect our team has identified the compound pterostilbene as a potent anti-inflammatory medication for the treatment of AD. To produce the compound our team turned to synthetic biology, by synthetically engineering E. coli cells to express four enzymes that mimic the biosynthetic pathway of pterostilbene within plants. Listed below are the basic and composite parts used for this goal.
Basic Parts
Table 1: Coding Sequences
Table 1. Depicting Coding sequences: BBa_K4388001 (RgTAL mutant), BBa_K4388002 (At4CL mutant),, BBa_K4388003 (VvSTS mutant), and BBa_K4388004 (VvRomt mutant)
Table 2: 5'UTR
Table 2. Depicting 5’ untranslated region (5’ UTR) sequences: BBa_K4388013 and BBa_K4388011,
Table 3: 3'UTR's
Table 3. Depicting 3’ untranslated region (3’ UTR) sequences:BBa_B0015 and BBa_K4388012,
Table 4: Backbones
Table 4. Depicting backbone sequences used: BBa_J428385, BBa_J428341, BBa_J428342, BBa_J428343, BBa_J428344, and BBa_J428361
Composite Parts
Table 5: Synthetic Operon Parts
Table 5. Depicting composite parts made: BBa_K4388005, (RgTAL-TU1), BBa_K4388006, (At4CL-TU2), BBa_K4388007,(VvSTS-TU3) BBa_K4388008,(VvROMT-TU4 and BBa_K4388010 (4-TU construct)
Rhodotorula glutinis Tyrosine Ammonia-Lyase
Tyrosine ammonia lyase (TAL) is the first enzyme necessary for the pathway we are using to synthesize pterostilbene from L-Tyrosine. TAL catalyzes the deamination of the amino acid L-tyrosine into p-coumaric acid (4-coumaric acid) (Halls & Yu, 2008; Pinto et al., 2015). Rhodotorula glutinis TAL (RgTAL) has been used mostly due to its much higher affinity towards L-tyrosine than L-phenylalanine (Camacho-Zaragoza et al., 2016), a desired characteristic to maximize utilization of L-tyrosine as a substrate. Consequently, engineering of E. coli for Resveratrol or Pterostilbene production from a tyrosine precursor often uses RgTAL for this step of the pathway (Feng et al., 2022). RgTAL was demonstrated to have greater catalytic efficiency than other TAL homologues (Cui et al., 2020), therefore we determined it to be the optimal enzyme for our first step in pterostilbene biosynthesis.
Arabidopsis thaliana 4-Coumarate:CoA Ligase Mutant
The 4-coumarate:CoA ligase (4CL) enzyme is a member of the ANL superfamily of CoA ligases which includes enzymes such as luciferase and long chain fatty acid CoA ligase (Gulick, 2009). 4CL enzymes work by taking in the substrate p-coumaric acid alongside ATP and coenzyme A, converting these substrates into 4-coumaroyl-CoA, diphosphate and AMP. Starting from an L-tyrosine precursor, this is the second step of biosynthesis in our modified E. coli. Many organisms produce homologues of the 4CL enzyme so to choose the optimal sequence, we looked into literature on synthetic stilbene synthesis.
The 4CL enzyme from Arabidopsis Thaliana was originally used by Heo et al (2017). Arabidopsis thaliana produces four 4CL enzymes (At4CL1-4) of which At4CL1 has been the most extensively used by researchers for producing pterostilbene within E. coli (Shin et al., 2011; Yechun et al., 2011; Yechun & Oliver, 2012; Zhang et al., 2006). Looking into literature we identified research supporting the effectiveness of the enzyme in producing resveratrol. In one paper by Lim et al. (2011), researchers investigated two 4CL enzymes from Arabidopsis thaliana and Pelargonium crispum. Researchers found that a combination of Vitis vinifera stilbene synthase and A. thaliana 4CL was best at synthesising resveratrol. Additionally, another paper by Wang et al. (2017) postulated that “resveratrol yield of At4CL (Arabidopsis thaliana) and AhSTS (Arachis hypogaea) is more than 6.25 times compared with Nt4CL and VvSTS” based off of two other papers (Beekwilder et al.,2006; Watts et al.,2006).
For the gene construct of pterostilbene within E. coli, the sequence was extracted from the NCBI database under the accession code NM_104046 and codon optimised to the codon bias of E. coli. Yan et al. (2021) detailed two mutations at positions L57I/L460H which increased the catalytic efficiency of At4CL by 1.7-fold. The L460H is at the entrance of the active site for At4CL so it may be that this mutation alters the accessibility of the substrate to the active site. L57I is distally located from the active site however and so its role is unclear. For the At4CL mutant, the wild type sequence was taken and converted at the appropriate locations to the mutant and then codon optimised.
Vitis vinifera Stilbene Synthase Mutant
Stilbene synthase (STS, EC 2.3.1.95) is a transferase of the polyketide synthase family that catalyses the C2-C7 aldol condensation of three malonyl-CoA derived acetyl units onto 4-coumaroyl-CoA, through its thioesterase activity followed by a specific cyclisation mechanism, to produce resveratrol, Coenzyme A, and carbon dioxide (Austin et al., 2004; Bhan et al., 2015). The main catalytically relevant structures within STS are the CoA binding tunnel, the substrate binding pocket, and the cyclization pocket (Austin & Noel, 2003). The thioesterase activity of STS is facilitated by a specialised hydrogen bonding network between Thr132, Ser338 and Glu192 with a water molecule (Austin et al., 2004).
The activity of STS has been explored and further engineered for the synthesis of novel compounds as well as the improvement of existing product yields. A well investigated application of the STS function is for resveratrol and its derivatives’ biosynthesis in microorganisms (Feng et al., 2022), with further improvements being demonstrated via mutagenesis (Bhan et al., 2015a, 2015b; Yan et al., 2021).
The STS from Vitis vinifera has shown the greatest resveratrol titre amongst the well studied STS homologues (Lim et al., 2011; Camacho-Zaragoza et al., 2016), with a recent study by Yan et al. (2021) increasing the activity from the VvSTS wild type via random mutagenesis. This involved generating a T50I/V170A mutant that increased resveratrol yield 2-fold (Yan et al., 2021), attributing this to the enlarged substrate-binding pocket upon the V170A mutation.
This basic part comprises the open reading frame of the VvSTS T50I/V170A mutant derived from the wild-type sequence (GenBank: DQ459351.1), which was codon-optimised for E. coli expression, altered with the according mutations, along with attached BioBrick prefixes and suffixes compatible with RFC10 assembly method. The part was then synthesised by IDT.
Vitis vinifera Resveratrol O-Methyltransferase Mutant
Enzymes with resveratrol-methylating activity have been mostly investigated in Oryza sativa (Katsuyama et al., 2007), Vitis vinifera (Wang et al., 2014; Herrera et al., 2021; Yan et al., 2021), Sorghum bicolor (Kang et al., 2014; Jeong et al., 2014, 2015), Vitis riparia (Jeong et al., 2014, 2015), and Arabidopsis thaliana (Heo, Kang & Hong, 2017). Different resveratrol O-methyltransferases (ROMT) vary in the product titre as well as the ratio amongst the methylated products pinostilbene (PIN) and pterostilbene (PTE).
After comparing distinct ROMT enzyme variations we concluded variants like OsROMT and SbROMT reported to produce PTE largely favor the production of PIN over PTE. In contrast, VvROMT favors the dimethylation of resveratrol (RES) into PTE (Jeong et al., 2014). Overall, VvROMT has demonstrated a preference for PTE production over PIN (Wang et al., 2014; Herrera et al., 2021), with higher efficiency and product yield compared to other ROMTs (Wang et al., 2014); therefore, this enzyme has been increasingly favored for PTE production.
The enzyme VvROMT has been explored and modified in literature through directed evolution to increase the catalytic performance of the enzyme (Yan et al., 2021). Via error-PCR, VvROMT was directly evolved to identify a strain with increased pterostilbene production. Strain ROMT18 showed the highest increase in pterostilbene production due to a four base VvROMT substitution (T18G/T85C/T294C/T47) resulting in one amino acid mutant S2P9. Both in vivo and in vitro, the mutant VvROMT enzyme showed more efficient production of pterostilbene in comparison to the wild type enzyme. Furthermore, after conducting homologous modeling the literature revealed the mutant enzyme is a homodimer and the S29P mutation was located on the interface. They identified 4 hydrogen bonds between S29 and H22 that were lost as a result of the mutation, making the N-terminal domain of the enzyme more flexible and thus improving substrate binding.
References
Camacho-Zaragoza, J. M., Hernández-Chávez, G., Moreno-Avitia, F., Ramírez-Iñiguez, R., Martínez, A., Bolívar, F., & Gosset, G. (2016). Engineering of a microbial coculture of Escherichia coli strains for the biosynthesis of resveratrol. Microbial cell factories, 15(1), 163. https://doi.org/10.1186/s12934-016-0562-z
Cui, P., Zhong, W., Qin, Y., Tao, F., Wang, W., & Zhan, J. (2020). Characterization of two new aromatic amino acid lyases from actinomycetes for highly efficient production of p-coumaric acid. Bioprocess and biosystems engineering, 43(7), 1287–1298. https://doi.org/10.1007/s00449-020-02325-5
Feng, C., Chen, J., Ye, W., Liao, K., Wang, Z., Song, X., & Qiao, M. (2022). Synthetic Biology-Driven Microbial Production of Resveratrol: Advances and Perspectives. Frontiers in bioengineering and biotechnology, 10, 833920. https://doi.org/10.3389/fbioe.2022.833920
Gulick, A. M. (2009). Conformational dynamics in the Acyl-CoA synthetases, adenylation domains of non-ribosomal peptide synthetases, and firefly luciferase. ACS Chem Biol, 4(10), 811-827. doi:10.1021/cb900156h
Halls, C., & Yu, O. (2008). Potential for metabolic engineering of resveratrol biosynthesis. Trends in biotechnology, 26(2), 77–81. https://doi.org/10.1016/j.tibtech.2007.11.002
Lim, C. G., Fowler, Z. L., Hueller, T., Schaffer, S. et Koffas, M. A. G.. (2011). High-Yield Resveratrol Production in Engineered Escherichia coli. Applied and environmental microbiology, 77(10), 3451‑3460. doi:10.1128/aem.02186-10
Pinto, G. P., Ribeiro, A. J., Ramos, M. J., Fernandes, P. A., Toscano, M., & Russo, N. (2015). New insights in the catalytic mechanism of tyrosine ammonia-lyase given by QM/MM and QM cluster models. Archives of biochemistry and biophysics, 582, 107–115. https://doi.org/10.1016/j.abb.2015.03.002
Shin, S.-Y., Han, N. S., Park, Y.-C., Kim, M.-D., & Seo, J.-H. (2011). Production of resveratrol from p-coumaric acid in recombinant Saccharomyces cerevisiae expressing 4-coumarate:coenzyme A ligase and stilbene synthase genes. Enzyme and microbial technology, 48(1), 48-53. doi:https://doi.org/10.1016/j.enzmictec.2010.09.004
Yechun, W., Coralie, H., Juan, Z., Michiyo, M., Yansheng, Z., & Oliver, Y. (2011). Stepwise increase of resveratrol biosynthesis in yeast Saccharomyces cerevisiae by metabolic engineering. Metabolic Engineering, 13(5), 455-463. doi:https://doi.org/10.1016/j.ymben.2011.04.005
Yechun, W., & Oliver, Y. (2012). Synthetic scaffolds increased resveratrol biosynthesis in engineered yeast cells. Journal of Biotechnology, 157(1), 258-260. doi:https://doi.org/10.1016/j.jbiotec.2011.11.003
Zhang, Y., Li, S.-Z., Li, J., Pan, X., Cahoon, R. E., Jaworski, J. G., . . . Yu, O. (2006). Using Unnatural Protein Fusions to Engineer Resveratrol Biosynthesis in Yeast and Mammalian Cells. Journal of the American Chemical Society, 128(40), 13030-13031. doi:10.1021/ja0622094