Team:USP-EEL-Brazil/Design
Common Parts
Our project can be divided into two major components, and that's how we are going to present it, but both segments have the following parts in its composition:
BBa_B0034, which is an RBS taken from Elowitz 1999's repressilator. It is commonly used and has a deffinition of efficiency of 1.0. Note that all RBS in our design is this same one. It was chosen because of its reliability and consistency.
BBa_J23100, which is a strong constitutive promoter. This part is commonly used and is one of the strongest promoters known. This represents both promoters used. It was chosen with the intent of having the highest yield of products.
Alkanes Complex
BBa_K2149020. This contains the LuxD gene, which encodes the Fatty Acyl Transferase enzyme. The linked biobrick has an RFP gene added, but the final construct contains just the gene itself.
BBa_K2149016. This part contains the LuxE gene, which encodes the Fatty Acyl Synthetase enzyme
This enzyme converts the fatty acid substrate into the acyl-protein thioester product.
The LuxE gene together with the LuxC, accounts for the main reactions of the fatty aldehyde synthesis.
LuxC. This part contains the LuxC gene, which encodes the Fatty Acyl Reductase enzyme.
This last unit converts the acyl-protein thioester product into the fatty aldehyde. As mentioned before, it represents the most important step for the fatty aldehyde production.
Together, these 3 parts compose the Fatty Acid Reductase complex, or FAR complex. All the Lux sequences were retrieved from Photoharbdus Luminescens organisms.
The Lux genes were synthetized in the form of gBlocks, which have a maximum of 2000bp in each sequence. Therefore, all of our Lux sequences were modified to fit the gBlocks. The RBS was included in each sequence for the for the Gibson Assembly. They were then joint in a single piece by using the Gibson Assembly method. The result was the BBa_K2149017, which is the Lux Operon.
BBa_K654058. This part contains the TesA gene, which encodes a Thioesterase enzyme. It was submitted by the Utah State 2011 iGEM Team.
This last gene adds the standardization ability to our cell. The enzyme cleaves the Fatty Acyl-Acp that is being elongated by the E.coli metabolism and force the elongation process to stop. This results in fatty acid and ACP, and because of the enzyme specificity, it produces all the FAs with the same chain length. This gene is also the one we rely on to have a bigger acid availability to the cell.
BBa_K590031 This part has been submitted by the Washington 2011 iGEM Team.
This gene encodes an Aldehyde Decarbonylase enzyme. The enzyme acts on the fatty aldehyde formed by the FAR complex and removes the carbonyl group, resulting in a alkane molecule, one carbon shorter than the used aldehyde. It is essential for our pathway.
Alkane Complex full representation. Source: Personal archive.
Tocopherol Complex
BBa_K214900. This part contains the Crte gene, which encodes a Geranylgeranylpyrophosphate synthase. Its function in our pathway is to take the substrates Farnesylpyrophosphate (FPP) and Isoprenylpyrophosphate (IPP) and catalyze their reaction, forming a Geranylgeranylpyrophosphate (GGPP), the first step to our four-stepped tocopherol synthesis.
GGH. This part contains the GGH gene, which encodes the Geranylgeranylpyrophosphate reductase enzyme. This enzyme acts on the GGPP formed and reduce the substrate. The reduced product, which loses 3 of its saturations, is called Phytylpyrophosphate (PPP), achieving 2 out of 4 steps until our desired tocopherol.
BBa_K2149002. This third part contains the HPT gene, encoding the Homogenisate-phytyltransferase enzyme. This enzyme has to ability to catalyze the reaction bewtween the recently formed PPP and homogentisic acid(HGA). The resulting assemble is a 2-methyl-6-phytyl-benzoquinol (MPBQ).
BBa_K2149001. This part contains the HPPD gene, which encodes the 4-hydroxyphenylpyruvate dioxygenase enzyme. Ok, you got us, this is not the last step... But it ain't the fourth either... So this would be the 3.5 optimization step. The produced enzyme, as its name suggests, remove oxygen from the 4-hydroxyphenylpyruvate (p-HPP) substrate. The oxidation results in homogentisic acid (HGA). This acid is really important because not only it is used with PPP to form MPBQ, but also because HGA presence is the limiting step to our synthesis. This optimization incresa the formation of the composite, enabling a better and more efficient synthesis.
VTE1. Okay, now we have the true last step. 4/4. This one is the VTE1 gene, which encodes the tocopherol cyclase enzyme. What it does? Well, it takes the MPBQ and closes a cycle in the molecule, turning it into delta-tocopherol. The delta-tocopherol is than methylated, finally forming our precious product, the alpha-tocopherol.
Just like the genes from the alkanes complex, these were also synthesized into gBlocks and assembled together using Gibson Assembly. This would result in a composite part, which we unfortunately were not able to finish in time.
The three GGH, HPT and Crte genes were taken from the Synechocystis sp genome. The VTE1 gene has Arabidopsis thaliana as its origin and the HPPD gene is derived from Pseudomonas putida.
Sources
ALBERMANN, C.; et al. Biosynthesis of the vitamin E compound delta-tocotrienol in recombinant Escherichia coli cells. ChemBioChem, v.9, n.15, p.2524-2533, 2008.
CHOI, Y. J.; LEE, S. Y. Microbial production of short-chain alkanes. Nature, v. 502, n. 7472, p. 571–4, 2013.
HOWARD, T. P. et al. Synthesis of customized petroleum-replica fuel molecules by targeted modi fi cation of free fatty acid pools in Escherichia coli. PNAS, p. 1–6, 2013.
RUFFING, A. M.; JONES, H. D. T. Physiological Effects of Free Fatty Acid Production in Genetically Engineered Synechococcus elongatus PCC 7942. Biotechnol. Bioeng., v. 109, n. 9, p. 2190–2199, 2013.
Utah State iGEM. CyanoBricks. Avaible at:https://2011.igem.org/Team:Utah_State
Washington iGEM. Make it or break it. Avaible at:https://2011.igem.org/Team:Washington
