Team:Virginia/Engineering
Index:
Engineering
Asking New Questions
Manifold introduces many significant improvements to the efficiency and capability of biosynthesis. Bacterial compartmentalization increases metabolic yield by decreasing the available reaction space and controlling metabolite passage. However, this advancement introduces a difficulty derived from the use of bacterial microcompartments for a role outside of their native application. The compartmentalization capabilities, which serve a crucial role in Manifold’s design, are due to the ability of BMCs to serve as selective barriers for metabolite diffusion. While this selective diffusion is crucial to the functionality of Manifold, it also requires all precursors and coenzymes be able to diffuse through the BMC pore and enter the reaction space. This requirement is an integral part of our continued development of Manifold and our proof of concept, resveratrol production system.
Our initial design utilized only the two enzymes responsible for resveratrol production, 4-coumarate-CoA ligase (4CL) and stilbene synthase (STS), from p-Coumaric. In order for this reaction to occur, malonyl-CoA must be present in the reaction space
[1]C. Lim, Z. Fowler, T. Hueller, S. Schaffer, and M. Koffas, “High-Yield Resveratrol Production in Engineered Escherichia coli,” Appl. Environ. Microbiol., vol. 77, pp. 3451–60, Mar. 2011, doi: 10.1128/AEM.02186-10.
. Thus, our original design required the diffusion of malonyl-CoA through the BMC pore. An investigation of this metabolite in relation to the pore (for more information see the Modeling page) introduced concerns about the ability of malonyl-CoA to freely diffuse into the BMC. Malonyl-CoA is much larger than metabolites which have previously been shown to move through the pore and has significant steric hindrances stemming from the bulky CoA group [2]C. Chowdhury, S. Sinha, S. Chun, T. O. Yeates, and T. A. Bobik, “Diverse Bacterial Microcompartment Organelles,” Microbiol. Mol. Biol. Rev. MMBR, vol. 78, no. 3, pp. 438–468, Sep. 2014, doi: 10.1128/MMBR.00009-14.
. Malonyl-CoA is also utilized in the fatty acid biosynthesis cycle; therefore, our system will be competing with native pathways for the limited molecule [1]C. Lim, Z. Fowler, T. Hueller, S. Schaffer, and M. Koffas, “High-Yield Resveratrol Production in Engineered Escherichia coli,” Appl. Environ. Microbiol., vol. 77, pp. 3451–60, Mar. 2011, doi: 10.1128/AEM.02186-10.
. The Manifold system is unlikely to create significant increases in yield if the availability of malonyl-CoA serves to limit the reaction rate. Thus, we were forced to take a step back and redesign our system to address this new need.
Engineering Solutions
We choose to focus on acetate, a molecule found two steps upstream of malonyl-CoA in the glucose metabolism pathway
[3]W. Zha, S. B. Rubin-Pitel, Z. Shao, and H. Zhao, “Improving cellular malonyl-CoA level in Escherichia coli via metabolic engineering,” Metab. Eng., vol. 11, no. 3, pp. 192–198, May 2009, doi: 10.1016/j.ymben.2009.01.005.
. The size of and functional groups present in acetate closely resemble 1,2-propanediol which exhibits free pore diffusion as it is the natural precursor of the reactions which occur in PDU BMCs. This led us to conclude that acetate will be able to diffuse through the pore more readily than malonyl-CoA.
Fig 1. Figure taken from iGEM Tainan 2019 for demo purposes. Notice how the figure is much longer than it is wide, and two images are coupled together to achive this. Try to do that as well so it looks good.
Additionally, protein linkers are usually present between this nucleic acid binding domain and the enzyme structure to prevent inhibition of enzyme activity. However the exact linker(s) used, if any, is(are) also dependent on the specific use case of the invention. These pathway enzymes are attached to the nucleic acid scaffolds via their nucleic acid binding domains. The nucleic acid recognition sequences (22) are unique or semi-unique sequences of nucleic acid monomers on the nucleic acid scaffolds to which the utilized nucleic acid binding domains have some degree of molecular complementarity. These nucleic recognition sequences comprise most of the scaffold and mark the locations to which the DNA binding domains of the pathway enzymes attach to the scaffolds. The nucleic acid spacers (32) are relatively short sequences of nucleic acid monomers that are also present on the nucleic acid scaffolds, between the recognition sequences. The linkage between the nucleic acid scaffolds (18) and protein shell (10) provides a means by which the nucleic acid scaffolds are bound to the protein shell through direct or multi-molecule complementarity. This linkage is found between the nucleic acid scaffolds and the protein shell. One example is through the addition of a nucleic acid binding domain (24) to one or more of the shell proteins forming a nucleic acid binding domain, shell protein fusion (14). Like the pathway enzymes, this nucleic acid binding-domain can be either internal to the shell protein structure or at its N or C terminus, where the exact placement depends on the shell protein being utilized. Alternatively, one or more intermediate proteins can be used to adhere the nucleic acid scaffolds to the shell, where the region of the protein interacting with the shell binds the shell via protein-protein complementarity (28) with a given shell protein, and the region of the protein interacting with the nucleic acid scaffold binds another recognition sequence on the nucleic acid scaffold through another nucleic acid binding domain (30). This forms a shell protein binding, nucleic acid domain fusion (26).
