Team:Virginia/Description
Index:
Description
Biosynthesis & Its Limitations
Many of the crowning achievements of biological engineering and synthetic biology have involved the use of bacterial and yeast systems to produce chemicals of medical and industrial value. This includes the production of anti-malarial drugs by yeast and stilbenoid natural-phenols such as resveratrol by Escherichia coli
[1]D.-K. Ro et al., “Production of the antimalarial drug precursor artemisinic acid in engineered yeast,” Nature, vol. 440, no. 7086, pp. 940–943, Apr. 2006, doi: 10.1038/nature04640.
, [2]M. Katz, H. P. Smits, J. Forster, and J. B. NIELSEN, “Metabolically engineered cells for the production of resveratrol or an oligomeric or glycosidically-bound derivative thereof,” US9404129B2, Aug. 02, 2016.
. These fields offer exciting possibilities to circumvent inconsistencies, dangers, or costs associated with plant-based or chemical production methods, and develop previously unseen chemicals with new functions. However, many limitations exist which prevent the implementation of biosynthetic production methods for efficient manufacturing of a wider array of chemicals.
Fig 1. The chemical structure of 1) the anti-malarial drug artemisinin and 2) the stilbenoid natural-phenolic chemical resveratrol.
Scaffolds & BMCs
Of these limitations, there are four large issues related to the need for a method of compartmentalization and organization of engineered biosynthesis pathways in cells. First, flux imbalances can occur when the amount of substrate available to an enzyme does not match the efficiency of the enzyme. In multi-enzyme pathways, this will result in an overabundance or lack of intermediates. While careful promoter and ribosome binding site choice can allow control of enzyme levels, it is still possible for flux imbalances to occur when enzyme ratios are uneven within a region of the cell. The second major constraint is the loss of intermediates as they cross a membrane or move to a region of the cell where pathway enzymes are not present. These intermediates can not act as substrates in future steps of the pathway and thus, overall yields are less than optimal. Biosynthetic yields can also be limited by pathway competition. If an enzyme, substrate, intermediate, or coenzyme of the pathway of interest is utilized by a process native to the cell it will be less likely to be available for biosynthesis. Finally, toxic intermediates can prevent any successful production of certain chemicals within biological systems. Intermediates which interfere with native processes and cause serious harm or cell death, significantly reduce chemical yields or make biosynthesis of the chemical impossible
[3]A. H. Chen and P. A. Silver, “Designing biological compartmentalization,” Trends Cell Biol., vol. 22, no. 12, pp. 662–670, Dec. 2012, doi: 10.1016/j.tcb.2012.07.002.
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Fig 2. Animations of the four major issues 1) flux imbalances 2) loss of intermediates 3) pathway competition 4) toxic intermediates which poison the cell are show from left to right.
Eukaryotic cells naturally possess compartmentalization capacity with membrane-bound organelles; however, eukaryotes generally have higher complexity which often decreases the efficiency of biosynthesis and ease of manipulation by metabolic engineering. Thus, a prokaryote-based method for addressing compartmentalization and organization of biosynthesis is more ideal.
Inspirations
Research on methods of reducing metabolic flux leakage has largely focused on two technologies: scaffolding and compartment creation.
Protein or nucleic acid-based scaffolds keep pathway enzymes and coenzymes in the same vicinity and spatially oriented to optimize pathway efficiency. Scaffolds also allow for control of enzyme ratios by selective binding of enzymes. Plasmids with
zinc-fingerZinc-fingers proteins are capable of binding to DNA by recognition of nine base pair sequences in the DNA.
binding site motifs has been shown to be effective scaffolds that can increase the production of resveratrol, 1,2-propanediol, and mevalonate when used in conjunction with zinc-finger fusion enzymes [4]R. J. Conrado et al., “DNA-guided assembly of biosynthetic pathways promotes improved catalytic efficiency,” Nucleic Acids Res., vol. 40, no. 4, pp. 1879–1889, Feb. 2012, doi: 10.1093/nar/gkr888.
. Additionally, it has been shown that short ssDNA fragments can be produced in vivo and consequently, used to create structures that may be able to serve as scaffolds [5]J. Elbaz, P. Yin, and C. A. Voigt, “Genetic encoding of DNA nanostructures and their self-assembly in living bacteria,” Nat. Commun., vol. 7, no. 1, p. 11179, Apr. 2016, doi: 10.1038/ncomms11179.
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Fig 3. Model of a DNA scaffold (green) with bound zinc-finger (purple) fusion enzymes (red).
Many different areas have been explored to design controllable compartmentalization within prokaryotes, most notably the use of bacterial microcompartments, BMCs. Native to 23 phyla of bacteria, BMCs are proteinaceous shells that sequester biological processes within the cell as analogs to membrane-bound organelles in eukaryotes
[6]S. D. Axen, O. Erbilgin, and C. A. Kerfeld, “A Taxonomy of Bacterial Microcompartment Loci Constructed by a Novel Scoring Method,” PLoS Comput. Biol., vol. 10, no. 10, Oct. 2014, doi: 10.1371/journal.pcbi.1003898.
. Notable classes of BMCs include carboxysomes, propanediol-utilizing (PDU), and ethanolamine-utilizing (EUT) BMCs, each of which encases unique pathway enzymes with functionally and genetically similar shell proteins [7]T. O. Yeates, C. S. Crowley, and S. Tanaka, “Bacterial Microcompartment Organelles: Protein Shell Structure and Evolution,” Annu. Rev. Biophys., vol. 39, pp. 185–205, Jun. 2010, doi: 10.1146/annurev.biophys.093008.131418.
. The shell-encoding genes can be separated from the rest of the operon and expressed in E. coli cells; when the protein ratios are adequately controlled, empty BMC can be synthesized [8]J. B. Parsons et al., “Synthesis of Empty Bacterial Microcompartments, Directed Organelle Protein Incorporation, and Evidence of Filament-Associated Organelle Movement,” Mol. Cell, vol. 38, no. 2, pp. 305–315, Apr. 2010, doi: 10.1016/j.molcel.2010.04.008.
. BMCs have pores that allow small polar molecules to diffuse across the shell while large or nonpolar molecules are prevented from moving into or out of the BMC; this control makes BMCs a good choice for biosynthesis pathways with polar starting materials and products and less polar intermediates [9]T. O. Yeates, J. Jorda, and T. A. Bobik, “The Shells of BMC-Type Microcompartment Organelles in Bacteria,” J. Mol. Microbiol. Biotechnol., vol. 23, no. 4–5, pp. 290–299, 2013, doi: 10.1159/000351347.
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Fig 4. Model of BMC showing various hexameric proteins which assemble to create the icosahedron shell
[10]Toyeates, English: Stylized view of the carboxysome and related bacterial structures such as the propanediol utilization (Pdu) and ethanolamine utilization (Eut) microcompartments. Distinct hexameric BMC shell proteins carrying out different functions in the shell are shown in different shades of blue. Pentameric vertex proteins are shown in magenta. Encapsulated enzymes are shown in green, organized in layers. [Image: T. Yeates]. 2013.
. Carboxysome BMCs visualized by electron micrography in the cell (A) and after isolation (B) with a scale bar representing 100 nm [11]Y. Tsai et al., “Structural Analysis of CsoS1A and the Protein Shell of the Halothiobacillus neapolitanus Carboxysome,” PLoS Biol., vol. 5, no. 6, Jun. 2007, doi: 10.1371/journal.pbio.0050144.
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Scaffolds and BMCs each address many of the issues previously mentioned. BMCs can be used to sequester pathway enzymes and intermediates, thus, significantly reducing the risk of intermediates loss by keeping them in the vicinity of pathway enzymes, preventing pathway competition by ensuring that pathway components do not interact with native processes outside of the BMC, and allowing for the production of toxic intermediates by placing a barrier between toxins and the cellular processes they interfere with. Scaffolds likewise localize pathway components which decreases the likelihood of intermediate loss and pathway competition. Scaffolds are also capable of minimizing flux imbalances by allowing for control of relative enzyme concentrations and spatial organization. Although each technology has many merits neither can solve the four problems discussed in isolation. There is currently no good method for targeting enzymes to the interior of the BMC which allows for control over the ratio of multiple enzymes within the shell. On the other hand, scaffolds do not fully sequester pathway components and thus, can only reduce, not prevent, the impact of lost and toxic intermediates, as well as, pathway competition. A device that combines the benefits of each of these technologies would effectively address each of the issues discussed.
Our Solution: MANIFOLD
Manifold is a platform technology designed to improve the efficiency of and introduce new functionalities to biosynthesis within prokaryotes, specifically Escherichia coli, by combining DNA scaffolds with BMC shells. A more full description of the technology can be found on the Design page but in short, DNA scaffolds with zinc-finger binding motifs are produced in vivo by reverse transcriptase and localized to the interior of a PDU BMC shell via an interaction with a zinc-finger fusion shell protein. As the shell assembles zinc-finger fusions of the pathway enzymes bind to the scaffolds allowing the enzymes to be targeted to the BMC interior.
The combination of both scaffolds and BMCs allows Manifold to serve as a more comprehensive solution to the compartmentalization and organization issues at hand. As previously discussed the BMC shell addresses lost intermediates, pathway competition, and toxic intermediates by sequestering the pathway, and the scaffolds provide a convenient way to target enzymes to the interior of the BMC in controllable ratios. Thus, Manifold can optimize metabolic flux by creating pathway orthogonality, which will actualize potential efficiency of existing biosynthetic pathways and allow for the development of previously impossible ones.
Fig 5. Animations of Manifold's ability to address the four major issues 1) flux imbalances 2) loss of intermediates 3) pathway competition 4) toxic intermediates are show from left to right. In 1 the light gray structure represents a DNA scaffold, and in 2-4 the light gray structures represent the proteinaceous shell of a BMC.
Manifold in 2020
This year the Virginia iGEM Team set out to develop a version of the Manifold system with the ability to synthesize resveratrol. Resveratrol was chosen for a proof of concept because it is produced by a relatively short, well understood two-step pathway, and resveratrol yields have been shown to be improved in E. coli through the utilization of zinc-finger fusion pathway enzymes bound to a plasmid scaffold
[4]R. J. Conrado et al., “DNA-guided assembly of biosynthetic pathways promotes improved catalytic efficiency,” Nucleic Acids Res., vol. 40, no. 4, pp. 1879–1889, Feb. 2012, doi: 10.1093/nar/gkr888.
. Although we were not able to access a lab space until September 8th, we were able to make the most of the summer to do extensive project development. This development is documented throughout our Wiki most notably our device design on the Design page, our computational modeling work to inform our promoter, RBS, and terminator choices on the Modeling page, our wet lab procedure planning on the Experiments page, our outreach to experts to better understand the possibilities, limitations, and needs of our project on the Human Practices page, and our patent filing and market research efforts on the Entrepreneurship page. Due to the COVID-19 pandemic, our iGEM experience was not what any of us originally expected; however, we are incredibly excited the share what we have been able to accomplish and to continue to develop Manifold into a platform technology with the ability to produce chemicals at competitive prices for medical and industrial uses.
Sources
[1] D.-K. Ro et al., “Production of the antimalarial drug precursor artemisinic acid in engineered yeast,” Nature, vol. 440, no. 7086, pp. 940–943, Apr. 2006, doi: 10.1038/nature04640.
[2] M. Katz, H. P. Smits, J. Forster, and J. B. NIELSEN, “Metabolically engineered cells for the production of resveratrol or an oligomeric or glycosidically-bound derivative thereof,” US9404129B2, Aug. 02, 2016.
[3] A. H. Chen and P. A. Silver, “Designing biological compartmentalization,” Trends Cell Biol., vol. 22, no. 12, pp. 662–670, Dec. 2012, doi: 10.1016/j.tcb.2012.07.002.
[4] R. J. Conrado et al., “DNA-guided assembly of biosynthetic pathways promotes improved catalytic efficiency,” Nucleic Acids Res., vol. 40, no. 4, pp. 1879–1889, Feb. 2012, doi: 10.1093/nar/gkr888.
[5] J. Elbaz, P. Yin, and C. A. Voigt, “Genetic encoding of DNA nanostructures and their self-assembly in living bacteria,” Nat. Commun., vol. 7, no. 1, p. 11179, Apr. 2016, doi: 10.1038/ncomms11179.
[6] S. D. Axen, O. Erbilgin, and C. A. Kerfeld, “A Taxonomy of Bacterial Microcompartment Loci Constructed by a Novel Scoring Method,” PLoS Comput. Biol., vol. 10, no. 10, Oct. 2014, doi: 10.1371/journal.pcbi.1003898.
[7] T. O. Yeates, C. S. Crowley, and S. Tanaka, “Bacterial Microcompartment Organelles: Protein Shell Structure and Evolution,” Annu. Rev. Biophys., vol. 39, pp. 185–205, Jun. 2010, doi: 10.1146/annurev.biophys.093008.131418.
[8] J. B. Parsons et al., “Synthesis of Empty Bacterial Microcompartments, Directed Organelle Protein Incorporation, and Evidence of Filament-Associated Organelle Movement,” Mol. Cell, vol. 38, no. 2, pp. 305–315, Apr. 2010, doi: 10.1016/j.molcel.2010.04.008. [9] T. O. Yeates, J. Jorda, and T. A. Bobik, “The Shells of BMC-Type Microcompartment Organelles in Bacteria,” J. Mol. Microbiol. Biotechnol., vol. 23, no. 4–5, pp. 290–299, 2013, doi: 10.1159/000351347.
[10] Toyeates, English: Stylized view of the carboxysome and related bacterial structures such as the propanediol utilization (Pdu) and ethanolamine utilization (Eut) microcompartments. Distinct hexameric BMC shell proteins carrying out different functions in the shell are shown in different shades of blue. Pentameric vertex proteins are shown in magenta. Encapsulated enzymes are shown in green, organized in layers. [Image: T. Yeates]. 2013.
[11] Y. Tsai et al., “Structural Analysis of CsoS1A and the Protein Shell of the Halothiobacillus neapolitanus Carboxysome,” PLoS Biol., vol. 5, no. 6, Jun. 2007, doi: 10.1371/journal.pbio.0050144.
[2] M. Katz, H. P. Smits, J. Forster, and J. B. NIELSEN, “Metabolically engineered cells for the production of resveratrol or an oligomeric or glycosidically-bound derivative thereof,” US9404129B2, Aug. 02, 2016.
[3] A. H. Chen and P. A. Silver, “Designing biological compartmentalization,” Trends Cell Biol., vol. 22, no. 12, pp. 662–670, Dec. 2012, doi: 10.1016/j.tcb.2012.07.002.
[4] R. J. Conrado et al., “DNA-guided assembly of biosynthetic pathways promotes improved catalytic efficiency,” Nucleic Acids Res., vol. 40, no. 4, pp. 1879–1889, Feb. 2012, doi: 10.1093/nar/gkr888.
[5] J. Elbaz, P. Yin, and C. A. Voigt, “Genetic encoding of DNA nanostructures and their self-assembly in living bacteria,” Nat. Commun., vol. 7, no. 1, p. 11179, Apr. 2016, doi: 10.1038/ncomms11179.
[6] S. D. Axen, O. Erbilgin, and C. A. Kerfeld, “A Taxonomy of Bacterial Microcompartment Loci Constructed by a Novel Scoring Method,” PLoS Comput. Biol., vol. 10, no. 10, Oct. 2014, doi: 10.1371/journal.pcbi.1003898.
[7] T. O. Yeates, C. S. Crowley, and S. Tanaka, “Bacterial Microcompartment Organelles: Protein Shell Structure and Evolution,” Annu. Rev. Biophys., vol. 39, pp. 185–205, Jun. 2010, doi: 10.1146/annurev.biophys.093008.131418.
[8] J. B. Parsons et al., “Synthesis of Empty Bacterial Microcompartments, Directed Organelle Protein Incorporation, and Evidence of Filament-Associated Organelle Movement,” Mol. Cell, vol. 38, no. 2, pp. 305–315, Apr. 2010, doi: 10.1016/j.molcel.2010.04.008. [9] T. O. Yeates, J. Jorda, and T. A. Bobik, “The Shells of BMC-Type Microcompartment Organelles in Bacteria,” J. Mol. Microbiol. Biotechnol., vol. 23, no. 4–5, pp. 290–299, 2013, doi: 10.1159/000351347.
[10] Toyeates, English: Stylized view of the carboxysome and related bacterial structures such as the propanediol utilization (Pdu) and ethanolamine utilization (Eut) microcompartments. Distinct hexameric BMC shell proteins carrying out different functions in the shell are shown in different shades of blue. Pentameric vertex proteins are shown in magenta. Encapsulated enzymes are shown in green, organized in layers. [Image: T. Yeates]. 2013.
[11] Y. Tsai et al., “Structural Analysis of CsoS1A and the Protein Shell of the Halothiobacillus neapolitanus Carboxysome,” PLoS Biol., vol. 5, no. 6, Jun. 2007, doi: 10.1371/journal.pbio.0050144.
