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Description
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The Problem
Introduction
Traditional methods of chemical manufacturing produce large volumes of chemical waste, release vast amounts of carbon dioxide into the atmosphere, and contribute to the soaring prices of many organic compounds and pharmaceuticals. In order to address these issues, a large-scale shift to more sustainable chemical production methods is needed. Fortunately, advances in metabolic engineering and biotechnology over the past two decades have shown that microbial biosynthesis is the greener, safer, and more sustainable approach that the planet desperately needs. But despite the advances made in the field, we still don’t possess the technology that is needed to enable a global shift toward biomanufacturing. The main reason for this is that metabolic engineering is complicated by four common problems: flux imbalances, intermediate loss, pathway competition, and toxic intermediates.
Limitations
The issue of flux imbalance occurs through a mismatch of enzyme substrate availability and enzyme efficiency. This can become an issue of overabundance or lack of intermediates in multi-enzyme pathways.
[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.
Despite pointed promoter and ribosome binding site selections, enzyme ratios can still become uneven, and modeling and tuning these ratios is often a difficult task that requires expensive and time consuming iterations of the engineering design cycle. The second issue is with the potential loss of intermediates as these intermediates cross membranes or migrate towards cell regions lacking pathway enzymes. Because of the inability of these intermediates to act as substrates for future steps, total yield decreases. The third issue arises with pathway competition, where pathway intermediates are utilized by native cellular processes, thereby reducing yield. While the fourth primary limitation lies with the presence of toxic intermediates.[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 toxic intermediates interfere with native processes of the cell through prevention of critical chemical production, leading to cell harm or death. Altogether, these issues significantly complicate the process of metabolic engineering, which limits the practicality and feasibility of producing complex compounds, thereby greatly impeding the shift away from traditional means of chemical production.
Fig 1. 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.
Bacterial Microcompartments
Bacterial Microcompartments
Bacterial microcompartments (BMCs) are organelle-like structures, consisting of a protein shell that encloses enzymes and other proteins. BMCs are typically about 40–200 nanometers in diameter and are entirely made of proteins. The shell functions like a membrane, as it is selectively permeable.
, or BMCs, have shown great promise for promoting compartmentalization and negating issues such as pathway competition. BMCs are proteinaceous shells that sequester biological processes within the cell as analogs to membrane-bound organelles in eukaryotes. Bacterial microcompartments (BMCs) are organelle-like structures, consisting of a protein shell that encloses enzymes and other proteins. BMCs are typically about 40–200 nanometers in diameter and are entirely made of proteins. The shell functions like a membrane, as it is selectively permeable.[3]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.
BMCs contain distinct pathway enzymes but maintain shell proteins that are similar functionally and genetically. [4]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.
These shell-encoding genes can be separated and expressed in E. coli cells and when the protein ratios are adequately controlled, synthesis of empty BMCs can begin. [5]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.
Pores within the BMCs selectively allow small polar molecules to diffuse through while preventing large or non-polar molecules from passing, making BMCs an ideal choice [6]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.
Fig 2. Electron micrographs showing alpha-carboxysomes (a type of BMC) from the chemoautotrophic bacterium Halothiobacillus neapolitanus: (A) arranged within the cell, and (B) intact upon isolation. Scale bars indicate 100 nm.
[7] Tsai Y, Sawaya MR, Cannon GC, et al. (June 2007). "Structural Analysis of CsoS1A and the Protein Shell of the Halothiobacillus neapolitanus Carboxysome". PLOS Biol. 5 (6): e144. doi:10.1371/journal.pbio.0050144. PMC 1872035. PMID 17518518.
Scaffolds
The reduction of flux leakage can be achieved through both scaffolding and compartmentalization. Specifically, scaffolds made from nucleic acids contain enzymes and their coenzymes locally and with the same spatial orientation for optimal pathway efficiency. These scaffolds selectively bind enzymes, allowing for successful control of enzyme ratios. Effective scaffolds include plasmids with
zinc-finger binding motifs
The zinc-finger domain is one of the most frequently utilized DNA-binding motifs found in eukaryotic transcriptional factors. The binding of a zinc-finger fusion protein to its target site on the DNA is made possible by its specific amino acid sequence. When Zinc Fingers are combined with enzymes, they serve as method of binding the enzyme to the DNA.
due to their increased production of resveratrol, 1,2-propanediol, and mevalonate when used together alongside zinc-finger fusion enzymes. The zinc-finger domain is one of the most frequently utilized DNA-binding motifs found in eukaryotic transcriptional factors. The binding of a zinc-finger fusion protein to its target site on the DNA is made possible by its specific amino acid sequence. When Zinc Fingers are combined with enzymes, they serve as method of binding the enzyme to the DNA.[8]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.
Further, scaffold structures can also be created through the in vivo production of short ssDNA [9] Geraldi, A.; Khairunnisa, F.; Farah, N.; Bui, L.M.; Rahman, Z. Synthetic Scaffold Systems for Increasing the Efficiency of Metabolic Pathways in Microorganisms. Biology 2021, 10, 216.
Fig 3. Spatial organization of pathway enzymes for efficient biosynthetic reactions. Note the DNA scaffold is the binding site for pathway enzymes.
[10] 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.
The combination of scaffolds and BMCs solve the four shortcomings of chemical manufacturing mentioned above. BMCs work to sequester pathway enzymes and potential intermediates within the vicinity, significantly decreasing chances for intermediate loss. As a result, pathway components are prevented from interacting with native processes outside of the BMC and toxic intermediate production is prevented through creating barriers between toxins and necessary cellular processes. Scaffolds localize pathway components to prevent intermediate loss and pathway competition while also controlling enzyme concentrations and spatial organization, thus minimizing flux imbalances.
Despite the advantages of each technology discussed, there is no singular, comprehensive method to address our four previously mentioned issues from chemical manufacturing. Although BMCs are promising, it is difficult to target enzymes to their interiors, limiting multi-enzyme ratios. Additionally, scaffolds only partially sequester pathway components, only reducing the presence of lost/toxic intermediates and pathway competition to a certain degree. Therefore, a device combining the advantages of each technology is urgently needed.
Our Solution
Manifold
Manifold is a device that can be used in a variety of scenarios to increase the flux of biosynthetic pathways. While the products and enzymes used in any given reaction may vary, the fundamentals of Manifold as a platform technology remain relatively the same.
Enzymes
Within an enzymatic pathway, enzymes are needed to convert precursor molecules to a final product. In our device, we combine necessary pathway enzymes with zinc fingers. When zinc fingers are combined with enzymes, they serve as a method of localizing the enzyme to a specific place on the DNA. The complex of an enzyme and zinc finger is known as zinc finger fusion protein.
Zinc Finger
Zinc finger fusion proteins bind to specific sites on the DNA scaffold, known as zinc finger fusion binding sites, and can be seen on the animated DNA strand as different colors separated by gray spacers. Once the DNA scaffolds have been created, they serve as a landing pad for zinc finger fusion proteins to bind. The binding motifs along the DNA scaffold are spacially located in a deliberate manner to allow for each intermediate to find its particular enzyme in a streamlined fashion, increasing the production of the desired product.
BMC
To isolate our device from the rest of the system, bacterial microcompartments (BMCs) are used. This allows for many similar enzyme-bound scaffolds to spatially localize without competitive inhibition inside the BMC, resulting in an increased production of the desired product. The use of BMCs in this fashion also contains any unwanted toxic intermediates inside the BMC and away from the lumen of the cell.
Manifold in Action
In our quest to increase the metabolic flux of a reaction, we chose to focus on the resveratrol pathway. The resveratrol pathway is a very well understood pathway that occurs in two simple steps, making it a perfect test-product for our devise. To increase pathway flux of resveratrol, we propose the use of zinc-finger-domain-containing DNA scaffolds localized to the lumen of bacterial microcompartments (BMCs), to which, upon translation and locationaztion, zinc finger fusion proteins capable of resveratrol biosynthesis are bound.
ENZYMES
Within the resveratrol pathway, two enzymes are needed to convert a precursor molecule (p-Coumoric acid), to the final product of resveratrol. Those two enzymes are 4-Coumarate-CoA ligase (4CL) and Stilbene Synthase (STS). In addition, two adjacent enzymes, acetyl-CoA Synthetase (ACS) and Acetyl-CoA Carboxylase (ACC) use acetate as a carbon source to create acetyl-CoA and malonyl-CoA, both of which are driving metabolites of the resveratrol pathway and needed for the continual recycling of necessary pathway intermediates.
Within the resveratrol pathway, two enzymes are needed to convert a precursor molecule (p-Coumoric acid), to the final product of resveratrol. Those two enzymes are 4-Coumarate-CoA ligase (4CL) and Stilbene Synthase (STS). In addition, two adjacent enzymes, acetyl-CoA Synthetase (ACS) and Acetyl-CoA Carboxylase (ACC) use acetate as a carbon source to create acetyl-CoA and malonyl-CoA, both of which are driving metabolites of the resveratrol pathway and needed for the continual recycling of necessary pathway intermediates.
Fig 4. Biosynthesis of the Resveratrol pathway
The final enzyme in our system encodes for PduD, a shell protein that bridges our DNA scaffold to the inside of the BMC. Each pathway enzyme in our system requires a Zinc-Finger protein to be bound and thus a subsequent zinc-finger fusion protein binding site on the DNA scaffold. When Zinc Fingers are combined with enzymes, they serve as a method of binding the enzyme to the DNA.
SCAFFOLDS
The creation of the dsDNA scaffolds themselves involves a unique process in which that we express RNA templates to then be converted into our dsDNA scaffolds. By doing it in this manner, we have control over the concentration of our scaffolds under different promoters, similar to the expression of a protein. The RNA templates are comprised of two complementary sequences of RNA known as r_oligos. Each stand consists of an HIV-RT (Human Immunodeficiency Virus - reverse transcriptase) promoter known as the HIV Terminator-Binding Site (HTBS). The enzyme HIV-RT processes a template from the 3’ to 5’ direction, meaning the promoter, HTBS, should be placed downstream of the resveratrol pathway enzyme’s binding domains. As the complementary RNA templates are created, they are transcribed into ssDNA complements by two enzymes, HIV reverse transcriptase (HIV-RT) and murine leukemia reverse transcriptase (ML-RT). While only HIV-RT is needed to produce the DNA scaffolds, it has been seen that the co-expression of ML-RT leads to an increase in DNA scaffold production. Once both ssDNA scaffold strands are created, they anneal into double-stranded DNA which contain the binding domain of zinc finger fusion proteins, completing the creation of our DNA scaffolds.
The creation of the dsDNA scaffolds themselves involves a unique process in which that we express RNA templates to then be converted into our dsDNA scaffolds. By doing it in this manner, we have control over the concentration of our scaffolds under different promoters, similar to the expression of a protein. The RNA templates are comprised of two complementary sequences of RNA known as r_oligos. Each stand consists of an HIV-RT (Human Immunodeficiency Virus - reverse transcriptase) promoter known as the HIV Terminator-Binding Site (HTBS). The enzyme HIV-RT processes a template from the 3’ to 5’ direction, meaning the promoter, HTBS, should be placed downstream of the resveratrol pathway enzyme’s binding domains. As the complementary RNA templates are created, they are transcribed into ssDNA complements by two enzymes, HIV reverse transcriptase (HIV-RT) and murine leukemia reverse transcriptase (ML-RT). While only HIV-RT is needed to produce the DNA scaffolds, it has been seen that the co-expression of ML-RT leads to an increase in DNA scaffold production. Once both ssDNA scaffold strands are created, they anneal into double-stranded DNA which contain the binding domain of zinc finger fusion proteins, completing the creation of our DNA scaffolds.
Fig 5. General overview of the parts that comprise the Manifold system. Within the BMC parts, PduA through PduT are shell proteins that allow for the close formation of the BMC shell components. The PduD (found under the enzymes list) part creates a PduD protein fused to a zinc finger binding protein, allowing the protein to bind to the DNA scaffolds and localize inside the forming BMC. Scaffold parts consist of reverse transcriptase p66/p55 and MLRT, which attach to transcribed scaffold parts containing zinc finger binding motifs and HTBS and convert them from RNA to DNA. Reverse transcribed scaffold parts can then bind to their complement, forming linear double-stranded DNA scaffolds. Enzyme parts consist of fusion proteins of the enzymes needed for the biosynthesis of Resveratrol and zinc finger binding proteins, allowing for their attachment to their respective DNA scaffolds.
BMC
To complete the system, we encapsulate our scaffolds into a bacterial microcompartment, where they may produce products unperturbed. The assembly of an empty Pdu microcompartment shell, requires PduA, PduB, PduJ, PduK, PduN, PduU, and PduT proteins, forming the complete construct, PduABJKNUT.
To complete the system, we encapsulate our scaffolds into a bacterial microcompartment, where they may produce products unperturbed. The assembly of an empty Pdu microcompartment shell, requires PduA, PduB, PduJ, PduK, PduN, PduU, and PduT proteins, forming the complete construct, PduABJKNUT.
Conclusion
Implication
Manifold is a platform technology designed to improve the efficiency of and introduce new functionalities to biosynthesis within prokaryotes such as Escherichia coli through the combination of DNA scaffolds with BMC shells. Specifically, DNA scaffolds containing 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. Enzymes bind to the scaffolds while the shell simultaneously assembles zinc-finger fusions of the pathway, allowing enzymes to be targeted to the interior of the BMC.
The combined solution of utilizing scaffolds and BMCs through Manifold provides a more comprehensive solution to our challenges of compartmentalization and organization. Manifold ensures enzymes are produced at the same rates, any intermediates are contained, there no is competition from interfering enzymes, and toxic intermediates are entirely prevented. In sum, Manifold optimizes metabolic flux through its creation of pathway orthogonality, increases the efficiency of existing biosynthetic pathways, and allows for development of previously impossible pathways.
Resources
[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] 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.
[4] 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.
[5] 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.
[6] 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.
[7] Tsai Y, Sawaya MR, Cannon GC, et al. (June 2007). "Structural Analysis of CsoS1A and the Protein Shell of the Halothiobacillus neapolitanus Carboxysome". PLOS Biol. 5 (6): e144. doi:10.1371/journal.pbio.0050144. PMC 1872035. PMID 17518518.. [8] 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. [9]Geraldi, A.; Khairunnisa, F.; Farah, N.; Bui, L.M.; Rahman, Z. Synthetic Scaffold Systems for Increasing the Efficiency of Metabolic Pathways in Microorganisms. Biology 2021, 10, 216. [10] 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.
[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] 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.
[4] 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.
[5] 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.
[6] 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.
[7] Tsai Y, Sawaya MR, Cannon GC, et al. (June 2007). "Structural Analysis of CsoS1A and the Protein Shell of the Halothiobacillus neapolitanus Carboxysome". PLOS Biol. 5 (6): e144. doi:10.1371/journal.pbio.0050144. PMC 1872035. PMID 17518518.. [8] 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. [9]Geraldi, A.; Khairunnisa, F.; Farah, N.; Bui, L.M.; Rahman, Z. Synthetic Scaffold Systems for Increasing the Efficiency of Metabolic Pathways in Microorganisms. Biology 2021, 10, 216. [10] 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.
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