Team:Virginia/Poster

Manifold: Protein Shells with Encapsulated DNA Scaffolds for Increasing Efficiency of Biosynthetic Pathways
Presented by Team Virginia 2020

Team: J. Ball, V. Gutierrez, C. Haws, A. Kola, S. Link, C. Marino, E. Micklovic, D. Patel, J. Polzin, A. Pradhan, P. Revelli

Advisors: K. G. Kozminski, J. A. Papin

Abstract:
The lack of a versatile and reliable way to improve metabolic flux channeling, pathway orthogonality, and product yields is a major impediment to the expanded utilization of biosynthesis for the production of drugs and industrially valuable chemicals. Manifold, a platform technology that addresses this problem, consists of bacterial microcompartments (BMCs) with encapsulated dsDNA scaffolds that sequester and spatially organize, at fixed concentrations, biosynthetic enzymes presented as zinc-finger fusion proteins. Here we deliver the designs for an E. coli cell capable of synthesizing resveratrol using the Manifold platform. The Manifold platform will help lower costs and expand the applications of chemical biosynthesis.
Inspiration
  • Prokaryotic biosynthesis capabilities suffer from a lack of methods for designing sufficiently controlled orthogonal pathways.
  • To address this issue two fields of research stand out: scaffolding and bacterial microcompartments (BMCs).
  • Scaffolds ensure enzymes are kept in proximity of pathway components and at appropriate relative concentrations.
    • Scaffolding has been shown to provide 5 fold increases in production of select chemicals in E. coli
  • BMCs are proteinaceous shells which mimic the compartmentalization capabilities of eukaryotic organelle.
    • Empty BMCs have been produced in E. coli suggesting their potential to serve as nanoreactors for biosynthesis.
4 Big Problems in Biosynthesis
  • Flux imbalances occur when the amount of substrate available does not match the efficiency of the enzyme. In multi-enzyme pathways, this results in an overabundance or lack of intermediates, even with careful promoter and ribosome binding site choice.
  • Loss of intermediates can occur as pathway intermediates cross a membrane or move to a region of the cell where pathway enzymes are not present and thus, overall yields are reduced.
  • Pathway competition also reduces yield. 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 the desired pathway.
  • Toxic intermediates can cause harm or cell death before any product can be made making biosynthesis highly inefficient or impossible for many pathways.
Proposed Solution
    Manifold is a platform technology designed to improve the efficiency of biosynthesis in prokaryotes by combining DNA scaffolds with BMC shells.
  • Short double-stranded DNA scaffolds with zinc-finger binding motifs are produced in vivo by reverse transcriptases.
  • Scaffolds localized to the lumen 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 for localization of the enzymes to the BMC interior.
  • The combination of scaffolds and BMCs creates a comprehensive solution to the compartmentalization and organization needs.
  • Proteinaceous BMC shells solve the problems of lost intermediates, pathway competition, and toxic intermediates by sequestering the pathway.
  • Scaffolds provide a convenient way to target enzymes to the interior of the BMC in controllable ratios to prevent flux imbalances.

    • 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.
Idea
    Resveratrol biosynthesis was chosen for a proof of concept due to its simplicity and capacity to be improved through scaffolding. (Cite)
  • Biosynthesis of resveratrol from p-Coumaric acid requires two enzymes, 4CL and STS, and the coenzyme malonyl-CoA.
    • Figure of resveratrol pathway
  • Steric hindrance of malonyl-CoA makes it unlikely to diffuse into the BMC through a pore.
    • Hindrance pore model
  • Malonyl-CoA must be available within the BMC to allow for resveratrol production.
  • ACC and ACS produce malonyl-CoA from acetate.
    • Figure of acetate pathway
  • Introduction of an additional scaffold with ACC and ACS fusion enzymes allows malonyl-CoA recycling within the BMC.
Modeling
    Part Expression Optimization - We used the Anderson RBS/Promoter libraries and the Salis Lab RBS Calculator to effectively control part expression.
  • Relevance - This process directly assisted our wet lab team by using quantification to determine the RBS\Promoter sequences for each part.
  • Model Validity – We utilized a mass action model to validate the RBS/Promoter combinations for parts required in creation of DNA Scaffolds.



    • BMC Pore Dynamics - We used PyMOL to visualize BMC pore interactions for acetate in comparison to natural BMC metabolites. These pore energies were then modeled using a mass action, three-compartment system to quantify BMC concentrations at quasi-steady state.
  • Relevance - This process generated a bounded estimation for the acetate concentration available for our compartmentalized pathway.
  • Model Validity - The compartment model is derived from Arrhenius and Fick’s Law equations and uses pore energies determined in silico. Further validation of this approach will require in vitro testing.



    • Reaction Kinetics - We used Michaelis-Menten equations to determine theoretical resveratrol concentration over time in our BMC system and a shell-free system.
  • Relevance - This process quantified the expected increase of resveratrol production in the BMC system compared to a shell-free system. MANIFOLD improves the resveratrol titer by a factor between 206.5 and 434.2 compared to a shell-free system after 12 hours.
  • Model Validity - Reaction kinetic models utilize parameters established in vitro and shell-free titer appears consistent with previous studies.(https://aem.asm.org/content/77/10/3451) The model relies on inputs from the pore dynamics methodology suggesting the necessity of in vitro studies for further model validation.

Achievements
  • Models validated that the Manifold design can provide a 206 fold increase in resveratrol production when compared to a free enzyme system.
  • A provisional patent has been filed and preliminary market research into resveratrol has been conducted to facilitate a future transition into the start-up space.
  • The “Resource Hub” contains tools for collaboration and organization and will be shared via the iGEM Foundation website after the competition.
  • To establish a company culture of respect and inclusion a Code of Ethical Conduct was constructed describing standards of behavior and just implementation of Manifold.
  • A proof of concept utilizing the resveratrol biosynthesis pathway was designed and is being implemented.
Future Directions
  • We will implement our extensive laboratory plans to develop and test our proof of concept.
  • Ideal enzymatic ratios will continue to be developed based on the model developed by the UChicago iGEM team to optimize enzymatic ratios for flux imbalance.
  • Following a successful in vivo proof-of-concept, a full patent application will be filed.
  • Manifold’s use will be expanded to new biosynthesis pathways through utilization of cutting edge BMC research namely controlled variation of pore size.
Integrated Human Practices
    Collaborating with University of Chicago
    UChicago’s R-based software utilizes reaction parameters from a multi-enzyme pathway to find the most efficient enzyme stoichiometry, which was used to optimize enzyme concentrations.

    Code of Ethical Conduct
    Our Code of Ethical Conduct establishes a framework for our projects implementation, and the guidelines we choose to abide by in our effort to maximize good and mitigate risk.

    Establishing an Entrepreneurship committee
    Industry research helped us contextualize the use and applications of Manifold in the chemical manufacturing industry. To further our vision, our team came up with an all-encompassing business plan to map out the steps required to establish a start-up. Most importantly, our Provisional Patent Application was reviewed and accepted by the USPTO.

    Understanding the content better
    None of this would have been possible without invaluable design and lab advice from Dr. Cheryl Kerfeld of UCLA and Michigan State, Dr. Martin Warren of Canterbury, UK, and Dr. George McArthur, founder of Virginia iGEM and current Head of Product at Ansa Biotechnologies, Inc. We also received tremendous guidance on building our models from Dr. Jason Papin, who is one of our advisors, and Ryan Taylor, a former Virginia iGEM captain.
References
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