Difference between revisions of "Team:CSU Fort Collins/Description"

Contents

1 Introduction
2 Breakdown
3 Terpenoid Production
4 Design Considerations
5 References

Introduction

Many valuable terpenoids are produced by plants in low concentrations which makes them costly to extract and limits commercial usage. Our project aims to engineer E. coli to produce a terpenoid via synthetic pathways. It has been shown to be more economic to produce terpenoids for commercial use in this manner. Our strains were first optimized to digest spent frying oil, a cheap carbon source. This was achieved by increasing beta-oxidation in the cell. The cells are then able to produce our product, a high-value plant steroid. This product can then be extracted from the cells and purified to produce a laboratory- and industrial-grade product.

Breakdown

Frying oil is mainly composed of triacylglycerols (TAGs). When heated to high temperatures during the frying process, TAGs breakdown into diacylglycerols (DAGs), monoacylglycerols (MAGs), and free fatty acids (FFAs). While this composition is no longer suitable for frying, the DAGs, MAGs and FFAs leftover are high-energy molecules, making the spent oil an ideal feedstock.

E. coli naturally breaks DAGs and MAGs down into their components, glycerol and fatty acids. These fatty acids and the ones already present in the oil are then broken down further by the beta-oxidation cycle. The rate limiting step of the beta-oxidation cycle is the synthesis of long-chain Acyl-CoA. Acyl-CoA synthetase (FadD) is transcriptionally regulated by FadR, slowing the rate of beta-oxidation when fatty acids are not present. By increasing the copy number of FadD under the control of a constitutive promoter our strain will be better able to utilize frying oil waste. It will also have excess energy and carbon to use for the creation of our product.

Terpenoid Production

Terpenoids are a large class of molecules which have a wide range of applications, including anti-malarial, aromatic, and coloration compounds. They are all derived from two molecules, isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). Because of the high cost and low yield of plant extraction to obtain these molecules, as well as the high cost of synthesis of these molecules, synthetic insertion of these pathways into bacterial hosts could provide an economical alternative to their current production.^[6]

Terpenoid production can be achieved through two pathways. The methylerythritol phosphate (MEP) pathway is endogenous to E. coli and other bacteria. Higher eukaryotes instead use the mevalonate (MVA) pathway to produce the necessary IPP and DMAPP. By increasing flux through the MEP pathway, we will increase product yields.

Design Considerations

As our project is based on creating a manufacturing process, our main focus is on its feasibility for use in industrial biotechnology. Even with the rigorous safety standards setup by organizations like OSHA, chemical spills are still a regular occurrence. When bioproduction reaches large industrial scale, cellular level biocontainment will need to become standard. Our idea is to create a kill switch which would add an extra level of protection for use in large-scale production and that can be easily used by other teams for years to come.

Our current kill switch utilizes a gene called KillerRed from Anthomedusae sp. DC-2005. It is called KillerRed because when the protein is produced it turns the cells red. When green light interacts with the KillerRed protein, it creates reactive oxygen species (ROS). ROS attack essential molecules like: the fatty acids in the cell’s membrane, proteins and DNA. As sunlight covers all wavelengths of visible light, simple exposure to sunlight will be enough to kill any escaped cell. While in our system the expression of KillerRed will be repressed. If the cells escape to the environment, KillerRed is produced and cells die when exposed to light.

References

1. Bulina, M., et. al. "A genetically encoded photosensitizer.” Nature Biotechnology, 95-99. 2005. October 2015.
2. Cabiscol, E., Tamarit, J., & Ros, J. (2010). Oxidative stress in bacteria and protein damage by reactive oxygen species. International Microbiology, 3(1), 3-8.
3. Dobarganes, M. Carmen. “Formation of New Compounds during Frying - General Observations.” AOCS Lipid Library. Web. 2 February 2009. June 2015.
4. Kameda, Kensuke, et al. “Purification and Characterization of Acyl Coenzyme A Synthase from Escherichia coli.” The Journal of Biological Chemistry, 11. Web. 10 June 1981. June 2015.
5. Martin, Vincent JJ, et al. "Engineering a mevalonate pathway in Escherichia coli for production of terpenoids." Nature Biotechnology; 21. Web. 1 June 2003. June 2015.
6. Pletnev, S., Gurskaya, N. G., et al. (2009). Structural basis for phototoxicity of the genetically encoded photosensitizer KillerRed. Journal of Biological Chemistry, 284(46), 32028-32039.
7. Zhang, Hanxing, et al. “Molecular effect of FadD on the regulation and metabolism of fatty acid in Escherichia coli.” FEMS Microbiology Letters, 2. Web. 16 May 2006. June 2015.

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