Team:CSU Fort Collins/Description

Project Description


Contents
1 Introduction
2 Breakdown
3 Trans-zeatin Production
4 Design Considerations
5 References


Introduction

Cytokinins are plant growth molecules which are currently produced by plant extraction. This method is inefficient and low-yield, driving up the cost of the products. Our project aims to engineer E. coli to produce a cytokinin via synthetic pathways. It is more economic to produce cytokinins for commercial use in this manner. Our strains were first optimized to digest spent frying oil, a cheap food source. This was achieved by increasing beta-oxidation in the cell. We hope to then be able to engineer the cells to produce trans-zeatin, a high-value plant steroid. This product can be extracted from the cells and purified to produce laboratory- and industrial-grade trans-zeatin.


Breakdown


fadD crystal structure / PyMol[10]
fadL crystal structure / PyMol[10]
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)[3]. 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.

In order to further breakdown the TAGs, DAGs, and MAGs, we expressed an extracellular lipase from Bacillus stearothermophilus L1[9]. E. coli can then naturally uptake glycerol and long chain fatty acids (LCFAs). In order to increase this uptake, we upregulated the expression of an LCFA outer membrane porin, coded for by the gene fadL.

Once the FFAs are in the cell, they undergo further breakdown by the endogenous beta-oxidation cycle. In order to increase the flux through this pathway we targeted the rate limiting step. Acyl-CoA synthetase is the first enzyme the beta oxidation pathway. It attaches Coenzyme A to long chain fatty acids, allowing them to continue through the pathway. Synthesis of long-chain Acyl-CoA from the gene fadD is transcriptionally regulated by fadR, slowing the rate of beta-oxidation when fatty acids are not present.[4][8] By increasing the copy number of fadD under the control of an inducible promoter we can produce more enzyme expression thereby increasing the metabolic flux through the entire pathway, increasing the utilization of frying oil waste. The cells will then have excess acyl-CoA that can used for energy to offset the creation of trans-zeatin.

View our breakdown results →


Trans-zeatin Production

Trans-zeatin BioReagent / Sigma-Aldrich
Trans-zeatin is a cytokinin which promotes lateral bud growth. Production of trans-zeatin currently relies on plant tissue extraction. Tissue extraction is notoriously difficult and low-yield, resulting in expensive products which are not feasible for use in industry[5]. Trans-zeatin is currently mainly used for tissue culture in research, but could be beneficial in increasing crop yields. In order to make widespread use of our product feasible, we will create trans-zeatin using E. coli.

The acetyl-CoA produced by the beta-oxidation breakdown of frying oil can be taken through gluconeogenesis to create the precursors to the methylerythritol phosphate MEP pathway. The MEP pathway is endogenous to E. coli, and increased flux through this pathway will result in isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), the universal precursors to terpenoids like trans-zeatin.

In order to produce trans-zeatin, we have inserted the Agrobacterium tumefaciens C58 gene tzS and the Oryza sativa gene LOG into E. coli. tzS produces zeatin riboside 5’-phosphate from 4-hydroxy-3-methyl-2-(E)-butenyl diphosphate and AMP. The LOG gene produces N6-dimethylallyladenine by removing the phosphoribosyl group from N6-(delta2-isopentenyl)-adenosine 5’-monophosphate. Hydroxylation of the N6-dimethylallyladenine produces biologically-active trans-zeatin[6].

View our trans-zeatin production results →


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 is an updated version of the part we submitted last year (BBa_K14950). It utilizes a gene called KillerRed from Anthomedusae sp. DC-2005[1][7]. 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[2]. 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.

As a proof of concept, our KillerRed construct is induced with IPTG. When IPTG is present, the cells should turn red and, if exposed to light, die.

View our kill switch results →


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. Kamada-Nobusada, Tomoe and Sakakibara, Hitoshi. "Molecular basis for cytokinin biosynthesis." Phytochemistry, 70:444-449. March 2009.
  7. 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.
  8. 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.
  9. Kim, Myung-Hee et al. "Thermostable Lipase of Bacillus stearothermophilus: High-level Production, Purification, and Calcium-dependent Thermostability." Bioscience, Biotechnology, and Biochemistry, 64(2):280-286. May 2014.
  10. Andersson, CS et al. "The Mycobacterium tuberculosis very-long-chain fatty acyl-CoA synthetase: structural basis for housing lipid substrates longer than the enzyme." Structure 20(6):1062-70. June 2012.
  11. van den Berg, B et al. "Crystal Structure of the long-chain fatty acid transporter FadL." Science 304(5676):1506-9. Jun 2004.

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