Team:Utah State/Project

Chlorophyllase

Chlorophyllase

Cellulase

Cellulose is a polymer of beta-linked glucose produced by plants, and can be broken down into its subunits by cellulase. Cellulose is not usable by most organisms because of the beta-linked glucose of which it is made up. Mammals use α-linked glucose to store their energy as glycogen while plants create starch the same way; cellulose is produced only for structural support. However, some organisms can enzymatically change beta links to alpha links, after breaking down a chain of cellulose. Our produced enzyme is only the enzyme that helps breakdown cellulose chains into smaller units called cellobiose, two glucose molecules linked by a β 1-->4 bond (Part:BBa_K118023). These smaller units of the large cellulose chain are more soluble in water, helping to remove the components of a plant stain on clothing.

Amylase

Starch is a polymer of alpha linked glucose, and is the primary energy storage in plants. This polymer can be broken down by amylase, an enzyme produced in our mouths to help break down food as soon as it enters our body. These long chains of glucose are broken down into single glucose molecules. Any organism that consumes starch produces amylase, including E. coli. The gene we used to produce our amylase is native to E. coli (Part:BBa_K523001). The breakdown of starch helps remove common food stains.

Bioplastic

What is bioplastic?

Bioplastics-as the name entails, means plastic that is biological in origin. The main focus of Utah State’s iGEM team in 2014 was to study polyhydroxybutyrates (PHBs), which are biodegradable polymers belonging to the group polyhydroxyalkanoates (PHAs). PHAs are naturally produced by some microorganisms in nature as a storage intermediate for energy and carbon. It has been reported that PHAs can accumulate up to 90% of dry cell weight under ideal conditions. PHAs are interesting plastics as they have similar physical and mechanical properties to their petroleum based counterparts such as polypropylene and polystyrene. PHB has a melting temperature of 179℃, a young’s modulus of 3.5 GPa, and a tensile strength of 40 MPa. This compares to polypropylene having a melting point of 170℃ a young’s modulus of 1.7 GPa and a tensile strength of 35 MPa (Khanna and Srivastava 2005). The similarity in properties between PHB and polypropylene is advantageous: 1) PHBs could replace polypropylene, 2) similar properties could mean mixing the two plastics leading to ‘hybrid’ materials, and 3) downstream processing of PHB could be carried out with the same equipment reducing processing costs. The potential applications for PHAs are so vast that many companies are starting to commercialize (Chanprateep 2010).

The figure above shows the standard structure of PHA. When R is methyl (CH3) the polymer is known as PHB. ‘n’ represents repeating subunits of the monomer. PHB polymers can up to 900-1000 kDa in size.

The genes from some of these PHA producing species of microbes can be expressed in laboratory based strains (e.g. E. coli) to overexpress the PHA polymer to obtain high yields.

Biosynthesis

Three genes are needed to produce PHB from acetyl-CoA in E. coli. These genes are phaA, phaB, and phaC. The pathway for PHB production is shown in the figure below, where 2 acetyl-CoAs are converted to acetoacetyl-CoA then 3-Hydoxybutyryl-CoA is made via phaB enzyme. Finally 3-Hydoxybutyryl-CoA is polymerized to make PHB (Agnew and Pfleger 2013). The PhaC enzyme remains covalently attached to PHB after polymerization (Rehm 2009).

Figure shows the biosynthesis of PHB from acetyl-CoA adapted from Rehm 2009.

Granule Formation

As mentioned earlier, the PhaC synthase remains covalently bound to the PHB polymer and during granule formation (Tomizawa, Hyakutake et al. 2011). Additionally other proteins, such as phasin, can also bind to the PHB granule and help granule structure and control granule size (York, Junker et al. 2001; York, Stubbe et al. 2001). Previous studies have fluorescently labeled PHB granules by tagging granule bound proteins, these studies helped in understanding granule formation and localization (Peters and Rehm 2005).

Traditional Purification

There are a variety of PHB purification strategies ranging from mechanical, chemical, and biological treatments to liberate PHB from cells. The different strategies are outlines in Jacquel et al. 2008 (Jacquel, Lo et al. 2008). The most common method of PHB purification is to use a chloroform/bleach extraction. The purpose of bleach is to lyse the cells and chloroform helps agglomerate the PHB together. The chloroform/bleach method has been reported to give extremely high purities with minimal cell contamination.

NMR-GC quantification of PHB

PHB concentrations were determined from a NMR-GC correlation as reported in Linton et al. 2012 (Linton, Rahman et al. 2012). Known concentrations of PHB were measured using GC to determine peak sizes. The same concentration of PHB samples were used in NMR to get NMR peak sizes. A correlation between NMR-GC peaks was established. Unknown experimental samples were freeze dried and weighed. Samples were then dissolved in a chloroform/bleach solution in a laboratory chemical hood, then samples were run in an NMR machine following the protocol outlined in Linton et al. 2012. Peak integration was carried out using NMR analysis software and calculations were carried out to determine %PHB as a measure of dry cell weight and concentration of PHB. NMR was also used to determine that the structure of PHB was correct and consistent with literature.

Polyhydroxyalkanoates at iGEM

Polyhydroxyalkanoates (PHAs) have a long established history at the iGEM competition. The first PHA project at iGEM was first carried out by the 2008 Utah State iGEM team (https://2008.igem.org/Team:Utah_State). In 2012 the Tokyo Tech team demonstrated a fully functional polyhydroxybutyrate (PHB) production system made out of BioBrick parts using a native promoter system (https://2012.igem.org/Team:Tokyo_Tech). Tokyo Tech’s complete operon was released to the iGEM community in the 2013 distribution kit. In 2013 the Imperial College team used a hybrid promoter system to improve upon Tokyo Tech’s 2012 design to get 11x more PHB production (https://2013.igem.org/Team:Imperial_College). Imperial’s project also explored degradation of PHB for carbon recycling.

References

Agnew, D. E. and B. F. Pfleger (2013). "Synthetic biology strategies for synthesizing polyhydroxyalkanoates from unrelated carbon sources." Chemical Engineering Science 103: 58-67. Chanprateep, S. (2010). "Current trends in biodegradable polyhydroxyalkanoates." Journal of Bioscience and Bioengineering.

Jacquel, N., C.-W. Lo, et al. (2008). "Isolation and purification of bacterial poly(3-hydroxyalkanoates)." Biochemical Engineering Journal 39(1): 15-27. Khanna, S. and A. K. Srivastava (2005). "Recent advances in microbial polyhydroxyalkanoates." Process Biochemistry 40(2): 607-619.

Linton, E., A. Rahman, et al. (2012). "Polyhydroxyalkanoate quantification in organic wastes and pure cultures using a single-step extraction and 1H NMR analysis." Water Science and Technology 66(5): 1000-1006.

Peters, V. and B. H. A. Rehm (2005). "In vivo monitoring of PHA granule formation using GFP-labeled PHA synthases." FEMS Microbiology Letters 248(1): 93-100.

Rehm, B. H. A. (2009). Microbial Production of Biopolymers and Polymer Precursors: Applications and Perspectives. Caister Academic Press, Norfolk, UK.

Tomizawa, S., M. Hyakutake, et al. (2011). "Molecular weight change of polyhydroxyalkanoate (PHA) caused by the PhaC subunit of PHA synthase from bacillus cereus YB-4 in recombinant Escherichia coli." Biomacromolecules 12(7): 2660-2666.

York, G. M., B. H. Junker, et al. (2001). "Accumulation of the PhaP phasin of Ralstonia eutropha is dependent on production of polyhydroxybutyrate in cells." Journal of Bacteriology 183(14): 4217-4226. York, G. M., J. Stubbe, et al. (2001). "New insight into the role of the PhaP phasin of Ralstonia eutropha in promoting synthesis of polyhydroxybutyrate." Journal of Bacteriology 183(7): 2394-2397.