The aim of our system is to increase the rate of breakdown of compost, by targeting and accelerating the breakdown of pectin. Pectin comprises a family of structurally complex polysaccharides found in the intercellular tissue and cell walls of vascular plants, as well as other organisms, including bryophytes, pteridophytes, gymnosperms and Chara . Pectin plays a role in increasing the rigidity of the cell walls, as it helps to crosslink cellulose and hemicellulose fibres . Why did we choose pectin as our substrate? The majority of upper fruits, plants, and similar organic matter which makes up a compost pile contains pectin, and, by targeting its breakdown, other organisms within a compost pile may degrade it with more ease. Thus, our project is based on the idea that an increase in the degradation rate of pectin will result in an increase of the rate of compost breakdown. We chose to target a specific pectin, known as homogalacturonan or HG, in its abbreviated form. This substrate’s breakdown will be accelerated by lyases strategically selected from the organism Paenibacillus amylolyticus; specifically, Pnl, PelB and PelC. We will then transform a host cell, likely E.coli or Bacillus subtilis, with plasmids containing constructs that will allow the cells to express the selected lyases. While the host in the end product of our system has yet to be decided between these two, we are currently running experiments in the lab with BL21 E.coli. We plan to deliver our end product in one of two cell-free systems; either in a liquid lysate form or with lyophilized purified enzymes, in a powder form. In order to incorporate our system into the targeted composting environment, the user would simply administer the liquid, or powdered enzymes directly to the compost mixture. Because the process of food breakdown and composting usually causes a substantial temperature increase in a compost pile, we are working on designing thermostable variants of our enzymes, so that they would remain effective whilst in the compost.
Homogalacturonan is the simplest and most abundant pectin that occurs in nature, often forming the backbone to all other pectins, excluding Rhamnogalacturonan I  and accounts for 65% of all pectin. Due to its commonality, as well as structural simplicity, it was clear that HG was an ideal target for our system.
Selected Lyases: Pnl, PelB and PelC
In finding the components that would accelerate the breakdown of HG, a paper published by Keggi and Doran-Peterson ,explores the qualities and discusses the industrial potential on an HG degradation system comprising a set of enzymes from the organism Paenibacillus Amylolyticus. The paper characterized various pectin and pectate lyases from this organism, finding a unique degradation property of one of the enzymes, pectate lyase C (PelC). The study found that PelC from this organism is able to degrade highly-methylated regions of homogalacturonan, which is very uncommon for a pectate lyase, which usually targets regions with lower methylation. In our system, we are utilizing this enzyme as well as two others characterized in the paper, pectin lyase (Pnl) and pectate lyase B (PelB). Each enzyme has a distinct role to play in our system, to break down HG with the most simple and effective way possible.
- As mentioned previously, PelC works to break down highly-methylated regions of homogalacturonan. This enzyme is extremely advantageous to our system, as its unique property allows us to utilize it without having to require a methylesterase; leading to a simpler system. According to Keggi and Doran-Peterson , this enzyme may also play a role in degrading junctions between methylated and unmethylated regions of the HG.
- PelB works synergistically with PelC, as PelC can free up internal regions of demethylated homogalacturonan, which PelB then degrades.
- Pnl, like PelC, works on highly methylated pectin. When working synergistically with PelB and PelC, these three enzymes show the highest amount of pectin breakdown over various methylated substrates .
Figure 1. Diagram demonstrating the mechanisms of pectin breakdown using enzymes, Pnl, PelB and PelC from P. amylolyticus. PelC first cleaves at methylated-unmethylated junctions within the HG substrate. Once cleaved, this allows PelB and Pnl to further cleave unmethylated and methylated pectin fragments respectively to achieve complete pectin degradation.
After we chose our enzymes, we needed to find hosts to transform with plasmids containing the DNA from our selected components. There were various considerations at play in choosing a host, primarily concerned around yield of the system, as well as ease of characterization, and advantage to our system. One of the major hosts we considered was E.coli, likely the common laboratory strains BL21 (DE3) or Rosetta. E. coli would be favourable as a host in our system as it is presently one of the most well characterized and understood in synthetic biology. Additionally, it provides high protein yields and rapid growth, allowing us to study metabolic pathways effectively. Additionally, pectin and pectate lyases are not present in these E.coli strain’s genomes, thus, providing a reliable host to characterize and validate our proposed system. Another host we have considered was the bacteria Bacillus subtilis; a bacteria classified as safe, non-toxic, and often used in probiotic supplements . This host would allow for a safer biological system. Additionally, as some strains may utilize pectin as its carbon source, pectinases may be present in its genome. This may allow for an increase in efficacy for our system, but also may make it harder to characterize our proposed system, because our results may also have to account for the naturally occurring pectinases in the host.
Delivery System- Lysate or Purified Enzymes
As mentioned previously, we have considered two delivery methods, both cell free systems. In the past, we have considered a live bacteria system for our project, however, upon reflection, realized the potential safety hazards of engineered bacteria in compost, which would eventually be used as fertilizer and be spread into the ecosystem. If we were to have such a system, we must include precautions such as a kill switch, and there are greater ethical and safety concerns. Thus, we decided on a cell free delivery method. Safety of our system is discussed in greater detail in the safety section of our wiki.
The two potential methods of delivery each have their benefits and drawbacks.
- The first method we considered was a lysate delivery method. This system would be extremely efficient to manufacture, requiring only that we grow our host cells and then lyse them afterwards.. However, there are potential concerns around this system, due to the risk of not being able to lyse the cells 100% effectively, which would bring up similar safety concerns to having a live system. In scaling up our system, lysate would likely be the way to go because it allows us to grow up huge batches of bacteria at once and lyse them all at once.
- The second method we considered was a purified enzyme system. This system would raise less concerns than the lysate, but would be considerably more expensive and less practical to manufacture; due to the intensive process of purification and lyophilization. Additionally, there may be concerns about the effectiveness of dried enzymes--whether they may still be active when placed within a compost pile.
Figure 2. Representation of lysate delivery method wherein cells are grown in a fermenter, lysed, then administered to the compost mixture. (Figure created with BioRender)
Figure 3. Overview of potential delivery method wherein enzymes Pnl, PelB and PelC are purified, then lyophilized in order to create a powder which would be administered to the compost directly. (Figure created with BioRender)
As discussed in the modeling section of our wiki, in order to optimize the effectiveness of our system, we aim to increase the thermostability of our enzymes so they remain effective in the high temperatures compost may reach. Keggi and Doran-Peterson  found that the enzymes Pnl and PelC worked best at a temperature of 55˚C, while PelB works best at a temperature of 70˚C. During the second stage of composting, the thermophilic stage, compost can reach temperatures of above 65˚C , Therefore, to allow our enzymes to remain active and not denature, we are working on the thermostability.
- Mohnen, D. (2008). Pectin structure and biosynthesis. Current Opinion in Plant Biology, 11(3), 266-277. doi:10.1016/j.pbi.2008.03.006
- Caspi, R. (2013). MetaCyc pathway: Homogalacturonan degradation. SRI International.
- Wolf, S., Mouille, G., & Pelloux, J. (2009). Homogalacturonan Methyl-Esterification and Plant Development. Molecular Plant, 2(5), 851-860. doi:10.1093/mp/ssp066
- Keggi, C., & Doran-Peterson, J. (2020). The Homogalacturonan Deconstruction System of Paenibacillus amylolyticus 27C64 Requires No Extracellular Pectin Methylesterase and Has Significant Industrial Potential. Applied and Environmental Microbiology, 86(12). doi:10.1128/aem.02275-19
- Lefrevre, M., Racedo, S., Denayrolles, M., Ripert, G., Desfougéres, T., Lobach, A., Simon, R., Pélerin, F., Justin, P., Urdaci, M. (2017) Safety assessment of Bacillus subtillus CU1 for use as a probiotic in humans. Regulatory Toxicology and pharmacology. 83: 54-65
- Beffa, T., Lyon, P., Marchiana, M., Fischer, J., and Aragno, M. (1996) Isolation of Thermus strains from hot composts (60 to 80 degrees C) Applied Environmental Microbiology. 65:1723-1727