Selectively binding and removing chlorophyll from canola oil gives our system a huge advantage over the current industry standard, acid-activated clays. With acid-activiated clays, chlorophyll cannot be recovered and ends up discarded as a waste product. The yOIL system allows chlorophyll to be recovered and repurposed into a valuable product. By enzymatically degrading chlorophyll into pheophorbide, we can create an end product valuable not only in photodynamic research, but also to prevent crop losses to fungal infestations.

In the canola oil processing industry, acid-activated clays are used to extract chlorophyll from canola oil to remove its bitter taste and prevent premature spoilage. However, the chlorophyll is discarded by processors because it is tightly bound to the clay particles. By using our chlorophyll extraction emulsion system described here we can capture and isolate the chlorophyll found in canola oil.

With the tools to capture chlorophyll, there was a new opportunity to turn this former waste product into a commercially valuable product. Our review of the current commercial uses of chlorophyll revealed that it is mainly used as a neutraceutical. With this knowledge, we wanted to dig deeper. To find an application for our system which goes beyond neutraceuticals and addresses a current issue. The conversion of recovered chlorophyll to a marketable product is imperative to the economic viability of our project as a whole and has the potential to create a market for what is now considered a waste product.

Examining the natural degradation pathway of chlorophyll we identified a catabolite of interest: Pheophorbide. Pheophorbide has been shown in recent research to be a strong photosensitizer producing cytotoxic effects on cancer cells and fungi when light activated. Particularly, we are interested in its potential as a new anti-fungal treatment that may slow down the rise of antibiotic resistance in fungal populations. Once light activated, pheophorbide expends its cytotoxins and is “irradiated”, unable to harm other fungi or humans, making it a safe crop.

While designing our parts (link part page(s) here) to recombinantly express native plant and algae genes in Escherichia coli, we decided to stick to a single expression system. We used BL21(DE3) E. coli cells as a chassis for our protein production, allowing for our parts to be controlled by IPTG induction, therefore we used a T7 promoter (part) in our constructs. To reduce the effect of leaky expression in our system we used a double terminator (part(s)). Our ribosome binding site was designed by us, based off of our prior knowledge of ribosome function in E. coli that would theoretically have the highest efficiency. In our background research, we found little work had been done on expressing our proteins of interest in E. coli. We were able, however, to find sequences which have been proven to have successful recombinant expression. Three of our four genes came from ____ and the fourth came from ____.

Figure 1: Blah Blah Blah

Of particular interest, in our preliminary literature review, we found that the 7-hydroxymethyl chlorophyll a reductase (HCAR) was catalytically inactive after being purified using a 6x His-tag, even though the protein was produced and functional from the whole cell lysate (HCAR paper). Further research was done to analyze the structure of our proteins and only HCAR had an available protein data-bank (.pdb) file. It was found that the resulting homo-hexamer of the folded HCAR protein has a highly electronegative binding pocket; as per our electrostatic interaction modelling detailed here. It was our assumption that due to this high electronegativity the 6x His-tag that was added to the protein from the aforementioned paper likely folded into the binding pocket, resulting in enzyme function inhibition. With this in mind we decided to try to design a spacer that would allow for non-disrupted functionality of both the protein and the 6x His-tag, allowing for the protein to be purified.

Initially, in attempting to create a suitable spacer, we attempted using standard methods of alternating alanine and glycine chains. However, the high GC content of these amino acids prevented the possibility of gene synthesis. To avoid this problem, we tested several combinations of alternating alanine amino acids with other “simple” amino acids, including threonine, serine, leucine, isoleucine, valine. By using homology modelling we thought the alternation of alanine and serine amino acids would be safest for our purposes. Further thinking about the ways in which proteins fold, we determined that it would be best to stabilize the spacer linkage between the large protein and the 6x His-tag by creating a helix-turn-helix-turn-helix structure. Allowing increased space between the two moieties, while also implementing a thrombin proteolytic site (turn #1) and a string of negatively charged aspartic acids (turn #2) causing repulsion between the problematic binding pocket and the spacer itself. By using homology modelling, explored in depth here, we determined that our designed spacer would form the desired helix-turn-helix-turn-helix structure with a confidence value of ____.

Additionally, we used Rosetta comparative and ab initio structural modeling to create predicted molecular structures for chlorophyll b reductase (CBR), Mg²⁺ dechelatase (SGR), and pheophytinase (PPH), which did not have pdb files, with our designed spacer attached. These models further solidified the hypothesized utility of the spacer with all of our enzymes, giving us confidence to use it as a universal spacer to increase the potential purification amount of our proteins in the lab. Therefore the spacer was implemented into all four of the genetic constructs, each containing an enzyme involved in the chlorophyll degradation pathway. To learn more about how Rosetta comparative and ab initio structural modelling was employed, read more here.

[ED. NOTE: WE CANT WRITE THIS] As seen here (results), it is clear that the spacer likely allowed for purification of our HCAR protein using a Nickel-NTA column (Protocol), an accomplishment we had not seen previously done in any published literature. We reproduced four seperate 10% SDS-PAGE gels showing that the HCAR and PPH proteins were successfully isolated and purified. Presence of HCAR and PPH proteins was demonstrated by the 58 kDa and 55.8 kDa bands, respectively, when compared to the control containing an empty vector (pSB1A3).

To gain further confidence in having purified our desired proteins, we performed a Western blot using a 6xHis-tag mouse antibody as depicted below/above that shows our proteins ______….. - *We need Dr. Marija to give us some insight on writing this part*

The isolated proteins underwent ammonium sulfate precipitation and were re-solubilized in our reaction buffer (25 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 0.1% Triton X-100) for storage. As our substrates were unable to be ordered from a chemical distributor we had to chemically produce them in order to characterize our recombinant proteins, this is described in the protocols section here

PPH (55.8 kDa) was confirmed to have been purified through 10% SDS-PAGE gel confirmation, staining with Coomassie blue (Figure _) and through Western blotting using a 6xHis-tag mouse antibody (Figure _). PPH characterization was done using thin layer chromatography (TLC) to track the rate at which PPH converts pheophytin into pheophorbide by visualising the relative amounts of pheophytin and pheophorbide in reaction solutions after given times. (Figure _) After optimizing the solvent system used for TLC visualisation it was determined that 70% Methanol - 30% Hexane mixture was the best to be used in order to clearly show the differential movement of pheophytin and pheophorbide on a silica plate. By comparing the relative concentration of the visualised spots across increasing reaction times, using photoshop, we were able to give our qualitative data, quantitative identity. This was done as per the protocol found here. The extrapolated data gave us a quantitative method with which we could study the rate of the reaction and determine the protein activity. Sample calculations: _____ As per the graph (Figure _) we can see “these” things about the protein’s activity and kinetics as well as how much pheophorbide is made over a given amount of time from starting substrate using this much PPH….

Overall, our recombinant PPH was shown to have the ability to produce pheophorbide given pheophytin. Pheophytin a and b, along with chlorophyll a and b, are the most prevalent pigments found within green oil. Therefore, PPH could be used in the future to capture and process pheophytin in an emulsion system to produce pheophorbide that can be extracted directly from the oil processing step, thus avoiding the need for in vitro degradation of chlorophyll a and b making it more economical.

HCAR (58 kDa) was confirmed to have been purified through 10% SDS-PAGE gel confirmation, staining with Coomassie blue (Figure _) and through Western blotting using a 6xHis-tag mouse antibody (Figure _). While HCAR was purified and available for protein characterization assays, we were unable to produce the substrate, “7-hydroxymethyl chlorophyll a” required for the assays, as we would have needed to use a highly reactive and explosive compound to chemically produce it.

Though the chlorophyll b reductase (CBR) and stay-green (SGR) proteins were not characterized due to time constraints, they were successfully cloned into BL21(DE3) E. coli cells and shown to be isolated and purified (___).

HCAR characterization can be done through spectrophotometry since its substrate, 7-hydroxymethyl chlorophyll a absorbs wavelengths at 659 and 664nm while its product, chlorophyll a absorbs wavelengths at 659nm and 664nm. Observing absorbances of the reaction at one minute intervals to a total of 15 minutes would be done to analyze the enzyme kinetics. High-Performance Liquid Chromatography (HPLC) analysis could also be done to quantify the amount of product made from the reaction. Alternatively, the equation from Porra et al. 1989 can be used to determine their concentrations.

To address the challenge of scaling up pheophorbide production using enzymatic degradation, CRISPR could be used to integrate the chlorophyll degradation enzymes as an operon into yeast. This will allow us to feed in chlorophyll and produce pheophorbide a in large quantities through batch fermentation, which is an established method for industrial-scale biosynthetic production. To improve this further, a multi-enzyme complex scaffold could be made within the yeast cells. The multi-enzyme complex increases the concentrations of produced chlorophyll catabolites being fed to the enzymes while preventing their accumulation to toxic levels. They also allow compound formation to have lower rates of intermediate losses.