Overview

The UNSW iGEM team explored the commercial potential of our Assemblase enzyme scaffold which acted to guide the direction of our project. We consulted with a number of industry experts to understand how they could see our scaffold fitting into industry and research practices. This resulted in numerous options for metabolic pathways that could be used in our system as commercialisation case studies; including the Taxol, Levofloxacin and Astaxanthin synthesis pathways. By integrating advice from industry experts, we were able to redesign our project to include a focus on scaffolding the enzymes involved in Taxol side-chain synthesis. We designed the relevant DNA sequences for the tagged enzyme fusion proteins that could be involved in this reaction. Furthermore, we applied our enzyme kinetics and diffusion model to theoretically test increased product titre that would be obtained through scaffolding with the Assemblase system.

Relevance

The foundational advance track exists to allow teams to come up with novel solutions to an observed need within the industrial and scientific research communities. For our solution to add value on a larger scale, the team realised that we would have to show how our Assemblase system could impact a metabolic pathway of commercial use. Therefore, we focused on consulting with industry experts to gain an understanding of the target market for our scaffold whilst compiling a short list of applications to which our system could be applied.

Research & Analysis

Indole Acetic Acid

Initially, in the design phase of our project, the indole-3-acetic acid (IAA) biosynthesis pathway was selected as a way of showing how our scaffold could increase product titre. IAA is a plant growth hormone and has been used by previous iGEM teams that have worked on enzyme scaffolding projects, such as ZJU-China 2012 and NUDT-China 2015. This is due to the fact that the pathway is made up of a simple, two-step reaction leading to the easy to quantify product, IAA. To expand on this, we decided to do our own research on the use of IAA in practice.

We contacted Amanda Rollason, a researcher at the PlantBank facillity, Australian Botanic Gardens, who uses tissue culture methods for rainforest species conservation. Rollason informed us their facility the exclusively use indole-3-butyric acid (IBA), another auxin, as they had found in previous experiments that IAA showed no benefits and that IBA was easier to work with. Amanda coordinated our visit to the PlankBank facilities to learn more about their tissue culturing methods with Lotte von Richter , a science facilities coordinator.

Lotte von Richter

Science Facilities Co-ordinator – PlantBank

During the tour of PlantBank, Lotte showed us all the equipment and explained the methods that they use for their tissue culture. We also received some advice as to how we should conduct our own plant experiments .

Figure 1: Pictured (Left to Right): Lotte von Richter, Emily Watson and Bec Schacht (UNSW iGEM members) in PlantBank’s tissue culture room.

After our visit to PlantBank, we decided that the synthesis of IAA using our scaffold was not a worthwhile pathway to commercialise. However, we still wished to validate our scaffold design by demonstrating IAA biosynthesis with our Assemblase system and showing its effects effects on plant growth and development . Applying Lotte’s advice on tissue culturing with aseptic techniques, we were able to more effectively conduct our future plant trials.

Stereoselective Synthesis

Next we approached Professor Paul Groundwater from the University of Sydney, to discuss the potential applications of our scaffold with regards to drug synthesis. His research into the synthesis of novel agents made him an excellent candidate to ask for suggestions on which aspects of the pharmaceutical synthesis industry our system could benefit.

Professor Groundwater highlighted the significance of stereogenic centres as a key candidate for metabolic engineering tools. Many drugs available to consumers have an inflated price due to the industrial synthesis of the compounds resulting in the costly and ineffective separation of the required enantiomers from those with undesirable or potentially toxic chiral structure. Synthesising these drugs through a biocatalytic pathway would remove the need for resolution of the desired compound, potentially reducing the cost of synthesis.

In particular, he mentioned the synthesis of paclitaxel (brand name Taxol) which is a semi-synthetic anti-cancer therapeutic, that is very costly to produce. He prompted us to look further into this pathway, which was significant in catalysing a turning point in the design of our project, away from the indole-3-acetic acid pathway, towards paclitaxel side-chain synthesis.

He also suggested that we look into the synthesis of the antimicrobial agent, Levofloxacin, which is also costly to produce, and therefore costly to purchase. Levofloxacin contains one stereocentre, and our complex could benefit its production in the same way it would benefit paclitaxel production, greatly reducing the final product's cost. Through discussing pharmaceuticals that are hard to synthesise the team was able to further brainstorm other pathways involve stereogenic centres, such as the metabolic synthesis of Astaxanthin, a pink pigment that is extensively used in the fisheries industry.

Prof. Paul Groundwater

School of Chemistry – University of Sydney

We approached Prof. Groundwater to discuss the potential future applications of our scaffold in regard to drug synthesis and possible drug delivery. He highlighted the significance of stereogenic centres as a candidate for enzymatic synthesis.

Figure 2: Pictured (Left to Right): Prof. Paul Groundwater; Tobias Gaitt and Rebecca Schacht (UNSW iGEM members); and Prof. Andrew McLachlan, Dean of Pharmacy at the University of Sydney.

Taxol Side-Chain Synthesis

Taxol, also known as paclitaxel, is an anticancer drug which is often used in chemotherapy for the treatment of ovarian and breast cancers (Croteau et al., 2006). Taxol is traditionally isolated from the Pacific yew tree (Taxus brevifolia) and research into cost-effectively producing Taxol has been ongoing since its isolation in 1967 (Li et al., 2015). Prof. Groundwater suggested that we look into the side-chain synthesis of Taxol, due to the production of a racemic mixture in industrial synthesis. To combat the production of a racemic mixture, biosynthetic pathways, such as the one shown for Taxol in figure 1, can be utilised to ensure the correct stereoisomers are produced (Croteau et al., 2006). However, these biosynthetic pathways are often slow and would benefit from co-localisation.

Figure 3: Overview of the Taxol biosynthetic pathway. Source: (Croteau et al., 2006).

The enzymes identified for the synthesis of the Taxol side-chain are Phenylalanine aminomutase (PAM) and Tyrocidine synthase I (S563A) (TycA-S563A). The enzymes have a slow turnover rate (Kcat of 0.015 /s and 0.05 /s respectively) which could be improved through co-localisation and altering the stoichiometry of the reaction, both of which can be achieved using our scaffold. We tested this assumption with our Enzyme Kinetics and Diffusion model, which showed…

Figure 4: Modelling Results

We have designed the synthetic DNA sequences that would be required to integrate the Taxol enzymes into our scaffold, but due to lack of time, we have not experimentally validated this section of our project. Insert sentence to wrap up taxol.

More case studies…

Integrate

Research into the commercialisation of our scaffold has guided the focus of our project and influenced the experiments we have performed. Through the team’s research into the use of IAA and plant hormones in industry, we have gained information about the commercial applications of IAA and the results of our research have directly influenced the experiments we have performed. This is demonstrated through the introduction of our plant experiments using IAA.

Although the prospects of commercialising our scaffold with a focus on IAA were bleak (due to the discovery that IAA was not widely used in industry), this information pushed the team to explore further applications. Our meeting with Prof. Paul Groundwater gave us a new perspective on the target market of our scaffold. The suggestion to concentrate our research to products in which chirality was an important factor in the synthesis (e.g. Taxol side-chain synthesis and Astaxanthin synthesis) directed our approach into future applications.

At our Symposium, we asked the audience for their opinion on which topic they would like to see advanced by synthetic biology. As 46 % of the respondents voted for pharmaceutical synthesis, we decided to make Taxol our preferred commercial case study. Following this decision, we tested the enzymes involved in Taxol side-chain synthesis with our model to determine the theoretical increase in reaction speed which would occur from co-localisation. We have additionally designed the synthetic DNA sequences that would be required to integrate the Taxol enzymes into our project, but did not get around to experimentally validating this section. We have put the effort into designing these future experiments for another team to be able to pick up from where we left off.

Resources

Guide to create new gBlock sequences for use with our scaffold

gBlock Sequence for 6xHis-PAM-SnoopTag

gBlock Sequence for 6xHis-TycA-S563A-SpyTag