Defining a Need

In industry and research there has been a growing interest in the construction of novel metabolic pathways, as alternatives to synthetic chemistry, for the production of cheap and renewable products.³ Yet, as these biocatalytic pathways gain complexity, the ability to gain viable productivity is diminished due to undesirable side reactions and low turnover rates.

One way that this is being addressed is through the development of scaffolds that will co-localise enzymes from such a pathway. By spatially organising enzymes to generate high local concentrations, this strategy aims to enhance metabolic flux and reduce the diversion of pathway intermediates into competing pathways that lead to undesirable side reactions. Ultimately, the use of scaffolds to create enzyme agglomerates will allow for the acceleration of reaction intermediate processing leading to increases in product titre.

This approach mimics enzyme clustering that is frequently observed in nature.⁴ However, previous attempts to replicate this natural enzyme organisation have fallen short, with no commercially viable modular tool available to facilitate enzyme clustering for the production of valuable metabolites. As such, the UNSW iGEM team decided to reflect on these past solutions and compile a list of requirements that would enable us to develop a versatile, commercially viable tool that could be used in laboratories and industry worldwide.

Functional Requirements

Having decided to develop an enzyme scaffold for use in biocatalytic systems, the team the worked to compiled a list of functional requirements that our system would have to meet. Given our research, it was evident that the main goal of our scaffold was to increase the product titre from a given synthetic enzyme pathway (FR₁). With previous studies showing that a considerable increase in titre could be achieved through the utilisation of synthetic scaffold proteins,^5,6 this seemed like an achievable function.

However, this main requirement of increasing yield was relatively broad, so breaking down this overall function into sub functions was necessary.⁷ For example, it has been found that engineered metabolic pathways often suffer from flux imbalances.⁵ But by increasing the concentration of enzymes around a localised amount of reaction intermediates would combat this (FR₁₂₁ and _FR122), resulting in increased metabolic flux rates of the system.⁸ Developing a system that allows for the reduction of enzyme concentration required to produce the same product titre would have significant cost saving outcomes in an industrial context.

Similarly, the solution must address the issue of undesirable intermediate reactions occurring (FR₁₁). Unintended interactions between synthetic pathways and the cellular environment of the host can have significant implications for the productivity of novel pathways.⁹ Considering the benefits observed by the natural channelling of intermediates in vivo,¹⁰ further lower level requirements can be identified such as limiting the diffusion of intermediates into the surroundings (FR₁₁₁) and facilitating the fast turnover of labile or toxic intermediates (FR₁₁₂). It was also agreed that optimising enzyme stoichiometry (FR₁₃) would additionally contribute to the overall function of increasing product titre, with Lee et al (2012)⁹ suggesting that the critical determinant of scaffold effectiveness was providing control over reaction stoichiometry.

On top of these key requirements, the system was also required to have commercial value and as such, would have to be able to be used in a variety of applications (FR₂). Previous enzyme agglomerates have been observed to only form under certain conditions, such as purinosomes discovered in HeLa cells that were found to cluster only in response to higher purine levels.¹¹ Yet, for the envisioned use of our system, the team determined that the scaffold would have to assemble independent of environmental conditions, ultimately being able to be used both in vivo and ex vivo (FR₂₂). Furthermore, the system would have to adapt to utilizing a range of different enzymes which, in turn, dictates some form of modularity (FR₂₁). This lower level requirement also stresses the need for the enzymes of a pathway to be exchanged without modification of the scaffold, with past designs, like that of the 2010 Slovenian iGEM team, requiring extensive protein modification, which impacted enzyme activity.

Finally, our scaffold system would have to be easy to produce and be utilised by laboratories and other interested parties in the future (FR₃). This includes having only a minimal number of subunits to reduce the complexity of the system and make it cost-effective to assemble (FR₃₁). Our design also involved needing to provide a thorough characterisation of DNA parts (FR₃₂) and standardised experimental protocols (FR₃₃) so that the process of developing the system could be replicated seamlessly.

INSERT FLOW CHART HERE (made by linda already)

Concept Generation

Make a carousel of the following components or drop downs - so that everything is nicely compartmentalised (BEC TO DO)

Scaffold

With the functional requirements of our system in mind, the team looked into how we could construct a scaffolding platform that could add modularity and stability to past enzyme clustering methods. We steered away from the concept of substrate channelling and physical tunnels that had been shown to unnecessary,¹ and instead focused on the idea of a synthetic molecular scaffold that would co-localise proteins. With research on the stable, modular, self-assembling chaperone proteins called prefoldin having already been conducted by our supervisor, Dominic Glover, we decided to investigate its use as a potential candidate for our scaffold.

Iteration 1 – Gamma Prefoldin

Prefoldins are generally hexameric molecular chaperone complexes that bind to non-native target proteins in a cell. They have a general role of de novo protein folding in Archaea and the biogenesis of cytoskeleton proteins, actin and tubulin, in eukaryotes.

A distinct variant of archaeal prefoldin (PFD), gamma prefoldin (gPFD), has a unique filamentous structure composed of hundreds of monomeric subunits.¹² This protein complex, which originates from hyperthermophilic Methanocaldococcus jannaschii, is involved in binding non-native proteins via distal regions of its highly flexible coiled coils. Our interest in this protein complex as a scaffold, stemmed from the fact that it has been well characterised by the lab our supervisor. Furthermore, Glover’s lab has shown that enzymes can be attached to gPFD sparking an interest in our team to replicate such attachment mechanisms.

However, gPFD had its limitations, including an inability to control the order of the subunits without using a heterogenous mix of filaments. This contradicts FR₃₁ which dictates that a minimal number of subunits is desired and as such, this protein complex was deemed inappropriate for our desired scaffold system.

Iteration 2 – Alpha and Beta Prefoldin

An elegant solution to this was found in a prefoldin variant, derived from archaea, which has just two subunits. This archaeal prefoldin complex is constructed from two alpha and four beta subunits which self-assemble into a jellyfish-like formation, creating a heterohexamer. Like gPFD, this complex originates from a thermophilic archaeon, Methanobacterium thermoautotrophicum, which affords the archaeal prefoldin complex with a relatively high thermostability, a function that falls under the required adaptability of our system (FR₂₂). Furthermore, the six components of this structure allow for the modification and expression of each subunit individually as desired, ensures that using this prefoldin would enable extensive adaptability of our scaffold to various scenarios.

Future developments

Our research also led us to consider how we could develop our scaffold into the future; in particular the use of eukaryotic Prefoldin. This is also a heterodimer, but unlike its archaeal partner, this variant is made up of six distinctly unique proteins. This would be beneficial as it would allow for us to easily incorporate six different enzymes onto the one scaffold, rather than just two. In accordance with FR₂, this would enhance the adaptability of our system to multiple bio catalytic pathways.

Attachment System

Having decided on the base scaffold, the team then began to look at the ways in which we could attach the desired enzymes onto the Prefoldin. Most fusion proteins are connected by non-covalent interactions such as dimerising linkers or streptavidin-biotin.¹³ Yet we wanted our system to be highly stable, with the enzymes in use being covalently bonded covalently to the scaffold to allow for continuing localisation. This could be achieved with covalent linkers such as inteins or sortases, however these limit molecular topologies, with sortase linkers only occurring between the N and C termini of target proteins.

Research instead led us to an alternative rapid covalent ligation strategy involving a short polypeptide tag (Spy- or Snoop-Tag) and its reactive domain partner (Spy- or Snoop-Catcher).¹⁴ This attachment mechanism involves the spontaneous formation of an isopeptide bond between the respective Tag and Catcher components, with Snoop only reacting with Snoop, and Spy only reacting with Spy. This bond has been shown to be robust under a whole range of reaction conditions,¹⁴ fulfilling the requirement of functional adaptability (FR₂) as the enzymes would remain attached to the scaffold irrespective of the metabolic pathway.

Future developments

If we moved to using the six-subunit eukaryotic prefoldin for our scaffold we would need to employ a further range of attachment systems that do not cross react. Although we could resort to using the afore mentioned systems, such as dimerizing linkers or sortase, sticking with similar Tag-Catcher arrangement would add value to the assembly and function of the system. This is feasible through the use of a third covalent ligation strategy, SdyCatcher.¹⁵ The team will look out for other developments in the Tag-Catcher space.

Enzymes

Having designed the scaffold and conjugation strategy, we then considered how to demonstrate the functionality of our approach. Mathematical modelling indicated that localising enzymes would result in an increase in product titre as required by our function specifications (FR₁). Yet, we needed to develop protocols that would allow us to demonstrate this in the lab. We used the KEGG database to search through a variety of pathways,¹⁶ looking at secondary metabolites and xenobiotic biodegradation to identify a set of enzymes that would produce a useful product. We also reviewed classic exemplars of synthetic pathways that are used in metabolic engineering, such as the production of terpanoids.¹⁷ However, through the requirement of reducing complexity (FR₁₃₁), our current design had two subunit types meaning that a most appropriate reaction pathway would be one that only required two enzymes.

Iteration 1 – Plastic Degrading Enzymes

Initially, it was proposed that we work with novel plastic degrading enzymes, PETase and MHETase. These enzymes were discovered in 2016 from a polyethylene terephthalate (PET) degrading bacterium, Ideonella sakaiensis201-F6.¹⁸ They were found to be involved extracellular enzymatic PET degradation, and their use in metabolic engineering could have wide spread impacts for our plastic dependent society. However, quantifying the enzymatic activity of PETase and MHETase was considered to be relatively difficult. A previous iGEM team, Tianjin 2016, attempted this with cell-free protein expression which does not directly and clearly show the alteration in product titre. An alternative suggestion was to use electron microscopy to physically measure plastic degradation, however this would have presented challenges for us regarding training and funding. So, although the benefits that could come from using such a metabolic pathway in our scaffold, it was evident that using this plastic degrading pathway would not be a smart way of highlighting the effectiveness of our scaffold. Instead the team decided to look for a more feasible pathway, that would involve a product that was easier to measure and quantify.

Iteration 2 – The IAA Pathway

This led us to consider using the indole-3-acetic acid (IAA) biosynthesis pathway:

Add image here of the pathway. (From Brian)

This metabolic pathway involves the conversion of tryptophan, a simple amino acid, to IAA using enzymes tryptophan 2-monooxygenase (IaaM) and indole-3-acetamide hydrolase (IaaH).¹⁹ In practice, IAA has a role in promoting plant root growth and has been shown to be chemically labile and easily metabolised by plants, by the 2011 iGEM Imperial College London team. Yet it also has a role in the lab, with previous iGEM teams who have worked on enzyme scaffolding projects, such as ZJU-China 2012 and NUDT-China 2015, using this two-step enzyme reaction. This meant that we would have access to protocols, standard curves to compare to and tips for its use.

On evaluation of this pathway, it was clear to us why previous teams had chosen to work with it, providing the ideal features to satisfy some of our functional requirements:

Input selection table here (From BEC)

Iteration 3 – Taxol Side-Chain Synthesis

Although, we decided to use the IAA biosynthesis pathway as a proof of concept for our experimental work, consultation with industry led us to realise that IAA itself was not widely used. Therefore, the team wanted to move on to using an enzyme pathway that would have commercial value while still demonstrating the prime functionality of our scaffold system. This led us to the Taxol side-chain synthesis pathway, which involves the synthesis of the side-chain of the anticancer drug Taxol, by the enzymes phenylalanine aminomutase (PAM) and tyrocidine synthase I (S563A) (TycA-S563A). These enzymes have a slow turnover rate which could be improved through co-localisation, making the pathway a great target for our scaffold system. It was therefore decided that these enzymes would be used in the place of IaaM and IaaH in our Enzyme Kinetics and Diffusion model when investigating the yield rates afforded by our scaffold system.

Experimental Design

Having developed our solution to the defined need, the UNSW iGEM team had to move on to building and testing the system.

Building

To demonstrate that our scaffold would assemble in solution, we first had to clone and express all of our components. This was achieved by designing specific G-Blocks for each of our constructs:

Add designs of gblocks

Testing

To physically test our assembled system we designed and set up experimental protocols.