Synthetic biology is commonly defined as the application of engineering design principles to biology in order to develop valuable methods and tools that benefit society and the world.¹ Teams pursuing such endeavours engage in the systematic forward-engineering of biological systems to effectively design systems that take into consideration physical, commercial and technological feasibility.

The UNSW iGEM team strived to follow suit, utilising literature and advice to appropriately follow an engineering design procedure.² Given that design is the first stage of the “Design-Build-Test” paradigm it was important that we considered all aspects of the system to avoid running into problems with building and testing. We started this process by researching and defining the need in the market for our tool, to ensure that our design was purpose-orientated. We then developed a list of requirements that our system must adhere to, a process that involved abstracting away the layers of the identified problem. Following this we went through design iterations, learning from modelling data and experimental issues to arrive upon our finalised scaffold system design.

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 at al (2012) 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

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.

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.