Team:UNSW Australia/Design

Overview

When developing our Assemblase system, we strived to follow an engineering design procedure. As design is the first step of the ‘Design-Build-Test’ engineering paradigm, we felt it was important to consider all aspects of the Assemblase system to minimise the occurrence of downstream problems during the building and testing phases. Thus, we started our design process by researching and defining the need for our scaffold tool in the market, which then allowed us to develop a list of requirements to which our system should adhere. Following this, we went through design iterations, learning from modelling data and experimental results to ultimately arrive upon our finalised Assemblase scaffold 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 products³. 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 nature⁴. 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 (FRs) 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 necessary⁷. 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 system⁸. 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 levels¹¹. 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 subunits¹². 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-biotin¹³. 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, SdyCatcher¹⁵. 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 terpanoids¹⁷. 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-F6¹⁸. 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.

Future Developments

Although most of our design iterations in this section were focused on identifying enzymes that would be attached to the Assemblase scaffold, it should be noted that the system has the potential to accommodate a range of different proteins. Our focus on enzyme attachment stemmed from the identified need to improve product yield efficiencies for metabolic pathways. However, it would be just as simple to fuse desired proteins to the Snoop or Spy Tags opening up a range of future applications for our scaffold. One way we did aim to show this, was through the attachment of fluorescent proteins for the FRET experiments

The Final Assemblase Scaffold

add IKEA image here?

Experimental Design

Having determined the protein components that make up the scaffold, the iGEM UNSW team then moved on to planning out how to experimentally characterise the Assemblase system. It was necessary to first determine the experimental tests we would perform before moving on to building the system, since experimental design informed the decisions we were to make regarding designing the DNA sequencesfor each part.

1. Assembly

Firstly, the team wanted to develop a series of practical experiments that would be able to show how well each of the components came together.

This included the SDS Page, an analytical method for separating charged molecules by their molecular masses. Using the known sizes of the prefoldin hexamer (87kDa)^{2^{and the enzymes as a baseline, this frequently used protocol would allow us to investigate the covalent interactions of our system, facilitated by the Tag-Catcher assembly.}}

In order to investigate assemblies in our system that were non-covalent, we decided to use size exclusion chromatography (SEC). This is a widely used characterization method for the analysis of large molecules such as protein complexes, separating them by size. Our team decided that this would be a good experiment to understand how the tagged hexamer would come together. This was especially important to see if the fusion of the Tag-Catcher components on to the prefoldin would affect its ability to self-assemble. We also considered using nativePage and MALS for the same reasons.

2. FRET

Fo&#776rster resonance energy transfer (FRET) is a mechanism that describes the energy transfer between two light sensitive molecules. It is widely used to study the dynamics of proteins, particularly by quantifying the distance between two subsequent proteins. In deciding that we wanted to use FRET, this dictated the use of fluorescent proteins in our system. We chose the proteins mVenus and mCerulean to be attached to the Assemblase scaffold in order to determine their distance apart once assembled on our structure. Not only would this enable us to get a good understanding of how the enzymes in our scaffold would be spatially organised, but would also highlight the fact that our Assemblase scaffold, although currently focused on enzymatic attachments, can easily accommodate a range of other proteins. As such this experiment would also serve to highlight the adaptability of our scaffold system.

3. Enzyme Assays

On top of being able to characterise the spatial organisation and assembly of our Assemblase scaffold, it was imperative that we also experimentally showed that our system satisfied our main functional requirement: FR₁ “Increase product titre”. We therefore planned to do assays that would measure the reaction kinetics of our IAA proof of concept pathway.

A simple yet effective way to achieve this was running the Salkowski assay. This colorimetric assay is standard for experiments involving the IAA pathwayand is an easy way to quantify the product, IAA²¹. It was also a method the ZJU-China 2012 iGEM team decided to use to show the effectiveness of their riboscaffold design, meaning we had a standard curve and experimental protocol as a reference. By creating a standard curve for the unbound enzymatic reaction of the IAA pathway we would then have a reference point to compare how co-localising IaaM and IaaH onto the scaffold would impact the product yield.

This could be further built on through the use of high-performance liquid chromatography (HPLC) or liquid chromatography mass spectrometry (LC-MS) which could be used to separate, identify and quantify each component the reaction mixture. This would not only give us an understanding of how the product yield would change when the enzymes were attached to our scaffold but also allow us to understand the changes in the concentration of the intermediate. This is important as it will enable us to consider the probability of undesired reactions occurring in our system, another key functional requirement; FR₁₁ “Reduce the probability of encountering undesired reactions”. Due to the more accessible nature of the HPLC protocol and its ability to produce results of higher accuracy, we decided to go with this enzyme assay first.

4. Plants

The team also wanted to consider how to show the effects of IAA that could relate to its practical use. From revision of 2011 iGEM Imperial College London it was decided that a similar plant based root growth assay would be performed. Using the model plant, Arabidopsis thaliana, we would be able to investigate how varying concentrations of IAA would affect the growth and development of the plants. As well as using this to connect the use of our scaffold to some sort of commercial plan, we wanted to develop a protocol for testing IAA which would enable us to observe whether the IAA produced from reaction by enzymes fused to our scaffold would have the same phenotypical effects as the IAA bought from the store.

In determining the experiments we wanted to conduct to conclusively show the merit of our scaffold, we were able to understand what gBlocks were necessary to clone and express these components. More about how we designed these specific gBlocks can be found here.