Team:UNSW Australia/Description
In industry and research there has been a growing interest in metabolically engineered pathways that use novel enzymes to produce cheap and renewable products of value. But as these biocatalytic pathways gain complexity, the ability of successfully achieving viable productivity becomes a challenge. That is where Assemblase can step in, offering a stable, self-assembling scaffold system that spatially organises enzymes from a multi-step pathway in order to increase product titre. The system will act as a foundational tool for research methods and industry applications worldwide, facilitating future developments that target many of the major global problems.
What is the Assemblase System?
Although cells naturally co-localise multi-step metabolic reactions to increase their efficiency of product formation, researchers running ex vivo reactions have struggled to replicate this. One way to introduce co-localisation is through substrate channelling, which spatially brings together the enzymes of a multi-step reaction, increasing the effective concentration of metabolic intermediates. The Assemblase system, a novel protein scaffold, has been designed to introduce substrate channelling into ex vivo reactions – in a modular and accessible way.
The Assemblase system is a heterohexameric complex of prefoldin, a thermostable archeal protein, which has been engineered to recruit enzymes using covalent protein-protein interactions. Spy Tag/Catcher and Snoop Tag/Catcher systems have been used to attach enzymes to the prefoldin ‘scaffold’, and were chosen because of their lack of cross-reactivity. The ‘Catchers’ have been fused to the prefoldin, and the ‘Tags’ (short peptide sequences) expressed on the enzymes. This modular design also allows a 2:1 stoichiometric ratio to be employed, which may further assist in increasing the efficiency of product formation.
Assemblase has undergone proof-of-concept testing using a two-step enzyme pathway for the production of a horticultural plant hormone, indole-3-acetic acid, from tryptophan. This pathway was chosen since its success can be quantified using a straightforward benchtop assay (the Salkowski Assay), as well as through the use of High Performance Liquid Chromatography (HPLC) and plant root growth testing. Furthermore, the relatively large size of the pathway’s enzymes will allow the inference to be drawn that smaller enzymes would also be accommodated by the Assemblase scaffold.
Verification of the proposed features was done through mathematical modelling, particularly in relation to the spatial arrangement of the proteins attached to the scaffold. Both MATLAB models and Molecular Dynamics models were used to support the science underlying the increased rate of product formation, particularly regarding intermediate diffusion and the scaffold’s movement in ‘real time and space’.
There are many potential applications of the Assemblase system given the wide usage of enzymes. Several exciting options arose during our testing and analysis, and a select few are explored below.
Who will use our tool?
We propose that the Assemblase system will be used worldwide for applications of metabolic engineering in fields such as pharmacy, agriculture, bioremediation and foundational research. The team engaged with the community to generate an understanding of areas they would like to see our tool having a positive influence. Discussions with experts also contributed to our repertoire of possible future applications. As a result, we have been able to compile examples of how we see our system making waves in the future:
Plastic Degradation
Assemblase offers a platform to maximise the potential of the plastic-degrading enzymes currently making waves in the synthetic biology community. Co-localising enzymes (like PET-ase and MHET-ase) using Assemblase could increase the overall efficiency of their multi-step plastic breakdown reaction, and thus helping more plastic be degraded more quickly.
Bioremediation
Oil Spills etc.
Industry
The use of Assemblase is targeted towards ex vivo enzymatic reactions, many of which are being developed by industry in lieu of synthetic methods. This trend is particularly prominent in pathways where products or intermediaries form significant quantities of unwanted enantiomers, which then usually become waste. Assemblase can tether enzymes together in a pathway that produces a single isomer in a specific stereochemical formation. There are many examples of such processes, including the synthesis of the Taxol sidechain for anti-cancer drugs.
Drug Delivery Systems
Assemblase, being based on the principles of protein attachment, is not limited to enzymes. It can actually co-localise most proteins, and so could be used for linking signalling proteins (like antibodies) and enzymes. There is a strong basis for further exploration of the Assemblase system as a way to target particular linked proteins to specific tissues as part of targeted drug delivery.
Pharmaceutical Production
Foundational Research
Bioremediation
Agriculture