In this blog post I will talk a little bit about how protein scaffoldings can be useful for controlling metabolic flux.
The metabolism of cells is essentially a large network of coupled chemical reactions that are (mostly) catalyzed by enzymes. Evolution has been very good at balancing when and where a reaction happens in order to optimize the various processes that a cell needs in order to function. This includes converting nutrients to energy, producing building block chemicals that are needed for growth, and enduring external stressors.
By necessity, metabolism is a dynamic process, continuously adapting in response to changes in the cell’s environment. However, this dynamic does not always happen as efficiently in engineered organisms. The enzymes we introduce are often foreign to the host and do neatly fit into the regulatory mechanisms evolved by the organism. Sometimes the foreign enzymes drain the host of the compounds it needs to stay healthy.
It is also possible that some functions that the cell performs are unnecessary for synthetic engineering purposes. Like treating the product (that we wish to extract) as a metabolite, converting it into another compound, or breaking it down its new building blocks. Some of these systems that can be critical for the cell to survive in its natural habitat may not even be necessary in the strictly controlled laboratory environment or factory setting in which we grow the cell.
Understanding and controlling when, where and how much a cell uses an enzyme is key to making it do the stuff we want it to do.
Metabolic flux can be described as the rate of turnover of molecules through a metabolic pathway(5). In any given pathway, a finite number of enzymes interact with a finite pool of metabolites, inside a finite set of space. Maintaining the right levels of flux is vital for regulating metabolic activity under different conditions, such as when we want to maximize titers of a certain compound. A useful analogue is presented by Dueber and colleagues. Imagine a pipeline, where each segment consists of pipes which vary in size. In the wild, the flow through each of these segments is maintained just right in order to deliver the specific concentration of products the cell needs under any given condition. But in an engineered organism the foreign enzymes may produce either too much, or too little, effectively introducing bottlenecks in the overall pathway. Potentially build-up of intermediates (like a pipe leaking due to too much pressure!) can be a burden, or even prove toxic, if the cell has no way of dealing with the increasing concentrations.
What are protein scaffolds?
Co-localizing enzymes through proteins scaffolding can help us fine-tune a whole pathway by keeping enzymes fixed in one space, and thus helping to regulate their stoichiometry.
Scaffolding proteins are common in nature and consist of multiple protein–protein interaction modules. Each protein is fused with a binding domain that can interact with a ligand domain on the scaffold. Some of these have been engineered to be suitable for synthetic biology.(1-4) This allows the scaffold to bring together two or more proteins in close vicinity. This can then be used to either concentrate the same type of enzymes in the same location (co-localize), or possibly allow a faster conversion of metabolites by quickly juggling any intermediates between different enzymes.
While more could be said on this topic, I hope that this blog post will have piqued the readers’ interest in using protein scaffolds for their own synthetic biology projects. What uses do you see for protein scaffolds?
(I would highly recommend a curious reader to investigate how they are used to regulate signals!) (6, 7)
References:
- Synthetic protein scaffolds provide modular control over metabolic flux: https://www.nature.com/articles/nbt.1557
- Synthetic scaffolds for pathway enhancement: https://www.sciencedirect.com/science/article/pii/S0958166915001081
- Metabolic engineering of Saccharomyces cerevisiae for efficient production of glucaric acid at high titer: https://microbialcellfactories.biomedcentral.com/articles/10.1186/s12934-018-0914-y
- Use of modular, synthetic scaffolds for improved production of glucaric acid in engineered E. coli: https://www.sciencedirect.com/science/article/pii/S1096717610000042
- Basic concepts and principles of stoichiometric modeling of metabolic networks: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4671265/
- Reprogramming Control of an Allosteric Signaling Switch Through Modular Recombination: https://science.sciencemag.org/content/301/5641/1904.long
- Scaffolding Proteins: Not Such Innocent Bystanders: https://www.sciencedirect.com/science/article/pii/S0960982213005654
