Yeast We Can Cut Costs By Optimizing Cell-Free Expression Systems

Choosing the right additives could help “cell-free” expression systems finally fulfill their potential and provide biopharma with a low-cost way of making protein drugs, according to a recent research report.

The new study looked at how cell-free systems, in which biochemical reactions occur independently of cells, could be fine-tuned to provide drug makers with alternatives for large-scale protein production.

And the potential of the approach is significant, says Karen Polizzi, PhD, a professor from the department of chemical engineering at Imperial College London, who adds, “Cell-free protein synthesis (CFPS) is a flexible manufacturing technology. It can be used for on-demand synthesis in low-resource environments or to make difficult-to-express products, especially medicines that are toxic to the cell. Cell-free reactions scale well across microliter to liter scale without needing adjustments.”

The Imperial team’s research focused on expression systems based on the yeast species Pichia pastoris, which, as Polizzi explains, “has machinery capable of post-translational modifications of proteins that can be necessary for function.”

As an expression host, P. pastoris combines elements of both prokaryotic and eukaryotic systems, such as a rapid growth rate and the ability to perform post-translational modifications (PTMs).

The problem is that current commercially available Pichia systems are only able to produce low amounts of protein. According to Polizzi and her co-authors, the productivity of P. pastoris-based cell-free systems usually ranges from 6 to 100 µg/mL, which is only approximately five percent of that achieved by comparable E. coli systems. In addition, the additives required by Pichia-based systems are more expensive than those required by equivalent platforms.

Additives to improve yields

To address this, Polizzi and co-authors systematically evaluated a variety of chemical additive combinations to identify the most effective stabilizers and crowding agents to be incorporated in the reaction.

The researchers also used a machine learning model to predict translation initiation rates and optimized the Kozak sequence—the protein translation initiation site in most eukaryotic mRNA transcripts—to enhance expression.

In addition, the Imperial team evaluated lower-cost glycolytic intermediates as substrates for ATP regeneration to reduce the cost of goods.

Polizzi says, “We focused on how to improve the yields and reduce the cost of production. We identified some additional additives that boost the yield without substantially increasing the cost. We also identified a different energy source that can be used.”

She adds, “This work underscores the importance of protein-stabilizing additives and the role of rationally designed DNA sequences with minimized mRNA structural complexity to enhance yield in CFPS. Our demonstration of glycolytic intermediates as a potential secondary energy system additionally provides the foundation for the development of a cost-effective P. pastoris CFPS.”

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