Engineering of Bottleneck Enzyme in a Metabolic Pathway

Increased Decarboxylase Activity in a Metabolic Pathway

An industry leader in bio-manufacturing developed a method for production of a nutritional supplement through fermentation. One of the enzymes in their biosynthetic cascade was found to be a bottleneck, limiting their product yield. After performing an internal enzyme engineering campaign that used rational edits to try and increase expression and solubility, they approached Allozymes to engineer more activity into the enzyme to increase yield of the desired product.

In the first stage of the project, Allozymes developed a fluorometric coupled-enzyme detection assay to enable screening of 10⁴ – 10⁶ variants in single libraries (Fig. 1).

Evaluation of Diverse Library Design Strategies

Using this assay, coupled with a high-throughput form of the customer’s assay, Allozymes screened libraries designed with several different concepts, including a “design-free” (Scanning) library, zero shot approaches, and libraries driven by large language models. Ultimately, several lead variants were identified using the different screening approaches (Fig. 2). These variants were expressed and shown to have improved performance in the customer’s E. coli strain backbone.

Media Engineering for Scaled-Up Bioprocess Efficiency

To improve scaled up production, Allozymes developed an alternative media formulation that enabled a more tightly controlled fermentation process. With this improved media formulation, Allozymes showed that its top performer produced 60% more product than the Parent strain in a scaled up (flask-based) bioprocess (Fig. 3).

Conclusion

Allozymes engineered a bottleneck enzyme in a biocatalytic pathway to produce 60% more of a desired product. Allozymes then aided in bioprocess development to enable scalable use of this strain.