Engineered Enzyme for Diagnostic Applications

‍uHTS with Pichia pastoris

A commercial partner engaged Allozymes to improve an enzyme for biosensing applications. They wanted to improve the sensitive toward specific compounds and establish a recombinant method to produce this enzyme. During the project scoping process, Pichia pastoris was identified as the most suitable expression organism. Although Pichia is not a common host for protein engineering campaign, we took on the challenge and successfully developed custom workflows to achieve both sensitivity and activity improvement in 6 months.

Pichia is an established platform for industrial-scale protein production; however, its low transformation efficiency and the tendency for multiple copy integration pose challenges for generating and screening protein variant libraries. To overcome these challenges, we developed a high efficiency Pichia transformation method and a deep sequencing workflow to identify hits (Fig. 1).

**Figure 1.** Pichia library generation and screening workflow

Low Expression

Since the enzyme is known to have very low expression in mammalian cells or Pichia, we first conducted expression optimization via cassette design, such as strain selection, secretion peptide screening, and promoter screening. 5-fold improvement in Pichia supernatant activity was achieved in 3 weeks.

For the screening method development, we collaborated with the partner to create a mid-throughput plate-based method (<1000 clones/day), which was directly translatable to their biosensing application. To accelerate the project, we also developed a proprietary microfluidic method to screen 50,000 cells in 1 day (Fig. 2).

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**Figure 2.** Microfluidic screening workflow

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Scanning Library Screening Results

We identified 10 hotspots based on the scanning library screening result, and then created 5 combinatorial libraries to consolidate these hotspots together. The resulting combinatorial libraries were screened via plate-based and microfluidic screening. In the end, we achieved 150-fold improvement in enzyme activity in Pichia supernatant, and 3-fold improvement in biomolecular inhibitory constant, ki (Fig.3).

**Figure 3.** (A) Activity of clones from different steps of the project. (B) Enzyme activity decreased over 20 mins of incubation with the inhibitor. Higher ki indicates higher sensitivity toward the inhibitor.

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Allozymes has developed a high-throughput Pichia protein engineering platform and successfully improved the activity and sensitivity of an enzyme within 6 months. Since there is no recombinant source of this enzyme currently available on the market, this achievement paves the way for commercialization opportunities.