"High-throughput screening of catalytically active inclusion bodies using laboratory automation and Bayesian optimization".

BACKGROUND: In recent years, the production of inclusion bodies that retain substantial catalytic activity was demonstrated. These catalytically active inclusion bodies (CatIBs) are formed by genetic fusion of an aggregation-inducing tag to a gene of interest via short linker polypeptides. The resulting CatIBs are known for their easy and cost-efficient production, recyclability as well as their improved stability. Recent studies have outlined the cooperative effects of linker and aggregation-inducing tag on CatIB activities. However, no a priori prediction is possible so far to indicate the best combination thereof. Consequently, extensive screening is required to find the best performing CatIB variant. RESULTS: In this work, a semi-automated cloning workflow was implemented and used for fast generation of 63 CatIB variants with glucose dehydrogenase of Bacillus subtilis (BsGDH). Furthermore, the variant BsGDH-PT-CBDCell was used to develop, optimize and validate an automated CatIB screening workflow, enhancing the analysis of many CatIB candidates in parallel. Compared to previous studies with CatIBs, important optimization steps include the exclusion of plate position effects in the BioLector by changing the cultivation temperature. For the overall workflow including strain construction, the manual workload could be reduced from 59 to 7 h for 48 variants (88%). After demonstration of high reproducibility with 1.9% relative standard deviation across 42 biological replicates, the workflow was performed in combination with a Bayesian process model and Thompson sampling. While the process model is crucial to derive key performance indicators of CatIBs, Thompson sampling serves as a strategy to balance exploitation and exploration in screening procedures. Our methodology allowed analysis of 63 BsGDH-CatIB variants within only three batch experiments. Because of the high likelihood of TDoT-PT-BsGDH being the best CatIB performer, it was selected in 50 biological replicates during the three screening rounds, much more than other, low-performing variants. CONCLUSIONS: At the current state of knowledge, every new enzyme requires screening for different linker/aggregation-inducing tag combinations. For this purpose, the presented CatIB toolbox facilitates fast and simplified construction and screening procedures. The methodology thus assists in finding the best CatIB producer from large libraries in short time, rendering possible automated Design-Build-Test-Learn cycles to generate structure/function learnings.

Construction and characterization of BsGDH-CatIB variants and application as robust and highly active redox cofactor regeneration module for biocatalysis.

BACKGROUND: Catalytically active inclusion bodies (CatIBs) are known for their easy and cost efficient production, recyclability as well as high stability and provide an alternative purely biological technology for enzyme immobilization. Due to their ability to self-aggregate in a carrier-free, biodegradable form, no further laborious immobilization steps or additional reagents are needed. These advantages put CatIBs in a beneficial position in comparison to traditional immobilization techniques. Recent studies outlined the impact of cooperative effects of the linker and aggregation inducing tag on the activity level of CatIBs, requiring to test many combinations to find the best performing CatIB variant. RESULTS: Here, we present the formation of 14 glucose dehydrogenase CatIB variants of Bacillus subtilis, a well-known enzyme in biocatalysis due to its capability for substrate coupled regeneration of reduced cofactors with cheap substrate glucose. Nine variants revealed activity, with highest productivity levels for the more rigid PT-Linker combinations. The best performing CatIB, BsGDH-PT-CBDCell, was characterized in more detail including long-term storage at -20 °C as well as NADH cofactor regeneration performance in repetitive batch experiments with CatIB recycling. After freezing, BsGDH-PT-CBDCell CatIB only lost approx. 10% activity after 8 weeks of storage. Moreover, after 11 CatIB recycling cycles in repetitive batch operation 80% of the activity was still present. CONCLUSIONS: This work presents a method for the effective formation of a highly active and long-term stable BsGDH-CatIB as an immobilized enzyme for robust and convenient NADH regeneration.