Accelerated production and screening of catalytically active inclusion body libraries via automated workflows

Küsters, Kira; Oldiges, Marco (Thesis advisor); Schwaneberg, Ulrich (Thesis advisor); Wiechert, Wolfgang (Thesis advisor)

Aachen : RWTH Aachen University (2022)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2022

Abstract

In recent years, the production of inclusion bodies that retained substantial catalytic activity was demonstrated. These catalytically active inclusion bodies (CatIBs) were formed by genetic fusion of an aggregation inducing tag to a gene of interest via short linker polypeptides and overproduction of the resulting gene fusion in Escherichia coli. The resulting CatIBs are known for their simple 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. However, no a priori prediction is possible to indicate the best linker/aggregation inducing tag combination. So, testing of many variations is required to find the best performing CatIB variant. Therefore, an accelerated CatIB processing is needed to allow a fast construction and screening of CatIB libraries. In a proof of concept study, a set of lysine decarboxylase CatIBs from E. coli (EcLDCc) were generated via Golden Gate Assembly and processed with an optimized purification protocol that would allow automation of the workflow in further processes. A total of ten EcLDCc variants consisting of combinations of two linker and five aggregation inducing tag sequences were generated. A flexible Serine/Glycine (SG)- as well as a rigid Proline/Threonine (PT)-Linker were tested in combination with artificial peptides (18AWT, L6KD and GFIL8) or coiled-coil domains (TDoT and 3HAMP) as aggregation inducing tags. Interestingly, most of the combinations with the rigid PT-Linker showed the highest conversions. EcLDCc-PT-L6KD was identified as the best of all variants allowing a specific volumetric productivity of 256 gDAP L 1 d 1 gCatIB-1. Furthermore, we demonstrated that microscopic analysis can serve as a tool to identify CatIB producing strains and thus allow for prescreening at an early stage to save time and resources. To accelerate the microscopy process, an automated microscopy workflow was implemented and used to observe the CatIB formation process. The automated and manual evaluated results showed that the CatIB formation started after 27 h. After the proof of concept study with the EcLDCc, a semi-automated cloning workflow was implemented to allow fast CatIB library construction with a time reduction of 83 %, resulting in only 11 h of manual work for the construction of 96 CatIB variants. The cloning workflow was used to generate 14 CgAHAS (acetolactate synthase of Corynebacterium glutamicum)-, 60 LbADH (alcohol dehydrogenase of Lactobacillus brevis)-, 63 BsGDH (glucose dehydrogenase of Bacillus subtilis)- and 30 BmMO (monooxygenase of Bacillus megaterium)-CatIB variants successfully. To test the constructed CatIB variants in parallel an acceleration of the manual CatIB analysis was needed. Therefore, a miniaturization of the cultivation and CatIB purification from a 10 mL shake flask scale to a 1 mL FlowerPlate® scale was realized. Furthermore, the enzymatic assay was performed in a microtiter plate (250 µL) instead of reaction tubes (1 mL). The accelerated workflow was used to screen the CgAHAS- and LbADH-CatIB libraries, which revealed 2 out of 14 (CgAHAS) and 8 out of 60 (LbADH) successful CatIB candidates. After process acceleration, the next step was to automate the CatIB screening workflow. The first step was to implement a robust manual workflow, which was transferred to a robotic platform subsequently. Therefore, 14 C-terminally tagged BsGDH variants were generated and analyzed in a manual approach. Similar as in the EcLDC study, SG- or PT-Linker were combined with one of eight aggregation inducing tags. Besides the five tested aggregation inducing tags, 18AWT, L6KD, GFIL8, TDoT and 3HAMP, the influence of ELK16, TorA and CBDCell on the CatIB activity were analyzed. The enzymatic assay showed that the highest conversions were again reached with PT-Linker variants. The most promising variant was BsGDH-PT-CBDCell with a specific volumetric productivity of 55.8 gNADH L 1 d 1 gCatIB-1. This variant 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 CatIBs only lost approx. 10 % activity after 8 weeks of storage. Furthermore, after 11 CatIB recycling cycles in repetitive batch operation, 80 % of the activity was still present. Not only NADH recycling was analyzed with BsGDH-CatIB, but also NADPH recycling. BmMO-CatIBs as NADPH consumer were constructed and used to prove the NADPH recyclability of the BsGDH. 13 out of 30 BmMO-CatIBs revealed activity in combination with NADPH recycling. After realizing the manual screening, BsGDH-PT-CBDCell was used to implement, optimize and validate the automated CatIB screening workflow to enhance the analysis of several CatIB candidates. Important optimization steps were for example the exclusion of a position effect in the BioLector® by enhancing the cultivation temperature to 25 °C and the provision of pipetting reproducibility by improving pipetting settings like velocity, mixing, air gaps and height. The validation of the optimized workflow with a relative standard deviation of 1.9 % revealed a high reproducibility for the whole workflow. After validation, the workflow was performed in combination with Thompson sampling, as a decision tool for strain selection, to analyze 63 BsGDH-CatIB variants. Three rounds with 48 cultivations in parallel each were realized. The screening showed that 24 BsGDH-CatIB out of 63 were successful CatIB variants. Already in the first screening round TDoT-PT-BsGDH was a promising CatIB candidate, resulting in a high likelihood of being the best BsGDH-CatIB performer. Therefore, the Thompson sampling algorithm has selected this variant in 50 biological replicates during the three screening rounds. With the implemented semi-automated CatIB construction, processing and screening workflow the manual work time could be reduced from 59 h to 7 h for 48 variants (-88 %). Moreover, an upscaling as shake flask cultivations of the best three BsGDH-CatIB variants as a validation study for the Thompson sampling revealed similar results as compared to the FlowerPlate® cultivation. These results showed that the Thompson sampling was successful for the BsGDH screening. Although the analysis of all CatIB enzymes revealed that for example the PT-Linker was more likely to form successful CatIB variants compared to the SG-Linker, still no a priori prediction is possible, because every enzyme requires different linker/aggregation inducing tag combinations for highest performance. Therefore, using the automated workflow is an important tool to enhance and simplify the screening to find the best CatIB producer from large CatIB libraries.