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

Küsters, Kira; Saborowski, Ronja; Wagner, Christian; Hamel, Rebecca; Spöring, Jan-Dirk; Wiechert, Wolfgang; Oldiges, Marco

doi:10.1186/s12934-022-01816-2

Cited by 4 publications

(12 citation statements)

References 26 publications

(31 reference statements)

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“…Moreover, the enzyme assay revealed that no activity was measured for the insoluble cell pellet fraction. These results indicate that BsGDH WT did not form natural CatIBs, which con rms previous results from a manual BsGDH study (11).…”

Section: Insights On Structure/function Relationshipsupporting

confidence: 90%

“…3a, see Automated protein production and protein puri cation). For further investigation of this effect, we cultivated 48 biological replicates of E. coli BL21(DE3) with BsGDH-PT-CBDCell, a variant that was previously identi ed as a positive CatIB producer under standard conditions in a manual study (11). Strikingly, the nal backscatter values across the FlowerPlate varied from approx.…”

Section: Optimization Of the Catib Work Ow -Biolector Positional Biasmentioning

confidence: 99%

“…Besides standardizing and optimizing the cultivation conditions, the enzymatic assay was adapted compared to a recent manual study (11). Previously, samples were taken after 0, 3, 6, 12 and 20 min from the BsGDH reaction and quenched with methanol to stop the reaction.…”

Section: Optimization Of the Catib Work Ow -Enzymatic Assay And Overa...mentioning

confidence: 99%

“…Catalytically active inclusion bodies (CatIBs) are inclusion bodies that were demonstrated to retain substantial catalytic activity (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11). CatIBs are known for their easy and cost-e cient production, recyclability as well as high stability.…”

Section: Introductionmentioning

confidence: 99%

“…The choice of linker and aggregation-inducing tag strongly in uences CatIB activities, as was previously shown for CatIBs of the lysine decarboxylase from E. coli (EcLDCc) or of the glucose dehydrogenase from Bacillus subtilis (BsGDH). For both enzymes, the rigid Proline/Threonine (PT) linker led to higher activities compared to the exible Serine/Glycine (SG) linker (10,11). Besides the linker and aggregation-inducing tag, also the terminus of the enzyme, which is used for fusion, in uences the activity of CatIBs.…”

Section: Introductionmentioning

confidence: 99%

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High-Throughput Screening of Catalytically Active Inclusion Bodies Using Laboratory Automation and Bayesian Optimization

Helleckes,

Küsters,

Wagner

et al. 2023

Preprint

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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 h 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.

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Section: Insights On Structure/function Relationshipsupporting

confidence: 90%

Section: Optimization Of the Catib Work Ow -Biolector Positional Biasmentioning

confidence: 99%

Section: Optimization Of the Catib Work Ow -Enzymatic Assay And Overa...mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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High-Throughput Screening of Catalytically Active Inclusion Bodies Using Laboratory Automation and Bayesian Optimization

Helleckes,

Küsters,

Wagner

et al. 2023

Preprint

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Carrier-free immobilized enzymatic reactor based on CipA-fused carbonyl reductase for efficient synthesis of chiral alcohol with cofactor self-sufficiency

Wang,

Liu,

Duan

et al. 2024

International Journal of Biological Macromolecules

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

Helleckes,

Küsters,

Wagner

et al. 2024

Microb Cell Fact

Self Cite

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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.

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

Cited by 4 publications

References 26 publications

High-Throughput Screening of Catalytically Active Inclusion Bodies Using Laboratory Automation and Bayesian Optimization

High-Throughput Screening of Catalytically Active Inclusion Bodies Using Laboratory Automation and Bayesian Optimization

Carrier-free immobilized enzymatic reactor based on CipA-fused carbonyl reductase for efficient synthesis of chiral alcohol with cofactor self-sufficiency

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

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