Enzyme cascade converting cyclohexanol into ε‐caprolactone coupled with NADPH recycling using surface displayed alcohol dehydrogenase and cyclohexanone monooxygenase on E. coli

Abstract

Summary The application of enzymes as biocatalysts in industrial processes has great potential due to their outstanding stereo‐, regio‐ and chemoselectivity. Using autodisplay, enzymes can be immobilized on the cell surface of Gram‐negative bacteria such as Escherichia coli. In the present study, the surface display of an alcohol dehydrogenase (ADH) and a cyclohexanone monooxygenase (CHMO) on E. coli was investigated. Displaying these enzymes on the surface of E. coli resulted in whole‐cell biocatalysts accessible for substrates without further purification. An apparent maximal reaction velocity VMAX(app) for the oxidation of cyclohexanol with the ADH whole‐cell biocatalysts was determined as 59.9 mU ml−1. For the oxidation of cyclohexanone with the CHMO whole‐cell biocatalysts a VMAX(app) of 491 mU ml−1 was obtained. A direct conversion of cyclohexanol to ε‐caprolactone, which is a known building block for the valuable biodegradable polymer polycaprolactone, was possible by combining the two whole‐cell biocatalysts. Gas chromatography was applied to quantify the yield of ε‐caprolactone. 1.12 mM ε‐caprolactone was produced using ADH and CHMO displaying whole‐cell biocatalysts in a ratio of 1:5 after 4 h in a cell suspension of OD578nm 10. Furthermore, the reaction cascade as applied provided a self‐sufficient regeneration of NADPH for CHMO by the ADH whole‐cell biocatalyst.

Combining lipid-mimicking-enabled transition metal and enzyme-mediated catalysis at the cell surface of E. coli

“…Combining alcohol dehydrogenase displaying cells with cyclohexanone monooxygenase displaying cells facilitated a cascade reaction of cyclohexanol to cyclohexanone and sequentially to ε -caprolactone in a two-cell approach. 37 We also reported that two functional enzymes were successfully combined by co-display in a single-cell approach. Here, truncated variants of human cytochrome P450 1A2 (CYP1A2) and human cytochrome P450 reductase (CPR) were co-displayed on E. coli .…”

A Novel Flow Cytometry-Based Assay for the Identification of HCN4 CNBD Ligands

“…To elucidate the affinity range of the assay, the amount of surface-displayed C-Linker-CNBD protein was determined by densitometry as described before by Tian et al, 2022 [ 50 ]. It was calculated to be 5.1 × 10 4 molecules per cell, which was in the same order of magnitude as reported before for other surface-displayed proteins [ 40 , 50 , 51 ].…”

“…To elucidate the affinity range of the assay, the amount of surface-displayed C-Linker-CNBD protein was determined by densitometry as described before by Tian et al, 2022 [ 50 ]. It was calculated to be 5.1 × 10 4 molecules per cell, which was in the same order of magnitude as reported before for other surface-displayed proteins [ 40 , 50 , 51 ]. Since a minimum number of 10 5 cells is required for a flow cytometry sample [ 48 ], a minimum number of approximately 10 9 –10 10 receptors can be calculated per sample.…”

“…However, it appears that the assay as described can be helpful as a prescreening method before more expensive and time-consuming eukaryotic cell experiments are conducted. It has been described before that proteins expressed as monomers using autodisplay can form dimeric [ 69 , 70 ] or tetrameric [ 50 , 71 ] structures at the cell surface. This is supposed to be due to the mobility of the anchoring β-barrel domain within the outer membrane after transport [ 41 ] and the affinity of the protein subunits to each other as displayed.…”

30th Annual GP2A Medicinal Chemistry Conference

“…1.12 mM ε-caprolactone was produced using ADH and CHMO displaying whole cell biocatalysts in a ratio of 1:5 after 4 h in a cell suspension of OD 578nm 10. Furthermore, the reaction cascade as applied provided a self-sufficient regeneration of NADPH for CHMO by the ADH whole cell biocatalyst [ 159 ].…”