An automated microscale platform for evaluation and optimization of oxidative bioconversion processes

Abstract

In this work an integrated robotic platform has been used for the development of a fully automated microscale process sequence comprising fermentation and bioconversion using E. coli TOP10 [pQR210] expressing cyclohexanone monooxygenase (CHMO). Ninety six-Deep Square Well (96-DSW) microtiter plates were used for microbial culture and enzyme-catalyzed conversion, where plate preparation, reagent addition, and sampling were all carried out without manual intervention. The adoption of automated robotic procedures has enabled the rapid collection of kinetic data for whole process optimization at the microscale. This high-throughput approach enabled a range of amino acid sources for media formulation and well fill volumes to be investigated highlighting when nutritional limitation and oxygen limitations took place. The automated process sequence has been applied to test six CHMO substrates including norcamphor and cycloheptanone all of which to the best of our knowledge have yet to be tested with E. coli TOP10 [pQR210]. Substrate specificity and product selectivity were effectively demonstrated and compared to both the natural substrate cyclohexanone and the model substrate bicyclo[3.2.0]hept-2-en-6-one used to demonstrate asymmetric synthesis. The results obtained using the developed process sequence could be reproduced at 75 L scale when a matched oxygen transfer coefficient k(L) a approach was used. The study demonstrates how automated microscale processing enables the rapid collection of kinetic quantitative data in a robust manner with clear implications for accelerating bioprocess development, optimization, and scale-up.