A toolbox approach for the rapid evaluation of multi-step enzymatic syntheses comprising a ‘mix and match’ E. coli expression system with microscale experimentation

This work describes an experimental ‘ toolbox ’ for the rapid evaluation and optimisation of multi-step enzymatic syntheses comprising a ‘ mix and match ’ E. coli -based expression system and automated microwell scale experimentation. The approach is illustrated with a de novo designed pathway for the synthesis of optically pure amino alcohols using the enzymes transketolase (TK) and transaminase (TAm) to catalyze asymmetric carbon-carbon bond formation and selective chiral amine group addition respectively. The E. coli expression system, based on two compatible plasmids, enables pairs of enzymes from previously engineered and cloned TK and TAm libraries to be evaluated for the sequential conversion of different initial substrates. This is complemented by the microwell experimentation which enables effi cient investigation of different biocatalyst forms, use of different amine donors and substrate feeding strategies. Using this experimental ‘ toolbox ’ , one-pot syntheses of the diastereoisomers (2 S ,3 S )-2-aminopentane-1,3-diol (APD) and (2 S ,3 R )-2-amino-1,3,4-butanetriol (ABT) were designed and performed, which gave fi nal product yields of 90% mol/mol for APD and 87% mol/mol for ABT (relative to the initial TK substrates) within 25 hours. For the synthesis of APD, the E coli TK mutant D469E was paired with the TAm from Chromobacterium violaceum 2025 while for ABT synthesis the wild-type E. coli TK exhibited the highest specifi c activity and ee ( enantiomeric excess) of 95%. For both reactions, whole-cell forms of the TK-TAm biocatalyst performed better than cell lysates while isopropylamine (IPA) was a preferable amine donor than methylbenzylamine (MBA) since side reactions with the initial TK substrates were avoided. The available libraries of TK and TAm enzymes and scalable nature of the microwell data suggest this ‘ toolbox ’ provides an effi cient approach to early stage bioconversion process design in the chemical and pharmaceutical sectors.