Process evaluation identifying bottlenecks

The flowchart is a helpful tool in identifying and evaluating the capacities of each element of the process and areas of the process that can be improved (e.g., areas that are causing bottlenecks for improved efficiency). A... 

In order to establish the two-step strategy, both en2ymatic steps were evaluated separately this included the engineering of the TK to accept the non-natural substrate propanal, and use of a bioinformatic-based strategy to identify a suitable co-TA with the ability to accept the ligation product. The final reaction was performed in two sequential steps, because preliminary experiments indicated that the co-TA also aminates the TK substrates in the presence of an amine donor. Notably, for the reductive amination (second step), cheap and achiral 2-PrNH2 could be used successfully as an alternative amine source. The final product was isolated after the two enzymatic steps, with a calculated overall isolated yield of 18% under nonoptimized conditions. However, while the TK-catalyzed C-C bond formation already displayed suitable conversions (23%), the cb-TA was identified to be the bottleneck of the cascade reaction further optimization, especially with respect to the reductive amination, should facilitate a more efficient process. 

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