Integrated Experimental and Mathematical Modeling to Guide Microbial Biocatalysis: <i>Pseudomonas putida</i> Conversion of L‐Phenylalanine to <i>trans</i>‐Cinnamic Acid

trans-Cinnamic acid (tCA) can be produced by the deamination of L-phenylalanine using phenylalanine ammonia-lyase (PAL). In bioprocesses, optimizing production processes to improve key performance metrics such as titer, rate, and yield has proved challenging. This study investigates tCA production in Pseudomonas putida KT2440 using a whole-cell biocatalyst expressing four different PAL genes. Among these, RmXAL showed the highest titer and rate. Biocatalysis at pH 8.5 and 37°C were identified as the best conditions, giving a tCA concentration of 2.65 g L^-1 and a production rate of 0.44 g L^-1 h^-1. To improve the metrics further, a decoupled bioprocess with various biocatalyst concentrations in the second stage was examined. With a whole-cell biocatalyst concentration of 30 g_DCW L^-1, optimal process parameters were achieved, giving a titer of 29.88 g L^-1, rate of 5.99 g L^-1 h^-1, yield on glucose of 0.27 g tCA g glucose^-1, and yield on L-phe of 0.75 g tCA g L-phe^-1. This study is the first report of a model that enables performance metrics to evaluate a suitable process for tCA production and provides valuable insights into tCA production using a decoupled bioprocess. This would appear applicable to larger-scale production, paving the way for an efficient and sustainable industrial process.