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Coniferol, as a natural monolignol, acts as a chemical building block for various high-value compounds. However, the current petrochemical synthesis of coniferol and its limited scalability raise environmental and cost concerns. A biocatalytic method for coniferol production from its natural precursor, ferulic acid, was previously developed using an engineered cell factory that co-expresses carboxylic acid reductase and aldo-keto reductase. The present study focused on investigating the limiting factors of coniferol production and optimizing the process using the design of experiments and fed-batch approaches. By introducing novel parameters, including an inoculum of 0.25 g/L cells and a starting concentration of 5.0 mM ferulic acid, followed by two fed-batch additions of 2.0 mM ferulic acid and 3.7 g/L glycerol, coniferol production was enhanced by a factor of 2 (reaching 1.6 g/L). The scale-up process in a 1 L …