Enhanced whole-cell biotransformation of carboxylic acid reductase enzymes via tandem expression of phosphopantetheinyl transferase

초록

Carboxylic acid reductases (CARs) function as versatile biocatalysts with a broad substrate range. However, CARs still struggled with relatively low activity in many enzyme reactions. While extensive efforts have focused on engineering CAR itself, the influence of phosphopantetheinyl transferase (Sfp), which is essential for CAR activation, has received significantly less attention. Consequently, information regarding the optimal ratio of CAR to Sfp for efficient biotransformation remains limited. To touch this issue, we investigated the effect of tandem sfp genes to CAR by introducing CAR-mediated 1,4-pentanediol (1,4-PDO) production with CAR of MSM5586, which is from Mycobacterium smegmatis. Different copy numbers of sfp were introduced in Escherichia coli and the introduction of three sfp genes (SU_MSM5586_3S was found to yield the highest 1,4-PDO, resulting more than the single-sfp system by 1.43-fold. Following the deletion of gabD to prevent aldehyde oxidation and the introduction of ppk2b for ATP regeneration, the optimized strain SU_M3Sd_p produced 60.64 mM 1,4-PDO after 36 h, achieving a 1.80-fold higher conversion rate. Tandem sfp expression also was applied to enhance the yield of other CAR, MabCAR (MAB4714) from Mycobacterium abscessus with different substrates such as 6-hydroxycaproic and cinnamic acids, increasing the production yield by 1.84- and 2.41-fold, respectively. These findings demonstrate that optimizing the sfp gene copy number is an effective strategy for CAR-mediated biotransformation. © 2026 Elsevier Inc.

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