Evaluation of protein and metabolic engineering strategies on (−)-α-bisabolol production by Escherichia coli

Abstract

(−)-α-bisabolol is a sesquiterpene compound found in various plants, with broad applications in pharmaceuticals, cosmetics, and personal care products due to its diverse pharmacological properties. Recombinant Escherichia coli offers an efficient and sustainable platform for its production. To further advance its industrial-scale synthesis, we enhanced the expression and catalytic activity of the key enzyme bisabolol synthase (CcBOS) and optimized the metabolic pathways of the host chassis. In this study, CcBOS from Cynara cardunculus was expressed in a previously engineered sesquiterpene-producing E. coli strain. By adjusting the fermentation temperature to 25 °C, the (−)-α-bisabolol titer reached 1.12 g/L in shake flask cultures. Protein engineering strategies—including optimization of the N-terminal sequence, increasing polar amino acid content, modifying non-conserved residues, and mutating histidine residues near the active site—significantly improved the solubility and catalytic efficiency of CcBOS. The triple mutant T258S/I364N/H479A increased (−)-α-bisabolol production by 154.5%. Metabolic engineering efforts, such as deleting atoDA to reduce fatty acid synthesis and attenuating gltA expression to limit tricarboxylic acid (TCA) cycle competition, further enhanced the titer by 63.1%. Integrating protein and metabolic engineering approaches resulted in a final titer of 3.08 g/L in shake flasks. When scaled up to a 5 L bioreactor, the titer reached 7.41 g/L, representing a high production of (−)-α-bisabolol using glucose as the sole carbon source. This study provides a novel integrated approach for the efficient microbial synthesis of (−)-α-bisabolol.

Keywords

(-)-α-bisabolol / Escherichia coli / Protein engineering / Metabolic engineering

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Funding

National Key Research and Development Program of China(2022YFA0912204)

Jiangsu Provincial Key Research and Development Program(BE2023685)

RIGHTS & PERMISSIONS

Jiangnan University