Construction of synthetic pathways for raspberry ketone production in engineered Escherichia coli

• Published in: Applied microbiology and biotechnology 2019-05, Vol.103 (9), p.3715-3725
• Main Authors: Wang, Chengcheng, Zheng, Pu, Chen, Pengcheng
• Format: Article
• Language: English
• Subjects: Acetone; Arabidopsis; Arabidopsis thaliana; Bacteria; Biomedical and Life Sciences; Biosynthetic Pathways; Biotechnological Products and Process Engineering; Biotechnology; Butanones - metabolism; Coumaric acid; E coli; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Essences and essential oils industry; Flavor; Fruits; Genes; Genetically modified organisms; Ketones; Life Sciences; Ligases; Metabolic Engineering; Microbial Genetics and Genomics; Microbiology; Microorganisms; Modules; p-Coumaric acid; Perfumes industry; Phenols (Class of compounds); Physiological aspects; Plant Proteins - genetics; Plant Proteins - metabolism; Plants - enzymology; Properties; Propionates - metabolism; Vegetables; Zingerone
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-019-09748-5

Raspberry ketone is an important ingredient in the flavor and fragrance industries. Due to its low content in fruits and vegetables, the production of natural raspberry ketone using heterologous synthesis in microbial strains is recently attracting increased attention. In this work, a heterologous pathway to produce raspberry ketone from p -coumaric acid, including 4-coumarate: CoA ligase (4CL), benzalacetone synthase (BAS), and raspberry ketone/zingerone synthase (RZS1) from plants, was successfully assembled in Escherichia coli . When the RZS1 gene was introduced into E. coli and co-expressed with two other genes, the intermediate 4-hydroxybenzylidene acetone in the pathway was almost completely transformed into a raspberry ketone. Substituting TB medium for M9 medium increased raspberry ketone titers by 3–4 times. Furthermore, the heterologous pathway was partitioned into two modules; module one produced p -coumaroyl-CoA from p -coumaric acid by 4CL, and module two produced raspberry ketone from coumaroyl-CoA by the action of BAS and RZS1. Optimizing the balanced expression of the two modules, it was shown that moderate expression of module one and high expression of module two was the best combination to enhance raspberry ketone production. The engineered strain CZ-8 reached 90.97 mg/l of raspberry ketone, which was 12 times higher than previously reported. In addition, the preferred approach of the heterologous pathway was related to the heterologous genes from different sources; for example, 4CL from Arabidopsis thaliana seemed to be more suitable for raspberry ketone production than that from Petroselinum crispum . This work paves an alternative way for future economic production of natural raspberry ketone.