Glucose-Derived Raspberry Ketone Produced via Engineered Escherichia coli Metabolism

Abstract

The demand for raspberry ketone (RK) as a plant-based natural flavoring agent is high, but natural RK is one of the most expensive flavor compounds due to its limited content in plants. Here, we produced RK de novo from simple carbon sources in Escherichia coli. We genetically engineered E. coli metabolism to overproduce the metabolic precursors tyrosine and p-coumaric acid and increase RK production. The engineered E. coli produced 19.3- and 1.9 g/L of tyrosine and p-coumaric acid from glucose, respectively. The p-coumaric acid CoA ligase from Agrobacterium tumefaciens and amino acid substituted benzalacetone synthase of Rhemu palmatum (Chinese rhubarb) were overexpressed in E. coli overproducing p-coumaric acid. The overexpression of fabF, encoding β-ketoacyl-acyl carrier protein synthetase II increased intracellular malonyl-CoA, the precursor of benzalacetone synthase for RK biosynthesis, and improved RK production. Fed-batch cultures given glucose as a carbon source produced 62 mg/L of RK under optimized conditions. Our production system is inexpensive and does not rely on plant extraction; thus, it should significantly contribute to the flavor and fragrance industries.

Machine learning-assisted medium optimization revealed the discriminated strategies for improved production of the foreign and native metabolites

“…The expression of papABC mediated the production of 4APhe. The E. coli strain BL21(DE3) harboring pET-tyrA/pACYC-aroG fbr was constructed to produce tyrosine (Tyr), as described previously [43]. Both products were induced by isopropyl β-D-1-thiogalactopyranoside (IPTG) (Fig.…”

Machine learning-assisted medium optimization revealed the discriminated strategies for improved production of the foreign and native metabolites

“…To test the availability of ML to medium optimization for synthetic construction, the genetically engineered E. coli strains producing 4-aminophenylalanine (4APhe) [35] or tyrosine (Tyr) [36] were used as a case study. The strains were previously developed and carried the synthetic pathways that were common from glucose to chorismate and differentiated from chorismate to the endpoint metabolites ( Fig.…”

“…A scaleup to 5 ML of culture was conducted to compare the production of 4APhe and Tyr in three different media, i.e., the fine-tuned, pre-fine-tuned, and M9-LB media. M9-LB was a commonly used rich medium optimized in the previous studies that constructed the two E. coli strains [35] , [36] . The highest yields of both 4APhe and Tyr were acquired in the fine-tuned media ( Fig.…”

“…The expression of papABC mediated the production of 4APhe. The E. coli strain BL21(DE3) harboring pET-tyrA/pACYC-aroG fbr was constructed to produce tyrosine (Tyr), as described previously [36] . The low-copy plasmids were used for the synthetic construction, and both products were induced by isopropyl β-D-1-thiogalactopyranoside (IPTG) ( Fig.…”

Transcriptome analysis of malate-induced Schizochytrium sp. FJU-512 reveals a novel pathway for biosynthesis of docosahexaenoic acid with enhanced expression of genes responsible for acetyl-CoA and NADPH accumulation

“…FabF is a key element in type II fatty acid synthesis, which is catalyzed by a “ping-pong” mechanism, and these enzymes have substrate-binding sites for acetyl-CoA and malonyl-ACP ( Abbadi et al, 2000 ). The overexpression of fabF increased intracellular malonyl-CoA ( Masuo et al, 2022 ). Therefore, malate addition resulted in the upregulation of fabF , which probably leading to PUFA-S exploit the improvement of malonyl-CoA and enhancement DHA production in Schizochytrium sp.…”