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.
Highlights
The raspberry phenylbutanoid ketone 4-(4-hydroxyphenyl)butan-2-one (RK) is a natural flavor in plants such as raspberries, grapes, peaches, and rhubarb
Escherichia coli BL21 (DE3) harboring either pET-FevV, pET28a-pal, pET-Cspal or pET-Lepal was cultured in 3 ml of LB medium, 2 ml portions were inoculated into 100 ml of LB medium and rotary-shaken at 120 rpm at 30°C under aerobic conditions
We cloned tyrosine ammonia lyase (TAL) or phenylalanine ammonia lyase (PAL) genes derived from yeast, plant or bacteria into the pRSFduet1 vector under the control of the T7 promoter and the resulting plasmids were introduced into BL21 (DE3)
Summary
The raspberry phenylbutanoid ketone 4-(4-hydroxyphenyl)butan-2-one (RK) is a natural flavor in plants such as raspberries, grapes, peaches, and rhubarb. The common approach converts p-coumaric acid as a starting material to RK in host cells producing heterogenous CL, BAS and BAR that originate from plants. Host E. coli and Corynebacterium glutamicum cells convert p-coumaric acid to RK with titers of 91.0 and 99.8 mg/l, respectively (Wang et al., 2019; Milke et al, 2020). One exception is a wine yeast that generates plant PAL, C4H, and the synthetic CL and BAS fusion enzyme, and this yeast produces 3.5 mg/l of RK in grape juice medium We showed that improving p-coumaric acid and malonyl-CoA availability significantly increased the productivity of microbial de novo RK synthesis. We constructed E. coli that generated abundant p-coumaric acid and malonyl-CoA by metabolic engineering and chemical stimulation. After gene selection and stepwise culture optimization, our recombinant E. coli produced RK biosynthesis enzymes and fermented glucose to produce 62 mg/L of RK
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