Efficient Biosynthesis of Raspberry Ketone by Engineered Escherichia coli Coexpressing Zingerone Synthase and Glucose Dehydrogenase

Abstract

Raspberry ketone (RK), the main aroma compound of raspberry fruit, has applications in cosmetics, food industry, and pharmaceutics. In this study, we biosynthesized RK via the catalytic reduction of 4-hydroxybenzylidenacetone using a whole-cell biocatalyst. Reductase RiRZS1 from Rubus idaeus and glucose dehydrogenase SyGDH from Thermoplasma acidophilum were expressed in Escherichia coli to regenerate NADPH for the whole-cell catalytic reaction. Following the optimization of balancing the coexpression of two enzymes in pRSFDuet-1, we obtained 9.89 g/L RK with a conversion rate of 98% and a space–time yield of 4.94 g/(L·h). The optimum conditions are 40 °C, pH 5.5, and a molar ratio of substrate to auxiliary substrate of 1:2.5. Our study findings provide a promising method of biosynthesizing RK.

High-yield ‘one-pot’ biosynthesis of raspberry ketone, a high-value fine chemical

“…For raspberry ketone, only tiny quantities are synthesized in the berries themselves and the process requires weeks of maturation. While the raspberry ketone intermediates in general inhibit microbial growth production, this can be bypassed using a E. coli whole-cell (stationary phase cells) biocatalyst system which is active to the g L −1 scale but requires the chemically derived precursor HBA ( 24 ). Alternatively, the substrates L-tyrosine or p -coumarate, which can be derived from a natural feedstock, can be converted by the type III polyketide described herein, in synthetically engineered E. coli and S. cerevisiae systems but yields are limited (up to 95 mg L −1 ) due to general toxicity ( 15 , 21 , 22 ).…”

“…Therefore, current microbial fermentation strategies, starting from p -coumarate, have produced limited yields in either E. coli or Saccharomyces cerevisiae , ranging from 0.2–95 mg L −1 ( 15 , 21–23 ), which largely rely upon growth media optimization and maximizing cell density to overpower general toxicity issues from ketone intermediates. To indirectly overcome growth inhibition, recently Yang et al used a E. coli whole-cell catalyst approach to make up to 9 g L −1 of raspberry ketone from HBA ( 24 ). However, this strategy bypasses the polyketide step and incubates E. coli cells containing a pre-expressed RZS1 and a glucose dehydrogenase for NADPH regeneration, in an optimized reaction buffer with HBA ( 24 ).…”

“…To indirectly overcome growth inhibition, recently Yang et al used a E. coli whole-cell catalyst approach to make up to 9 g L −1 of raspberry ketone from HBA ( 24 ). However, this strategy bypasses the polyketide step and incubates E. coli cells containing a pre-expressed RZS1 and a glucose dehydrogenase for NADPH regeneration, in an optimized reaction buffer with HBA ( 24 ).…”

Efficient Asymmetric Synthesis of (S)-N-Boc-3-hydroxypiperidine by Coexpressing Ketoreductase and Glucose Dehydrogenase

“…In addition, when asymmetric reduction reaction is carried out using two different whole cells, the reaction efficiency will be significantly affected due to the cell membrane barrier effect. The expression of multiple enzymes in the same host cell can avoid the shortcomings of the above reaction process and significantly improve the catalytic efficiency of an enzyme-coupled coenzyme regeneration system [11][12][13].…”

An ADH toolbox for raspberry ketone production from natural resources via a biocatalytic cascade

“…In literature, different routes for the natural production of raspberry ketone are stated: on the one hand, this can be achieved by heterologous pathways in microorganisms incorporated via metabolic engineering. Approaches with engineered microorganisms like E. coli, S. cerevisiae, or C. glutamicum yielded product titers between 5 and 9.89 g/l raspberry ketone either starting from expensive p-coumaric acid (Beekwilder et al 2007;Lee et al 2016;Wang et al 2019;Milke et al 2020;Paulino 2021), lower-priced tyrosine (Farwick et al 2019), 4-hydroxybenzylidene acetone (Yang et al 2021), or fatty acids as alternative low-cost feedstock (Chang and Liu 2021). Furthermore, the de novo production of raspberry ketone was achieved by genetically modified E. coli or C. glutamicum strains that produce tyrosine by themselves yielding 19 mg/l (Cankar et al 2019) or even up to 780 mg/l raspberry ketone (Schloesser and Lambert 2018).…”