Heterologous production of raspberry ketone in the wine yeast Saccharomyces cerevisiae via pathway engineering and synthetic enzyme fusion

Abstract

Background: Raspberry ketone is the primary aroma compound found in raspberries and naturally derived raspberry ketone is a valuable flavoring agent. The economic incentives for the production of raspberry ketone, combined with the very poor yields from plant tissue, therefore make this compound an excellent target for heterologous production in synthetically engineered microbial strains.

Methods: A de novo pathway for the production of raspberry ketone was assembled using four heterologous genes, encoding phenylalanine/tyrosine ammonia lyase, cinnamate-4-hydroxlase, coumarate-CoA ligase and benzalacetone synthase, in an industrial strain of Saccharomyces cerevisiae. Synthetic protein fusions were also explored as a means of increasing yields of the final product.

Results: The highest raspberry ketone concentration achieved in minimal media exceeded 7.5 mg/L when strains were fed with 3 mM p-coumaric acid; or 2.8 mg/L for complete de novo synthesis, both of which utilized a coumarate-CoA ligase, benzalacetone synthase synthetic fusion protein that increased yields over fivefold compared to the native enzymes. In addition, this strain was shown to be able to produce significant amounts of raspberry ketone in wine, with a raspberry ketone titer of 3.5 mg/L achieved after aerobic fermentation of Chardonnay juice or 0.68 mg/L under anaerobic winemaking conditions.

Conclusions: We have shown that it is possible to produce sensorially-relevant quantities of raspberry ketone in an industrial heterologous host. This paves the way for further pathway optimization to provide an economical alternative to raspberry ketone derived from plant sources.

Fig. 1

Engineering the raspberry ketone biosynthetic pathway in S. cerevisiae. a The phenylpropanoid pathway begins with the conversion of phenylalanine to p-coumaric acid via cinnamate or directly from tyrosine to p-coumaric acid (pink box). Conversion of p-coumaric acid to raspberry ketone requires three additional enzymatic steps including a condensation reaction between coumaroyl-CoA and malonyl-CoA. Heterologous production of raspberry ketone can be accomplished by the final three enzymatic reactions, if microbial cells are supplied with exogenous p-coumaric acid (blue box). The heterologous enzymes used for each reaction in this study are also listed. b Expression constructs used in this study for the production of raspberry ketone. Rigid and flexible linker sequences that were used for the protein fusions are represented by bold black lines (straight and wavy, respectively)

Fig. 2

Biosynthesis of raspberry ketone from p-coumaric acid during anaerobic fermentation. Codon optimized genes encoding coumarate CoA ligase (4CL) from either A. thaliana (At4CL1, pink) or P. crispum (Pc4CL2, blue) and benzalacetone synthase (BAS) from R. palmatum (RpBAS) were integrated at the HO locus of S. cerevisiae as either two independent genes or as a single ORF fused by either a flexible (f) or rigid (r) amino acid linker. Levels of raspberry ketone were assessed following 5 days growth at 22 °C in air-lock flasks in synthetic grape juice medium, supplemented with 3 mM p-coumaric acid and assessed for raspberry ketone production via LC/MS

Fig. 3

The Effect of oxygen on the production of raspberry ketone. Strains containing the P. crispum coumarate CoA ligase 2 (Pc4CL2) and benzalacetone synthase (BAS) from R. palmatum (RpBAS) ORFs fused by rigid (r) amino acid linker were fermented in either airlock flasks (anaerobic) or standard flasks (aerobic) in synthetic grape juice medium, supplemented with 3 mM p-coumaric acid and assessed for raspberry ketone production via LC/MS

Fig. 4

Full de novo biosynthesis of raspberry ketone. a Codon optimized genes encoding phenylalanine ammonia lyase from Rhodosporidium toruloides PAL (RtPAL) and cinnamate-4-hydroxylase from Arabidopsis thaliana (AtC4H) were integrated at the HO locus of S. cerevisiae as either two independent genes or as a single ORF fused by either a flexible (f) or rigid (r) amino acid linker. All strains also contained the P. crispum coumarate CoA ligase 2 and benzalacetone synthase from R. palmatum ORFs fused by a flexible linker (Pc4CL2-f-RpBAS), positioned adjacently in the HO locus. Levels of raspberry ketone were assessed following five days growth at 22 °C in air-lock flasks in synthetic grape juice medium and assessed for raspberry ketone production via LC/MS. b A strain containing the Rhodosporidium toruloides PAL (RtPAL) and cinnamate-4-hydroxylase from Arabidopsis thaliana (AtC4H) as separate ORFs in addition to the P. crispum coumarate CoA ligase 2 and benzalacetone synthase from R. palmatum ORFs fused by rigid linker (Pc4CL2-r-RpBAS) were fermented in either airlock flasks (anaerobic) or standard flasks (aerobic) in synthetic grape juice medium and assessed for raspberry ketone production via LC/MS

Fig. 5

De novo production of raspberry ketone during wine fermentation. A strain containing the Rhodosporidium toruloides PAL (RtPAL) and cinnamate-4-hydroxylase from Arabidopsis thaliana (AtC4H) as separate ORFs in addition to the P. crispum coumarate CoA ligase 2 and benzalacetone synthase from R. palmatum ORFs fused by rigid linker (Pc4CL2-r-RpBAS) was fermented in Chardonnay grape juice in either airlock flasks (anaerobic) or standard flasks (aerobic) until dryness and assessed for raspberry ketone production via LC/MS