Abstract
BackgroundRaspberry 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.MethodsA 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 Saccharomycescerevisiae. Synthetic protein fusions were also explored as a means of increasing yields of the final product.ResultsThe 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.ConclusionsWe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0446-2) contains supplementary material, which is available to authorized users.
Highlights
Raspberry ketone is the primary aroma compound found in raspberries and naturally derived raspberry ketone is a valuable flavoring agent
Biosynthesis of raspberry ketone from p‐coumaric acid The production of raspberry ketone from p-coumaric acid requires the action of two heterologous enzyme activities, a coumarate-CoA ligase (4CL) and benzalacetone synthase (BAS), as yeast has been shown to natively display efficient benzalacetone reductase (BAR) activity [5] (Fig. 1a)
Codon optimized versions of the heterologous genes were synthesized and the two different 4CL and BAS pairs (At4CL1 RpBAS and Pc4CL2 RpBAS) were integrated in the HO locus of AWRI2975 (TDH3p:ACC1) in a tandem arrangement, with each gene driven from a separate copy of the highly-fermentation expressed promoter of FBA1 [10, 11] (Fig. 1b)
Summary
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, make this compound an excellent target for heterologous production in synthetically engineered microbial strains. Plant natural compounds represent a large, chemicallydiverse collection of secondary metabolites, this diversity is generated from a limited number of conserved pathways (reviewed in Marienhagen et al [1]). One such class of plant compounds are the phenylpropanoids, which like flavonoids, stilbenes and lignans, are formed. The economic incentives for the production of raspberry ketone, combined with the very poor yields from plant tissue, make this compound an excellent target for production via the use of synthetically engineered microbial strains. De novo production of raspberry ketone, without the need for precursor addition, has not yet been demonstrated
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