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

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.

Synthetic genome engineering forging new frontiers for wine yeast

“…To construct a biosynthetic pathway for the de novo production of raspberry ketone in a wine strain of S. cerevisiae, the following codon-optimized genes were synthesized and integrated into the yeast's HO locus: the phenylalanine ammonia lyase from an oleaginous yeast, Rhodosporidium toruloides; the cinnamate-4-hydroxylase from the well-characterized model plant, A. thaliana; and the coumarate CoA ligase 2 gene from parsley, Petroselinum crispum, fused by a rigid linker to the benzalacetone synthase from rhubarb, Rheum palmatum. [4] This "synthetic" wine yeast is capable of synthesizing raspberry ketone at concentrations almost two orders of magnitude above its predicted sensory threshold in Chardonnay grape juice under standard wine fermentation conditions, while retaining the ability to ferment the must to dryness. This first small step marks a giant leap forward for wine yeast into an unwritten future with synthetic DNA.…”

“…In a world-first, a wine strain of S. cerevisiae was equipped with a biosynthetic pathway -comprising four separate enzymatic activities -to produce the highly desirable raspberry ketone, 4-[4-hydroxyphenyl] butan-2-one ( Figure 20). [4] This phenylpropanoid is the primary aroma compound found in raspberries and it is also present in other fruits, vegetables and berries, such as blackberries, grapes, and rhubarb.…”

“…In fact, the use of Synthetic Biology techniques are already spilling over into the development of improved wine yeast strains. [4] To engage all stakeholders of the wine industry in a sensible debate over the complexities surrounding this controversial and disruptive new science, this paper briefly reviews the state-of-play in Synthetic Biology, illuminates the critically important role of the global Synthetic Yeast Genome (Sc2.0) Project (more detail available from www.syntheticyeast.org) in advancing the frontiers of synthetic genomics and proposes future directions and guiding principles for future wine yeast research.…”

“…Development of the world's first synthetically engineered wine strain of Saccharomyces cerevisiae for the de novo biosynthesis of the highly desirable raspberry ketone aroma compound, 4-[4-hydroxyphenyl]butan-2-one. [4] have to carefully consider the complex gravitational forces of perception and control every step of the way across the gorge caused by this emerging science. The pioneering "extreme tightrope-daredevils" in Synthetic Biology cannot afford to lose their footing even once.…”

“…The recent de novo production of raspberry ketone in a haploid wine strain (AWRI1631) of S. cerevisiae was achieved via metabolic pathway engineering and synthetic enzyme fusion. [4] The phenylpropanoid pathway starts with the conversion of phenylalanine to p-coumaric acid via cinnamate or directly from tyrosine to p-coumaric acid. Conversion of p-coumaric acid to raspberry ketone requires three additional enzymatic steps including a condensation reaction between coumaroyl-CoA and malonyl-CoA.…”