The Australian Wine Research Institute

Genomics innovation initiative

Project summary

Synthetic biology and metabolic engineering are frontier areas of biological research. These cutting-edge technologies represent exciting new opportunities in areas such as the creation of plant-derived pharmaceuticals, production of economically feasible sources of biofuels and major improvements in current food and beverage production.

The yeast Saccharomyces cerevisiae represents an obvious target for synthetic engineering. It is an established and prominent industrial microorganism, used to produce a diversity of high-value food, beverage and biotechnology products such as biofuels, pharmaceuticals, wine and beer. In addition, yeast is a key model organism for the development of new technologies in fundamental research, including genomics, transcriptomics, metabolomics and systems biology. The existing fundamental knowledge provides an extensive base on which to build a synthetic biology strategy that can be applied in an industrial setting to address key opportunities. Specific objectives of this project are to engineer Saccharomyces cerevisiae with new metabolic pathways to enable biosynthesis of compounds such as monoterpenes, raspberry ketone, anthocyanins and lactic acid during fermentation.

In partnership with Macquarie University, the AWRI is also a member of the international Sc2.0 collaboration that is building the world’s first synthetic yeast. Macquarie and the AWRI are responsible for chromosome XIV. Other collaborating institutions on the project are located in the USA, China, the UK and Singapore.

Latest information

Production of raspberry ketone by wine yeast
Raspberry ketone is the primary aroma compound found in raspberries and naturally derived raspberry ketone is a valuable flavouring 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 production by synthetically engineered microbial strains.

A de novo pathway for the production of raspberry ketone was assembled using four genes from other species, encoding phenylalanine/ tyrosine ammonia lyase, cinnimate-4-hydroxlase, coumarate-CoA ligase 2 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.

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 utilised a coumarate-CoA ligase 2, benzalacetone synthase synthetic fusion protein that increased yields more than sixfold 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 concentration of 3.5 mg/L achieved after aerobic fermentation of Chardonnay juice or 0.68 mg/L under anaerobic winemaking conditions. This paves the way for further pathway optimisation to provide an economic alternative to raspberry ketone derived from plant sources.

Sc2.0 international collaboration
Work is in progress on the synthesis of the chromosome allocated to the Macquarie/AWRI partnership; two chromosomes are being prepared, with both due for completion during the next 12 months. The AWRI’s focus is on additional work needed to ensure that the new knowledge gained about yeast through the project has relevance to yeasts used in industries such as wine, beer, sake, baking or biofuels. A pan genome for the new Yeast 2.0 platform is now approximately 50% complete and due for completion in the next 12 months. Efforts to engineer the metabolic pathways of yeast to produce flavours and aromas are also yielding results, with laboratory strains of yeast able to undertake novel transformations and/or produce novel compounds.