Engineering de novo anthocyanin production in Saccharomyces cerevisiae

Abstract

Background: Anthocyanins are polyphenolic pigments which provide pink to blue colours in fruits and flowers. There is an increasing demand for anthocyanins, as food colorants and as health-promoting substances. Plant production of anthocyanins is often seasonal and cannot always meet demand due to low productivity and the complexity of the plant extracts. Therefore, a system of on-demand supply is useful. While a number of other (simpler) plant polyphenols have been successfully produced in the yeast Saccharomyces cerevisiae, production of anthocyanins has not yet been reported.

Results: Saccharomyces cerevisiae was engineered to produce pelargonidin 3-O-glucoside starting from glucose. Specific anthocyanin biosynthetic genes from Arabidopsis thaliana and Gerbera hybrida were introduced in a S. cerevisiae strain producing naringenin, the flavonoid precursor of anthocyanins. Upon culturing, pelargonidin and its 3-O-glucoside were detected inside the yeast cells, albeit at low concentrations. A number of related intermediates and side-products were much more abundant and were secreted into the culture medium. To optimize titers of pelargonidin 3-O-glucoside further, biosynthetic genes were stably integrated into the yeast genome, and formation of a major side-product, phloretic acid, was prevented by engineering the yeast chassis. Further engineering, by removing two glucosidases which are known to degrade pelargonidin 3-O-glucoside, did not result in higher yields of glycosylated pelargonidin. In aerated, pH controlled batch reactors, intracellular pelargonidin accumulation reached 0.01 µmol/g_CDW, while kaempferol and dihydrokaempferol were effectively exported to reach extracellular concentration of 20 µM [5 mg/L] and 150 µM [44 mg/L], respectively.

Conclusion: The results reported in this study demonstrate the proof-of-concept that S. cerevisiae is capable of de novo production of the anthocyanin pelargonidin 3-O-glucoside. Furthermore, while current conversion efficiencies are low, a number of clear bottlenecks have already been identified which, when overcome, have huge potential to enhance anthocyanin production efficiency. These results bode very well for the development of fermentation-based production systems for specific and individual anthocyanin molecules. Such systems have both great scientific value for identifying and characterising anthocyanin decorating enzymes as well as significant commercial potential for the production of, on-demand, pure bioactive compounds to be used in the food, health and even pharma industries.

Keywords: Anthocyanin; Flavonoids; Metabolic engineering; Natural products; Pelargonidin; Plant secondary metabolites; Saccharomyces cerevisiae.

Fig. 1

Representation of the integrated pelargonidin 3-O-glucoside biosynthesis pathway. Nine A. thaliana genes were overexpressed: PAL phenylalanine ammonia lyase, C4H cinnamate 4-hydroxylase, CPR cytochrome P450 reductase, 4CL 4-coumaric acid-CoA ligase, CHS chalcone synthase, CHI chalcone isomerase, F3H flavanone 3-hydroxylase, ANS anthocyanidin synthase, and 3GT anthocyanin 3-O-glucosyltransferase; one gene from G. hybrida: DFR dihydroflavonol 4-reductase; and one gene from R. capsulatus: TAL tyrosine ammonia lyase. Enzyme names are in green. Bold dark blue arrows indicate the pelargonidin 3-O-glucoside biosynthesis pathway. Dashed lines indicate routes to side-products produced by the catalytic activity of ANS, 3GT or the endogenous TSC13 (a very long chain fatty acid enoyl reductase)

Fig. 2

Extracts of cell pellets from S. cerevisiae strains PATW002, PATW011 and PATW012. These strains express coGhDFR, coMtDFR1 and coAaDFR respectively in combination with coF3H, coANS, co3GT genes. The strains were grown in SMNar (1.5 mM naringenin) and the cell pellets were lyophilized and extracted with acidified methanol

Fig. 3

Growth of IMK393 & PATW076 and flavonoids formation by PATW076 in bioreactors. Growth and extracellular metabolite formation were studied in duplicate pH controlled (pH 5.0) and aerobic controlled batch cultures of IMK393 and PATW076 on glucose and auxotrophic supplements. a Concentrations of glucose (White circle), ethanol (Black circle), acetate (White square), glycerol (Black square), and optical density (OD₆₆₀) (Triangle) for IMK393. b Concentrations of glucose (White circle), ethanol (Black circle), acetate (White square), glycerol (Black square), and optical density (OD₆₆₀) (Triangle) for PATW076. c Concentrations of kaempferol (Black down-pointing triangle), and kaempferol 3-O-glucoside (K3G) (Black up-pointing triangle) for PATW076. d Concentrations of coumaric acid (Times), naringenin (Inverted triangle), dihydrokaempferol (White diamond), and the sum of all flavonoids (Black diamond) for PATW076. Results are shown from a single representative experiment