Engineering de novo anthocyanin production in Saccharomyces cerevisiae

Abstract

BackgroundAnthocyanins 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.ResultsSaccharomyces 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/gCDW, while kaempferol and dihydrokaempferol were effectively exported to reach extracellular concentration of 20 µM [5 mg/L] and 150 µM [44 mg/L], respectively.ConclusionThe 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.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-0951-6) contains supplementary material, which is available to authorized users.

Expression and function of an HAC1-regulated multi-copy xylanase gene in Saccharomyces cerevisiae

“…Moreover, compared with the 24-h and 48-h time points, gene expression levels in strain S22-H were increased at 72 h post-inoculation. This suggested that at 72 h post-inoculation, the synthesis of heterologous proteins required the participation of protein folding and assembly genes, which was consistent with the results of xylanase gene and enzyme production analyses for this strain 4…”

“…Approved by the Food and Drug Administration of the United States as a safe species, Saccharomyces cerevisiae is an ideal host for the expression of enzymes used in food and feed production [4] . With its clear biological and genetic background, S. cerevisiae boasts the advantageous features of prokaryotes, including fast growth and reproduction and simple genetic manipulation procedures, as well as post-translational modification of heterologous proteins that is common to eukaryotes.…”

Promotion of compound K production in Saccharomyces cerevisiae by glycerol

“…Lastly, the production levels in the 5-L bioreactor reached 1.7 g/L. Use of S. cerevisiae in synthesis of glycosylated natural products has been reported widely [16,28,29]. The findings from this study may be used to synthesize other glycosylated natural products using S. cerevisiae, and the cost of production can be reduced by using glycerin to synthesize natural products.…”

Engineering Plant Secondary Metabolism in Microbial Systems

“…The flavonoid pathway has been diversified in E. coli and yeast to yield the dihydrochalcone phloretin and its derivatives, the flavanone liquiritigenin, and the flavonols kaempferol, quercetin, and fisetin, among others (Table 1). This pathway was extended to produce anthocyanin pigments using E. coli polycultures (Jones et al, 2017) and a single engineered yeast strain (Eichenberger et al, 2018;Levisson et al, 2018).…”

“…The same pathway has been reconstructed within a single S. cerevisiae strain (Galanie et al, 2015), albeit at lower titers, again highlighting the potential advantages of eukaryotic hosts for synthesizing complex plant secondary metabolites. Analogously, de novo anthocyanin synthesis in E. coli employed four engineered strains (Jones et al, 2017), which again contrasts strategies using S. cerevisiae in which a single strain hosts the full anthocyanin pathway (Eichenberger et al, 2018;Levisson et al, 2018). Although yeast is clearly favored for harboring downstream pathways, precursor titers are often higher in E. coli (e.g., for production of tyrosine, p-coumaric acid, dopamine, reticuline, and taxadiene; Table 1).…”