Metabolic engineering of Saccharomyces cerevisiae for de novo production of dihydrochalcones with known antioxidant, antidiabetic, and sweet tasting properties

Abstract

Dihydrochalcones are plant secondary metabolites comprising molecules of significant commercial interest as antioxidants, antidiabetics, or sweeteners. To date, their heterologous biosynthesis in microorganisms has been achieved only by precursor feeding or as minor by-products in strains engineered for flavonoid production. Here, the native ScTSC13 was overexpressed in Saccharomyces cerevisiae to increase its side activity in reducing p-coumaroyl-CoA to p-dihydrocoumaroyl-CoA. De novo production of phloretin, the first committed dihydrochalcone, was achieved by co-expression of additional relevant pathway enzymes. Naringenin, a major by-product of the initial pathway, was practically eliminated by using a chalcone synthase from barley with unexpected substrate specificity. By further extension of the pathway from phloretin with decorating enzymes with known specificities for dihydrochalcones, and by exploiting substrate flexibility of enzymes involved in flavonoid biosynthesis, de novo production of the antioxidant molecule nothofagin, the antidiabetic molecule phlorizin, the sweet molecule naringin dihydrochalcone, and 3-hydroxyphloretin was achieved.

Production of (S)-2-aminobutyric acid and (S)-2-aminobutanol in Saccharomyces cerevisiae

“…Our entry vectors contain different combinations of promoters and terminators, which are flanked by 60 bp homologous regions (“linkers”) that enable the assembly of multiple expression cassettes in vivo by homologous recombination [57, 58]. Accessory entry vectors dispose of autonomously replicating sequences, centromere regions, and auxotrophic markers as described in Eichenberger et al [59]. One-pot digestion of entry vectors by Asc I releases the expression cassettes composed of (1) promoter-gene-terminator flanked by 60 bp linkers at the 5′ and 3′ end; (2) selection marker URA3 flanked by 60 bp linkers at the 5′ and 3′ end; and (3) replication origin ARS/CEN flanked by 60 bp linkers at the 5′ and 3′ end); or, for integration, two 560 bp homologous regions flanked by 60 bp linkers at the 5′ and 3′ sites.…”

Advances in Metabolic Engineering of Saccharomyces cerevisiae for the Production of Industrially and Clinically Important Chemicals

“…Glutathione Overexpression of YAP1 [58] Manipulation of the sulphate assimilation pathway by overexpressing MET14 and MET16 [59] Improved oxidized glutathione production by overexpression of GSH1, GSH2, and ERV1 and the deletion of GLR1 [60] Adaptive laboratory evolution in the presence of increasing levels of acrolein and screening for enhanced glutathione production [61] Whole-genome engineering via genome shuling and screening for enhanced glutathione production [62] Artemisinin/artemisinic acid Reconstruction of the complete biosynthetic pathway of artemisinic acid, including the three-step oxidation of amorphadiene to artemisinic acid by expression of CYP71AV1, CPR1, CYB5, ADH1 and ALDH1 from Artemisia annua [48] Taxol/taxadiene Expression of a truncated version of the endogenous tHMG1 and GGPPS from Taxus chinensis or Sulfolobus acidocaldarius together with TDC1 from T. chinensis [66] Prediction of the eiciency of diferent GGPPS enzymes via computer aided protein modelling [67] Forskolin Expression of a promiscuous cytochrome P450 from Salvia pomifera [68] Polyketides Heterologous expression of 6-MSA synthase gene from Penicillium patulum together with PPTases from either Bacillus subtilis or Aspergillus nidulans [69] Construction of polyketide precursor pathways by expressing prpE from Salmonella typhimurium and PCC pathway from Streptomyces coelicolor [70] Enhanced cofactor supply by expressing 2-PS from Gerbera hybrida [71] Resveratrol Reconstruction of a de novo pathway by expressing TAL from Herpetosiphon aurantiacus, 4-CL1 from Arabidopsis thaliana and VST1 from Vitis vinifera [49] Expression of 4CL1 from A. thaliana and STS from Arachis hypogaea [73] Expression of PAL from Rhodosporidium toruloides, C4H and 4-CL1 from A. thaliana, and STS from A. hypogaea [74] Expression of 4-coumaroyl-coenzyme A ligase (4CL1) from A. thaliana and stilbene synthase (STS) from V. vinifera [75] Overexpression of the resveratrol biosynthesis pathway, enhancement of P450 activity, increasing the precursor supply for resveratrol synthesis via phenylalanine pathway [76] Dihydrochalcones Expression of the heterologous pathway genes in a TSC13-overexpressing S. cerevisiae strain [78] Alkaloids Expression of 14 monoterpene indole alkaloid pathway genes from Catharanthus roseus and enhanced secondary metabolism to produce strictosidine de novo [79] Construction of the complete de novo biosynthetic pathway to norcoclaurine by expressing a mammalian TyrH enzyme and DODC from Pseudomonas putida, along with four genes required for biosynthesis of its electron carrier cosubstrate …”

“…Recently, de novo synthesis of DHCs via phloretin intermediate has been reported in S. cerevisiae [78]. First, phloretin biosynthesis was achieved with the aid of a side activity of an endogenous double-bond reductase, in combination with heterologous pathway enzymes.…”

Towards Metabolic Engineering of Podophyllotoxin Production

“…The full biosynthetic pathway toward coniferyl alcohol has not been tested for expression in S. cerevisiae yet. However, production of ±100 mg/L coumaric acid has been shown [76]. To convert coumaric acid to coniferyl alcohol in S. cerevisiae, four or five additional genes have to be expressed; therefore, in order to produce coniferyl alcohol levels similar to E. coli, further optimization of coumaric acid production is necessary.…”