Systematic Engineering of Saccharomyces cerevisiae for the De Novo Biosynthesis of Genistein and Glycosylation Derivatives

Abstract

Isoflavones are predominantly found in legumes and play roles in plant defense and prevention of estrogen-related diseases. Genistein is an important isoflavone backbone with various biological activities. In this paper, we describe how a cell factory that can de novo synthesize genistein was constructed in Saccharomyces cerevisiae. Different combinations of isoflavone synthase, cytochrome P450 reductase, and 2-hydroxyisoflavone dehydratase were tested, followed by pathway multicopy integration, to stably de novo synthesize genistein. The catalytic activity of isoflavone synthase was enhanced by heme supply and an increased intracellular NADPH/NADP⁺ ratio. Redistribution of the malonyl-CoA flow and balance of metabolic fluxes were achieved by adjusting the fatty acid synthesis pathway, yielding 23.33 mg/L genistein. Finally, isoflavone glycosyltransferases were introduced into S. cerevisiae, and the optimized strain produced 15.80 mg/L of genistin or 10.03 mg/L of genistein-8-C-glucoside. This is the first de novo synthesis of genistein-8-C-glucoside in S. cerevisiae, which is advantageous for the green industrial production of isoflavone compounds.

Keywords: genistein; glycosylation; heme; isoflavones; malonyl-CoA.

Figure 1

Systematic engineering for the de novo biosynthesis of genistein and glycosylated derivatives. The black lines indicate the biosynthetic pathways of the two isoflavone glycosides. The outermost six modules demonstrate the strategies in this paper. They are represented sequentially as a regulation of fatty acid metabolic pathways, multicopy site integration of the naringenin pathway, heme supply, intracellular NADPH production, multicopy site integration of the genistein pathway, and UDPG supply (clockwise). Notes on abbreviations: E4P, erythrose-4-phosphate; PEP, phosphoenolpyruvate; _L-Phe, _L-phenylalanine; _L-Tyr, _L-tyrosine; AtPAL2, phenylalanine ammonialyase from Arabidopsis thaliana; CYB5, yeast native cytochrome b5; AtATR2, cytochrome P450 reductase from A. thaliana; AtC4H, cinnamic acid-4-hydroxylase from A. thaliana; FjTAL, transaldolase from Flavobacterium johnsoniae; Pc4CL, 4-coumarate-coenzyme A ligase from Petroselinum crispum; PhCHS, chalcone synthase from Petunia hybrida; MsCHI, chalcone isomerase from Medicago sativa; AtCPR, cytochrome P450 reductase from A. thaliana; GmIFS, isoflavone synthase from Glycine max; GmHID, 2-hydroxyisoflavanone dehydratase from G. max; GmUGT4, isoflavone-7-O-glucosyltransferase from G. max; PlUGT43, isoflavone-8-C-glucosyltransferase from Pueraria lobate; FAS1, β-subunit of yeast fatty acid synthetase; Suc-CoA, succinyl-CoA; 5-ALA, 5-aminolevulinic acid; HEM2, 5-aminolevulinic acid dehydratase; HEM3, 4-porphobilinogen deaminase; G-6-P, glucose-6-phosphate; STB5, yeast native transcriptional factor; ALD6, cytoplasmic NADP⁺-dependent aldehyde dehydrogenase; EcPntAB, membrane-bound transhydrogenase from E. coli; YEF1, ATP-NADH kinase; HIS, histidine; PGM1/2, phosphoglucomutase 1/2; UGP1, UDP-glucose pyrophosphorylase; UDP-glucose, uridine diphosphate-glucose.

Figure 2

Construction of a synthetic plate for genistein. (a) The mechanism of naringenin-to-genistein conversion catalyzed by IFS and HID. (b) To screen the effect of different IFS on genistein synthesis with 400 mg/L naringenin addition. (c) Comparison of the effects on genistein synthesis by different HIDs expression with 400 mg/L naringenin addition. (d) Effect of different CPRs on genistein synthesis with 400 mg/L naringenin addition. Data are presented as mean ± SD.

Figure 3

Building the de novo biosynthetic pathway for genistein. (a) Naringenin biosynthetic pathway. (b) Effect of expression of different genes on p-coumaric acid synthesis. (c) Schematic of multicopy site integration in the naringenin and genistein synthesis pathways. (d) Analysis of multicopy site integration of the naringenin synthesis pathway. (e) Shake flask rescreen fermentation by multiple-copy site-integrated strains of the naringenin pathway. (f) Comparison of differences between plasmid expression and single-copy integrated expression of the genistein synthesis pathway. (g) Analysis on the integration of the genistein synthesis pathway at the Ty1 or Ty2 transposons in S. cerevisiae. (h) Shake flask rescreen fermentation by multiple-copy site-integrated strains of the genistein pathway. Asterisks denote the statistical significance of a two-tailed t-test. *** p < 0.001, **** p < 0.0001. Data are presented as mean ± SD.

Figure 4

Enhancement of IFS catalytic efficiency and yeast growth-promoting modification. (a) Schematic diagram of the strategy for NADPH production and heme supply. (b) Effect of NADPH production and heme supply on genistein titer. (c) A positive correlation between OD₆₀₀ and genistein titer. Blue samples indicate the relationship between genistein titers and OD₆₀₀ for strains fermented with 400 mg/L naringenin addition. r² = 0.6336, p < 0.0001. Pink samples indicate the relationship between titers and OD₆₀₀ for the de novo synthesis of genistein by fermentation using glucose as the sole carbon source. r² = 0.7956, p < 0.0001. (d) Mechanism of OCA5 action in the intracellular compartment. Hxk1/2, hexokinase; 5-InsP7, 5-diphosphoinositol 1,2,3,4,6-pentakisphosphate; InsP6, inositol hexakisphosphate. (e) Effect of OCA5 deletion on genistein synthesis and OD₆₀₀. (f) Effective strategies to integrate into the genome of NHG12. Asterisks denote the statistical significance of a two-tailed t-test. * p  < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Data are presented as mean ± SD.

Figure 5

Optimization of the malonyl-CoA supply. (a) Demonstration of titers in NHGO01 for genistein, naringenin, and p-coumaric acid. (b) Schematic diagram of FAS1, which regulates the fatty acid synthesis pathway, to redistribute the malonyl-CoA flow. (c) Effect of different promoters for FAS1 expression on genistein titers. Asterisks denote the statistical significance of a two-tailed t-test. **** p < 0.0001. Data are presented as mean ± SD.

Figure 6

Synthesis of glycosylated derivatives of genistein. (a) Demonstration of the isoflavone glycoside synthesis pathway and the principle of glycosylation optimization strategy. (b) Effect of the expression of two glycosyltransferases, PGM1, PGM2, and UGP1 on the synthesis of isoflavone glycosides. (c) LC-MS for genistein and genistein-8-C-glucoside. Asterisks denote the statistical significance of a two-tailed t-test. * p <  0.05. Data are presented as mean ± SD.