Manipulation of sterol homeostasis for the production of 24-epi-ergosterol in industrial yeast

Abstract

Brassinolide (BL) is the most biologically active compound among natural brassinosteroids. However, the agricultural applications are limited by the extremely low natural abundance and the scarcity of synthetic precursors. Here, we employ synthetic biology to construct a yeast cell factory for scalable production of 24-epi-ergosterol, an un-natural sterol, proposed as a precursor for BL semi-synthesis. First, we construct an artificial pathway by introducing a Δ²⁴⁽²⁸⁾ sterol reductase from plants (DWF1), followed by enzyme directed evolution, to enable de novo biosynthesis of 24-epi-ergosterol in yeast. Subsequently, we manipulate the sterol homeostasis (overexpression of ARE2, YEH1, and YEH2 with intact ARE1), maintaining a balance between sterol acylation and sterol ester hydrolysis, for the production of 24-epi-ergosterol, whose titer reaches to 2.76 g L^-1 using fed-batch fermentation. The sterol homeostasis engineering strategy can be applicable for bulk production of other economically important phytosterols.

Fig. 1. Construction of engineered yeast strains for de novo biosynthesis of 24-epi-ergosterol, serving as a synthetic precursor for BL.

a Semi-synthesis of 24-epi-brassinolide (EBL), 28-homo-brassinolide (HBL), and brassinolide (BL) from ergosterol, stigmasterol, and crinosterol, respectively. In comparison with crinosterol, 24-epi-ergosterol, the diastereoisomer to ergosterol, could be produced on a large scale using yeast strains constructed in the present study. b Manipulation of sterol homeostasis for the production of 24-epi-ergosterol in yeast. The introduction of a Δ²⁴⁽²⁸⁾-sterol reductase (DWF1) from plants enabled de novo biosynthesis of 24-epi-ergosterol. Afterward, the catalytic activity of DWF1 and, accordingly, the production of 24-epi-ergosterol was enhanced by directed evolution. The sterol fluxes towards 24-epi-ergosterol were further strengthened by the engineering of sterol homeostasis, maintaining a balance between sterol acylation (for storage in LDs) and steryl ester hydrolysis (for releasing free sterols in cellular membranes) via overexpression of YEH1, YEH2, and ARE2 with intact ARE1. DWF1 Δ²⁴⁽²⁸⁾-sterol reductase, ARE1 sterol O-acyltransferase 1, ARE2 sterol O-acyltransferase 2, YEH1 yeast steryl ester hydrolase 1, YEH2 yeast steryl ester hydrolase 2, TGL1 triglyceride lipase.

Fig. 2. Design and optimization of an artificial pathway for de novo biosynthesis of 24-epi-ergosterol.

a De novo biosynthesis of 24-epi-ergosterol using an artificial pathway. In yeasts, ergosta-5,7,22,24(28)-tetraene-3β-ol was converted to ergosterol by ERG4; In plants, 24-methylenecholesterol was catalyzed by DWF1 to synthesize campesterol. Based on the chiral preference and structural similarity, 24-epi-ergosterol was expected to be produced in yeast by introducing DWF1. ERG4, Δ²⁴⁽²⁸⁾-sterol reductase in yeasts; DWF1, Δ²⁴⁽²⁸⁾-sterol reductase in plants. b Development of an HTS method for directed evolution of DWF1. YQP1 (BY4741 harboring pRS42H), YQP2 (YQE101 harboring pRS42H), YQP3 (YQE101 harboring pRS42H-AtDWF1), YQP4 (YQE101 harboring pRS42H-ArDWF1) were constructed to examine the relationship between the growth under SDS with HygB stressed conditions and the ability to synthesize ergosterol (YQP1) or 24-epi-ergosterol (YQP3 and YQP4). SDS with different concentrations was used to screen higher 24-epi-ergosterol producing strains with positive DWF1 mutants. All yeast strains were cultivated in YPD medium at 30 °C for 72 h. SDS sodium dodecyl sulfate; Hyg hygromycin. c The activity of DWF1 single mutants. Six mutants with a total of ten mutations (Supplementary Table 2) were obtained in the first round of directed evolution. The corresponding single mutants were constructed by site-directed mutagenesis and transformed into YQE101 as episomal plasmids for activity assays. To exclude the effect of HygB on the growth of ergΔ yeast strains (YQE101), the ratio of 24-epi-ergosterol over ergosta-5,7,22,24(28)-tetraene-3β-ol was employed to represent the enzymatic activity of DWF1 mutants. WT wild-type. d Activity of DWF1 combinatorial mutants. The second round of directed evolution via DNA shuffling resulted in the construction of a series of combinatorial mutants, whose activity was evaluated by genome integration into YQE102 (for 24-epi-ergosterol production). b–d Data were presented as mean values ± SD from three independent biological replicates (n = 3), the circles represent individual data points. Significance (p value) was evaluated by a two-sided t-test, no significance (n.s.) presents p > 0.05. Source data are provided as a Source Data file.

Fig. 3. Manipulation of sterol homeostasis for the production of 24-epi-ergosterol in yeast.

a Scheme of sterol homeostasis for maintaining a balance between sterol acylation (ARE1 and ARE2) and steryl ester hydrolysis (YEH1 and YEH2). LD lipid droplet. b The effect of sterol homeostasis engineering strategies on the production of 24-epi-ergosterol. The sterol acylation pathway (ARE1 and ARE2) and steryl ester hydrolysis pathway (YEH1, YEH2, and TGL1) genes were overexpressed and/or knocked out, and their effects on the production of 24-epi-ergosterol and other late sterols were evaluated both individually and combinatorially. c Comparison of the fed-batch fermentation performance for YQE231, YQE717, and YQE722. Fermentation profiles of cell growth, titer of 24-epi-ergosterol, and titer of late sterols (here defined as the total amount of ergosta-5,7,24(28)-trien-3β-ol, ergosta-5,7,22,24(28)-tetraen-3β-ol, ergosta-5,7-dien-3β-ol, 24-epi-ergosta-5,7-dien-3β-ol, ergosterol, and 24-epi-ergosterol) were obtained by analyzing samples every 4–8 h. b, c Data were presented as mean values ± SD from three independent biological replicates (n = 3). b The circles represent individual data points. Significance (p value) of the titer of total late sterols (red) and 24-epi-ergosterol (black) were evaluated by a two-sided t-test, no significance (n.s.) presents p > 0.05. Source data are provided as a Source Data file.

Fig. 4. Biosynthetic pathway engineering for the production of 24-epi-ergosterol.

a Enhancing 24-epi-ergosterol production by regulating the expression level of Ar207, ACC1, and ERG5. The expression level of Ar207 was regulated by various promoters, including P_TEF1, P_TDH3, P_ERG4, P_ERG5, P_CIT2, and P_GAL1. The expression of ACC1 and ERG5 was enhanced by replacing the endogenous promoter with P_GAL10 and P_CIT2, respectively. P-Ar207 the promoter chosen for Ar207, P_TEF1 the promoter of TEF1 (encoding translation elongation factor 1), P_TDH3 the promoter of TDH3 (encoding triose-phosphate dehydrogenase), P_ERG4 the promoter of ERG4 (encoding Δ²⁴⁽²⁸⁾-sterol reductase), P_ERG5 the promoter of ERG5 (encoding C-22 sterol desaturase), P_CIT2 the promoter of CIT2 (encoding citrate synthase), P_GAL1 the promoter of GAL1 (encoding galactokinase), ACC1 acetyl-CoA carboxylase, ERG5 C-22 sterol desaturase. b Fermentation profiles of YQE729 and YQE734 in fed-batch bioreactors. Time courses of cell growth, titer of 24-epi-ergosterol, and the ratio of 24-epi-ergosterol to late sterols were obtained by analyzing samples every 4–8 h. a, b Data were presented as mean values ± SD from three independent biological replicates (n = 3). a The circles represent individual data points. Significance (p value) was evaluated by a two-sided t-test, no significance (n.s.) presents p > 0.05. Source data are provided as a Source Data file.