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. 2023 Jan 27;14:437. doi: 10.1038/s41467-023-36007-z

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — 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.