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24-Methylene-cholesterol is a necessary substrate for the biosynthesis of physalin and withanolide, which show promising anticancer activities. It is difficult and costly to prepare 24-methylene-cholesterol via total chemical synthesis. In this study, we engineered the biosynthesis of 24-methylene-cholesterol in Saccharomyces cerevisiae by disrupting the two enzymes (i.e., ERG4 and ERG5) in the yeast's native ergosterol pathway, with ERG5 being replaced with the DHCR7 (7-dehydrocholesterol reductase) enzyme. Three versions of DHCR7 originating from different organisms-including the DHCR7 from Physalis angulata (PhDHCR7) newly discovered in this study, as well as the previously reported OsDHCR7 from Oryza sativa and XlDHCR7 from Xenopus laevis-were assessed for their ability to produce 24-methylene-cholesterol. XlDHCR7 showed the best performance, producing 178 mg/L of 24-methylene-cholesterol via flask-shake cultivation. The yield could be increased up to 225 mg/L, when one additional copy of the XlDHCR7 expression cassette was integrated into the yeast genome. The 24-methylene-cholesterol-producing strain obtained in this study could serve as a platform for characterizing the downstream enzymes involved in the biosynthesis of physalin or withanolide, given that 24-methylene-cholesterol is a common precursor of these chemicals.
Figure 1. Schematic diagram illustrating the construction of the campesterol and 24-methylene-cholesterol biosynthesis pathways, based on the native ergosterol biosynthesis pathway in Saccharomyces cerevisiae. The campesterol biosynthesis pathway was constructed by disrupting ERG5 and expressing the heterologous 7-dehydrocholesterol reductase gene (DHCR7). The 24-methylene-cholesterol biosynthetic pathway was constructed from the campesterol biosynthesis pathway via the disruption of ERG4. Sterol biosynthesis uses a common acetyl-CoA precursor.
Figure 2. Full-length amino acid sequence alignment of DHCR7s from Physalis angulata, Xenopus laevis, and Oryza sativa. Blue triangles represent the putative NADPH binding sites; red pentagrams represent the putative binding sites for the hydroxyl groups of sterol acceptors.
Figure 3. Identification of fermentation products in the recombinant strains via gas chromatography–mass spectrometry (GC–MS): (A) GC–MS-extracted ion profiles of the control strain YS5 producing ergosterol and YS6–8 strains producing the campesterol product. (B) Mass-fragmented patterns of the campesterol and ergosterol products. (C) Quantification of the campesterol product extracted from the strains of YS6, YS7, and YS8. Error bars represent standard deviations (n = 3). Asterisks indicate significant differences compared to YS6 and YS7; Student’s t-test, * p < 0.05.
Figure 4. Identification of fermentation products in recombinant yeast strains via gas chromatography–mass spectrometry (GC–MS): (A) GC–MS patterns of the parent strain YS5 producing ergosterol, and YS9–11 strains producing the 24-methylene-cholesterol product. (B) Mass chromatography of the 24-methylene-cholesterol product as well as its authentic standard. (C) Quantification of the 24-methylene-cholesterol produced by the strains YS9, YS10, and YS11. Error bars represent standard deviations (n = 3). Asterisks indicate significant differences compared to YS9 and YS10; Student’s t-test, * p < 0.05.
Figure 5. Real-time PCR analysis of XlDHCR7 in strains YS11 and YS12, with different 24-methylene-cholesterol yields: (A) YS12 has 1.55-fold higher mRNA levels of XlDHCR7 compared to YS11. (B) 24-Methylene-cholesterol content in the strains with heterologous expression of XlDHCR7—YS12 compared with YS11. An additional copy of XlDHCR7 increased 24-methylene-cholesterol production by 23%. Error bars represent standard deviations (n = 3). Asterisks indicate significant differences compared to YS11; Student’s t-test, * p < 0.05.
Figure 6. Characteristics of the optimal strain YS12 in shake-flask fermentation with glucose. Error bars represent standard deviations (n = 3).
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