Engineering of Saccharomyces cerevisiae for 24-Methylene-Cholesterol Production

Abstract

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.

RNAi of Sterol Δ24-Isomerase Implicated Its Involvement in Physalin Biosynthesis in Physalis angulata L.

“…This study is the first to isolate the Pa24ISO gene from P. angulata. Upon expression of Pa24ISO in a 24-methylenecholesterol-producing yeast strain YS11 (Yang et al, 2021), 24-methylenecholesterol was isomerized to 24-methyldesmosterol (Figure 3), implying that it is a functional 24ISO in P. angulata. The silencing of Pa24ISO was significant, with its transcript level being almost halved, as compared to the empty vector-transferred plants (Figure 5A).…”

“…To optimize the expression of Pa24ISO in yeast, the full-length sequences of Pa24ISO were manually synthesized following the S. cerevisiae codon usage preference (see the optimized Pa24ISO cDNA sequence in Supplementary Figure 5), and inserted into a yeast expression vector pESC-HIS3 at BamHI/SalI sites, yielding the construct pESC-HIS3-Pa24ISO. The plasmid pESC-HIS3-Pa24ISO was transferred into a 24-methylenecholesterol-producing yeast strain YS11, which was recently developed by our group (Yang et al, 2021). As a negative control, the empty vector pESC-HIS3 was also transformed.…”

“…As a positive control, the known gene Ws24ISO was also synthesized, and expressed in the same way as Pa24ISO. Pa24ISO or Ws24ISO was expressed in a 24-methylenecholesterol-producing yeast strain YS11, which was previously made by Yang et al (2021). The yeast extracts were subsequently prepared, and analyzed by GC-MS for the presence of 24-methyldesmosterol.…”

Deuterated squalene and sterols from modifiedSaccharomyces cerevisiae

“…Furthermore, the engineering of Saccharomyces cerevisiae to produce 24-methylenecholesterol has been reported, in which it is co-produced with campesterol. 36…”

Structures and molecular mechanisms of action of the cholesterol C17 side-chain-degrading enzymes

“…The recently developed recombinant Saccharomyces cerevisiae strains for 24-methylene-cholesterol [82] and 7-dehydrocholesterol [83] production offer new opportunities to produce steroid drugs, such as progesterone, DHEA, and AD, in S. cerevisiae using glucose as the carbon source. The production of steroid hormones can be achieved by biotransformation of cholesterol with S. cerevisiae expressing the cholesterol sidechain degradation enzymes or introducing the cholesterol C17 side-chain degradation pathways into the cholesterol-producing S. cerevisiae.…”