Engineering of Saccharomyces cerevisiae for 24-Methylene-Cholesterol Production.

Jiao Yang,Changfu Li,Yansheng Zhang

doi:10.3390/biom11111710

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.

Highlights

This study is the first report on cloning and functional analysis of a DHCR7 gene (PhDHCR7) from P. angulate, which is well known to accumulate abundant 24-methylenecholesterol-derived compounds, such as physalin and withanolide
To the best of our knowledge, PhDHCR7 is the second DHCR7 gene isolated from plant species to date, with the first being OsDHCR7 from Oryza sativa [26]
Successful production of campesterol (Figure 3) or 24-methylene-cholesterol (Figure 4) in the yeast strains expressing the PhDHCR7 demonstrated that PhDHCR7 could accept the yeast’s native metabolite 5-dehydroepisterol as a substrate (Figure 1)

Summary

Introduction

Phytosterols are an important class of plant secondary metabolites, with great potential in the healthcare and food industries. The predominant sterols in plants are β-sitosterol, stigmasterol, and campesterol [1]. Phytosterols have been shown to lower cholesterol levels, and have antioxidant effects; they are widely used as additives in foods and cosmetics [2]. Phytosterols serve as critical pharmaceutical precursors of hydrocortisone, progesterone, and pregnenolone [3]. 24-Methylene-cholesterol was first isolated from honeybees by Barbier, and was later detected in the pollen tubes of various land plants [4]. Some marine organisms—such as mussels and brown seaweeds—were reported to contain 24-methylene-cholesterol [5]. Recent studies have demonstrated that 24-methylene-cholesterol has moderate antioxidant activity [6]

Methods

Results

Conclusion