Production of Meiosis-Activating Sterols from Metabolically Engineered Yeast

Abstract

Meiosis-activating sterols (MAS), a class of potent regulators of reproductive processes, are difficult to obtain by chemical synthesis or isolation from natural sources. We demonstrate the development of metabolically engineered strains of Saccharomyces cerevisiae that accumulate MAS as the predominant sterol product. Homologous recombination was used to construct an erg24Delta erg25Delta hem1Delta mutant RXY4.3, which lacked sterol Delta14 reductase, C-4 oxidase, and delta-aminolevulinate synthase. The HEM1 deletion allowed sterol import and rendered RXY4.3 viable under aerobic conditions. This mutant accumulated 4,4-dimethyl-5alpha-cholesta-8,14,24-trien-3beta-ol (FF-MAS), and a similar erg25Delta hem1Delta mutant produced 4,4-dimethyl-5alpha-cholesta-8,24-dien-3beta-ol (T-MAS). Based on consistent yields of approximately 5 mug of FF-MAS per mL of culture, fermentation of genetically modified yeast compares favorably with other approaches to produce MAS.

Interactions of the ergosterol biosynthetic pathway with other lipid pathways

“…They concluded that oxidation products of ergosterol are responsible for this effect. Xu et al [7] described the production of meiosisactivating methylated sterol intermediates. For an overview on the biosynthetic pathway of the post-squalene ergosterol biosynthesis, see Scheme 1.…”

Sterol biosynthesis in the harmful marine dinoflagellate, Karenia brevis: Identification of biosynthetic intermediates produced during exposure to the fungicide fenpropidine

“…Both the C 29 and C 28 intermediates displayed a one hydrogen singlet at 5.34 and 5.28 ppm, respectively (Fig. 2); this has been identified previously by Xu et al . (2002) and Wilson et al .…”

“…Fourth, the presence of a fragment at m/z 397, rather than a fragment at m/z 411, for the C 28 intermediate suggests that this compound possesses the same side‐chain as the C 29 intermediate (and gymnodinosterol); this is further confirmed by the multiplet at 5.21 ppm for both the C 29 and C 28 intermediates, which indicated a side‐chain with a Δ 22 unsaturation. Fifth, Xu et al . (2002) have published the proton NMR spectrum of 4,4‐dimethylcholesta‐8,14,24‐trien‐3β‐ol; like that compound, the C 29 and C 28 intermediates of K. brevis have a singlet at approximately 5.3 ppm (Fig.…”

Biosynthesis of isoprenoids, polyunsaturated fatty acids and flavonoids in Saccharomyces cerevisiae

“…FF-MAS is the substrate of Erg24p and T-MAS is the substrate of Erg25p. While supplying exogenous ergosterol or cholesterol, engineered strains with abolished heme biosynthesis and deleted ERG25 gene accumulated T-MAS (1.3 mg/L) while deleting both ERG24 and ERG25 genes accumulated FF-MAS (5.4 mg/L) [ 40 ]. Zymosterol is another intermediate of interest as it is the precursor of cholesterol lowering substances.…”