title: Enhancing production of ergosterol in Pichia pastoris GS115 by over-expression of 3-hydroxy-3-methylglutaryl CoA reductase from Glycyrrhiza uralensis.
abstract: The rate-limiting enzyme in the mevalonic acid (MVA) pathway which can lead to triterpenoid saponin glycyrrhizic acid (GA) is 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR). In order to reveal the effect of copy number variation in the HMGR gene on the MVA pathway, the HMGR gene from Glycyrrhiza uralensis Fisch. (GuHMGR) was cloned and over-expressed in Pichia pastoris GS115. Six recombinant P. pastoris strains containing different copy numbers of the GuHMGR gene were obtained and the content of ergosterol was analyzed by HPLC. The results showed that all the recombinant P. pastoris strains contained more ergosterol than the negative control and the strains with 8 and 44 copies contained significantly more ergosterol than the other strains. However, as the copy number increased, the content of ergosterol showed an increasing-decreasing-increasing pattern. This study provides a rationale for increasing the content of GA through over-expressing the GuHMGR gene in cultivars of G. uralensis.
authors.affiliation: School of Chinese Pharmacy, Beijing University of Chinese Medicine, Beijing 100102, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Liu
authors.affiliation: School of Chinese Pharmacy, Beijing University of Chinese Medicine, Beijing 100102, China.
authors.forename: Xiaoqing
authors.initials: X
authors.lastname: Zhu
authors.affiliation: Department of Antibiotics, Beijing Institute for Drug Control, Beijing 100102, China.
authors.forename: Wendong
authors.initials: W
authors.lastname: Li
authors.affiliation: School of Chinese Pharmacy, Beijing University of Chinese Medicine, Beijing 100102, China.
authors.forename: Hao
authors.initials: H
authors.lastname: Wen
authors.affiliation: School of Chinese Pharmacy, Beijing University of Chinese Medicine, Beijing 100102, China.
authors.forename: Ya
authors.initials: Y
authors.lastname: Gao
authors.affiliation: School of Chinese Pharmacy, Beijing University of Chinese Medicine, Beijing 100102, China.
authors.forename: Yong
authors.initials: Y
authors.lastname: Liu
authors.affiliation: School of Chinese Pharmacy, Beijing University of Chinese Medicine, Beijing 100102, China.
authors.forename: Chunsheng
authors.initials: C
authors.lastname: Liu
keywords: 3-Hydroxy-3-methylglutaryl-CoA reductase gene; BMGY, buffered glycerol-complex medium; BMMY, buffered methanol-complex medium; CNV, copy number variation; Copy number variation; Glycyrrhiza uralensis Fisch.; HMGR, 3-hydroxy-3-methylglutaryl-CoA reductase; LLOQ, lower limit of quantitation; LOD, limit of detection; MD, minimal dextrose medium; MM, minimal medium; MVA, mevalonic acid; Over-expression; PCR, polymerase chain reaction; Pichia pastoris; RSD, relative standard deviation; YPD, yeast peptone dextrose medium