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. 2014 Apr;4(2):161-6.
doi: 10.1016/j.apsb.2014.02.007. Epub 2014 Apr 2.

Enhancing production of ergosterol in Pichia pastoris GS115 by over-expression of 3-hydroxy-3-methylglutaryl CoA reductase from Glycyrrhiza uralensis

Ying Liu  1 , Xiaoqing Zhu  1 , Wendong Li  2 , Hao Wen  1 , Ya Gao  1 , Yong Liu  1 , Chunsheng Liu  1
Affiliations

Enhancing production of ergosterol in Pichia pastoris GS115 by over-expression of 3-hydroxy-3-methylglutaryl CoA reductase from Glycyrrhiza uralensis

Ying Liu et al. Acta Pharm Sin B. 2014 Apr.

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.

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.

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Figures

None
Graphical abstract
Figure 1
Figure 1
Structure of pPIC9K.
Figure 2
Figure 2
PCR analysis of the construction of recombinant plasmid pPIC–GuHMGR. Lane 1: marker; lanes 2 and 3: fragments obtained by PCR.
Figure 3
Figure 3
SDS-PAGE analysis of the expression of the GuHMGR gene. Lane 1: marker; lanes 2–4: recombinant P. pastoris containing the GuHMGR gene; lane 5: negative control.
Figure 4
Figure 4
Semi-quantitative RT-PCR analysis of the expression of the GuHMGR gene in different recombinant P. pastoris strains. (a) RT-PCR results of the GAP gene; (b) RT-PCR results of the GuHMGR gene (the numbers are the copy numbers of the GuHMGR gene in the recombinant P. pastoris strains); (c) relative expression of the GuHMGR gene in recombinant P. pastoris strains with different copy numbers.
Figure 5
Figure 5
Content of ergosterol in recombinant P. pastoris strains with different copy numbers of the GuHMGR gene (n=3).
None
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