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

Liu Y, Zhu X, Li W, Wen H, Gao Y, Liu Y, Liu C - Acta Pharm Sin B (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: School of Chinese Pharmacy, Beijing University of Chinese Medicine, Beijing 100102, China.

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.

No MeSH data available.


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.
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f0015: 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.

Mentions: The linearized pPIC–GuHMGR was transformed to P. pastoris GS115. Most single colonies of recombinant P. pastoris simultaneously growing on MM and MD media were shown by PCR to have the correct fragment length. After inducing for 96 h, the negative control was dark yellow while the recombinant P. pastoris was yellowish-white. SDS-PAGE (Fig. 3) showed a band between 86 and 47 kDa in samples from recombinant P. pastoris which was not present in the negative control. These results demonstrate that the construction and inducible expression of recombinant P. pastoris strains containing the GuHMGR gene were successful.


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

Liu Y, Zhu X, Li W, Wen H, Gao Y, Liu Y, Liu C - Acta Pharm Sin B (2014)

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.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4590296&req=5

f0015: 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.
Mentions: The linearized pPIC–GuHMGR was transformed to P. pastoris GS115. Most single colonies of recombinant P. pastoris simultaneously growing on MM and MD media were shown by PCR to have the correct fragment length. After inducing for 96 h, the negative control was dark yellow while the recombinant P. pastoris was yellowish-white. SDS-PAGE (Fig. 3) showed a band between 86 and 47 kDa in samples from recombinant P. pastoris which was not present in the negative control. These results demonstrate that the construction and inducible expression of recombinant P. pastoris strains containing the GuHMGR gene were successful.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: School of Chinese Pharmacy, Beijing University of Chinese Medicine, Beijing 100102, China.

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.

No MeSH data available.