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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.


PCR analysis of the construction of recombinant plasmid pPIC–GuHMGR. Lane 1: marker; lanes 2 and 3: fragments obtained by PCR.
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f0010: PCR analysis of the construction of recombinant plasmid pPIC–GuHMGR. Lane 1: marker; lanes 2 and 3: fragments obtained by PCR.

Mentions: A 1745 bp fragment was shown by PCR and BLAST analysis to have a 99% identical sequence to that of the GuHMGR gene (GenBank accession number: GQ345405.1). It was successfully inserted at the SnaBI–NotI site of pPIC9K to give the recombinant plasmid pPIC–GuHMGR shown in Fig. 2 where lane 1 is the marker and lanes 2 and 3 are fragments obtained by PCR with the correct length.


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)

PCR analysis of the construction of recombinant plasmid pPIC–GuHMGR. Lane 1: marker; lanes 2 and 3: fragments obtained by PCR.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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

f0010: PCR analysis of the construction of recombinant plasmid pPIC–GuHMGR. Lane 1: marker; lanes 2 and 3: fragments obtained by PCR.
Mentions: A 1745 bp fragment was shown by PCR and BLAST analysis to have a 99% identical sequence to that of the GuHMGR gene (GenBank accession number: GQ345405.1). It was successfully inserted at the SnaBI–NotI site of pPIC9K to give the recombinant plasmid pPIC–GuHMGR shown in Fig. 2 where lane 1 is the marker and lanes 2 and 3 are fragments obtained by PCR with the correct length.

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.