Increase of copy number of HMG-CoA reductase and FPP synthase genes improves the amorpha4,11-diene production in engineered yeast.
- Author:
Jian-qiang KONG
1
;
Ke-di CHENG
;
Li-na WANG
;
Xiao-dong ZHENG
;
Jun-gui DAI
;
Ping ZHU
;
Wei WANG
Author Information
1. Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China.
- Publication Type:Journal Article
- MeSH:
Alkyl and Aryl Transferases;
biosynthesis;
genetics;
Artemisia annua;
enzymology;
genetics;
Fermentation;
Gene Dosage;
Genes, Plant;
Genetic Engineering;
methods;
Genetic Vectors;
Geranyltranstransferase;
genetics;
metabolism;
Hydroxymethylglutaryl CoA Reductases;
genetics;
metabolism;
Saccharomyces cerevisiae;
genetics;
metabolism;
Sesquiterpenes;
metabolism
- From:
Acta Pharmaceutica Sinica
2007;42(12):1314-1319
- CountryChina
- Language:Chinese
-
Abstract:
The gene encoding amorpha-4, 11-diene synthase was cloned from Artemisia annua L. Other two genes encoding the FPP synthase (FPPS) and HMG-CoA reductase (HMGR) were cloned from Saccharomyces cerevisiae. The cloned cDNAs were confirmed by DNA sequencing. Two expression vectors were constructed, one is named pGBT9/A/HMG/FPP harboring genes for HMG-CoA reductase and FPP synthase and the other is pYeDP60/G/AS, containing the gene encoding amorpha-4,11-diene synthase. Two kinds of engineered yeast were constructed: the first was named WHT [AS], which contained the plasmid pYeDP60/G/AS; the second was WHT [HMG + FPP + AS], in which the vectors pGBT9/A/ HMG/FPP and pYeDP60/G/AS were introduced by cotransformation mediated with LiOAc and PEG4000. The positive clones were identified for further fermentations. The samples from fermentations were analyzed by GC-MS for amorpha-4,11-diene. The results show that engineered yeasts could produce amorpha-4,11-diene. Moreover, the amorpha-4,11-diene production of WHT[ HMG + FPP + AS] and WHT[ AS] were 23.6 mg x L(-1) and 10 microg x L(-1), respectively. Its concentrations were reported as equivalents of valencene. The results showed the copy number increase of HMGR and FPPS genes can improve the production of amorpha-4, 11-diene in the fermentation of engineered yeasts.
