[Increase of copy number of HMG-CoA reductase and FPP synthase genes improves the amorpha4,11-diene production in engineered yeast].
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.
Kong
Jian-qiang
JQ
Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China.
Cheng
Ke-di
KD
Wang
Li-na
LN
Zheng
Xiao-dong
XD
Dai
Jun-gui
JG
Zhu
Ping
P
Wang
Wei
W
D019883:Alkyl and Aryl Transferases / Q000096:biosynthesis* / Q000235:genetics; D031172:Artemisia annua / Q000201:enzymology / Q000235:genetics; D005285:Fermentation; D018628:Gene Dosage; D017343:Genes, Plant; D005818:Genetic Engineering / Q000379:methods; D005822:Genetic Vectors; D051228:Geranyltranstransferase / Q000235:genetics* / Q000378:metabolism; D006903:Hydroxymethylglutaryl CoA Reductases / Q000235:genetics* / Q000378:metabolism; D000081223:Polycyclic Sesquiterpenes; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D012717:Sesquiterpenes / Q000378:metabolism*
D000081223:Polycyclic Sesquiterpenes; D012717:Sesquiterpenes; C515348:amorpha-4,11-diene; D006903:Hydroxymethylglutaryl CoA Reductases; D019883:Alkyl and Aryl Transferases; C403714:amorpha-4,11-diene synthase; D051228:Geranyltranstransferase