High-level production of amorpha-4,11-diene, a precursor of the antimalarial agent artemisinin, in Escherichia coli.
BACKGROUND
Artemisinin derivatives are the key active ingredients in Artemisinin combination therapies (ACTs), the most effective therapies available for treatment of malaria. Because the raw material is extracted from plants with long growing seasons, artemisinin is often in short supply, and fermentation would be an attractive alternative production method to supplement the plant source. Previous work showed that high levels of amorpha-4,11-diene, an artemisinin precursor, can be made in Escherichia coli using a heterologous mevalonate pathway derived from yeast (Saccharomyces cerevisiae), though the reconstructed mevalonate pathway was limited at a particular enzymatic step.


RESULTS
By combining improvements in the heterologous mevalonate pathway with a superior fermentation process, commercially relevant titers were achieved in fed-batch fermentations. Yeast genes for HMG-CoA synthase and HMG-CoA reductase (the second and third enzymes in the pathway) were replaced with equivalent genes from Staphylococcus aureus, more than doubling production. Amorpha-4,11-diene titers were further increased by optimizing nitrogen delivery in the fermentation process. Successful cultivation of the improved strain under carbon and nitrogen restriction consistently yielded 90 g/L dry cell weight and an average titer of 27.4 g/L amorpha-4,11-diene.


CONCLUSIONS
Production of >25 g/L amorpha-4,11-diene by fermentation followed by chemical conversion to artemisinin may allow for development of a process to provide an alternative source of artemisinin to be incorporated into ACTs.
Tsuruta
Hiroko
H
Amyris Biotechnologies, Emeryville, California, United States of America.
Paddon
Christopher J
CJ
Eng
Diana
D
Lenihan
Jacob R
JR
Horning
Tizita
T
Anthony
Larry C
LC
Regentin
Rika
R
Keasling
Jay D
JD
Renninger
Neil S
NS
Newman
Jack D
JD
D000085:Acetates / Q000378:metabolism; D000641:Ammonia / Q000378:metabolism; D000890:Anti-Infective Agents / Q000378:metabolism* / Q000627:therapeutic use; D000962:Antimalarials / Q000378:metabolism* / Q000627:therapeutic use; D037621:Artemisinins / Q000378:metabolism*; D002675:Child, Preschool; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005285:Fermentation; D005947:Glucose / Q000378:metabolism; D006801:Humans; D016778:Malaria, Falciparum / Q000188:drug therapy; D008798:Mevalonic Acid / Q000378:metabolism; D009876:Operon; D000081223:Polycyclic Sesquiterpenes; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D012717:Sesquiterpenes / Q000378:metabolism*
D000085:Acetates; D000890:Anti-Infective Agents; D000962:Antimalarials; D037621:Artemisinins; D000081223:Polycyclic Sesquiterpenes; D012717:Sesquiterpenes; C515348:amorpha-4,11-diene; D000641:Ammonia; C031327:artemisinin; D005947:Glucose; D008798:Mevalonic Acid