Engineering cotton (+)-delta-cadinene synthase to an altered function: germacrene D-4-ol synthase.
The combined approaches of rational design and random mutagenesis were applied to generate a sesquiterpene synthase with an altered activity. Due to the lack of a convenient screen for sesquiterpene synthase activity, a high-throughput dual-activity screen was used by fusing (+)-delta-cadinene synthase to chloramphenicol acetyltransferase (CAT). The gene encoding (+)-delta-cadinene synthase was mutagenized using error-prone PCR. The resulting mutant fusion proteins were screened for CAT activity and altered sesquiterpene selectivity. Twenty-one clones producing (+)-delta-cadinene and germacrene D-4-ol in different ratios were isolated from the library. Analysis using a homology model of (+)-delta-cadinene synthase suggested that the G helix plays a very important role in (+)-delta-cadinene formation. Reconstruction of the G helix using site-directed, saturation mutagenesis yielded a mutant, N403P/L405H, that maintained its specific activity and showed higher selectivity to germacrene D-4-ol in vivo (up to 93%).
Yoshikuni
Yasuo
Y
UCSF/UCB Joint Graduate Group in Bioengineering, University of California, Berkeley, 94720, USA.
Martin
Vincent J J
VJ
Ferrin
Thomas E
TE
Keasling
Jay D
JD
D019883:Alkyl and Aryl Transferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D003500:Cyclization; D003368:Gossypium / Q000201:enzymology* / Q000235:genetics; D057927:Hydrophobic and Hydrophilic Interactions; D007535:Isomerases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D013058:Mass Spectrometry; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012717:Sesquiterpenes / Q000737:chemistry / Q000378:metabolism
D011994:Recombinant Proteins; D012717:Sesquiterpenes; D015500:Chloramphenicol O-Acetyltransferase; D019883:Alkyl and Aryl Transferases; C470416:germacrene D synthase; D007535:Isomerases; C097608:delta-cadinene synthase