Engineering a Polyketide Synthase for In Vitro Production of Adipic Acid.
Polyketides have enormous structural diversity, yet polyketide synthases (PKSs) have thus far been engineered to produce only drug candidates or derivatives thereof. Thousands of other molecules, including commodity and specialty chemicals, could be synthesized using PKSs if composing hybrid PKSs from well-characterized parts derived from natural PKSs was more efficient. Here, using modern mass spectrometry techniques as an essential part of the design-build-test cycle, we engineered a chimeric PKS to enable production one of the most widely used commodity chemicals, adipic acid. To accomplish this, we introduced heterologous reductive domains from various PKS clusters into the borrelidin PKS' first extension module, which we previously showed produces a 3-hydroxy-adipoyl intermediate when coincubated with the loading module and a succinyl-CoA starter unit. Acyl-ACP intermediate analysis revealed an unexpected bottleneck at the dehydration step, which was overcome by introduction of a carboxyacyl-processing dehydratase domain. Appending a thioesterase to the hybrid PKS enabled the production of free adipic acid. Using acyl-intermediate based techniques to "debug" PKSs as described here, it should one day be possible to engineer chimeric PKSs to produce a variety of existing commodity and specialty chemicals, as well as thousands of chemicals that are difficult to produce from petroleum feedstocks using traditional synthetic chemistry.
Andrew
A
Hagen
Sean
S
Poust
Tristan de
Td
Rond
Jeffrey L
JL
Fortman
Leonard
L
Katz
Physical Bioscience Division, Lawrence Berkeley National Laboratory , Berkeley, California 94270, United States.
Christopher J
CJ
Petzold
Physical Bioscience Division, Lawrence Berkeley National Laboratory , Berkeley, California 94270, United States.
Jay D
JD
Keasling
D000272:Adipates / Q000378:metabolism*; D002853:Chromatography, Liquid; D060847:Metabolic Engineering / Q000379:methods*; D048630:Polyketide Synthases / Q000737:chemistry / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D053719:Tandem Mass Spectrometry
D000272:Adipates; C029900:adipic acid; D048630:Polyketide Synthases
adipic acid; polyketide synthase; tandem mass-spectrometry