Microbial Production of Curcuminoids

Curcuminoids, which are produced specifically by Zingiberales, have been used as food additives because of their aromatic, stimulant, and coloring properties and as traditional Asian medicines because of their anti-tumor, antioxidant, and hepatoprotective activities.   We have discovered a novel type III polyketide synthase catalyzing the synthesis of curcuminoids from Oryza sativa.  Curcuminoid synthase synthesizes bisdemethoxycurcumin via a unique mechanism from two 4-coumaroyl-CoAs and one malonyl-CoA.  The reaction begins with the thioesterification of the thiol moiety of Cys174 by a starter molecule, 4-coumaroyl-CoA.  Decarboxylative condensation of the first extender substrate, malonyl-CoA, onto the thioester of 4-coumarate results in the formation of a diketide-CoA intermediate.  Subsequent hydrolysis of the intermediate yields a beta keto acid, which in turn acts as the second extender substrate.  The beta keto acid is then joined to the Cys174-bound 4-coumarate by decarboxylative condensation to form bisdemethoxycurcumin.  This reaction violates the traditional head-to-tail model of polyketide assembly; the growing diketide intermediate is hydrolyzed to a beta keto acid, which subsequently serves as the second extender to form curcuminoids.

An artificial curcuminoid biosynthetic pathway, including reactions of 4-coumarate:CoA ligase and curcuminoid synthase was constructed in Escherichia coli for the production of curcuminoids.  Cultivation of the recombinant E. coli cells led to production of bisdemethoxycurcumin, dicinnamoylmethane and cinnamoyl-p-coumaroylmethane from exogenously supplemented phenylpropanoid acids, such as p-coumaric acid, cinnamic acid and ferulic acid. 

Malonyl-CoA is an important intermediate in the pathway of the de novo fatty acid biosynthesis and fatty acid elongation.  The rate of fatty acid synthesis is strictly controlled by the regulation of acetyl-CoA carboxylase, which catalyzes carboxylation of acetyl-CoA to yield malonyl-CoA.  In E. coli, it has been shown that the cellular availability of malonyl-CoA is limited due to its direct association with cell growth and the biosynthesis of fatty acids.  Besides being a rate-limiting compound of the central metabolic pathway, malonyl-CoA is a major building block of secondary metabolites, such as polyketides, in plants and microbes.   For example, naringenin chalcone, a key intermediate of the biosynthetic pathway of flavonoids, is synthesized from p-coumaroyl-CoA and three molecule of malonyl-CoA by chalcone synthase, which is a plant specific type III polyketide synthase.  In addition, RppA, a type III polyketide synthase from Streptomyces griseus, catalyzes the condensation of 5 malonyl-CoAs to synthesize 1,3,6,8-tetrahydroxynapthalene, which is an intermediate of DHN melanin.  E. coli is a genetically tractable microorganism that can express multiple genes simultaneous by using expression vectors with different replication origins and selectable markers.  Moreover, the fermentation process of E. coli is manageable and reproducible.   Although, E.coli has been used for a host for the large-scale production of natural products, the reconstitution of the plant biosynthetic pathways with type III polyketide synthases in E. coli often resulted in a low production rate.  To overcome the obstacle impeding the use of type III polyketide synthase for the large-scale production of polyketides, several approaches have been shown to improve the cellular concentration of malonyl-CoA.  The activation of the pathways leading to the synthesis of malonyl-CoA and the elimination of the competing pathways reducing the precursors of malonyl-CoA are the major targets of the metabolic engineering. Inactivation of fatty acid biosynthesis will be presented.