Modular pathway engineering for resveratrol and piceatannol production in engineered Escherichia coli

Resveratrol and its ortho-hydroxylated derivative piceatannol were biosynthesized by modular pathway engineering in Escherichia coli. The biosynthetic pathway was divided into three different modules. Module I includes polyketide biosynthetic genes; module II genes include acetyl-CoA and malonyl-CoA pool-enhancing genes from three different organisms; and module III genes are regiospecific 3'-hydroxylating enzymes. E. coli BL21(DE3) with module I produced 8.6 mg/L of resveratrol from exogenously fed 1 mM p-coumaric acid after 72 h. Combination of module I and acetyl-CoA supplementing module IIb genes from N. farcinica IFM10152 produced 2.5-fold higher (60 mg/L) titer of resveratrol than the module IIa genes from E. coli. The exogenous supplementation of sodium acetate further enhanced production to 64 mg/L. Furthermore, module I with module IIc harboring matBC from S. coelicolor A3(2) produced 73 mg/L of resveratrol, which was elevated to 151 mg/L upon supplementing disodium malonate exogenously. This increment is 17.5-fold higher than module I harboring E. coli BL21(DE3). The combination of module I and two different module II genes yielded 137 mg/L resveratrol when supplemented with both sodium acetate and disodium malonate. The high resveratrol-producing combination module was further modified with incorporation of hpaBC for the ortho-hydroxylation of resveratrol to produce piceatannol. The engineered strain harboring modules I, IIc and III produced 124 mg/L of piceatannol, the highest titer after 72 h in disodium malonate-supplemented strain, which is 2-fold higher than in non-supplemented strain. The remaining resveratrol was about 30 mg/L. Furthermore, caffeic acid (85.5 mg/L) was also produced in the same strain. Resveratrol and piceatannol were biosynthesized along with caffeic acid by three different modules overexpressing acetate and malonate assimilation pathway genes from three different sources. The production titer of both resveratrol and piceatannol could be achieved higher upon blocking acetyl-CoA and malonyl-CoA utilizing pathway genes in host strain.