Metabolic engineering of Escherichia coli for the synthesis of the plant polyphenol pinosylvin

Abstract

Plant polyphenols are of great interest for drug discovery and drug development since many of these compounds have health-promoting activities as treatments against various diseases, such as diabetes, cancer, or heart diseases. However, the limited availability of polyphenols represents a major obstacle to clinical applications that must be overcome. In comparison to the quantities of these compounds obtained by isolation from natural sources or costly chemical synthesis, the microbial production of these compounds could provide sufficient quantities from inexpensive substrates. In this work, we describe the development of an Escherichia coli platform strain for the production of pinosylvin, a stilbene found in the heartwood of pine trees which could aid in the treatment of various cancers and cardiovascular diseases. Initially, several configurations of the three-step biosynthetic pathway to pinosylvin were constructed from a set of two different enzymes for each enzymatic step. After optimization of gene expression and evaluation of different construct environments, low pinosylvin concentrations up to 3 mg/liter could be detected. Analysis of the precursor supply and a comparative analysis of the intracellular pools of pathway intermediates and product identified the limited malonyl coenzyme A (malonyl-CoA) availability and low stilbene synthase activity in the heterologous host to be the main bottlenecks during pinosylvin production. Addition of cerulenin for increasing intracellular malonyl-CoA pools and the in vivo evolution of the stilbene synthase from Pinus strobus for improved activity in E. coli proved to be the keys to elevated product titers. These measures allowed product titers of 70 mg/liter pinosylvin from glucose, which could be further increased to 91 mg/liter by the addition of l-phenylalanine.

FIG 1

Biosynthetic pathway from l-phenylalanine to pinosylvin. Abbreviations: PAL, phenylalanine ammonia lyase; 4CL, 4-coumarate-CoA ligase; STS, stilbene synthase.

FIG 2

Pinosylvin production of E. coli BL21(DE3)/pR-HisPstrsts2-Sc4cl-Pcpal1 with and without supplementation of l-phenylalanine and/or addition of cerulenin. The experiments were performed in triplicate.

FIG 3

Relative intracellular levels of trans-cinnamic acid, trans-cinnamoyl-CoA, and pinosylvin determined by LC-MS/MS. The experiments with E. coli BL21(DE3)/pR-HisPstrsts2-Sc4cl-Pcpal1 were performed in duplicate with supplementation of 3 mM l-phenylalanine and 0 μM or 200 μM cerulenin. The relative levels are given as the area (percent), with the areas for the intermediates determined with 0 μM cerulenin being set equal to 100%.

FIG 4

Comparison of the pinosylvin titers of E. coli BL21(DE3)/pR-HisPstrsts2-Sc4cl-Pcpal1 expressing either wild-type HisPstrsts2 (light gray bars), HisPstrsts2 with the Q361R substitution (dark gray bars), or HisPstrsts2 with the T248A substitution (black bars) without any supplementation, with addition of 200 μM cerulenin, or with addition of 200 μM cerulenin and supplementation of 3 mM l-phenylalanine. The experiments were performed in duplicate.

FIG 5

(A) Cartoon representation of a close-up of the pinosylvin synthase STS2 from P. strobus. The structure model was calculated using the model of the pinosylvin synthase of P. sylvestris as the template. The monomers are shown in green and purple, and the resveratrol (Res) and coenzyme A (CoA) highlighted in one monomer are shown in the stick mode in yellow and cyan/orange, respectively. T248 is located at the dimer interface, whereas Q361 is located on the protein surface. Both amino acids are shown in the stick mode. (B) Both T248 and Q361, shown in orange, interact with the C-terminal β strand. The β strand in which T248 is located is adjacent to this C-terminal β strand, and the α helix of Q361 interacts indirectly with the same C-terminal β strand via a loop.