Biosynthesis of plant-specific stilbene polyketides in metabolically engineered Escherichia coli

Abstract

Background: Phenylpropanoids are the precursors to a range of important plant metabolites such as the cell wall constituent lignin and the secondary metabolites belonging to the flavonoid/stilbene class of compounds. The latter class of plant natural products has been shown to function in a wide range of biological activities. During the last few years an increasing number of health benefits have been associated with these compounds. In particular, they demonstrate potent antioxidant activity and the ability to selectively inhibit certain tyrosine kinases. Biosynthesis of many medicinally important plant secondary metabolites, including stilbenes, is frequently not very well understood and under tight spatial and temporal control, limiting their availability from plant sources. As an alternative, we sought to develop an approach for the biosynthesis of diverse stilbenes by engineered recombinant microbial cells.

Results: A pathway for stilbene biosynthesis was constructed in Escherichia coli with 4-coumaroyl CoA ligase 1 4CL1) from Arabidopsis thaliana and stilbene synthase (STS) cloned from Arachis hypogaea. E. coli cultures expressing these enzymes together converted the phenylpropionic acid precursor 4-coumaric acid, added to the growth medium, to the stilbene resveratrol (>100 mg/L). Caffeic acid, added in the same way, resulted in the production of the expected dihydroxylated stilbene, piceatannol (>10 mg/L). Ferulic acid, however, was not converted to the expected stilbene product, isorhapontigenin. Substitution of 4CL1 with a homologous enzyme, 4CL4, with a preference for ferulic acid over 4-coumaric acid, had no effect on the conversion of ferulic acid. Accumulation of tri- and tetraketide lactones from ferulic acid, regardless of the CoA-ligase expressed in E. coli, suggests that STS cannot properly accommodate and fold the tetraketide intermediate to the corresponding stilbene structure.

Conclusion: Phenylpropionic acids, such as 4-coumaric acid and caffeic acid, can be efficiently converted to stilbene compounds by recombinant E. coli cells expressing plant biosynthetic genes. Optimization of precursor conversion and cyclization of the bulky ferulic acid precursor by host metabolic engineering and protein engineering may afford the synthesis of even more structurally diverse stilbene compounds.

Figure 1

Engineered route to stilbene biosynthesis in E. coli. Stilbene biosynthesis takes place by sequential addition of acetate units, derived from the decarboxylation of malonyl-CoA, to a CoA activated phenylpropionic acid. Polyketide intermediates (in brackets), if not cyclized properly, can be released from stilbene synthase (STS) and spontaneously form lactone derailment products. Acceptable starter units are extended to a linear tetraketide intermediate, which then undergoes aldol condensation and decarboxylation, followed by aromatization, to produce the proper stilbene structure. E. coli was engineered to produce stilbenes through the biotransformation of phenylpropionic acids by the enzymes 4-coumaroyl:CoA ligase 1 (4CL1) or 4-coumaroyl:CoA ligase 4 (4CL4) and STS. 4-coumaric acid and caffeic acid were transformed to the expected stilbene compounds, resveratrol and piceatannol, using this pathway. Ferulic acid was not converted to the corresponding stilbene using either 4CL1 or 4CL4, but lactone derailment products were detected, indicating that this substrate can be used as a starter unit in vivo.

Figure 2

Expression of Arachis hypogaea STS in E. coli. SDS-PAGE gel (12%) showing the overexpressed STS from peanut roots. The sts gene was cloned into the plasmid pUCMod as described in Materials and Methods. E. coli expressing STS, and a control culture with empty plasmid, were grown overnight in modified M9 medium at 30°C containing glycerol (lanes 1–4) or glucose (lanes 5–7). Cell lysates were prepared as described in Material and Methods. Samples are as follows: Lane 1: empty pUC control, Lane 2: pUC-STS grown in glycerol crude cell lysate, Lane 3: pUC-STS grown in glycerol soluble protein fraction, Lane 4: pUC-STS grown in glycerol insoluble protein fraction, Lane 5: pUC-STS grown in glucose crude cell lysate, Lane 6: pUC-STS grown in glucose soluble protein fraction and Lane 7: pUC-STS grown in glucose insoluble protein fraction, Lane 8: molecular weight standard.

Figure 3

Effect of increasing 4-coumaric acid concentrations on E. coli growth. E. coli was grown in modified M9 medium containing glycerol with increasing concentrations of 4-coumaric acid to determine growth inhibition. Concentrations of added 4-coumaric acid were as follows: (●) 0 mM, (■) 2 mM, (▲) 6 mM, (▼) 12 mM and (◆) 20 mM. Growth was determined by OD measurement at 600 nm. Cultures were supplemented with varying concentrations of 4-coumaric acid once the starter culture reached an OD of 0.1. Data points represent the means of three independent measurements.

Figure 4

HPLC analysis of resveratrol. E. coli expressing 4CL1 and STS was cultured in modified M9 medium with glycerol, and containing 1 mM 4-coumaric acid, at 30°C for 24 hrs prior to extraction. Upper chromatogram shows extracted E. coli culture and bottom chromatogram shows resveratrol standard run under identical HPLC conditions. At right are the UV spectra for both recombinant resveratrol (1) (m/z 227.1) and resveratrol standard (2) (m/z 227.1). No residual 4-coumaric acid was detected by HPLC or LC-MS.

Figure 5

Growth and production curves for resveratrol biosynthesis. Culture of E. coli pAC-4CL1 + pUC-STS was supplemented with 1 mM 4-coumaric acid and grown for approximately 48 hours in modified M9 medium with glycerol at 30°C. Samples were removed periodically for extraction to determine the consumption of the substrate 4-coumaric acid (○) and the formation of the product resveratrol (●). Growth (■) was monitored spectrophotometrically by measuring OD at 600 nm. Data points represent the mean of three independent measurements.

Figure 6

HPLC analysis of cultures supplemented with caffeic acid or ferulic acid. E. coli expressing 4CL1 and STS was supplemented with 1 mM substrate and grown in modified M9 medium at 30°C for 24 hrs prior to extraction. Panel A: Production of piceatannol from caffeic acid. Upper chromatogram shows extracted E. coli culture, bottom chromatogram shows piceatannol standard run under identical HPLC conditions. At right are the UV spectra for both recombinant piceatannol (1) (m/z 243.1) and piceatannol standard (2) (m/z 243.1). Peak 3 is residual caffeic acid (m/z 179.2). Panel B: E. coli expressing 4CL1 and STS supplemented with ferulic acid. Peak 1 corresponds to residual ferulic acid (m/z 193.1). Peak 2 corresponds to the ferulic acid derived tetraketide lactone (m/z 301.1) and peak 3 is the corresponding triketide lactone (m/z 259.1).