- Open Access
Artificial de novo biosynthesis of hydroxystyrene derivatives in a tyrosine overproducing Escherichia coli strain
© Kang et al. 2015
- Received: 8 April 2015
- Accepted: 25 May 2015
- Published: 10 June 2015
Styrene and its derivatives as monomers and petroleum-based feedstocks are valuable as raw materials in industrial processes. The chemical reaction for styrene production uses harsh reaction conditions such as high temperatures or pressures, or requires base catalysis with microwave heating. On the other hand, production of styrene and its derivatives in Escherichia coli is an environmental friendly process to produce conventional petroleum-based feedstocks.
An artificial biosynthetic pathway was developed in E. coli that yields 4-hydroxystyrene, 3,4-dihydroxystyrene and 4-hydroxy-3-methoxystyrene from simple carbon sources. This artificial biosynthetic pathway has a codon-optimized phenolic acid decarboxylase (pad) gene from Bacillus and some of the phenolic acid biosynthetic genes. E. coli strains with the tal and pad genes, the tal, sam5, and pad genes, and the tal, sam5, com, and pad genes produced 4-hydroxystyrene, 3,4-dihydroxystyrene and 4-hydorxy-3-methoxystyrene, respectively. Furthermore, these pathways were expressed in a tyrosine overproducing E. coli. The yields for 4-hydroxystyrene, 3,4-dihydroxystyrene and 4-hydorxy-3-methoxystyrene reached 355, 63, and 64 mg/L, respectively, in shaking flasks after 36 h of cultivation.
Our system is the first to use E. coli with artificial biosynthetic pathways for the de novo synthesis of 3,4-dihydroxystyrene and 4-hydroxy-3-methoxystyrene in a simple glucose medium. Similar approaches using microbial synthesis from simple sugar could be useful in the synthesis of plant-based aromatic chemicals.
- de novo Biosynthesis
Styrene is one of the most important aromatic chemicals produced industrially. It has many uses including in the manufacture of polystyrenes, plastics, and styrene-butadiene rubbers. Hydroxystyrene is also a monomer used in the production of numerous polymers and in petroleum-based feedstocks for resins, elastomers, and adhesives. Poly-hydroxystyrene, also called polyvinylphenol (PVP), is a plastic structurally similar to polystyrene. PVP is used in electronics as a dielectric layer in organic transistors of organic thin-film-transistor liquid–crystal display. Its ability to form linear polymers and its excellent solubility in organic solvents make hydroxystyrene a good reagent in the chemical synthesis of various coatings for electronic devices, such as a photoresist . Currently, styrene production predominantly comes from the energy-intensive chemocatalytic dehydrogenation of petroleum-derived ethylbenzene [2, 3]. Because of concerns over depleting feedstock availability and deleterious environmental impacts, a bio-based method could be a low energy, renewable alternative to petroleum-derived styrene . Thus, an artificial pathway for styrene and hydroxystyrene biosynthesis from glucose in Escherichia coli was previously engineered [5–7]. 4-Hydroxystyrene is also produced in the solvent-tolerant Pseudomonas putida strains, originally designed for phenol and 4-coumarate production . In addition, styrene production in Saccharomyces cerevisiae was recently reported combining metabolic evolution with systematic strain and pathway engineering [9, 10].
In recent years, several of artificial biosynthetic pathways have been engineered in microorganisms to produce useful, functionalized phenolic compounds from glucose [4, 6, 11–15]. In our laboratory, we investigate artificial biosynthetic pathways in microorganisms to produce a number of useful phenylpropanoids from plants [16–18]. Many of these substances in the phenylpropanoid pathway is phenolic acids, e.g., cinnamic, 4-coumaric, caffeic, ferulic, and sinapic acids. Their abundance has garnered much interest in their use to produced novel flavors, fragrances, pharmaceuticals and other chemicals .
This study constructed an artificial biosynthesis pathway to produce hydroxystyrene with the tyrosine ammonia lyase gene (tal) from Saccharothrix espanaensis and phenolic acid decarboxylase gene (pad) from Bacillus amyloliquefaciens. Additionally, serial artificial biosynthetic gene expression sets were developed and used to produce 3,4-dihydroxystyrene and 4-hydorxy-3-methoxystyrene by adding 4-coumarate 3-hydroxylase gene (sam5) and caffeic acid methyltransferase gene (com), respectively. Then, an E. coli strain capable of high-level tyrosine production was constructed with the feedback-inhibition 3-deoxy-d-arabinoheptulosonate-7-phosphate (DAHP) synthase gene (aroG) and the chorismate mutase/prephenate dehydrogenase gene (tyrA) by modifying a previously reported expression system. This strain was highly optimized to produce phenolic acids with a heavily increased metabolic flux toward l-tyrosine. Production of both 4-hydroxystyrene and 3,4-dihydroxystyrene from glucose was about 20-fold higher in the engineered tyrosine overproducing E. coli strain compared to that of the wild type E. coli. Finally, the yields for 4-hydroxystyrene, 3,4-dihydroxystyrene and 4-hydorxy-3-methoxystyrene were 355, 63, and 64 mg/L, respectively, in shaking flasks after 36 h of cultivation. This is the first report of a de novo biosynthesis yielding 3,4-dihydroxystyrene and 4-hydroxy-3-methoxystyrene using a single vector system combining phenolic acid biosynthetic genes and phenolic acid decarboxylase gene in E. coli. In this study, the production of 4-hydroxystyrene and its derivatives is accomplished with a carbohydrate feedstock through serial artificial biosynthetic pathways in E. coli strains.
Bioconversion of phenolic acids to hydroxystyrenes through phenolic acid decarboxylase
Construction of artificial biosynthetic pathways in E. coli to produce hydroxystyrene derivatives
To produce hydroxystyrene derivatives in E. coli with a simple sugar as a substrate, a series of plasmids were constructed containing artificial biosynthetic pathways that included the pad gene (Figure 2). We previously reported the production of 4-coumaric acid, caffeic acid, and ferulic acid in E. coli harboring artificial biosynthetic gene clusters in which the tyrosine ammonia lyase (tal) and 4-coumarate 3-hydroxylase (sam5) from S. espanaensis and caffeic acid methyltransferase (com) from Arabidopsis thaliana . The artificial biosynthetic plasmids containing the additional pad gene for the hydroxystyrene derivatives were constructed as previously described methods . In this study, the pad gene was under the control of the T7 promoter. A DNA fragment containing the promoter, the pad coding region, and the terminator was amplified using the pET22-baPAD plasmid as a template. The amplified pad fragment was ligated into the pET-opTAL, -opT5 and -opT5M plasmids containing the tal gene, the tal and sam5 genes, and the tal, sam5, and com genes and designated as pET-opTD, -opT5D and -opT5MD plasmids, respectively (Figure 2). The genes each have their own T7 promoter, ribosome-binding site (RBS), and terminator sequence same as the parental vector.
Improved production of l-tyrosine-derived phenolic acids in a tyrosine overproducing E. coli strain
Because tyrosine serves as an immediate endogenous precursor to the hydroxystyrene pathway, its over production in E. coli is essential for hydroxystyrene biosynthesis. To develop a tyrosine overproducing E. coli strain, a classical metabolic engineering strategy was used. In E. coli, the aromatic amino acid biosynthesis pathway starts with the condensation of phosphoenolpyruvate and erythrose-4-phosphate, catalyzed by 3-deoxy-D-arabinoheptulosonate-7-phosphate (DAHP) synthase present in three isoforms with each feedback-regulated by aromatic amino acids. Subsequently, l-tyrosine biosynthesis from chorismate is catalyzed by the bifunctional enzyme chorismate mutase/prephenate dehydrogenase and the aromatic amino acid transaminase. Recently, l-tyrosine excreting E. coli strains were produced by deregulating the aromatic amino acid biosynthesis pathway . A high-copy number vector with feedback-inhibition resistant (fbr) derivatives of DAHP synthase (aroG fbr ) and chorismate mutase/prephenate dehydrogenase (tyrA fbr ) genes  was overexpressed in an E. coli ΔtyrR strain . In this study, the l-tyrosine producer, E. coli ΔCOS1, was engineered on the genome to also overexpress the aroG fbr and tyrA fbr genes in a ΔtyrR strain background. The aroG fbr and tyrA fbr gene cassette with a strong inducible T7 promotor was inserted into the tyrR gene region to make a stable strain for fermentation (Additional file 1: Figures S4, S5). Overall, greater improvements in l-tyrosine production were achieved initially in wild type E. coli (Additional file 1: Figure S6). A maximum yield of ~450 mg/L of l-tyrosine was produced by the E. coli ΔCOS1 strain in shaken flask experiments. The tyrosine yield was comparable to the tyrosine overproducing E. coli strain with the aroG fbr and tyrA fbr gene cassette vector (400 mg/L) as previously reported . This ΔCOS1 strain was optimized for the production of aromatic compounds resulting in a heavily increased metabolic flux towards l-tyrosine. Therefore, it is suitable platform strain for the production of other l-tyrosine-derived phenolic acids including 4-coumaric acid, caffeic acid and ferulic acid, and hydroxystyrene derivatives using the phenolic acids as precursors.
Improved production of hydroxystyrene derivatives in tyrosine overproducing strains
The system in this study converts 4-coumaric acid to 4-hydroxystyrene through pad (phenolic acid decarboxylase), which is eventually converted caffeic acid and ferulic acid and then to 3,4-dihydroxystyrene and 4-hydorxy-3-methoxystyrene, respectively. E. coli strains expressing tal (tyrosine ammonia lyase) and pad, and tal, sam5 (4-coumarate 3-hydroxylase), and pad, and tal, sam5, com (caffeic acid methyltransferase), and pad produced 4-hydroxystyrene, 3, 4-dihydroxystyrene and 4-hydroxy-3-methoxystyrene, respectively, using a simple sugar medium without precursor feeding. Furthermore, these pathways were extended in an E. coli strain with the biosynthesis machinery for overproducing tyrosine. Finally, the titers for 4-hydroxystyrene, 3,4-dihydroxystyrene and 4-hydroxy-3-methoxystyrene in the tyrosine-overproducing E. coli strains reached 355, 63, and 64 mg/L, respectively, in shaking flasks after 36 h of cultivation.
Bacterial strains, plasmids, and chemicals
Plasmids and strains used in this study
Plasmid or strain
f1 ori, T7 promoter, AmpR
f1 ori, T7 promoter, KanR
pET-28a(+) carrying codon-optimized tyrosine ammonia lyase gene (tal)
Kang et al. 
pET-28a(+) carrying 4-coumarate 3-hydroxylase gene (sam5)
Choi et al. 
pET-28a(+) carrying caffeic acid methyltransferase gene (com)
Choi et al. 
pET-28a(+) carrying tal and sam5 gene
Kang et al. 
pET-28a(+) carrying tal, sam5, and com gene
Kang et al. 
pET-28a(+) carrying feedback-inhibition resistant chorismate mutase/prephenate dehydrogenase gene (tyrAfbr)
Kang et al. 
pET-22b(+) carrying feedback-inhibition resistant DAHP synthase gene (aroGfbr)
Kang et al. 
pET-28a(+) carrying tyrAfbr and aroGfbr gene
pET-22b(+) carrying codon-optimized phenolic acid decarboxylase gene (pad)
pET-28a(+) carrying tal and pad gene
pET-28a(+) carrying tal, sam5 and pad gene
pET-28a(+) carrying tal, sam5, com and pad gene
E. coli DH5α
E. coli C41(DE3)
Derivative strain of E. coli BL21(DE3)
Miroux and Walker 
tryR gene in-frame deletion mutant of E. coli C41(DE3)
Kang et al. 
E. coli C41(DE3); ΔtyrR::tyrA fbr, aroG fbr; tyrosine overproducing strain
The restriction enzymes (NEB; Takara), Ex Taq polymerase (Takara), pfu Taq polymerase (Enzynomics, Korea), an AccuPower Ligation kit (Bioneer, Korea), and a Quick & Easy E. coli gene deletion kit (Gene Bridges, German) were used according to the manufacturers’ instructions. The optimized tyrosine ammonia lyase gene (tal) and 4-coumarate 3-hydroxylase gene (sam5) from S. espanaensis and the caffeic acid methyltransferase gene (com) from A. thaliana were synthesized previously by DNA 2.0 . Codon optimization and synthesis of the phenolic acid decarboxylase gene (pad) from B. amyloliquefaciens DSM7 (GenBank FN597644) were performed with the GeneGPS™ program (DNA2.0).
Construction of phenolic acid decarboxylase expression vectors and assembly of the artificial biosynthetic pathways
In order to construct an expression vector containing the phenolic acid decarboxylase gene (pad) that was under the control of independent T7 promoter, a 0.5-kb DNA fragment, which contained the synthetic pad coding region, was cloned into the NdeI and XhoI sites on pET-22b(+), which resulted in pET22-baPAD. The three genes (tal, sam5, and com) were independently cloned into pET-22b(+) or pET-28a(+) vectors [17, 20]. Using the tal, sam5, com, and pad genes as templates, four DNA fragments were amplified by PCR with the appropriate pairs of primers. In order to assemble the pET-opTD vector, the tal coding region was amplified using pET-opTAL as a template with the primer opTAL-F (5′-CATATGACCCAGGTGGTTGAACGCC-3′) and Cpac (the sequence is located downstream of the T7 terminator region of the pET vector and contains the designed PacI site: TTAATTAATGCGCCGCTACAGGGCGCGTCC), also the pad coding region was amplified using pET22-baPAD as a template with the primer Npac (the sequence is located upstream of the T7 promoter region of the pET vector and contains the designed PacI site: TTAATTAATCGCCGCGACAATTTGCGACGG) and baPAD-R (the sequence contains the designed XhoI site 5′-CTCGAGTTACTTCAGTTTACC-3′). Each of the amplified fragments were digested with corresponding sites and cloned between the NdeI and XhoI digested pET-28a(+) via ligation, which resulted in pET-opTD. A 2.5-kb PacI fragment containing the sam5 gene was PCR-amplified with the NPac and CPac primers using pET22-Sam5 as a template. The amplified fragment was digested with PacI and cloned between the PacI digested pET-opTD, which resulted in pET-opT5D. Finally, a 2.5-kb PacI/SpeI fragment containing the com gene was PCR-amplified with the NPac and Cspe (the sequence is located downstream of the T7 terminator region of the pET vector and contains the designed SpeI site: ACTAGTTCCTCCTTTCAGCAAAAAACCCCTC) primers using pET22-COM as a template. A 2.5-kb PacI fragment containing the sam5 gene was PCR-amplified with the NPac and CPac primers using pET22- Sam5 as a template. Each of the two amplified fragments were digested with corresponding sites and cloned into PacI digested pET-opTD, which resulted in pET-opT5MD (Figure 2).
Construction of the l-tyrosine overproducing strain
An l-tyrosine over-producing strain of E. coli (ΔCOS1) was achieved by extra gene insertion of aroG and tyrA, feedback-inhibition resistance (fbr) genes on the tyrR gene locus. The genetic design of the aroG and tyrA feedback-inhibition resistance (fbr) genes was followed as previously described by Lütke-Eversloh and Stephanopoulos [25, 30] and used our previously made constructs . The PCR product was generated using the tyrA fbr -aroG fbr -FRT-neo-FRT fragment as a template for pET-AG and FRT-neo-FRT fragment (Gene Bridges). We made a fragment containing both the tyrA fbr and aroG fbr gene cassette, in which the RBS and T7 promoter were positioned in front of each gene, through PCR with the following primers, IF-N1(5′-CGGTACCCGGGGATCACTAGTTGATCGGCGCGAGATTTAATCGCCGCGCAA T-3′) and IF-C1(5′-GTTAATTAAACTAGTCACGCTGCGCGTAACCACCACACCCGC CGCGCT-3′), and another fragment containing the FRT-neo-FRT using the following primers, IF-FRT1(5′-ACTAGTTTAATTAACCCTCACTAAAGGGCGGCCGCGAAGTTCCTATT-3′) and IF-FRT2(5′-CGACTCTAGAGGATCACTAGTAATACGACTCACTATAGGGCTCGAG GAAGTTCC-3′). These two fragments were connected between the SpeI site of pUC19 using the In-fusion kit (Clontech Laboratories, Inc., USA), resulting in pUC-AGFRT (Additional file 1: Figure S4). The 5.9-kb insertion PCR product was generated using pUC-AGFRT as a template and the following primers, tyrRr (5′-ATCAGGCATATTCGCGCTTACTCTT CGTTCTTCTTCTGACTCAGACCATATAATACGACTCACTATAGGGCTC-3′) and Inf-tryRfAG (5′-GTCATATCATCATATTAATTGTTCTTTTTTCAGGTGAAGGTTCCCATGC GTACTAGTCGTTCTACCATCGACACC-3′), and this gene cassette was inserted between the tyrR gene for gene insertional inactivation, which was done as previously reported using RED/ET recombination with a Quick & Easy E. coli Gene deletion kit (Gene Bridges). The clones growing on the kanamycin plate still contained the selection marker cassette, while all other clones containing insertional inactivation lost the selection marker. The kanamycin selection marker was removed from the chromosome by transforming the cells with an FLP recombinase expression plasmid, 707-FLPe (Gene Bridges). The insertional inactivation mutant (ΔCOS1) was verified through PCR using the following primers: tyrA-F (5′- CCATGGTTGCTGAATTGACCGCATTACG-3′) and aroG-R (5′- AAGCTTAACCACGA CGCGCTTTCACAGC-3′). The PCR product was sequenced and verified (Additional file 1: Figure S5).
Culture conditions for production
Recombinant E. coli C41 (DE3) strains  harboring plasmids were grown at 37°C in a Luria–Bertani (LB) medium containing 50 μg/mL kanamycin. The overnight culture was inoculated (1.5%) into fresh LB medium supplemented with the same concentration of kanamycin. The culture was grown at 37°C to an optical density at 600 nm (OD600) of 0.6, and IPTG was added to the final concentration of 1 mM, and the culture was incubated for 6 h. The cells were harvested by centrifugation, suspended, and incubated at 26°C for 36 h in a modified synthetic medium (3 g/L KH2PO4, 7.3 g/L K2HPO4, 8.4 g/L MOPS, 2 g/L NH4Cl, 0.5 g/L NaCl, 0.1 ml/L Trace elements, 5 g/L (NH4)2SO4, 5 g/L MgSO4, and supplemented with 15 g/L glucose, 1 mM IPTG and appropriate antibiotics) . For the feeding experiments, the cultures were supplemented with cinnamic acid, 4-coumaric acid, caffeic acid, ferulic acid, or sinapic acid (final concentration: 2 mM), respectively. The samples were collected after 36 h and analyzed by HPLC.
Detection and quantification of the products
To quantify 4-hydroxystyrene, 3,4-dihydroxystyrene and 4-hydroxy-3-methoxystyrene, 1 mL of cell-free culture supernatants was filtered through a 0.2 μm cellulose membrane syringe filter (Sartorius) and used for HPLC analysis with a Dionex Separations module connected with a Photodiode Array detector (Dionex) set. Twenty microliters of the samples were applied to a J’sphere ODS-H80 column (4.6 × 150 mm i.d., 5 μm; YMC, Japan) in a high-performance liquid chromatography (HPLC) system [CH3CN-H2O (0.05% trifluoroacetic acid), 20–60% acetonitrile (CH3CN) for 25 min at flow rate of 1 mL/min; Dionex, USA] equipped with a photodiode array detector. Quantification of the three above-mentioned compounds was based on the peak areas of absorbance at 259 nm. Purchased 3,4-dihydroxystyrene and 4-hydroxy-3-methoxystyrene as a standard contains the impurity. Therefore, all the hydroxystyrene derivatives were measured as equivalent to 4-hydroxystyrene. For the quantification of 4-coumaric acid, caffeic acid, and ferulic acid, the HPLC analysis was followed as our previously described methods . The data shown in this study were generated from triplicate independent experiments. The titers for each production were compared with single-factor ANOVA (P < 0.05) using the single-factor ANOVA tool.
SK and OC performed the experiments and wrote the manuscript. JL co-performed the experiments on the metabolite analysis. BH, JOA and JSA contributed general advice, particularly on the metabolite analysis and resource support. YH designed all the experiments and wrote the manuscript. All authors read and approved the final manuscript.
This work was supported in part by Global R&D Center program, NRF and by the Next-Generation BioGreen 21 Program (SSAC, PJ011084012015), RDA Republic of Korea. We thank the Gyeonggi Bio Center for the GC/MS measurements.
Compliance with ethical guidelines
Competing interests The authors declare that they have no competing interests.
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