Heterologous synthesis of 4-ethylphenol in engineered Escherichia coli

Highlights

• An artificial biosynthesis pathway of 4-ethylphenol was constructed in E. coli.

• This is the first report of 4-ethylphenol biosynthesis from renewable substrates.

• Optimization was performed for improving enzyme expression and 4-ethylphenol production.

• 110 mg/L 4-ethylphenol was produced in Terrific Broth (TB) medium with glycerol in flask cultivation.

Abstract

4-Ethylphenol (4-EP) is an industrially versatile commodity chemical widely applied in the pharmaceutical and food industries. In this study, an artificial biosynthetic pathway was constructed in Escherichia coli for production of 4-ethylphenol from simple sources of carbon. The pathway consists of the tal, pad and vpr genes, which encode tyrosine ammonia lyase (TAL), phenolic acid decarboxylase (PAD) and vinylphenol reductase (VPR), respectively. Our results confirmed that the TAL from Saccharothrix espanaensis possessed higher catalytic activity than the TAL from Rhodobacter sphaeroides for biosynthesis of p-hydroxycinnamic acid. The low solubility of Lactobacillus plantarum VPR (LpVPR) in E. coli was a critical factor limiting its availability in the biosynthetic pathway. The solubility of LpVPR was improved by E. coli strain and induction condition optimization. Under the optimized conditions, the engineered E. coli TransB-TPV produced as high as 110 mg/L 4-EP at 37 ℃ in Terrific Broth (TB) medium with glycerol as carbon source after cultivation of 48 h. This study provided a new and feasible strategy for biosynthesis of 4-EP from simple sugars, which may provide a basis for future large-scale industrial application.

Introduction

Biorenewable fuels and chemicals are receiving more and more attention owing to the decline of fossil fuel resources and concerns about sustainable development of conventional petrochemical industry [1]. Along with the development of metabolic engineering and the discovery of new metabolic routes, compounds that are previously difficult to produce from nature are becoming available through biosynthesis in microorganisms. In recent years, several artificial biosynthetic pathways have been engineered to produce useful aromatic compounds including cinnamic acid [2,3], p-hydroxycinnamic acid [4,5], caffeic acid [[6], [7], [8]], ferulic acid [4], vanillin [[9], [10], [11]], and others [[12], [13], [14]] from renewable substrates such as glucose and glycerol. The use of these simple sugars as fermentation substrates has the potential to improve the production economics of biotechnology-derived products.

4-Ethylphenol (4-EP) is widely used as a flavoring [15], phenolic resin [16], antioxidant [17], etc. It is also an important organic synthesis intermediate and chemical reagent [18]. 4-EP can be extracted from biomass by pyrolysis [19]. Current industrial production of 4-EP solely relies on the petrochemical industry but the process is complex and environmentally unfriendly, consisting of three primary steps which include sulphonation of ethylbenzene to produce an ethylbenzenesulfonic acid mixture, separation of para-ethylbenzenesulfonic acid, andalkali fusion to obtain 4-EP [20]. This 4-EP manufacture process is one of the most energy-intensive among commodity chemical production methods [20,21]. It was reported previously that 4-ethylphenol can be produced in the tryptone–yeast extract (TY) broth containing 10 mmol/L p-hydroxycinnamic acid by a Lactobacillus sp. isolated from a swine waste lagoon [22]. In addition, 4-EP can be naturally produced from p-hydroxycinnamic acid by the yeast Dekkera, which is responsible for wine spoilage [23]. Provided this background, a biotechnological approach would be an attractive alternative for 4-EP production from renewable and sustainable resources. Although 4-EP has been observed as a trace metabolite in some microbial culture broths or fermentation food, no natural metabolic pathway has been identified from microbes for biosynthesis of 4-EP from simple carbon sources [24,25].

Recently, two corresponding genes encoding vinylphenol reductase (VPR), which catalyzes the reduction of vinylphenols (4-vinylphenol, 4-vinylcatechol, and 4-vinylguaiacol) into ethylphenols (including 4-ethylphenol, 4-ethylcatechol, and 4-ethylguaiacol), were identified from the bacterium Lactobacillus plantarum [26] and yeast Dekkera bruxellensis [23], respectively. This novel reductase provides the possibility for us to design an artificial 4-EP biosynthetic pathway in E. coli, since it was well known that vinylphenol could be efficiently transformed from p-hydroxycinnamic acid, as a natural metabolite, through enzymatic decarboxylation using phenolic acid decarboxylase (PAD) [[27], [28], [29]].

In this study, we described the heterologous synthesis of functional 4-EP from glucose or glycerol by an engineered E. coli strain. For this, an artificial 4-EP biosynthetic pathway was constructed in E. coli, which involved co-expression of three genes encoding tyrosine ammonia lyase (TAL), phenolic acid decarboxylase (PAD) and vinylphenol reductase, respectively (Fig. 1). l-tyrosine (L-Tyr) was synthesized through the shikimate pathway, then converted to p-hydroxycinnamic acid (pHCA) by the action of the recombinant tyrosine ammonia lyase (TAL). pHCA was then converted to 4-vinylphenol (4-VP) by the recombinant phenolic acid decarboxylase (PAD). The 4-VP was converted to 4-EP by the action of the recombinant vinylphenol reductase (VPR).

Compared to PAD, TAL and VPR usually suffer from low catalytic activity and insoluble expression issues, which may reduce their effectiveness in the artificial 4-EP biosynthetic pathway [23,26,30]. With this in mind, two TAL candidates from different source organisms were compared for their ability to catalyze the l-Tyr conversion to pHCA. In addition, the recombinant enzyme induction conditions for enhancing the soluble expression of LpVPR were also optimized in this study. With systematic optimization of 4-EP biosynthesis in E. coli, we obtained a relatively high titer of 4-EP from simple carbon sources, showing the feasibility of this artificial biosynthetic pathway. To our knowledge, this is the first report of 4-EP biosynthesis from renewable substrates.