Screening PAL isoenzymes and codon optimization

In a previous study, Nielsen et al. confirmed that PAL was the rate-limiting enzyme in the styrene biosynthesis because the tCA titers remained low throughout [11]. In this study, to screen for a more efficient PAL, three candidate isoenzymes from Petroselinum crispum (PcPAL), Fagopyrum tataricum (FcPAL), and Artemisia annua (AaPAL), were screened with AtPAL2 as a control. It has been reported that recombinant PcPAL exhibits activity towards its natural substrate l-Phe, with a Km value of 116 ± 4 and Kcat value of 1 ± 0.05, and might have better catalytic properties [26]. Both FtPAL and AaPAL come from medicinal and nutrient-rich plants with high levels of flavonoids, and recombinant FtPAL protein has been found to be specific to l-Phe, with an activity of up to 35.7 IU/g [27, 28]. In the present study, to improve expression level, codon-optimized versions of all the above-mentioned genes were synthesized. FDC1 from Saccharomyces cerevisiae and optimized PALs were cloned into the pACYCDuet-1 vector and introduced into E. coli BL21(DE3), and the obtained transformants were inoculated into M9 medium for 24 h. As shown in Fig. 2a, the expression of FDC1 with AtPAL2 led to production of 55 mg/L styrene, which was higher than that achieved with FtPAL (44 mg/L) or PcPAL (36 mg/L), respectively, whereas the strains containing AaPAL rarely produced styrene. Although no enzyme with higher activity was detected, both FtPAL and PcPAL were confirmed to be specific to L-Phe because gas chromatography–mass spectrometry (GC–MS) did not detect 4-Vinylphenol in the fermentation products (Additional file 1: Figure S1). Based on these results, the expression of AtPAL2 combined with FDC1 was used for the production of styrene in subsequent experiments.

Selection of a suitable plasmid for increasing styrene production

Production of recombinant proteins in E. coli cells is affected by the number of plasmids, as well as their structural and segregational stability, which have essential impacts on productivity [29]. To achieve a high styrene producing capability, a two-step pathway was incorporated into four plasmids with different copy numbers and different promoters to assess any effect on styrene production. All the strains produced styrene from an initial glucose concentration of 15 g/L under aerobic conditions in 600-mL flasks. As shown in Fig. 2b, the strains with high-copy-number plasmids achieved higher styrene titers. E. coli BL06 harboring high-copy-number plasmid pET-28a-AtPAL2-FDC1 produced 70 mg/L styrene, which had a 123 and 507% improvement compared with that achieved in E. coli BL01 harboring the medium-copy-number plasmid pACYCDuet-AtPAL2-FDC1 and E. coli BL07 harboring the low-copy-number plasmid pColADuet-AtPAL2-FDC1, respectively. In contrast, plasmid copy number had little effect on cell growth during the process of styrene fermentation, with OD₆₀₀ of all strains were around 2.0 (Fig. 2b). In addition to the effect of plasmid copy number, the promoter also had a significant influence on styrene production. E. coli BL05 harboring trc promoter plasmid pTrcHis2B-AtPAL2-FDC1 produced 103 mg/L styrene under the same conditions, which was a 146% higher than that produced by E. coli BL06 harboring T7 promoter plasmid pET-28a-AtPAL2-FDC1. Based on these results, E. coli BL05 harboring plasmid pTrcHis2B-AtPAL2-FDC1 was chosen as the parent strain for further styrene production optimization.

Effect of co-expression of upstream genes aroF and pheA on styrene production

Aromatic amino acids such as l-Phe are naturally produced mainly from the shikimate pathway. The first rate-limiting step in this pathway is the condensation reaction between PEP and E4P to form 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP). This reaction is catalyzed by three DAHPS isoenzymes encoded by three genes aroF, aroG, and aroH, respectively. The second rate-limiting step is the conversion of chorismate into phenylpyruvate, via prephenate, catalyzed by CM-PDT, which is encoded by pheA. In a previous study, Backman et al. utilized a genetically modified E. coli strain with pheA^fbr and aroF^fbr genes to improve the metabolic flux towards l-Phe biosynthesis and achieved 50 g/L l-Phe with a yield of 0.25 (mol l-Phe/mol glucose) [30], which was the highest l-Phe production reported thus far. In our study, to enhance upstream pathway flux, aroF and pheA, were overexpressed in E. coli BL05 and the resultant strain E. coli BL0501 was evaluated for its ability to produce styrene in shake flask. After 24 h of cultivation, the styrene titers produced by E. coli BL0501 reached 210 mg/L, while the control strain E. coli BL0500 produced 100 mg/L styrene (Fig. 3a). These results confirmed that aroF and pheA genes are the key factors determining the biosynthesis of endogenous l-Phe and co-expression of CM-PDT and DAHPS could significantly improve l-Phe and l-Phe derivatives produced by E. coli, similar to that reported in previous studies [31, 32].

Effect of co-expression of central metabolic pathway genes tktA and ppsA on styrene production

To produce one molecule of l-Phe, two molecules of PEP and one molecule of E4P, which are both involved in the central metabolic pathways, are required. PEP is predominantly utilized in the phosphotransferase system (PTS), which is responsible for the translocation and phosphorylation of glucose, converting one PEP molecule to pyruvate (Fig. 1) [33]. Enhancing the expression level of PEP synthase (encoded by ppsA), which recycles pyruvate generated by PTS-mediated glucose transport to PEP, is an important approach for increasing the carbon flux from PEP to the aromatic amino acids pathway [33]. E4P can be directly produced by transketolase (encoded by tktA) or transaldolase (encoded by talB), and tktA have been demonstrated to be more effective in directing the carbon flux to the aromatic pathway than talB [34].

In our study, to investigate the effect of overexpression of ppsA and tktA on styrene production, styrene production of engineered E. coli strains harboring different constructs in shake-flask fermentation was investigated. As shown in Fig. 3b, E. coli BL0801 (harboring pTrc-AtPAL2-FDC1-ppsA-tktA and pACYC-aroF-pheA) accumulated 275 mg/L styrene after 24 h of fermentation with the consumption of 6.7 g/L glucose, which represents a 131 and 268% improvement over styrene production by E. coli BL08 (harboring pTrc-AtPAL2-FDC1 and pACYC-aroF-pheA) and E. coli BL05 (harboring pTrc-AtPAL2-FDC1), respectively. When compared with the previous study, which overexpressed AtPAL2 and FDC1 in an L-Phe overproduction strain E. coli NST74 (aroH367, tyrR366, tna-2, lacY5, aroF394(fbr), malT384, pheA101(fbr), pheO352, aroG397(fbr)) and the resulting strain was able to produce 260 mg/L styrene from 15 g/L glucose [11], it seemed that the styrene titer achieved in E. coli BL0801 was not improved as much as expected. Several reasons might be responsible for this result. Different genetic backgrounds between E. coli NST74 (K-12) and E. coli BL21(DE3) may result in different styrene yields. Furthermore, aroF ^fbr , aroG^fbr, tyrR, pheA^fbr, and pheO were overexpressed in E. coli NST74, while aroF^wt, pheA^wt, tktA, and ppsA were introduced into E. coli BL21(DE3). Multiple isozymes encoding genes, aroF and aroG, pheA, and pheO were co-expressed, which may significantly increase the flow of central metabolic carbon to phenylalanine biosynthesis. In the case, our engineered strain only had a slightly higher styrene production than the strain using E. coli NST74 as a host. Therefore, different host including E. coli NST74 (K-12) and more genes would be considered to increase the production of styrene in the subsequent research.

During the fermentation, the growth of engineered E. coli strains was very slow, with OD₆₀₀ reaching 2.0 after 24 h of induction. Therefore, the effect of induction time on styrene production was examined. As shown in Fig. 2c, styrene production and OD₆₀₀ did not increase along with the elongation of induction time, which could possibly be owing to the inhibition of cell growth by styrene produced by the host strains. This result reconfirmed that product toxicity is a limiting factor that must be addressed in addition to metabolic regulations.

Styrene toxicity assay

To evaluate styrene toxicity on E. coli BL21(DE3), the effect of exogenous addition of styrene at different concentrations (100, 200, 300, and 400 mg/L) on growing cultures was investigated. As shown in Fig. 4a, an OD₆₀₀ of 3.4 was reached in the absence of styrene. When the concentration of styrene was less than 300 mg/L, no significant growth inhibition was observed. However, when styrene concentration was increased to more than 300 mg/L, a remarkable cell growth inhibitory effect to cell growth was detected at 0–10 h. Interestingly, after 10 h, the values of OD₆₀₀ started to increase, which could possibly be due to the adaptation of cells to cultivation conditions with styrene or evaporation of styrene due to the insolubility of styrene in water. These findings are consistent with the previously reported styrene toxicity thresholds for E. coli NST74 [11].

After systematic optimization, the styrene output achieved 275 mg/L in the present study, which was close to the inhibitory threshold (300 mg/L). However, for economically viable and sustainable production of microbial-derived renewable styrene, the styrene titers and productivity must be ultimately improved, in other words, styrene toxicity must be overcome or effectively circumvented [11]. The accumulation of de novo synthesized biofuels or other solvent-like compounds within the cytoplasmic membrane has been shown to disrupt membrane integrity, resulting in the leakage of ions, metabolites, lipids, and proteins, as well as affecting the cells ability to maintain its internal pH and an appropriate trans-membrane proton gradient [35, 36]. Efforts have been made to improve host strains for better solvent tolerance, including introduction of efflux pumps or transporters, heat shock proteins, membrane modifications, genome engineering, random mutation, adaptive evolution, and approaches that integrated multiple-tolerance strategies [24]. In addition, ISPR and medium supplements can help to ease the burden of end-product toxicity and may be used in combination with genetic approaches. Therefore, in the present study, ISPR was used in combination with genetic approaches to increase styrene tolerance and production capacity of the host strains.

In situ product removal (ISPR) and solvent selection

For successful application of this approach, the selection of a suitable solvent is important. Ideal solvents should be biocompatible yet non-bioavailable and display high equilibrium partitioning of the target compound over water [37]. Various kinds of solvents, such as oleic acid, oleyl alcohol, miglyol, isopropyl myristate, and polypropylene glycol have been tested for their ability to improve the production of aromatic compounds [23–25, 38]. In our study, the effect of oleyl alcohol, n-dodecane, and isopropyl myristate on cell growth and styrene production of the engineered strain E. coli BL0801 was investigated, with no-solvent culture as a control. After 48 h of shake-flask fermentation, in the presence of n-dodecane, oleyl alcohol, and isopropyl myristate, the styrene titers reached 304, 340, and 350 mg/L, representing 110, 124, 125% improvements, respectively, when compared with that achieved in single-phase cultures (275 mg/L) (Fig. 4b). In addition, the cell growth curves of the E. coli BL0801 in biphasic and in single-phase cultures were also examined. The results revealed that the cell growth was significantly increased in biphasic culture when compared with that in single-phase culture (Fig. 4c), it is presumed that isopropyl myristate could selectively remove the styrene from the reaction system, thereby maintaining the styrene concentration around the cells below the inhibitory threshold, and allowing the strains to continue styrene production, resulting in higher biphasic culture than that in single-phase culture.

Furthermore, maximum theoretical yield coefficients ^maxY _Phe /Glc were calculated from the known stoichiometry of l-Phe biosynthesis from glucose, in an engineered strain where either the PTS was inactive or PYR was being recycled back to PEP, and the maximum theoretical yield(^{eng. max}Y _Phe /Glc) was 0.55 g/g [20, 39, 40]. Moreover, based on the hypothesis that complete conversion of all endogenously produced l-Phe to styrene is possible (e.g., if the pathway engineered in the present study could achieve a particularly high flux), the maximum theoretical yield (^{eng. max}Y _styrene /Glc) was calculated to be 0.35 g/g. According to this value, in single-phase culture, E. coli BL0801 strains reached yields 0.041 g/g, corresponding to 12% of the ^{eng. max}Y _styrene /Glc, while in biphasic culture, E. coli BL0801 strains reached yields 0.048 g/g, corresponding to 14% of the ^{eng. max}Y _styrene /Glc. The above data indicate that it is possible to achieve higher yield efficiency. Major challenges may come from low enzymatic activity and flux imbalance.

In the present study, methods to improve activity of PAL, which is a rate-limiting enzyme in the styrene biosynthesis, were first considered. Significant achievements were made by enzyme engineering, such as screening of enzymes with high activity and specificity [11, 41, 42], mutating the coding sequence in the regulatory domain [12, 43, 44], and family shuffling recombines natural proteins with high sequence identity [45–47]. Currently, these strategies have not yet been applied to attain PAL with higher activity. Feedback inhibition is one of the fundamental mechanisms that regulates the synthesis of amino acids and avoids their excessive accumulation which may cause imbalanced metabolism [48, 49]. To improve the metabolic flux towards l-Phe biosynthesis, overexpression of feedback-resistant pheA (pheA^fbr) and aroF (aroF^fbr) genes may be effective strategies. However, mutant enzymes may decrease thermostability and catalytic efficiency. For example, the use of the aroF^wt produced much more l-Phe than aroF^fbr (Asn8-Lys) due to the decreasing thermostability of aroF^fbr [50, 51].

To overcome flux imbalances, rational strategies to regulate gene expression were developed, such as application of inducible promoters, use of non-native RNA polymerase [52], the replacement of the ribosome binding site [53], as well as multivariate modular metabolic engineering [54]. Recently, biosensors have been employed to regulate metabolic flux. Biosensor application is an effective strategy that could dynamically detect pathway flux or the levels of pathway intermediates or products and regulators that respond to sensor input and accordingly regulate enzyme expression [55]. In a report, transcription factor (TF)-based sensor, a mutated transcriptional activator NahR from Pseudomonas putida, was used to detect benzoate and 2-hydroxybenzoate accumulation in E. coli [56]. In another report, researchers utilized a lysine riboswitch (RNA-based) to regulate the expression of citrate synthase and control the metabolic flux of the tricarboxylic acid cycle in a lysine-producing strain Corynebacterium glutamicum LP917, which increased the lysine production by 63% [57]. However, most applications have been limited to natural sensor-regulators [55]. Modular scaffold strategies are also effective approaches to improve metabolic flux. A scaffold protein carrying multiple protein–protein interaction domains is used to co-localize sequential pathway enzymes that have been tagged with peptide ligands specific for the domains on the scaffold [58]. The combined use of these multi-functional enzymes might increase the yield and titer of aromatic compounds from glucose. However, a major drawback of this method is that it often results in decreased activity of one enzyme or both the enzymes [59].

Based on the above reason, our subsequent work would focus on finding feedback-resistant pheA (pheA^fbr) and aroF (aroF^fbr) genes with improving thermostability and catalytic efficiency. Furthermore, screening an efficient method to overcome flux imbalances would lay the foundation for industrialized production of styrene.

Strains and media

Construction of plasmids

Cultivation conditions

Shake-flask fermentation: Seed culture was prepared by cultivating the strain in 5 mL of LB medium with appropriate antibiotics at 37 °C and 250 rpm overnight. Then, 1 mL of the seed culture was then transferred into 600-mL salt water bottle containing 100 mL of fermentation medium at 37 °C and 200 rpm. When the cell density reached OD₆₀₀ of 0.6–0.8, the cultures were induced with 0.4 mM of isopropyl-β-d-thiogalactopyranoside (IPTG), with plug added onto the bottle, and then incubated at 30 °C for an additional 48 h.

In situ extraction of styrene in shake flask: A total of 500 μL of the seed culture were inoculated into a 250 mL shake flask containing 50 mL of M9 medium and incubated aerobically at 37 °C and 200 rpm. When the cell density reached an OD₆₀₀ of 0.6–0.8, 0.4 mM IPTG and 10 mL of desired solvent were added to the cultures, and then incubated at 30 °C for an additional 48 h.

Styrene toxicity assay

A total of 1 mL of the seed culture was transferred into 600 mL salt water bottle containing 100 mL of fermentation medium containing styrene with different concentrations of styrene (0, 100, 200, 300, and 400 mg/L, respectively) and cultivated at 30 °C and 200 rpm. The cell growth was determined by OD₆₀₀ measurements using a UV/Vis spectrophotometer.

Evaluation of styrene production in engineered E. coli strains

After fermentation, the cultures were centrifuged at 12,000 rpm for 1 min, and 50 mL of the supernatant was extracted with 10 mL of ethyl acetate. Then, the extract was filtrated with 0.22 μm nylon membranes and analyzed by GC–MS to confirm the biosynthesis of styrene. M9 medium without inoculation and treated with the same procedures was used as a control.

GC-MS analysis was performed using Agilent 5975C system [7]. The following conditions were used: 50 °C for 1 min, which was increased at 20 °C/min to 240 °C and held for 10 min. Peak identification was based on the relative retention time and total ion mass spectral comparison with the external standard. Styrene production was quantified by GC, with following program: 50 °C for 1 min, which was increased at 20 °C/min to 240 °C and held for 1 min.