- Open Access
Enhanced production of styrene by engineered Escherichia coli and in situ product recovery (ISPR) with an organic solvent
© The Author(s) 2019
- Received: 15 March 2019
- Accepted: 27 April 2019
- Published: 3 May 2019
Styrene is a large-volume commodity petrochemical, which has been used in a wide range of polymer industry as the main building block for the construction of various functional polymers. Despite many efforts to produce styrene in microbial hosts, the production titers are still low and are not enough to meet the commercial production of styrene.
Previously, we developed a high l-phenylalanine producer (E. coli YHP05), and it was used as a main host for de novo synthesis of styrene. First, we introduced the co-expression system of phenylalanine-ammonia lyase (PAL) and ferulic acid decarboxylase (FDC) genes for the synthesis of styrene from l-phenylalanine. Then, to minimize cell toxicity and enhance the recovery of styrene, in situ product recovery (ISPR) with n-dodecane was employed, and culture medium with supplementation of complex sources was also optimized. As a result, 1.7 ± 0.1 g/L of styrene was produced in the flask cultures. Finally, fed-batch cultivations were performed in lab-scale bioreactor, and to minimize the loss of volatile styrene during the cultivation, three consecutive bottles containing n-dodecane were connected to the air outlet of bioreactor for gas-stripping. To conclude, the total titer of styrene was as high as 5.3 ± 0.2 g/L, which could be obtained at 60 h.
We successfully engineered E. coli strain for the de novo production of styrene in both flask and fed-batch cultivation, and could achieve the highest titer for styrene in bacterial hosts reported till date. We believe that our efforts in strain engineering and ISPR strategy with organic solvent will provide a new insight for economic and industrial production of styrene in a biological platform.
- Escherichia coli
- Fed-batch culture
- In situ product recovery (ISPR)
Styrene is a large-volume commodity petrochemical whose global production exceeded 26.4 million tons in 2012 . Particularly, styrene has been used in a wide range of polymer industry as it is the main building block for the construction of various functional polymers including styrene butadiene rubber (SBR), expanded polystyrene (EPS), and acrylonitrile butadiene styrene (ABS). Since these functional co-polymers are commonly used in construction fields as insulating materials, consistent increase in demand for styrene production is expected in the near future [2, 3]. The conventional production of styrene solely relies on the petrochemical industry, where petroleum-derived ethylbenzene is converted into styrene through a catalytic dehydrogenation . However, such typical process requires extensive thermal energy of more than 200 trillion BTU of steam annually in the US alone . Therefore, the chemical synthesis of styrene has been regarded as one of the most energy-intensive process among various petrochemical synthesis.
As an alternative, a biological approach was suggested to provide an eco-friendly and sustainable platform for styrene production [6–9]. An economic evaluation revealed that the bio-based styrene production could be competitive with the current petroleum-based platforms . The natural production of styrene has been found in various hosts including microorganisms, such as Penicillium camemberti and certain specific plant species [6, 11]. However, their extremely low productivities suggested the need for a suitable host for an economically feasible production. Instead of natural producers, several recombinant hosts including Escherichia coli and Saccharomyces cerevisiae have been engineered for the production of styrene. McKenna et al.  were successful in incorporating phenylalanine-ammonia lyase (PAL) from Arabidopsis thaliana and ferulic acid decarboxylase (FDC) from S. cerevisiae into a high l-phenylalanine producer strain (E. coli NST74) to demonstrate a styrene biosynthesis pathway in E. coli, where 260 mg/L of styrene was produced in flask cultures. The production titer of styrene was further enhanced to 836 mg/L in flasks by adopting a two-phase partitioning method using organic solvents . Meanwhile, Liu et al.  optimized the biosynthesis pathway to l-phenylalanine in E. coli BL21(DE3) based on enzyme screening and a metabolic flux analysis, in combination with in situ product removal (ISPR), resulting in a styrene titer of 350 mg/L of styrene production in flask cultivation. Same pathway has been incorporated into S. cerevisiae to produce styrene, where PAL and FDC genes were overexpressed along with the down-regulation of a competing pathway . However, the titer reached only up to a maximum of 29 mg/L in flask cultivation. Despite these efforts to increase the production titer of styrene in microbial hosts, the current titers are not high enough for the commercial production and it is necessary to develop more potential host and efficient bioprocess.
Construction of styrene biosynthesis pathway in E. coli
Optimization of the enzyme expression levels
Together with ScFDC, the expression of SmPAL gene is highly critical in the production of styrene, and both gene expression levels should be optimized. We noted that tCA barely remained in supernatant (data not shown), suggesting that tCA has been converted to styrene immediately after it was synthesized from l-phenylalanine by SmPAL-mediated reaction. This result implied that the production of styrene can be further increased by more supplementation of tCA and it is necessary to increase the expression level of SmPAL gene. For this purpose, we decided to use the original plasmid (pHC-CA) instead of pHC-CA-FDC, and ScFDC gene was moved to the other plasmid (pYHP), yielding pYHP-FDC in which gene expression was under a constitutive promoter (BBa_J23100) (Additional file 1: Fig. S1b). E. coli YHP05 harboring pHB-CA and pYHP-FDC was cultivated in LB medium, and the expression levels of both genes were analyzed by SDS-PAGE. We found that the expression of SmPAL gene in pHB-CA increased and maintained sufficient level (Additional file 2: Fig. S2b). In contrast, the expression level of ScFDC in pYHP-FDC decreased slightly compared to the previous construct (pHC-CA-FDC) due to the plasmid copy number: The backbone of pYHP-FDC is a pTac15K, which has lower copy number than pHC-CA-FDC . In flask cultivation with this strain, the styrene titer was 248.9 ± 22.1 mg/L, which was approximately 2-fold higher compared with the previous system (Fig. 2b). In this cultivation, cells showed a little decrease of cell density (OD of 5.3) compared with that of cell harboring pYHP and pHB-CA-FDC (OD of 6.2), but higher expression of biosynthesis genes (Additional file 2: Fig. S2) could drive more supplementation of main precursor (tCA) and consequently more production of styrene.
Optimization of culture media
In addition to culture media, we also examined the effect of culture temperatures on styrene production. In the previous work for production of tCA [12, 13], we examined two temperatures 30 °C and 37 °C, and cultivation at high temperature gave higher production of tCA. E. coli YHP05 harboring pHB-CA and pYHP-FDC was cultivated in the PHE medium with supplementation of yeast extract at 37 °C, and after induction, cells were cultivated at both temperatures (30 °C or 37 °C). Similar as previous results [12, 13], we confirmed that styrene production was higher by cultivation at 37 °C than 30 °C (Additional file 3: Fig. S3). Those conditions (37 °C and supplementation of yeast extract) was employed in the following fed-batch cultivations.
Fed-batch cultivation for styrene production
Overview on de novo production of styrene in bacterial hosts
Solvent for extraction
Max. titers (mg/L)
E. coli YHP05
E. coli NST74
E. coli NST74
E. coli BL21(DE3)
Bacterial strains and plasmids
Bacterial strains and plasmids used in this study
recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F′ proAB lacIqZ ΔM15 Tn10 (Tetr)]
F− l− rph-1 INV(rrnD, rrnE)
W3110 Δcrr ΔtyrR ΔtrpE ΔtyrA ΔpykA
KmR, p15A origin, Ptac promoter, 4.0 kb
AmpR, pBR322 origin, Ptrc promoter, 4.2 kb
pTac15k, Ptac–aroG8/15–ydiB–aroK–pheAfbr, dm, SacII region modification, Ppc113-glk-Tlpp, Ppc113-galP-Tlpp
pYHP derivative, PBBa_J23100-ScFDC (N-term 6×His-tag)
pTrc99A derivative, Ptrc–SmPAL (C-term FLAG-tag)
pTrc99A derivative, Ptrc-SmPAL (C-term FLAG-tag)-ScFDC (N-term 6×His-tag)
Primer sequences used in this study
Sequence (5′ → 3′)
Cells were inoculated in LB liquid medium (10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl) with 2% (w/v) glucose or PHE medium . PHE medium composition is described below: 20 g/L glucose, 5 g/L (NH4)2SO4, 3 g/L KH2PO4, 3 g/L MgSO4·7H2O, 3 g/L yeast extract, 1.5 g/L sodium citrate, 1 g/L NaCl, 0.3 g/L l-tyrosine, 0.3 g/L l-tryptophan, 0.075 g/L thiamine-HCl, 0.015 g/L CaCl2·2H2O, 0.01125 g/L FeSO4·7H2O, and 1.5 mL/L Trace Metals Solution (TMS) at pH 6.8. TMS is composed of 15 g/L ZnSO4·7H2O, 14.64 g/L MnSO4·H2O, 12 g/L CaCO3, 3 g/L Na2MoO4·2H2O, 2.5 g/L NiSO4·6H2O, 2.5 g/L CuSO4·H2O, 2 g/L Al2(SO4)3·18H2O, 0.75 g/L CoSO4·7H2O, 0.5 g/L H3BO3, and 10 mL/L HCl. After overnight cultivation in LB or PHE medium, 2.5 mL of cells were transferred to 250 mL flasks containing 50 mL of fresh LB or PHE medium and 10 mL of n-dodecane. All flask cultivations were performed at 37 °C shaking at 200 rpm. Two antibiotics (100 μg/mL ampicillin and 40 μg/mL kanamycin) were added for plasmid maintenance, if necessary. When cells reached mid-exponential phase (OD600 of 1.0–1.2), IPTG (Sigma-Aldrich, St. Louis, MO, USA) was added to the final concentration of 1 mM to induce gene expression. After induction, cells were further cultivated in the same conditions (at 37 °C shaking with 200 rpm) which were previously optimized for higher production of precursors (l-phenylalanine and tCA) [12, 13].
Fed-batch cultivations were performed in a 5 L bioreactor (BioCNS, Daejeon, Republic of Korea). For seed culture (200 mL), cells were cultivated in PHE medium containing 3 g/L yeast extract at 37 °C and 200 rpm. After overnight cultivation, the seed was transferred into 1.8 L of same fresh medium in the bioreactor. Furthermore, 100 μg/mL ampicillin and 40 μg/mL kanamycin were also added for plasmid maintenance. An aerobic condition of 40% saturated dissolved oxygen (DO) concentration was maintained by automatically increasing the agitation speed up to 1000 rpm and by mixing pure oxygen during the cultivation . The temperature was maintained at 37 °C, and the pH was kept at 6.8 by the automatic addition of 25% (v/v) ammonia solution, when the pH was lower than 6.77. When the pH value was greater than 6.86, an appropriate volume of feeding solution (500 g/L glucose, 100 g/L casamino acid, and 20 g/L MgSO4·7H2O) was automatically added to avoid glucose depletion. When cell density reached an OD600 of 45, IPTG was added to the final concentration of 1 mM. Moreover, 400 mL of n-dodecane was also added to the culture medium just before induction.
In both flask and fed-batch cultivations, culture samples were periodically collected for the analysis of cell concentration and protein expression. Furthermore, to determine the styrene concentration during the cultivation, organic phases were acquired by phase separation via centrifugation (13,000 rpm for 10 min) of the culture broth. Styrene concentrations in the organic solvent (n-dodecane) was determined using gas chromatography (YL6500; YL instruments, Anyang, Korea) equipped with an HP-5 ms capillary column (30 m × 0.25 mm; Agilent Technology Inc., Santa Clara, CA, USA) . After injection, the samples were detected and analyzed using a flame ionization detector (FID). Column temperature was set to increase from 40 to 135 °C at a rate of 20 °C/min. Styrene concentrations were determined by the standard curves.
KL, HBB, and YHL designed and performed most experiments. KL, HBB, YHL, and KJJ analyzed data. KJJ initiated and coordinated the study and contributed to the experimental design and data interpretation. KL, HBB, and KJJ mainly wrote the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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This work was supported by the Intelligent Synthetic Biology Center of Global Frontier Project (Grant No. 2014M3A6A8066443) funded by the Ministry of Science and ICT (MSIT).
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