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Jun 13, 2019 - 2-Phenylethanol (2-PE) is a fragrance widely used in food and cosmetics. Natural 2-PE is preferred in these applications but with high ...
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Research Article Cite This: ACS Sustainable Chem. Eng. 2019, 7, 12231−12239

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Production of Natural 2‑Phenylethanol from Glucose or Glycerol with Coupled Escherichia coli Strains Expressing L‑Phenylalanine Biosynthesis Pathway and Artificial Biocascades Balaji Sundara Sekar, Benedict Ryan Lukito, and Zhi Li* Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, 117585 Singapore

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S Supporting Information *

ABSTRACT: 2-Phenylethanol (2-PE) is a fragrance widely used in food and cosmetics. Natural 2-PE is preferred in these applications but with high price and very limited availability. Microbial synthesis from glucose or glycerol is an attractive way to produce natural 2-PE, but suffers from low product titer. Escherichia coli NST74-Phe-Sty was engineered to coexpress the L-phenylalanine (L-Phe) biosynthesis pathway and enzyme cascade of L-Phe to 2-PE, producing only 0−0.17 g/L 2-PE from glucose at 22−37 °C due to the incompatibility of the temperature for enzyme expression and activity. Enhanced production of 2-PE (8.4−9.1 g/L) from glucose or glycerol was achieved by coupling of E. coli NST74-Phe expressing the L-Phe biosynthesis pathway for L-Phe production at the optimal temperature of 37 °C with E. coli T7-Sty expressing enzyme cascades of L-Phe to 2-PE at the optimal expression temperature of 22 °C and the optimal biotransformation temperature of 30 °C. The 2-PE titer is 4.7-fold higher than the best reported 2-PE concentration produced from glucose. The coupled strains approach could be generally applicable for enhancing microbial production of useful chemicals from sugars or glycerol. KEYWORDS: Biocatalysis, Biotransformation, Coupled strains approach, 2-Phenylethanol production, Cascade reaction, Sustainable production

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INTRODUCTION

An alternative way of producing natural 2-PE is the microbial synthesis from glucose or glycerol which are green and sustainable sources. Glucose can be obtained from nonedible lignocellulosic biomass such as sugarcane bagasse.10,11 Glycerol can be also considered as a sustainable source when obtained as a byproduct from second generation biodiesel synthesis using nonedible sources such as waste cooking oil12,13 and waste grease.14,15 In addition, biocatalysts are environmental-friendly, nontoxic and biodegradable. Several microorganisms including yeast were reported for the fermentative production of 2-PE from glucose,5,16 but at very low concentration. While the conversion of L-phenylalanine (LPhe) to 2-PE was achieved via the well-known Ehrlich pathway by 3 enzymes,17 the wild-type microorganisms could not efficiently biosynthesize L-Phe. Thus, L-Phe was supplemented to Saccharomyces cerevisiae to enhance the microbial production of 2-PE to 3.60 g/L.18 Overexpression of the Ehrlich pathway in S. cerevisiae or Escherichia coli further increased 2-PE production to 6.119 or 6−9 g/L20,21 from LPhe. Coexpression of the native L-Phe biosynthesis pathway and Ehrlich pathway in E. coli was achieved, but produced only

2-Phenylethanol (2-PE), a high-value aromatic alcohol responsible for the signature rose smell found in rose, tomato, etc.,1 is widely used as a fragrance in the production of perfumes and cosmetics and as an organoleptic enhancer in food.23 2-PE also has antifungal and antibacterial properties,4 making it useful for long-term storage of cosmetics and food without spoilage. The global demand of 2-PE reached 10 000 tons in 20105 with an expected market of $700 million by 2019.6 Most of the commercially available 2-PE are produced by chemical synthesis from nonrenewable, petroleum-based chemicals such as styrene oxide.1,5,7 The catalysts used in chemical synthesis includes ruthenium8 or palladium7 which are highly toxic, carcinogenic chemicals that create health hazard and environmental issues. In addition, chemical synthesis of 2-PE also requires high temperature (300 °C) and pressure which are energy intensive processes.7,8 Moreover, chemically synthesized flavor compounds are less preferred in the food and cosmetics industry and restricted to use as flavoring agents in Europe and U.S.5,9 On the other hand, natural 2-PE is favored for such applications. Currently, natural 2-PE is produced by isolation from plants such as rose and jasmine, with very limited availability and very high price ($1000/kg).5 © 2019 American Chemical Society

Received: March 19, 2019 Revised: May 29, 2019 Published: June 13, 2019 12231

DOI: 10.1021/acssuschemeng.9b01569 ACS Sustainable Chem. Eng. 2019, 7, 12231−12239

Research Article

ACS Sustainable Chemistry & Engineering 0.29−1.2 g/L of 2-PE from glucose.22,23 Phenylacetaldehyde synthase (PAAS) is the enzyme present in plants, catalyzing the conversion of L-Phe to phenylacetaldehyde which can be further enzymatically reduced to 2-PE. Heterologous expression of PAAS in E. coli and yeast was explored, which resulted in very low activity due to the difficulty in functional expression of plant enzyme in microbes.24,25 The development of new enzyme cascades could provide with new methods for one-pot production of useful and valuable chemicals from easily available starting materials in a sustainable way.26−34 Recently, we reported novel artificial enzyme cascades for the synthesis of 2-PE from styrene via epoxidation−isomerization−reduction.35−38 We also developed the cascade biotransformation of natural amino acid LPhe to 2-PE via deamination−decarboxylation−epoxidation− isomerization−reduction39−41 (Figure 1A). Compared to the microbial production of 2-PE from L-Phe via the Ehrlich pathway, our L-Phe to 2-PE artificial enzyme cascades are more efficient and achieved a higher product titer within a shorter time: 8.8−10.4 g/L of 2-PE were produced from L-Phe in 6h

with 85−89% conversion by using E. coli cells expressing the cascades.39−41 A recombinant E. coli expressing the L-Phe biosynthesis pathway and L-Phe to 2-PE artificial cascades was engineered23 to give 1.9 g/L of 2-PE from glucose. Although the product concentration is higher than that with the E. coli expressing the L-Phe biosynthesis and Ehrlich pathway (1.9 vs 1.2 g/L), it is necessary to improve further the product concentration. It is known that engineered E. coli expressing the L-Phe biosynthesis pathway could produce L-Phe from glucose42,43 or glycerol44,45 reaching up to 62 g/L L-Phe46 and cascade conversion of L-Phe gave 10.4 g/L of 2-PE.39 Thus, in principle, the production of 2-PE from glucose could be further improved by efficient combination of the two pathways. Many factors are suspected to be the causes for low 2-PE concentration with an E. coli strain expressing all necessary enzymes.47,48 Here, we report the identification of incompatibility of optimal temperatures for the expression and activity of individual enzymes of the L-Phe biosynthesis pathway and artificial cascade of L-Phe to 2-PE as the key factor for low 2-PE production from glucose using a single recombinant strain. We also report a new strategy by separately expressing the incompatible enzyme systems at their own optimal temperatures in two E. coli strains and coupling the two strains for the production of the final product from glucose or glycerol at the optimal temperature. By using this concept, we successfully demonstrated the enhanced production of 2-PE from sustainable sources such as glucose or glycerol through the coupling of E. coli strains expressing the L-Phe biosynthesis pathway and the artificial L-Phe to 2-PE cascade, respectively (Scheme 1). Scheme 1. Coupled Strains Approach for the Production of 2-PE from Glucose or Glycerol

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Figure 1. (A) Artificial cascade for the production of 2-PE from L-Phe in E. coli T7-Sty. (B) Biosynthesis pathway for the production of LPhe from glucose or glycerol in E. coli NST74-Phe. Dashed arrows indicate multiple enzymatic steps. The genes coding for the proteins highlighted in bold were overexpressed using a T7 expression system. Enzymes: PAL, phenylalanine ammonia lyase; Pad1, phenylacrylic acid decarboxylase; Fdc1, ferulic acid decarboxylase; StyA, styrene monooxygenase; StyB, flavin oxidoreductase; StyC, styrene oxide isomerase; PAR, phenylacetaldehyde reductase; AroG/AroF/AroH, phospho-2-dehydro-3-deoxyheptonate aldolase; AroB, 3-dehydroquinate synthase; AroD, 3-dehydroquinate dehydratase; YdiB/AroE, shikimate dehydrogenase; AroK/AroL, shikimate kinase; AroA, 3phosphoshikimate 1-carboxyvinyltransferase; AroC, chorismate synthase; PheA, bifunctional chorismate mutase/prephenate dehydratase; TyrB/AspC/IlvE, aromatic amino acid aminotransferase. Abbreviations of pathway intermediates: Trans-CA, trans-cinnamic acid; Sty, styrene; StyO, styrene oxide; PA, phenylacetaldehyde; G6P, glucose6-phosphate; G3P, glycerol-3-phosphate; DHAP, dihydroxyacetone phosphate; E4P, erythrose-4-phosphate; PEP, phosphoenolpyruvate; DAHP, 3-deoxy-D-arabino-heptulosonate-7-phosphate; DHQ, 5-dehydro-quinate; DHS, 5-dehydro-shikimate; SHIK, shikimate; S3P, shikimate-5-phosphate; EPSP, 3-enolpyruvylshikimate-5-phosphate; CHO, chorismate; PPA, prephenate; PPY, phenylpyruvate.

EXPERIMENTAL SECTION

Construction and Growth of E. coli NST74-Phe for the Production of L-Phenylalanine. The genes for overexpression of the necessary enzymes to produce L-Phe were amplified by polymerase chain reaction (PCR) from E. coli MG1655 genomic DNA using the primers stated in Table S1 (Supporting Information), and the pictorial representation of the cloning approach is presented in Figure S1 (Supporting Information). Initially, aroG and pheA were mutated to aroG* (AroG-D146N) and pheA* (truncated version of PheA with 1−300 AA) based on the literature.42,49 aroG* was digested with NcoI/BamHI and cloned in the multiple cloning site (MCS)-1 of pCDFDuet. The genes ydiB and aroK were amplified, overlapped using PCR, digested with BamHI/EcoRI, and cloned in the MCS-1 of pCDFDuet. The BamHI-ydiB-aroK-EcoRI fragment was subcloned in the MCS-1 of pCDFDuet-aroG*. The genes pheA* and tyrB were amplified, overlapped using PCR, digested with NdeI/BglII, and cloned in the MCS-2 of pCDFDuet. The NdeI- pheA*- tyrB-BglII fragment was subcloned in the MCS-2 of pCDFDuet-aroG*-ydiBaroK, and the recombinant plasmid was named as pCDF-Phe. PCDFPhe was then transformed into E. coli NST74 (DE3), and the recombinant strain was named as E. coli NST74-Phe. 12232

DOI: 10.1021/acssuschemeng.9b01569 ACS Sustainable Chem. Eng. 2019, 7, 12231−12239

Research Article

ACS Sustainable Chemistry & Engineering E. coli NST74-Phe was inoculated from glycerol stock into 2 mL of LB with streptomycin (50 mg/L) and grown at 37 °C and 220 rpm for 6h. 0.4 mL of culture was inoculated to 20 mL of seed medium (see Supporting Information for medium composition) containing streptomycin (50 mg/L), and the mixture was shaken at 37 °C and 220 rpm. At 2 h, isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 0.1 mM to initiate the expression of enzymes. At 24 h of growth, the culture reached an OD600 of 15. 1 mL of culture was taken, the cells were removed by centrifugation at 10,000g for 10 min, and the supernatant was used for highperformance liquid chromatography (HPLC) analysis to measure LPhe concentration (Figure S3) (see Supporting Information for the analytical method). Construction and Growth of E. coli NST74-Phe-Sty for the Production of 2-PE from Glucose. The two recombinant plasmids, pRSFDuet overexpressing pad1, fdc1, and pal genes as well as pETDuet overexpressing styA, styB, styC,35,39 were transformed into E. coli NST74-Phe. The recombinant strain was named as E. coli NST74Phe-Sty. For the production of 2-PE from glucose, E. coli NST74-Phe-Sty was cultured in 20 mL of seed medium containing streptomycin (50 mg/L), kanamycin (50 mg/L), and ampicillin (100 mg/L) at 37 °C. At 2 h, IPTG was added to a final concentration of 0.1 mM and the growth temperature was set at 22, 30, and 37 °C, respectively. At 24 h, 1 mL of culture was taken and centrifuged at 10,000g for 10 min, and the aqueous phase was analyzed in HPLC to measure L-Phe and 2-PE concentration (see Supporting Information for the analytical method). Production of L-Phenylalanine with E. coli NST74-Phe in Fed-Batch Bioreactor. E. coli NST74-Phe was inoculated from glycerol stock into 100 mL of LB containing streptomycin (50 mg/L) and grown in a 250 mL flask at 37 °C for 12 h to reach an OD600 of 3−4. The culture was transferred to a 3 L bioreactor with 1 L of fermentation medium (for media composition, see Supporting Information). 600 g/L glucose or 800 g/L glycerol solution was fed to maintain the concentration of glucose or glycerol as 5−10 g/L. The pH was controlled at 6.8 by addition of conc. ammonium hydroxide solution. The dissolved oxygen (DO) was maintained between 20 and 30% by supplying air or pure oxygen at 0.5−3 L/min and varying the agitation speed between 500 and 1500 rpm. IPTG was added to a final concentration of 0.1 mM when the cell density reached an OD600 of 15−20. The fermentation was stopped when the rate of consumption of the carbon source decreased to reach a stationary phase. A OD600 values of 80 (24 h) and 145 (28 h) were reached with glucose and glycerol as the carbon source, respectively, and 70−78 mM L-Phe was produced (Figure S5). The fermentation culture containing biosynthesized L-Phe and E. coli NST74-Phe cells was stored at 4 °C or used directly in biotransformation experiments. Production of 2-Phenylethanol from Glucose with Coupled E. coli NST74-Phe and E. coli T7-Sty in Shaking Flask. E. coli NST74-Phe was grown from glucose to produce 70.2 ± 0.2 mM LPhe. 0.2 g of glucose, 0.32 g of K2HPO4, and 0.02 g of KH2PO4 were added to 10 mL of a fermented mixture of E. coli NST74-Phe, to give 2% glucose and 200 mM potassium phosphate, respectively. To the mixture was added 0.6 g of wet cells of E. coli T7-Sty, which was obtained by growing the cells according to previously published procedure35,39 (see Supporting Information for growth procedure), to give a cell concentration of 15 g cdw/L. 10 mL of n-hexadecane, ethyl oleate, or biodiesel was added, and the biotransformation was performed at 30 °C and 220 rpm for 6 h. The reaction mixture was centrifuged at 10,000g for 10 min to separate the aqueous and organic phase. The aqueous samples were analyzed by using reverse phase HPLC (Figure S3, S6), whereas the organic samples were analyzed by using gas chromatography (GC) (Figure S4, S7), to quantify the concentration of 2-PE. Production of 2-Phenylethanol from Glucose or Glycerol with Coupled E. coli NST74-Phe and E. coli T7-Sty in Bioreactor. Production of 2-PE from Glucose via Fermentation with E. coli NST74-Phe and Cascade Reactions with Wet Cells of E. coli T7-Sty. E. coli T7-Sty cells were prepared as described in

Supporting Information. Fed-batch fermentation of E. coli NST74-Phe from glucose was performed as mentioned before to produce 69.6 mM L-Phe in a final volume of 1.61 L. To the fermented media was added 100 g of wet cells of E. coli T7-Sty to give 15 g cdw/L, followed by the addition of 32.3 g of glucose (2% final concentration) as well as 52.7 g of K2HPO4 and 2.6 g of KH2PO4 (to form 200 mM buffer at pH 8.0). 1.61 L of biodiesel was finally added, and the biotransformation was performed at 30 °C and 500 rpm for 6 h. The reaction mixture were centrifuged at 10,000g for 10 min to separate the aqueous buffer and biodiesel, followed by reverse-phase HPLC and GC analysis, respectively. 67.7 mM 2-PE was produced. Production of 2-PE from Glycerol via Fermentation with E. coli NST74-Phe and Cascade Reactions with Wet Cells of E. coli T7-Sty. Similarly, fed-batch fermentation of E. coli NST74-Phe from glycerol was performed as mentioned before to produce 78.2 mM L-Phe in a final volume of 1.43 L. To the mixture was added 85 g of wet cells of E. coli T7-Sty for a final concentration of 15 g cdw/L; 28.6 g of glucose (2% final concentration) as well as 46.8 g of K2HPO4 and 2.3 g of KH2PO4 (to form 200 mM buffer at pH 8.0) were added. 1.43 L of biodiesel was then added, and the biotransformation was performed at 30 °C and 500 rpm for 6 h. The reaction mixture were centrifuged at 10,000g for 10 min to separate the aqueous buffer and biodiesel. The aqueous phase analyzed by reverse-phase HPLC showed no 2-PE and L-Phe, whereas the biodiesel analyzed by GC showed 73.7 mM 2-PE. Production of 2-PE from Glycerol via Fermentation with E. coli NST74-Phe and Cascade Reactions with Culture of E. coli T7-Sty. 480 mL of E. coli T7-Sty (41 g cdw/L) culture was prepared as described in the Supporting Information and added to 1.43 L of fermentation culture from glycerol with E. coli NST74-Phe containing 78.2 mM L-Phe. To that were added 38.2 g of glucose (2% final concentration) as well as 62.5 g of K2HPO4 and 3.1 g of KH2PO4 (to form 200 mM buffer with pH 8.0) were added. 1.91 L of biodiesel was finally added, and biotransformation was performed at 30 °C and 500 rpm for 9 h. The reaction mixture was centrifuged at 10,000g for 10 min to separate the aqueous buffer and biodiesel. The aqueous phase and organic phase were analyzed by reverse-phase HPLC and GC, respectively, giving 55.4 mM 2-PE produced in the organic phase. Isolation of Synthesized 2-Phenylethanol from Fermentation. At the end of biotransformation of glucose or glycerol to 2-PE, the biodiesel was separated from the reaction mixture by centrifugation at 10,000g for 10 min, and 2-PE was obtained from biodiesel by extraction. As a representative example, 10 mL of biodiesel containing 67.7 mM 2-PE was extracted with 5 × 10 mL of water (1:1 v/v), and the aqueous phase was separated and subsequently extracted with 5 × 50 mL ethyl acetate (1:1 v/v). The ethyl acetate was collected and dried using anhydrous sodium sulfate. After filtration, ethyl acetate was evaporated at reduced pressure in a rotary evaporator to give crude 2-PE. Purification by flash chromatography on a silica gel column with n-hexane:ethyl acetate (9:1; Rf ≈ 0.3) as an eluent and subsequent removal of solvents by evaporation at reduced pressure gave 2-PE as a light-yellow liquid with 81.8% yield (69 mg) from LPhe and >98% purity (GC) (Figure S8). 1H nuclear magnetic resonance (NMR) (400 MHz, CDCl3):35 δ = 7.39−7.31 (m, 2H), 7.29−7.23 (m, 3H), 3.89 (t, J = 6.6 Hz, 2H), 2.90 (t, J = 6.6 Hz, 2H) ppm. 13C NMR (100 MHz, CDCl3): δ = 138.67, 129.24, 128.81, 126.70, 63.90, 39.41 ppm (Figure S9, S10).

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RESULTS AND DISCUSSION Engineering of E. coli NST74-Phe-Sty Expressing LPhe Biosynthesis Pathway and Cascade of L-Phe to 2-PE To Produce 2-PE at Different Temperatures. Improvement of L-Phe production from glucose or glycerol was attempted in E. coli NST74 which was shown as a good host for L-Phe production.41,50 Based on the literature,42,49,51 the key enzymes for improving L-Phe production were identified as phospho-2-dehydro-3-deoxyheptonate aldolase (AroG), shikimate kinase (AroK), shikimate dehydrogenase (YdiB), bifunctional chorismate mutase/prephenate dehydratase (PheA), and 12233

DOI: 10.1021/acssuschemeng.9b01569 ACS Sustainable Chem. Eng. 2019, 7, 12231−12239

Research Article

ACS Sustainable Chemistry & Engineering aromatic amino acid aminotransferase (TyrB) (Figure 1B). As AroG and PheA activities are inhibited by L-Phe and/or Ltyrosine, feedback inhibition resistant mutants of AroG (AroG15 renamed as AroG*) and PheA (PheAfbr renamed as PheA*)42 were used. As L-Phe transporter (AroP) is a highly active membrane transporter protein52 and constitutively expressed from the chromosome,53 additional overexpression of AroP was not performed in this study. The genes of the five enzymes were cloned in pCDFduet-1 and transformed to E. coli NST74 to give the recombinant E. coli NST74-Phe. The expression of TyrB (43 kDa), AroG* (38 kDa), PheA* (33 kDa), YdiB (31 kDa), and AroK (19 kDa) in E. coli NST74Phe is evidenced by their protein bands shown in Figure S2 (Supporting Information). L-Phe production from glucose or glycerol was studied using E. coli NST74-Phe. Overexpression of the key enzymes significantly improved L-Phe production from glucose or glycerol. E. coli NST74-Phe produced 3.0−3.4fold higher concentration of L-Phe from glucose and glycerol compared to E. coli NST74 (Figure S2, Supporting Information). After successful improvement of L-Phe production in E. coli NST74-Phe, the genes of the artificial cascade of L-Phe to 2PE35,39 were cloned in E. coli NST74-Phe to give E. coli NST74-Phe-Sty expressing the two pathways for the production of 2-PE directly from glucose. In our previous study,39 it was identified that expression of PAL, Fdc1, and Pad1 from pRSFDuet-1 and StyA, StyB, StyC, and PAR from pETDuet-1 showed the highest conversion of L-Phe to 2-PE. Therefore, the same plasmids were used to construct E. coli NST74-Phe-Sty. As low temperatures are preferred for the regulation of protein production and promotion of active expression of overexpressed enzymes,54−56 cell growth on glucose and expression of the key enzymes in E. coli NST74Phe-Sty were studied at 22, 30, and 37 °C. As shown in the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in Figure 2A, the expression levels of all key enzymes of L-Phe biosynthesis were similar at the tested temperatures. On the other hand, significant amounts of PAL, Fdc1, and StyA, the enzymes of the artificial cascade, were expressed in particulate fractions at 37 °C. 2-PE and L-Phe production from glucose by E. coli NST74Phe-Sty was also studied by growing the cells at 22, 30, and 37 °C for 24 h, respectively (Figure 2B). The OD600 of E. coli NST74-Phe-Sty grown at 22, 30, and 37 °C is provided in Table S2. 1.4, 1.1, and 0 mM 2-PE were produced at 22, 30, and 37 °C, respectively. On the other hand, L-Phe was obtained in 2.2, 2.7, and 5.1 mM at 22, 30, and 37 °C, respectively. These results indicate that the optimal conditions for expression and production of L-Phe is 37 °C while 2-PE production from L-Phe prefers 22 °C. Thus, it is impossible to provide the optimal temperature for both L-Phe biosynthesis and artificial cascade at the same time in a single recombinant E. coli strain. Investigation of Temperature Influence on L-Phe Production with E. coli NST74-Phe and 2-PE Production from L-Phe with E. coli T7-Sty. As the optimal temperatures for L-Phe fermentation from glucose or glycerol and cascade reaction for L-Phe to 2-PE were different, it is necessary to use two different E. coli strains to express the necessary enzymes and catalyze the two parts of the reactions, at their corresponding optimal temperatures. To investigate further the optimal temperature for two different strains, E. coli NST74-Phe was grown at 22, 30 °C,

Figure 2. Production of 2-PE from glucose with E. coli NST74-PheSty. (A) SDS-PAGE: Lane M, Protein marker; Lane C, E. coli NST74; Other lanes, soluble and particulate fractions of E. coli NST74-Phe-Sty grown at 22, 30, and 37 °C for 12 h. (B) Production of L-Phe and 2PE at 24 h with E. coli NST74-Phe-Sty grown on glucose at 22, 30, and 37 °C. The data presented in panel B are the mean values with standard deviation from triplicate experiments.

and 37 °C for 12 h, respectively, and the enzyme expression was studied by SDS-PAGE analysis (Figure 3A). Except for YdiB, all enzymes showed similar expression levels at 22, 30, and 37 °C. Further, the cells were cultured for 24 h, and L-Phe production was studied. Higher L-Phe production was observed at 37 °C, giving 4.2, 8.5, and 10.4 mM L-Phe at 22, 30, and 37 °C, respectively (Figure 3C). This result indicated that 37 °C is the optimal temperature for both the expression of the key enzymes of L-Phe biosynthesis and the production of L-Phe among the tested temperatures. The production of L-Phe with E. coli NST74-Phe was performed in a bioreactor at the optimal temperature of 37 °C using fed-batch fermentation (Figure S2, Supporting Information). L-Phe formation was started after the addition of IPTG at 6 h, and increased in parallel with cell growth on glucose until 15 h and on glycerol until 24 h, respectively. Continuous production of L-Phe was observed even after the cell growth reached the stationary phase. 11.5 g/L (69.6 mM) and 12.9 g/L (78.2 mM) L-Phe were produced at 24 h from glucose and at 28 h from glycerol, respectively. The yield of L-Phe was 0.06 and 0.05 g/g from glucose and glycerol, respectively. Metabolic engineering approaches reported by Ding et al.46 and Liu et al.49 could be applied in the future to improve further L-Phe yield. With continuous monitoring of aeration and feeding, overflow metabolism was prevented, and lower than 1 g/L values of 12234

DOI: 10.1021/acssuschemeng.9b01569 ACS Sustainable Chem. Eng. 2019, 7, 12231−12239

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ACS Sustainable Chemistry & Engineering

Figure 3. (A) SDS-PAGE: Lane M, Protein marker; Lane C, E. coli NST74; Other lanes, soluble and particulate fractions of E. coli NST74-Phe grown at 22, 30, and 37 °C for 12 h. (B) SDS-PAGE: Lane M, Protein marker; Lane C, E. coli T7; Other lanes, soluble and particulate fractions of E. coli T7-Sty grown at 22, 30, and 37 °C for 12h. (C) Effect of growth temperature on the production of L-Phe by E. coli NST74-Phe. The strain was grown at 22, 30, and 37 °C, and production of L-Phe was analyzed at 24 h. (D) Effect of growth temperature of E. coli T7-Sty on the production of 2-PE from L-Phe. E. coli T7-Sty was grown at 22, 30, and 37 °C for 16 h, and the cells were used for biotransformation of 70 mM L-Phe at 30 °C in aqueous buffer and ethyl oleate (1:1, v/v). (E) Effect of biotransformation temperature on the production of 2-PE from L-Phe by E. coli T7-Sty. The strain was grown at 22 °C, and the cells were used for biotransformation of 70 mM L-Phe at 22, 30, and 37 °C in aqueous buffer and ethyl oleate (1:1, v/v). The data presented in panels C, D, and E are the mean values with standard deviation from triplicate experiments.

Table 1. Coupling of the Culture of E. coli NST74-Phe with the Cells of E. coli T7-Sty for the Production of 2-PE from Glucosea 2-PE conc. (mM) fermentation time of E. coli NST74-Phe (h)

conc.b (mM)

L-Phe

cells of E. coli T7-Styc (g cdw/L)

organic solvent

aqueous phased

organic phasee

total

conv. of L-Phe to 2PE (%)

− nhexadecane ethyl oleate biodiesel

18.2 ± 1.1 32.8 ± 1.3

− 11.9 ± 0.7

18.2 ± 1.1 44.7 ± 2.0

25.9 ± 1.5 63.7 ± 2.6

9.7 ± 0.4 6.1 ± 0.8

57.1 ± 1.2 59.3 ± 1.4

66.8 ± 1.6 65.4 ± 2.2

95.2 ± 2.1 93.2 ± 3.1

24 24

70.2 ± 0.1 70.2 ± 0.2

15 15

24 24

70.2 ± 0.1 70.1 ± 0.1

15 15

a

The aqueous reaction medium is 10 mL of cell culture of E. coli NST74-Phe cells containing biosynthesized L-Phe, 200 mM potassium phosphate buffer (pH 8.0), and 2% (w/v) glucose with or without 10 mL organic solvent. bL-Phe in the cell culture of E. coli NST74-Phe. cE. coli T7-Sty was grown at 22 °C for 14 h, and the cells were harvested and added to the reaction medium. The biotransformation was performed for 6 h at 30 °C. d 2-PE conc. in the aqueous phase analyzed by using reverse-phase HPLC. e2-PE conc. in the organic phase analyzed by using GC. The data are the mean value with standard deviation from triplicate experiments.

of L-Phe to 2-PE, were performed by the growth of E. coli T7Sty and biotransformation at 22, 30, and 37 °C, respectively (see Supporting Information for procedure). As shown in Figure 3B, significant differences in expression levels of the enzymes were observed among 22, 30, and 37 °C. Enzymes such as PAL, Fdc1, StyA, and Pad1 were expressed strongly in soluble fractions at 22 °C, whereas at 37 °C most of the enzymes were observed in particulate fractions as inclusion bodies. These results clearly showed that 22 °C is the best temperature for the functional expression of the enzymes. As shown in Figure 3D, the cells grown at 22 °C showed the

other metabolites such as acetate, lactate, and succinate were produced. E. coli T7-Sty was engineered to express PAL, PAD, StyABC, and PAR for the conversion of L-Phe to 2-PE.35,39 These enzymes originated from different sources (PAL, Arabidopsis thaliana; PAD, Aspergillus niger; StyABC, Pseudomonas sp.; PAR, Solanum lycopersicum). The optimal temperature for expression of PAL in E. coli was reported as 22 °C,57 whereas the activity of styrene monooxygenase (StyAB) was studied at 30 °C.58,59 The optimal temperature for functional expression of these enzymes in E. coli T7-Sty and the cascade conversion 12235

DOI: 10.1021/acssuschemeng.9b01569 ACS Sustainable Chem. Eng. 2019, 7, 12231−12239

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ACS Sustainable Chemistry & Engineering

Scheme 2. Flow Diagram for the Production of 2-PE from Glucose or Glycerol by Coupling E. coli NST74-Phe Strain with E. coli T7-Sty Strain

Table 2. Production of 2-PE from Glucose or Glycerol by Coupled Strains Approach in Bioreactor strain

carbon source

fermentation time (h)

cell density (g cdw/L)

final volumea (L)

E. coli NST74-Phe E. coli NST74-Phe E. coli NST74-Phe

glycerol glucose glycerol

24 28 24

49.1 ± 0.5 28.6 ± 0.3 49.1 ± 0.5

1.43 1.61 1.43

(mM)

strain

cell density (g cdw/L)

78.2 ± 2.2 69.6 ± 0.3 78.2 ± 2.2

E. coli T7-Sty E. coli T7-Sty E. coli T7-Sty

16c 15c 10d

L-Phe

b

volume of biodiesel (L)

reaction time (h)

2-PE conc. (mM)

1.45 1.63 1.91

6 6 9

74.8 ± 2.5 68.6 ± 0.4 55.4 ± 0.5e

a

The initial volume of the fed-batch fermentation was 1 L. 800 g/L glycerol or 600 g/L glucose solution was fed intermittently to maintain the concentration at 5−10 g/L during the fermentation. bThe concentration of biosynthesized L-Phe in fermentation medium. cE. coli T7-Sty was grown on glucose in bioreactor for 24 h. The culture was centrifuged, and the cells were added to the fermentation medium of E. coli NST74-Phe. d 480 mL of E. coli T7-Sty culture with a cell density of 41 g cdw/L were added to the fermentation medium of E. coli NST74-Phe. eDue to the addition of the T7-Sty culture, the L-Phe conc. in the reaction was diluted from 78.2 to 58.3 mM. The data are the mean value with standard deviation from triplicate experiments.

biotransformation of L-Phe to 2-PE. For in situ extraction of 2-PE, n-hexadecane, ethyl oleate, and biodiesel were examined. Among them, high 2-PE conversion was observed with ethyl oleate and biodiesel producing 66.8 mM (95.2% conversion) and 65.4 mM (93.2% conversion) 2-PE, respectively. Though first generation biodiesel obtained from edible sources such as palm oil are not sustainable due to its competition for food and fertile land,12 researchers have developed second generation biodiesel from sustainable, nonedible sources such as waste cooking oil, waste grease etc.12−15 Second generation biodiesel (B100) prepared from waste cooking oil was used in this study as a greener and sustainable solvent12,13 for in situ extraction of 2-PE. Production of 2-PE from Glucose or Glycerol by Using Coupled Strains Approach in Bioreactor. The coupled strains approach for the production of 2-PE from glucose or glycerol was attempted as shown in Scheme 2. Production of L-Phe with E. coli NST74-Phe using glucose or glycerol as the carbon source was performed at 37 °C in a bioreactor for 24−28 h. 69.6 and 78.2 mM L-Phe were produced by fermentation of glucose and glycerol, respectively. In parallel to L-Phe production, E. coli T7-Sty was cultured in a bioreactor at 22 °C for 24 h to produce 41 g cdw/L of active cells containing the artificial cascade enzymes (see Supporting Information for procedure). 1.43−1.61 L of L-Phe fermentation mixture containing 69.6−78.2 mM L-Phe and E. coli NST74-Phe cells were added to a 5 L bioreactor and the wet cells of E. coli T7-Sty were added to the fermentation mixture to a final concentration of 15−16 g cdw/L. The temperature was set at 30 °C, biodiesel was added in 1:1 v/v, and the biotransformation was performed for 6 h. 74.8 mM (9.1 g/L) 2-PE could be produced from the glycerol-fermented media containing 78.2 mM L-Phe with conversion efficiency of 95.7% from L-Phe in 6

highest conversion of L-Phe to 2-PE (68 mM; 97.1% conversion). The cells grown at 37 °C were unable to produce any 2-PE (Figure 3D), which is similar to the result obtained with E. coli NST74-Phe-Sty grown at 37 °C (Figure 2B). To investigate the optimal temperature for catalyzing the cascade bioconversion of L-Phe to 2-PE, the biotransformation with resting cells produced at 22 °C was performed at 22, 30, and 37 °C, respectively (see Supporting Information for procedure). As shown in Figure 3E, 41.6, 68, and 25.8 mM 2-PE were obtained at 22, 30, and 37 °C, respectively. Accordingly, 30 °C is the best temperature for cascade reactions. Coupling of E. coli NST74-Phe and E. coli T7-Sty for the Production of 2-PE from Glucose. As the optimal temperatures for the enzyme expression and reaction of the LPhe biosynthesis pathway and artificial cascade enzymes for 2PE synthesis are different, a coupled strains approach was explored (Scheme 1). With this system, the optimal temperature for enzyme expression and reaction for each of the two parts can be provided to achieve high performance. E. coli NST74-Phe was grown on glucose at 37 °C to produce 70.2 mM (11.6 g/L) L-Phe in a bioreactor. In the meantime, E. coli T7-Sty was grown at 22 °C to produce cells containing the L-Phe to 2-PE enzyme cascades. According to our previous study,39 15 g cdw/L of cells is optimal for the conversion of 50−80 mM L-Phe to 2-PE. Therefore, 15 g cdw/ L of active E. coli T7-Sty cells was obtained by centrifugation and added to the 10 mL of fermentation culture containing 70.2 mM L-Phe. The biotransformation was performed for 6 h at 30 °C to give 18.2 mM 2-PE with 25.9% conversion (Table 1). The inhibitory effect of 2-PE in biotransformation performed in buffer systems was well-reported.60 To solve this problem, an equal amount of high logP organic solvent was added to form a 2-liquid phase system for the 12236

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ACS Sustainable Chemistry & Engineering

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h (Table 2). Similarly, with glucose-fermented media containing 69.6 mM L-Phe, 68.6 mM (8.4 g/L) 2-PE can be produced with 98.6% conversion efficiency from L-Phe. Based on our knowledge, this is the highest titer reported thus far on 2-PE production from glucose or glycerol without any external addition of L-Phe. The two parts of the process was completed in 30−34 h with 24−28 h for L-Phe production from glucose or glycerol and 6 h for the conversion of biosynthesized L-Phe to 2-PE, which is also faster than other reported 2-PE production from glucose.22,23,61 As centrifugation is tedious and energy-consuming on an industrial scale, 2-PE production by addition of E. coli T7-Sty culture without centrifugation, to the glycerol-fermented mixture was attempted (Scheme 2). To limit the dilution of L-Phe concentration by the addition of E. coli T7-Sty culture, the culture was added to a final concentration of 10 g cdw/L to the fermentation mixture containing 78.2 mM L-Phe and E. coli NST74-Phe cells. Due to the increase in volume by the addition of E. coli T7-Sty culture, the initial L -Phe concentration is diluted to 58.3 mM whereas the absolute quantity did not change in the reaction. With biodiesel as the organic phase, biotransformation for 9 h gave 55.4 mM (6.8 g/ L) 2-PE with 95.2% conversion from L-Phe (Table 2). In principle, 2-PE could be easily isolated with high purity from biodiesel using fractional distillation in industries.62 As a laboratorial scale demonstration, we used an extraction method to isolate 2-PE from the reaction mixture. As an example, from a 20 mL reaction mixture, 10 mL of biodiesel was separated which contained 69−75 mM (84−91 mg) 2-PE. Extraction of 2-PE from biodiesel with water followed by extraction of 2-PE from water with ethyl acetate gave 70−73 mg of isolated 2-PE with >98% purity (GC) and 77−82% isolated yield based on LPhe.

Research Article

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.9b01569.

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Detailed information about materials used in this study, experimental methods, supporting data and chromatograms (PDF)

AUTHOR INFORMATION

Corresponding Author

*Z. Li. E-mail: [email protected]. ORCID

Zhi Li: 0000-0001-7370-2562 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was financially supported by National Research Foundation (NRF) Singapore by through AME IRG Grant (Project iGrants number A1783c0014; Project No. 279-000511-305) and Ministry of Education of Singapore by through AcRF Tier 1 Grant (Project No. 279-000-477-112).

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CONCLUSIONS The production of 2-PE from glucose with a single E. coli strain overexpressing both the L-Phe biosynthesis pathway and enzyme cascade of L-Phe to 2-PE resulted in low 2-PE concentration (0−0.17 g/L) at 22−37 °C, due to the incompatibility of the optimal temperatures for the enzyme expression and catalysis of the two pathways. Enhanced production of 2-PE from glucose and glycerol was achieved by coupling two E. coli strains expressing the L-Phe biosynthesis pathway and artificial cascade of L-Phe to 2-PE, respectively. E. coli NST74-Phe expressing the L-Phe biosynthesis pathway produced 12.9 g/L of L-Phe from glucose or glycerol at the optimal temperature of 37 °C in 28 h. E. coli T7-Sty expressing the artificial enzyme cascades at the optimal temperature of 22 °C followed by biotransformation at the optimal temperature of 30 °C gave 97.1% conversion of L-Phe to 2-PE (8.3 g/L). Coupling of E. coli NST74-Phe at 37 °C for enzyme expression and L-Phe production with E. coli T7-Sty at 22 °C for enzyme expression and at 30 °C for biotransformation produced 8.6 and 9.1 g/L of 2-PE from glucose and glycerol, respectively, with second generation biodiesel as an in situ extraction solvent. The product titer is 4.7-fold higher than the best result reported thus far for 2-PE production from glucose. The coupled strains approach could be generally applicable for enhancing microbial synthesis of valuable chemicals from sustainable carbon sources such as sugars and glycerol by avoiding the incompatibility of optimal temperatures for expression and function of different enzymes in a single recombinant strain. 12237

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