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Production of rosmarinic acid with ATP and CoA double regenerating system
Enzyme and Microbial Technology 131 (2019) 109392
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Enzyme and Microbial Technology journal homepage: www.elsevier.com/locate/enzmictec
Production of rosmarinic acid with ATP and CoA double regenerating system ⁎
Yi Yana,1, Pu Jiab,1, Yajun Baib, Tai-Ping Fanc, Xiaohui Zhengb, , Yujie Caia,
T
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a
The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China College of Life Sciences, Northwest University, Xi’an, Shaanxi 710069, China c Department of Pharmacology, University of Cambridge, Cambridge CB2 1T, UK b
A R T I C LE I N FO
A B S T R A C T
Keywords: Rosmarinic acid Cell-Free ATP CoA Regeneration system
Rosmarinic acid (RA), as a hydroxycinnamic acid ester of caffeic acid (CA) and 3,4-dihydroxyphenyllactic acid (3,4-DHPL), is a phenylpropanoid-derived plant natural product and has diverse biological activities. This work acts as a modular platform for microbial production using a two-cofactor (ATP and CoA) regeneration system to product RA based on a cell-free biosynthetic approach. Optimal activity of the reaction system was pH 8 and 30 °C. Total turnover number for ATP and CoA was 820.60 ± 28.60 and 444.50 ± 9.65, respectively. Based on the first hour data, the RA productivity reached 320.04 mg L−1 h−1 (0.889 mM L−1 h−1).
1. Introduction
lyase (PAL), 4-coumarate:coenzyme A ligase (4CL), tyrosine aminotransferase (TAT), 4-hydroxyphenylpyruvate reductase (HPPR), rosmarinic acid synthase (RAS) and P450 monooxygenases, catalyze the precursors to form RA [1]. However, the RA yield of the engineered E. coli was very low. In the present report, we focused on building the adenosine-5′-triphosphate (ATP) regeneration system for RA biosynthesis through a four-enzyme cascade (Fig. 1). A two-cofactor double-cycle system was established, which produced tiny amounts of by-product, and produced RA with high efficiency. The At4CL catalyzes CA and CoA, leading to the production of caffeoyl-CoA. During this reaction, costly cofactors such as ATP and CoA are continuously required. Direct addition of ATP is not only expensive but high concentrations inhibit enzyme activity and also lead to the accumulation of inhibitory by-products such as ADP or AMP [18]. Therefore, AjPPK2 and SmPPK2 are added to enable the ATP to be recycled. The CbRAS is the enzyme for the end of the synthesis pathway, which combines caffeoyl-CoA and 3,4-DHPL into RA, and CoA is released for recycling. Additionally, the optimum reaction conditions for RA biosynthesis were investigated.
Rosmarinic acid (RA), as defense compounds, is an ester of caffeic acid (CA) and 3,4-dihydroxyphenyllactic acid (3,4-DHPL). Recent research showed that RA is widely distributed in plants - from one of the oldest groups of terrestrial plants (hornworts) to the highly evolved species of the monocotyledonous and eudicotyledonous plants [1]. It was isolated as a pure compound from the plant Rosmarinus officinalis for the first time and named accordingly [2]. The RA and its derivatives have diverse biological functions, including anti-inflammatory [3,4], anti-oxidant [5,6], anti-tumor [7,8], anti-microbial and anti-viral properties [9] and in immune regulation [10]. These attributes have increased demands for biotechnological production and application of RA and its derivatives. However, due to the limited availability of raw material in nature and the complexity of chemical synthesis, the RA supply is unable to meet the growing demand. Chemical synthesis of RA was first achieved by Albrecht [11]. Subsequently, chemical syntheses of RA and its derivatives have been described, such as for the methyl ester, lithospermic acid, different stereoisomers, yunnaneic acid and the less hydroxylated isorinic acid [12]. Recently, biosyntheses of RA and derivatives in Escherichia coli were also achieved [13–16]. The biosynthetic pathway of RA from the precursors aromatic amino acid L-phenylalanine and L-tyrosine was established for Coleus blumei (Lamiaceae) [17]. These enzymes, including cinnamic acid 4-hydroxylase (CAH), phenylalanine ammonia-
2. Materials and methods 2.1. Bacterial strains, plasmids, and reagents The E. coli strains JM109 and BL21 (DE3) were used as host strains
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Corresponding authors. E-mail addresses: [email protected] (X. Zheng), [email protected] (Y. Cai). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.enzmictec.2019.109392 Received 2 May 2019; Received in revised form 31 July 2019; Accepted 4 August 2019 Available online 05 August 2019 0141-0229/ © 2019 Elsevier Inc. All rights reserved.
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Fig. 1. Scheme of four-enzyme cascade for synthesis of RA with the ATP and CoA-regeneration system. The involved enzymes are: SmPPK2 = polyphosphate kinase from Sinorhizobium meliloti; AjPPK2 = polyphosphate kinase from Acinetobacter johnsonii; At4CL = 4coumarate: coenzyme A ligase from Arabidopsis thaliana; CbRAS = rosmarinic acid synthase from Coleus blumei.
molecular weights of pure enzymes were close to those expected based on the putative amino acid sequences, see the Supplementary Material (Figure S1). Protein concentration was determined by BCA Protein Concentration Assay Kit (TaKaRa).
for cloning and expression, respectively. Plasmid pRSFduet-1 was purchased from TaKaRa (Dalian, China), and kanamycin (50 μg/mL) was used for the positive strain. All restriction enzymes and DNA ligase were purchased from TaKaRa. The IPTG, ATP and CoA were purchased from Shuangjin chemical., LTD (Wuhan, China); MgCl2 from Shanghai Sangon Biological Engineering Technology (Shanghai, China); CA and 3,4-DHPL from Sigma–Aldrich (Shanghai, China); and polyP from Wangan Fine Chemical Ltd. (Suzhou, China).
2.4. Product detection and quantification via high-performance liquid chromatography-mass spectrometry (HPLC–MS) All samples were analyzed using a Waters MALDI SYNAPT Q-TOF MS in negative-ion electrospray mode. The instrument was tuned for a range of 20–2000 m/z. The HPLC conditions follow: solvent A = H2O with 0.1% methanoic acid; solvent B = acetonitrile; 0.5 min at 10% acetonitrile, a gradient to 100% acetonitrile over 15 min. The flow rate was 1.0 mL⋅ min–1 and the absorption was determined at 280 nm. Standard calibration curves were generated from a series of standard compounds of known concentrations. If not otherwise stated, all reactions were performed and analyzed at least in triplicate.
2.2. Plasmid construction The AjPPK2 from Acinetobacter johnsoni [19], SmPPK2 from Sinorhizobium meliloti [20], At4CL from Arabidopsis thaliana [21] and CbRAS from Coleus blumei [22] were overexpressed in E. coli BL21 (DE3). Four genes were codon-optimized for E. coli expression and submitted to National Center for Biotechnology Information (ajppk2, GenBank ID: MK609539; smppk2, GenBank ID: MK605595; 4 cl, GenBank ID: MK676011; and ras, GenBank ID: MK609538). The gene syntheses, primer syntheses and DNA sequencing were performed by Shanghai Talen-bio Biological Technology Co. Ltd (Shanghai, China). Target genes were amplified by PCR using the appropriate primer sets, see the Supplementary Material (Table S1). Genes were cloned into pRSFduet-1 to construct plasmids pR-AjPPK2, pR-SmPPK2, pR-At4CL and pR-CbRAS, respectively. The E. coli BL21 (DE3) was transformed with the above plasmids, generating recombinant strains BLPPK2-I, BLPPK2-II, BL4CL, and BLRAS.
2.5. Optimization of pH and temperature The reaction velocity was determined by measuring the conversion of CA under different reaction conditions. The reaction mixture consisted of 10 mM MgCl2, 1 mM CA, 1 mM 3,4-DHPL, 2 μM CoA, 50 μM ATP, 10 mM polyP (calculated as single phosphate residues), 0.5 mg mL–1 At4CL, 0.5 mg mL–1 CbRAS, 0.05 mg mL–1 AjPPK2 and 0.05 mg mL–1 SmPPK2. The reactions started upon addition of enzymes, and the enzymes were added in order of the sequential cascade reaction. Reactions were terminated by adding 2.5% (v/v) HClO4. The optimal pH for the four-enzyme cascade was determined by experiments using a pH range of 3–10at 30 °C; similarly the optimal temperature was determined using the temperature range of 20–50 °C at optimal pH.
2.3. Enzymes used in cascades (cloning, expression and purification) The engineered E. coli strain was routinely cultivated in 3 mL of Luria–Bertani (LB) medium with incubation overnight at 37 °C and 200 rpm. The medium was supplemented with kanamycin (50 μg⋅ mL−1) for selection when necessary depending on the vectors harbored by E. coli. Subsequently, cultures were diluted 1 mL into 50 mL fresh LB medium, followed by incubation at 37 °C and 200 rpm. When the OD600 of the culture reached 0.6–0.8, it was induced with 0.4 mM IPTG to trigger recombinant protein expression at 200 rpm and 15 °C for 24 h. The cells were isolated viacentrifugation at 8000 × g and 4 °C for 10 min and washed twice with 50 mM Tris−HCl (pH 8.0). After the cells had been disrupted by sonication, the soluble proteins were purified by HiTrap desalting column and His Trap HP column. Crude and pure enzymes were analyzed by SDS-PAGE, and the apparent
2.6. Comparing conversion efficiency in the presence or absence of ATP regeneration system Reactions contained 50 mM Tris−HCl pH 8.0, 10 mM MgCl2, 1 mM CA, 1 mM 3,4-DHPL, 2 μM CoA, 50 μM ATP and 10 mM polyP (calculated as single phosphate residues). The final reaction volume was 3 mL. Samples were incubated at 30 °C and reactions were started upon addition of enzymes (presence of ATP regeneration: 0.5 mg mL–1 At4CL, 0.5 mg mL–1 CbRAS, 0.05 mg mL–1 AjPPK2 and 0.05 mg mL–1 SmPPK2; absence of ATP regeneration: 0.5 mg mL–1 At4CL and 0.5 mg mL–1 2
Enzyme and Microbial Technology 131 (2019) 109392
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Fig. 2. Bioconversion of CA and 3,4-DHPL to RA by the four-enzyme cascade. Transformants were cultured at 30 °C for 30 min. Supplemented with 2 μM CoA, 1 μM ATP, 10 mM MgCl2, 10 mM polyP (calculated as single phosphate residues), 1 mM CA and 1 mM 3,4-DHPL and processed for HPLC analysis. Negative controls were performed analogously except that no enzyme was added. An analytical standard of RA was used to confirm identity of the RA peak, and mass spectra confirmed the presence of RA.
For pH < 4 or pH > 11, the cascade reaction rate was practically halted. Because the four enzymes are associated with the whole reaction, the pH and temperature of different enzymes should match each other. Fortunately, the results were consistent with expectations. The AjPPK2 was stable for pH 7–11 and optimum pH was 8.0–9.0; the optimum temperature for AjPPK2 was 50 °C, and the relative activity at 30 and 37 °C represented 31.2% and 55.6%, respectively; furthermore, AjPPK2 was stabilized in the presence of polyP [19]. The SmPPK2 had a broad pH optimum (pH 8.0–9.5) and showed no activity in the absence of a divalent metal cation [20]. The temperature optimum of CbRAS was 40–45 °C, and the pH optimum was pH 7.8 [23]. Although the optimum pH and temperature of these enzymes were inconsistent, they effectively worked together.
CbRAS). The reaction was terminated by adding 2.5% (v/v) HClO4. Negative controls were performed analogously except that no enzyme was added. 2.7. Conversions and number of cofactor regeneration cycles The reactions of calculated conversions and number of cofactor regeneration cycles were performed analogously to the above. This corresponds to the total turnover number (TTN) calculated as the amount of RA produced divided by the catalytic amount of CA. The main changes were concentrations of CoA and ATP: calculated CoA regeneration cycles, 2 μM CoA and 50 μM ATP; and calculated ATP regeneration cycles, 2 μM CoA and 1 μM ATP. 3. Results
3.3. One-pot RA synthesis with two-cofactor regeneration system 3.1. Identifying RA production The time curve of the reaction with or without AjPPK2 and SmPPK2 enzymes for different ATP concentrations is shown in Fig. 4. Interestingly, conversion yield at both 1 mM and 1 μM ATP concentrations was very low without ATP regeneration. The main reason may be that RAS had reserve activity to split RA into caffeoyl-CoA and 3,4-DHPL [24]. The ATP regeneration system greatly improved the productivity of RA, even at ATP concentration of 1 μM. There were changes in each substrate concentration in the presence or absence of ATP regeneration, with different concentrations of ATP, and with other conditions (i.e.2 μM CoA, pH 8, 30 °C) unchanged (Fig. 5). When 1 mM ATP was added in the absence of ATP regeneration, after incubation for 2 h, we detected the highest RA concentration of 0.05 mM (Fig. 5a); in addition, concentrations of CA and 3,4-DHPL decreased over time. Very little RA was produced in the reaction when 1 μM ATP was added in the absence of ATP regeneration, and CA and 3,4-DHPL also decreased with time (Fig. 5b). When 50 μM ATP was added with ATP regeneration, the initial reaction rate was very fast; after about 1 h of reaction, the conversion rate of substrate reached a maximum of 88.9% (Fig. 5c). When 1 μM ATP was added with ATP
After biotransformation for 30 min, the metabolites were subjected to HPLC analysis (Fig. 2). Starting materials were 10 mM MgCl2, 1 mM CA, 1 mM 3,4-DHPL, 2 μM CoA, 50 μM ATP and 10 mM polyP (calculated as single phosphate residues). Negative controls were performed analogously except that no enzyme was added. The profile of the negative controls showed that Rt of 3,4-DHPL and CA were 5.26 and 7.73 min, respectively, see the Supplementary Material (Table S2). A new peak at 9.17 min was observed in the reaction supplied with enzymes, which conformed to the standard; the molecular mass ion of this peak was 359.07 [M−H]−, suggesting that RA was produced successfully. 3.2. Effects of pH and temperature Temperature and pH profiles of the four-enzyme cascade for RA production are shown in Fig. 3. The optimum temperature and pH for the cascade reaction was 30 °C and 8.0, respectively. The results showed a relatively narrow pH and temperature ranges for the entire reaction. 3
Enzyme and Microbial Technology 131 (2019) 109392
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Fig. 3. Effect of pH and temperature on the four-enzyme cascade for RA production. (a) Effect of temperature on RA production. Productivity was assayed in 50 mM Tris−HCl buffer (pH 8.0) at 10–55 °C. Maximum productivity at 30 °C was defined as 100%. (b) Effect of pH on RA production. Productivity activity was measured in buffers with various pH (4.0–11.0) at 30 °C. The maximum productivity at pH 8.0 was defined as 100%. Results are means ± SD of three parallel replicates.
concentrations cannot provide enough energy for the reaction. Bloch et al redesigned chimeric RA biosynthetic pathway for microbial production of RA [15]. However, the activity of hydroxylase cloned from E. coli is very low, and RA cannot be produced quickly. Instead, it produced large amounts of isorinic acid, fourfold in relation to RA. The yield of RA was only 1.8 ± 0.3 μM after 72 h. Li et al. constructed two or three strains to make the pathway modular [16]. The study has achieved that maintained a delicate balance of the RA synthesis pathway as a non-linear biosynthetic pathway under optimal production conditions to achieve a yield of 172 mg L−1 at 48 h. However, 4hydroxyphenylacetate 3-hydroxylase as a key enzyme has a lower enzyme activity and the substrates pass through too many cell membranes before the reaction, which has a great impact on production. To overcome these problems, we designed and developed an approach to generate RA using an in vitro ATP regeneration system. The approach addresses the growing sustainability concerns associated with the ester bond. To facilitate the uptake and release of substrates and products for the regeneration process, pure enzymes were used. The improved methodology utilizes cell-free metabolic engineering which does not require the addition of numerous cofactors. To exploit this type of cascade chemistry, a powerful ATP regeneration system would be a breakthrough for corresponding industrial processes. The previous conversion ratio of 3,4-DHPL and CA was low at 9.7 ± 0.1%, and 3,4DHPL could be condensed with caffeoyl coenzyme A by RAS to produce 4.33 mg L−1 h−1 RA [14]. In comparison, a more than 70-fold productivity of RA of about 320 mg L−1 h−1 was achieved with our doublecycle regeneration system. By combined action of AjPPK2 and SmPPK2 enzymes, AMP can be successfully converted to ATP. Because polyP was continuously consumed in the process of AMP conversion to ATP, coupled with PPK, use of commercial grade polyP as a low-cost phosphorylating agent can significantly reduce cost [18]. Because of the advantages of high stability and low cost, ATP regeneration demonstrates the power of the reaction using ATP-dependent enzymes. In fact, CoA is not consumed in most applications. The CoA only acts as an acyl carrier that is released after the acyl-transfer reaction and can be reused for activation of another carboxylic acid molecule [26]. The cyclic cascade represents a breakthrough toward a universally applicable cofactor regeneration system. Our research group has carried out long-term research on production of 3,4-DPHL by using L-DOPA as the substrate [27], and the production cost of 3,4-DPHL is about $200/kg. Currently, chemically synthesized CA is already very cheap, about $120/kg. Since the market price of RA is now as high as $3000/kg, it is easier to provide a technically and economically viable production method with this enzyme synthesis method at this stage. In order to save costs, crude enzymes or whole cells are usually used in large-scale production. In addition, more work needs to be done to disrupt endogenous enzymes in E. coli that degrade phenolic compounds, such as HpaD and MhpB [28]. Glucose as the substrate is usually the most economical solution. However, due to
Fig. 4. Time curve of the reaction with or without AjPPK2 and SmPPK2 enzymes under different different ATP concentrations. Negative controls were performed analogously with added 50 μM ATP and 2 μM CoA except that no enzyme was added. Results are means ± SD of three parallel replicates.
regeneration, the reaction was similar to that shown in Fig. 5c, but the amount of RA produced was slightly lower (Fig. 5d); however, this experiment used the lowest amount of ATP, demonstrating that the number of ATP cycles could be higher. The conversion of the substrate into the product was monitored and quantified by HPLC. The reaction with 1 μM ATP (ATP/CoA = 1:2) added resulted in conversions of 82.06 ± 2.86%; corresponding to 820.60 ± 28.60 regeneration cycles for ATP (TTN). The cascade started using a catalytic amount of CoA (2 μM, ATP/CoA = 25:1) for 1 mM CA, and 88.90 ± 1.93% of CA was converted into RA, corresponding to 444.50 ± 9.65 regeneration cycles of CoA (TTN). In the first hour, the substrates were almost converted to RA and the productivity of RA reached 320 mg L−1 h−1 with the double-cycle regeneration system of ATP and CoA in vitro. 4. Discussion Engineered synthesis of RA in E. coli by whole cell biotransformation has resulted in low conversions, and was explained by probable slow transport of 3,4-DHPL into E. coli cells [14]. Cell-free biosynthesis offers advantages over in vivo production, enabling plug-and-play assembly of separately produced enzymes and to determine the optimal reaction mode, and allowing the substrate to enter the reaction environment directly without passing through a membrane [25]. Our experiments showed that not only do membranes hinder the progress of the reaction, but also the amount of ATP is extremely important. High concentrations of ATP inhibit reaction development, and low 4
Enzyme and Microbial Technology 131 (2019) 109392
Y. Yan, et al.
Fig. 5. Time course of biotransformation of CA and 3,4-DHPL into RA in different reaction situations. (a) Starting material: 1 mM CA, 1 mM 3,4-DHPL, 2 μM CoA, 1 mM ATP and 10 mM MgCl2 without ATP regeneration system. (b) Starting material: 1 mM CA, 1 mM 3,4-DHPL, 2 μM CoA, 1 μM ATP and 10 mM MgCl2 without ATP regeneration system. (c) Starting material: 1 mM CA, 1 mM 3,4-DHPL, 2 μM CoA, 1 μM ATP, 10 mM MgCl2 and 10 mM polyP (calculated as single phosphate residues) with ATP regeneration system. (d) Starting material: 1 mM CA, 1 mM 3,4-DHPL, 2 μM CoA, 50 μM ATP, 10 mM MgCl2 and 10 mM polyP (calculated as single phosphate residues) with ATP regeneration system. Results are means ± SD of three parallel replicates.
online version, 109392.
the extremely complex metabolic pathways, it is difficult to obtain an efficient engineered E. coli. In conclusion, we developed a cell-free biotransformation system with a double-cycle regeneration system of ATP and CoA for the production of RA from CA and 3,4-DHPL. To improve reaction sustainability and minimize production costs, it is necessary to perform transformations in vitro to alleviate the requirement to supply stoichiometric ATP and costly CoA cofactors. This approach also creates the opportunity to integrate ester bond formation into the ‘synthetic biology toolbox’ for production of high-value compounds by designer enzyme factories. Cell-free catalysis and the double-cycle regeneration system of ATP and CoA in vitro are very promising in such cases.
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doi:https://doi.org/10.1016/j.enzmictec.2019.
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Compliance with ethical standards This article does not contain any studies with human participants or animals performed by any of the authors. Declaration of Competing Interest The authors declare that they have no conflict of interest. Acknowledgments We thank the National Key Scientific Instrument and Equipment Development Project of China (2013YQ17052504), the Program for Changjiang Scholars and Innovative Research Team in the University of Ministry of Education of China (IRT_15R55), and the seventh group of Hundred-Talent Program of Shanxi Province (2015) for financial support. Appendix A. Supplementary data Supplementary material related to this article can be found, in the 5
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Production of rosmarinic acid with ATP and CoA double regenerating system ⁎
Yi Yana,1, Pu Jiab,1, Yajun Baib, Tai-Ping Fanc, Xiaohui Zhengb, , Yujie Caia,
T
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a
The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China College of Life Sciences, Northwest University, Xi’an, Shaanxi 710069, China c Department of Pharmacology, University of Cambridge, Cambridge CB2 1T, UK b
A R T I C LE I N FO
A B S T R A C T
Keywords: Rosmarinic acid Cell-Free ATP CoA Regeneration system
Rosmarinic acid (RA), as a hydroxycinnamic acid ester of caffeic acid (CA) and 3,4-dihydroxyphenyllactic acid (3,4-DHPL), is a phenylpropanoid-derived plant natural product and has diverse biological activities. This work acts as a modular platform for microbial production using a two-cofactor (ATP and CoA) regeneration system to product RA based on a cell-free biosynthetic approach. Optimal activity of the reaction system was pH 8 and 30 °C. Total turnover number for ATP and CoA was 820.60 ± 28.60 and 444.50 ± 9.65, respectively. Based on the first hour data, the RA productivity reached 320.04 mg L−1 h−1 (0.889 mM L−1 h−1).
1. Introduction
lyase (PAL), 4-coumarate:coenzyme A ligase (4CL), tyrosine aminotransferase (TAT), 4-hydroxyphenylpyruvate reductase (HPPR), rosmarinic acid synthase (RAS) and P450 monooxygenases, catalyze the precursors to form RA [1]. However, the RA yield of the engineered E. coli was very low. In the present report, we focused on building the adenosine-5′-triphosphate (ATP) regeneration system for RA biosynthesis through a four-enzyme cascade (Fig. 1). A two-cofactor double-cycle system was established, which produced tiny amounts of by-product, and produced RA with high efficiency. The At4CL catalyzes CA and CoA, leading to the production of caffeoyl-CoA. During this reaction, costly cofactors such as ATP and CoA are continuously required. Direct addition of ATP is not only expensive but high concentrations inhibit enzyme activity and also lead to the accumulation of inhibitory by-products such as ADP or AMP [18]. Therefore, AjPPK2 and SmPPK2 are added to enable the ATP to be recycled. The CbRAS is the enzyme for the end of the synthesis pathway, which combines caffeoyl-CoA and 3,4-DHPL into RA, and CoA is released for recycling. Additionally, the optimum reaction conditions for RA biosynthesis were investigated.
Rosmarinic acid (RA), as defense compounds, is an ester of caffeic acid (CA) and 3,4-dihydroxyphenyllactic acid (3,4-DHPL). Recent research showed that RA is widely distributed in plants - from one of the oldest groups of terrestrial plants (hornworts) to the highly evolved species of the monocotyledonous and eudicotyledonous plants [1]. It was isolated as a pure compound from the plant Rosmarinus officinalis for the first time and named accordingly [2]. The RA and its derivatives have diverse biological functions, including anti-inflammatory [3,4], anti-oxidant [5,6], anti-tumor [7,8], anti-microbial and anti-viral properties [9] and in immune regulation [10]. These attributes have increased demands for biotechnological production and application of RA and its derivatives. However, due to the limited availability of raw material in nature and the complexity of chemical synthesis, the RA supply is unable to meet the growing demand. Chemical synthesis of RA was first achieved by Albrecht [11]. Subsequently, chemical syntheses of RA and its derivatives have been described, such as for the methyl ester, lithospermic acid, different stereoisomers, yunnaneic acid and the less hydroxylated isorinic acid [12]. Recently, biosyntheses of RA and derivatives in Escherichia coli were also achieved [13–16]. The biosynthetic pathway of RA from the precursors aromatic amino acid L-phenylalanine and L-tyrosine was established for Coleus blumei (Lamiaceae) [17]. These enzymes, including cinnamic acid 4-hydroxylase (CAH), phenylalanine ammonia-
2. Materials and methods 2.1. Bacterial strains, plasmids, and reagents The E. coli strains JM109 and BL21 (DE3) were used as host strains
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Corresponding authors. E-mail addresses: [email protected] (X. Zheng), [email protected] (Y. Cai). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.enzmictec.2019.109392 Received 2 May 2019; Received in revised form 31 July 2019; Accepted 4 August 2019 Available online 05 August 2019 0141-0229/ © 2019 Elsevier Inc. All rights reserved.
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Fig. 1. Scheme of four-enzyme cascade for synthesis of RA with the ATP and CoA-regeneration system. The involved enzymes are: SmPPK2 = polyphosphate kinase from Sinorhizobium meliloti; AjPPK2 = polyphosphate kinase from Acinetobacter johnsonii; At4CL = 4coumarate: coenzyme A ligase from Arabidopsis thaliana; CbRAS = rosmarinic acid synthase from Coleus blumei.
molecular weights of pure enzymes were close to those expected based on the putative amino acid sequences, see the Supplementary Material (Figure S1). Protein concentration was determined by BCA Protein Concentration Assay Kit (TaKaRa).
for cloning and expression, respectively. Plasmid pRSFduet-1 was purchased from TaKaRa (Dalian, China), and kanamycin (50 μg/mL) was used for the positive strain. All restriction enzymes and DNA ligase were purchased from TaKaRa. The IPTG, ATP and CoA were purchased from Shuangjin chemical., LTD (Wuhan, China); MgCl2 from Shanghai Sangon Biological Engineering Technology (Shanghai, China); CA and 3,4-DHPL from Sigma–Aldrich (Shanghai, China); and polyP from Wangan Fine Chemical Ltd. (Suzhou, China).
2.4. Product detection and quantification via high-performance liquid chromatography-mass spectrometry (HPLC–MS) All samples were analyzed using a Waters MALDI SYNAPT Q-TOF MS in negative-ion electrospray mode. The instrument was tuned for a range of 20–2000 m/z. The HPLC conditions follow: solvent A = H2O with 0.1% methanoic acid; solvent B = acetonitrile; 0.5 min at 10% acetonitrile, a gradient to 100% acetonitrile over 15 min. The flow rate was 1.0 mL⋅ min–1 and the absorption was determined at 280 nm. Standard calibration curves were generated from a series of standard compounds of known concentrations. If not otherwise stated, all reactions were performed and analyzed at least in triplicate.
2.2. Plasmid construction The AjPPK2 from Acinetobacter johnsoni [19], SmPPK2 from Sinorhizobium meliloti [20], At4CL from Arabidopsis thaliana [21] and CbRAS from Coleus blumei [22] were overexpressed in E. coli BL21 (DE3). Four genes were codon-optimized for E. coli expression and submitted to National Center for Biotechnology Information (ajppk2, GenBank ID: MK609539; smppk2, GenBank ID: MK605595; 4 cl, GenBank ID: MK676011; and ras, GenBank ID: MK609538). The gene syntheses, primer syntheses and DNA sequencing were performed by Shanghai Talen-bio Biological Technology Co. Ltd (Shanghai, China). Target genes were amplified by PCR using the appropriate primer sets, see the Supplementary Material (Table S1). Genes were cloned into pRSFduet-1 to construct plasmids pR-AjPPK2, pR-SmPPK2, pR-At4CL and pR-CbRAS, respectively. The E. coli BL21 (DE3) was transformed with the above plasmids, generating recombinant strains BLPPK2-I, BLPPK2-II, BL4CL, and BLRAS.
2.5. Optimization of pH and temperature The reaction velocity was determined by measuring the conversion of CA under different reaction conditions. The reaction mixture consisted of 10 mM MgCl2, 1 mM CA, 1 mM 3,4-DHPL, 2 μM CoA, 50 μM ATP, 10 mM polyP (calculated as single phosphate residues), 0.5 mg mL–1 At4CL, 0.5 mg mL–1 CbRAS, 0.05 mg mL–1 AjPPK2 and 0.05 mg mL–1 SmPPK2. The reactions started upon addition of enzymes, and the enzymes were added in order of the sequential cascade reaction. Reactions were terminated by adding 2.5% (v/v) HClO4. The optimal pH for the four-enzyme cascade was determined by experiments using a pH range of 3–10at 30 °C; similarly the optimal temperature was determined using the temperature range of 20–50 °C at optimal pH.
2.3. Enzymes used in cascades (cloning, expression and purification) The engineered E. coli strain was routinely cultivated in 3 mL of Luria–Bertani (LB) medium with incubation overnight at 37 °C and 200 rpm. The medium was supplemented with kanamycin (50 μg⋅ mL−1) for selection when necessary depending on the vectors harbored by E. coli. Subsequently, cultures were diluted 1 mL into 50 mL fresh LB medium, followed by incubation at 37 °C and 200 rpm. When the OD600 of the culture reached 0.6–0.8, it was induced with 0.4 mM IPTG to trigger recombinant protein expression at 200 rpm and 15 °C for 24 h. The cells were isolated viacentrifugation at 8000 × g and 4 °C for 10 min and washed twice with 50 mM Tris−HCl (pH 8.0). After the cells had been disrupted by sonication, the soluble proteins were purified by HiTrap desalting column and His Trap HP column. Crude and pure enzymes were analyzed by SDS-PAGE, and the apparent
2.6. Comparing conversion efficiency in the presence or absence of ATP regeneration system Reactions contained 50 mM Tris−HCl pH 8.0, 10 mM MgCl2, 1 mM CA, 1 mM 3,4-DHPL, 2 μM CoA, 50 μM ATP and 10 mM polyP (calculated as single phosphate residues). The final reaction volume was 3 mL. Samples were incubated at 30 °C and reactions were started upon addition of enzymes (presence of ATP regeneration: 0.5 mg mL–1 At4CL, 0.5 mg mL–1 CbRAS, 0.05 mg mL–1 AjPPK2 and 0.05 mg mL–1 SmPPK2; absence of ATP regeneration: 0.5 mg mL–1 At4CL and 0.5 mg mL–1 2
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Fig. 2. Bioconversion of CA and 3,4-DHPL to RA by the four-enzyme cascade. Transformants were cultured at 30 °C for 30 min. Supplemented with 2 μM CoA, 1 μM ATP, 10 mM MgCl2, 10 mM polyP (calculated as single phosphate residues), 1 mM CA and 1 mM 3,4-DHPL and processed for HPLC analysis. Negative controls were performed analogously except that no enzyme was added. An analytical standard of RA was used to confirm identity of the RA peak, and mass spectra confirmed the presence of RA.
For pH < 4 or pH > 11, the cascade reaction rate was practically halted. Because the four enzymes are associated with the whole reaction, the pH and temperature of different enzymes should match each other. Fortunately, the results were consistent with expectations. The AjPPK2 was stable for pH 7–11 and optimum pH was 8.0–9.0; the optimum temperature for AjPPK2 was 50 °C, and the relative activity at 30 and 37 °C represented 31.2% and 55.6%, respectively; furthermore, AjPPK2 was stabilized in the presence of polyP [19]. The SmPPK2 had a broad pH optimum (pH 8.0–9.5) and showed no activity in the absence of a divalent metal cation [20]. The temperature optimum of CbRAS was 40–45 °C, and the pH optimum was pH 7.8 [23]. Although the optimum pH and temperature of these enzymes were inconsistent, they effectively worked together.
CbRAS). The reaction was terminated by adding 2.5% (v/v) HClO4. Negative controls were performed analogously except that no enzyme was added. 2.7. Conversions and number of cofactor regeneration cycles The reactions of calculated conversions and number of cofactor regeneration cycles were performed analogously to the above. This corresponds to the total turnover number (TTN) calculated as the amount of RA produced divided by the catalytic amount of CA. The main changes were concentrations of CoA and ATP: calculated CoA regeneration cycles, 2 μM CoA and 50 μM ATP; and calculated ATP regeneration cycles, 2 μM CoA and 1 μM ATP. 3. Results
3.3. One-pot RA synthesis with two-cofactor regeneration system 3.1. Identifying RA production The time curve of the reaction with or without AjPPK2 and SmPPK2 enzymes for different ATP concentrations is shown in Fig. 4. Interestingly, conversion yield at both 1 mM and 1 μM ATP concentrations was very low without ATP regeneration. The main reason may be that RAS had reserve activity to split RA into caffeoyl-CoA and 3,4-DHPL [24]. The ATP regeneration system greatly improved the productivity of RA, even at ATP concentration of 1 μM. There were changes in each substrate concentration in the presence or absence of ATP regeneration, with different concentrations of ATP, and with other conditions (i.e.2 μM CoA, pH 8, 30 °C) unchanged (Fig. 5). When 1 mM ATP was added in the absence of ATP regeneration, after incubation for 2 h, we detected the highest RA concentration of 0.05 mM (Fig. 5a); in addition, concentrations of CA and 3,4-DHPL decreased over time. Very little RA was produced in the reaction when 1 μM ATP was added in the absence of ATP regeneration, and CA and 3,4-DHPL also decreased with time (Fig. 5b). When 50 μM ATP was added with ATP regeneration, the initial reaction rate was very fast; after about 1 h of reaction, the conversion rate of substrate reached a maximum of 88.9% (Fig. 5c). When 1 μM ATP was added with ATP
After biotransformation for 30 min, the metabolites were subjected to HPLC analysis (Fig. 2). Starting materials were 10 mM MgCl2, 1 mM CA, 1 mM 3,4-DHPL, 2 μM CoA, 50 μM ATP and 10 mM polyP (calculated as single phosphate residues). Negative controls were performed analogously except that no enzyme was added. The profile of the negative controls showed that Rt of 3,4-DHPL and CA were 5.26 and 7.73 min, respectively, see the Supplementary Material (Table S2). A new peak at 9.17 min was observed in the reaction supplied with enzymes, which conformed to the standard; the molecular mass ion of this peak was 359.07 [M−H]−, suggesting that RA was produced successfully. 3.2. Effects of pH and temperature Temperature and pH profiles of the four-enzyme cascade for RA production are shown in Fig. 3. The optimum temperature and pH for the cascade reaction was 30 °C and 8.0, respectively. The results showed a relatively narrow pH and temperature ranges for the entire reaction. 3
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Fig. 3. Effect of pH and temperature on the four-enzyme cascade for RA production. (a) Effect of temperature on RA production. Productivity was assayed in 50 mM Tris−HCl buffer (pH 8.0) at 10–55 °C. Maximum productivity at 30 °C was defined as 100%. (b) Effect of pH on RA production. Productivity activity was measured in buffers with various pH (4.0–11.0) at 30 °C. The maximum productivity at pH 8.0 was defined as 100%. Results are means ± SD of three parallel replicates.
concentrations cannot provide enough energy for the reaction. Bloch et al redesigned chimeric RA biosynthetic pathway for microbial production of RA [15]. However, the activity of hydroxylase cloned from E. coli is very low, and RA cannot be produced quickly. Instead, it produced large amounts of isorinic acid, fourfold in relation to RA. The yield of RA was only 1.8 ± 0.3 μM after 72 h. Li et al. constructed two or three strains to make the pathway modular [16]. The study has achieved that maintained a delicate balance of the RA synthesis pathway as a non-linear biosynthetic pathway under optimal production conditions to achieve a yield of 172 mg L−1 at 48 h. However, 4hydroxyphenylacetate 3-hydroxylase as a key enzyme has a lower enzyme activity and the substrates pass through too many cell membranes before the reaction, which has a great impact on production. To overcome these problems, we designed and developed an approach to generate RA using an in vitro ATP regeneration system. The approach addresses the growing sustainability concerns associated with the ester bond. To facilitate the uptake and release of substrates and products for the regeneration process, pure enzymes were used. The improved methodology utilizes cell-free metabolic engineering which does not require the addition of numerous cofactors. To exploit this type of cascade chemistry, a powerful ATP regeneration system would be a breakthrough for corresponding industrial processes. The previous conversion ratio of 3,4-DHPL and CA was low at 9.7 ± 0.1%, and 3,4DHPL could be condensed with caffeoyl coenzyme A by RAS to produce 4.33 mg L−1 h−1 RA [14]. In comparison, a more than 70-fold productivity of RA of about 320 mg L−1 h−1 was achieved with our doublecycle regeneration system. By combined action of AjPPK2 and SmPPK2 enzymes, AMP can be successfully converted to ATP. Because polyP was continuously consumed in the process of AMP conversion to ATP, coupled with PPK, use of commercial grade polyP as a low-cost phosphorylating agent can significantly reduce cost [18]. Because of the advantages of high stability and low cost, ATP regeneration demonstrates the power of the reaction using ATP-dependent enzymes. In fact, CoA is not consumed in most applications. The CoA only acts as an acyl carrier that is released after the acyl-transfer reaction and can be reused for activation of another carboxylic acid molecule [26]. The cyclic cascade represents a breakthrough toward a universally applicable cofactor regeneration system. Our research group has carried out long-term research on production of 3,4-DPHL by using L-DOPA as the substrate [27], and the production cost of 3,4-DPHL is about $200/kg. Currently, chemically synthesized CA is already very cheap, about $120/kg. Since the market price of RA is now as high as $3000/kg, it is easier to provide a technically and economically viable production method with this enzyme synthesis method at this stage. In order to save costs, crude enzymes or whole cells are usually used in large-scale production. In addition, more work needs to be done to disrupt endogenous enzymes in E. coli that degrade phenolic compounds, such as HpaD and MhpB [28]. Glucose as the substrate is usually the most economical solution. However, due to
Fig. 4. Time curve of the reaction with or without AjPPK2 and SmPPK2 enzymes under different different ATP concentrations. Negative controls were performed analogously with added 50 μM ATP and 2 μM CoA except that no enzyme was added. Results are means ± SD of three parallel replicates.
regeneration, the reaction was similar to that shown in Fig. 5c, but the amount of RA produced was slightly lower (Fig. 5d); however, this experiment used the lowest amount of ATP, demonstrating that the number of ATP cycles could be higher. The conversion of the substrate into the product was monitored and quantified by HPLC. The reaction with 1 μM ATP (ATP/CoA = 1:2) added resulted in conversions of 82.06 ± 2.86%; corresponding to 820.60 ± 28.60 regeneration cycles for ATP (TTN). The cascade started using a catalytic amount of CoA (2 μM, ATP/CoA = 25:1) for 1 mM CA, and 88.90 ± 1.93% of CA was converted into RA, corresponding to 444.50 ± 9.65 regeneration cycles of CoA (TTN). In the first hour, the substrates were almost converted to RA and the productivity of RA reached 320 mg L−1 h−1 with the double-cycle regeneration system of ATP and CoA in vitro. 4. Discussion Engineered synthesis of RA in E. coli by whole cell biotransformation has resulted in low conversions, and was explained by probable slow transport of 3,4-DHPL into E. coli cells [14]. Cell-free biosynthesis offers advantages over in vivo production, enabling plug-and-play assembly of separately produced enzymes and to determine the optimal reaction mode, and allowing the substrate to enter the reaction environment directly without passing through a membrane [25]. Our experiments showed that not only do membranes hinder the progress of the reaction, but also the amount of ATP is extremely important. High concentrations of ATP inhibit reaction development, and low 4
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Fig. 5. Time course of biotransformation of CA and 3,4-DHPL into RA in different reaction situations. (a) Starting material: 1 mM CA, 1 mM 3,4-DHPL, 2 μM CoA, 1 mM ATP and 10 mM MgCl2 without ATP regeneration system. (b) Starting material: 1 mM CA, 1 mM 3,4-DHPL, 2 μM CoA, 1 μM ATP and 10 mM MgCl2 without ATP regeneration system. (c) Starting material: 1 mM CA, 1 mM 3,4-DHPL, 2 μM CoA, 1 μM ATP, 10 mM MgCl2 and 10 mM polyP (calculated as single phosphate residues) with ATP regeneration system. (d) Starting material: 1 mM CA, 1 mM 3,4-DHPL, 2 μM CoA, 50 μM ATP, 10 mM MgCl2 and 10 mM polyP (calculated as single phosphate residues) with ATP regeneration system. Results are means ± SD of three parallel replicates.
online version, 109392.
the extremely complex metabolic pathways, it is difficult to obtain an efficient engineered E. coli. In conclusion, we developed a cell-free biotransformation system with a double-cycle regeneration system of ATP and CoA for the production of RA from CA and 3,4-DHPL. To improve reaction sustainability and minimize production costs, it is necessary to perform transformations in vitro to alleviate the requirement to supply stoichiometric ATP and costly CoA cofactors. This approach also creates the opportunity to integrate ester bond formation into the ‘synthetic biology toolbox’ for production of high-value compounds by designer enzyme factories. Cell-free catalysis and the double-cycle regeneration system of ATP and CoA in vitro are very promising in such cases.
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doi:https://doi.org/10.1016/j.enzmictec.2019.
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Compliance with ethical standards This article does not contain any studies with human participants or animals performed by any of the authors. Declaration of Competing Interest The authors declare that they have no conflict of interest. Acknowledgments We thank the National Key Scientific Instrument and Equipment Development Project of China (2013YQ17052504), the Program for Changjiang Scholars and Innovative Research Team in the University of Ministry of Education of China (IRT_15R55), and the seventh group of Hundred-Talent Program of Shanxi Province (2015) for financial support. Appendix A. Supplementary data Supplementary material related to this article can be found, in the 5
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