High-level soluble expression of Serratia marcescens H30 lipase in Escherichia coli Erzheng Sua,*, Jingjing Xub, Xiangping Wub a
Enzyme and Fermentation Technology Laboratory, College of Light Industry Science and Engineering, Nanjing Forestry University, Nanjing 210037, P. R. China.
b
State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, P.R .China.
* Corresponding author: Erzheng Su Tel. +86 25 85428906
Fax: +86 25 85428906
E-mail address: [email protected]
Running Title：High-level soluble expression of lipase
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/bab.1248. This article is protected by copyright. All rights reserved.
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Abstract Serratia marcescens lipase (SmL) is an important biocatalyst used to enantioselectively hydrolyze (±)-trans-3-(4-methoxyphynyl) glycidic acid methyl ester. However, the economically justified level recombinant soluble expression of SmL in Escherichia coli has not been established. Thus, fusion genes of lipase from S. marcescens H30 with different fusion tags were constructed and expressed in E. coli. The effects of fusion tags were revealed. A significant increase in recombinant lipase solubility showed that E. coli BL21 (DE3)/pET32a-SmL was a suitable choice for SmL production. To optimize the performance of recombinant SmL production, changes in culture medium compositions and induction conditions were systematically tested. Finally, the recombinant SmL activity and productivity reached approximately 23,000 U/L and 1278 U L−1 h−1 in shake flasks, respectively. This value is the highest SmL activity attained by heterogeneous recombinant expression in E. coli. Lipase activity and productivity reached 19,650 U/L and 1228 U L−1 h−1, respectively, by scaling up SmL production in a 7.0 L fermenter. The existence of the Trx tag did not influence the chiral selectivity of recombinant SmL. These findings indicate a possibility for soluble and economical SmL expression in E. coli to meet industrial needs. Keywords: Serratia marcescens H30; Lipase; Fusion tags; Solubility; Culturing medium; induction conditions.
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1. Introduction Lipases (triacylglycerol ester hydrolases, EC 3.1.1.3) are ubiquitous in animals, plants, and microorganisms. They can catalyze the hydrolysis of medium- and long-chain triglycerides into fatty acids and glycerol at the oil–water interface, as well as ester synthesis and transesterification reactions with high regioselectivity and stereoselectivity in non-aqueous medium systems [1]. As of this writing, lipases have become excellent biocatalysts in a broad range of industrial applications, including pharmaceutical, fine chemical, and other industrial areas, such as detergents, oil/fats, cheese making, hard-surface cleaning, and leather and paper processing [2]. Among many successful examples of lipase applications, the Serratia marcescens lipase (SmL) is well-known in the pharmaceutical industry for its excellent enantioselectivity in the biocatalytic hydrolysis of trans-3-(4-methoxyphynyl) glycidic acid methyl ester [(±)-MPGM] to produce (2R, 3S)-MPGM [(−)-MPGM], which is a key intermediate for the synthesis of diltiazem hydrochloride [3, 4]. Given the importance of SmL, a productive bioprocess for SmL production should be developed. To increase enzyme production, the effects of medium compositions and cultivation conditions on the accumulation of S. marcescens ECU1010 lipase were optimized in shake flasks, with lipase production only being 4,780 U/L [5]. The strain S. marcescens ECU1010 was further subjected to UV irradiation. A mutant strain (UV-01) has only given a 2.3-fold increase in lipase activity compared with a wild-type strain [6]. SmL genes from different S. marcescens have also been cloned and expressed in recent years. An SmL gene from S. marcescens SM6 was expressed in Escherichia coli, and the lipase activity only reached wild-type level. This gene was further subcloned into the expression vector pSE420, providing a strong trc promoter and containing a bacteriophage T7 gene 10 translational enhancer. However, the level of lipase activity has remained very low [7]. An SmL gene was cloned into pET11d and overexpressed in E. coli JM109/DE3. The overexpressed protein was not secreted but formed insoluble inclusion bodies inside the bacterial cytoplasm, which were refolded to produce
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enzymatically active lipase [8]. The SmL gene from S. marcescens ECU1010 has also been cloned and ligated to the plasmid pET-24a to create an overexpression plasmid. An overproducing strain has been constructed by transforming E. coli BL21 (DE3) with this recombinant plasmid. After the optimization of many physical and chemical factors, lipase production has only increased to 5000–6000 U/L [9]. The aforementioned review of previous studies revealed that an economically justified level expression of SmL in E. coli for industrial purposes has not been achieved. Most SmL genes have been expressed in the form of inclusion bodies; for example, the SmL gene from S. marcescens ECU1010 expressed in soluble fraction is estimated to be only 30% in cell-free extract after optimization [9]. Refolding of inclusion bodies is highly complicated that it wastes considerable money and time [10]. In the current study, a lipase gene from S. marcescens H30, which is a Gram-negative enteric organism previously used for the efficient production of 2,3-butanediol in our laboratory [11], was ligated to three plasmids with different fusion tags to overexpress active lipase in soluble form. Changes in culture medium compositions and conditions were investigated to optimize the performance of SmL production in E. coli. Scale-up cultivation was conducted in a 7.0 L fermenter to determine the feasibility of our strategy.
2. Materials and methods 2.1. Materials Restriction enzymes, T4 DNA ligase, and Taq polymerase premix were supplied by MBI Fermentas (Germany). DNA gel and plasmid extracts were purchased from Generay Biotech (Shanghai) Co., Ltd. Molecular markers (DNA and protein markers) were bought from Thermo Fisher Scientific Inc. (Shanghai, China). Tryptone and yeast extract were obtained from Oxoid Co., Ltd., UK. Carbon sources (glycerol, sucrose, glucose, sorbitol, mannitol, starch, and dextrin), nitrogen sources [fish tryptone, Angel yeast extract, NH4Cl, (NH4)2SO4, and NH4NO3], and inorganic salt (Na2HPO4) were employed for medium composition screening and This article is protected by copyright. All rights reserved.
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optimization. All these sources were of industrial grade and purchased from the Chinese market with the lowest possible price. All other chemicals were of reagent grade and obtained from commercial sources. 2.2. Bacterial strains, plasmids, and culture conditions S. marcescens H30 was deposited in our laboratory. Genomic DNA from S. marcescens H30 was used as the source of SmL gene for PCR amplification. E. coli DH5α and BL21(DE3) were employed as host strains in gene manipulation and protein expression, respectively. The plasmids used for cloning and protein expression were pMD19-T, pET-28a, pET-32a, and pGEX-4T-1. S. marcescens H30 was grown at 30 °C in a nutrient broth (3 g/L beef extract, 5 g/L peptone, and 5 g/L NaCl). Wild-type and recombinant E. coli cells were regularly cultured in Luria–Bertani (LB) medium (10 g/L tryptone, 5 g/L yeast extract, and 5 g/L NaCl) at 37 °C. An appropriate amount of ampicillin or kanamycin was added. 2.3. Cloning and expression of S. marcescens H30 lipase gene in E. coli To obtain the full-length lipase gene from S. marcescens H30, total genome was extracted. Two specific primers, namely, forward 5'-GGTTGAATTCGGCATCTTTAGCTATAAGGA-3' (EcoRI cutting site is underlined) and reverse 5'-CCTTGCGGCCGCTAGGCCAACACCACCTGATC-3' (NotI cutting site is underlined), were designed according to the reported SmL gene sequence [7]. PCR amplifications were performed at 94 °C for 4 min, followed by 30 cycles of 94 °C for 30 s, 58 °C for 1 min, and 72 °C for 1 min, with a final extension step of 8 min at 72 °C. The PCR product was separated, cloned into pMD19-T, and sequenced. The DNA and protein sequence similarities were assessed using BLASTN and BLASTP programs [12]. Three plasmids, namely, pET28a (6 × His tag), pET32a (Trx tag), and pGEX-4T-1 (GST tag), with different fusion tags were chosen as recombinant expression plasmids. The obtained SmL gene and plasmids were digested with EcoRI and NotI restriction enzymes simultaneously, and then ligated to construct the SmL
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expression vectors pET28a-SmL, pET32a-SmL, and pGEX-4T-1-SmL. These expression plasmids were transformed into E. coli BL21 (DE3). A single colony was inoculated into 3 mL of LB medium in a 10 mL test tube supplemented with kanamycin (50 μg/mL) or ampicillin (100 μg/mL) according to the different selection markers of plasmids and hosts, and cultivated at 37 °C and 200 rpm overnight as seed. Subsequently, 1% (v/v) seed was inoculated into 100 mL of LB medium in a 500 mL Erlenmeyer flask, and grown for 2.5–3.0 h at 37 °C and 200 rpm until OD600 reached 0.6–0.8. IPTG was added to a final concentration of 0.5 mM for inducing SmL expression. Cultivation continued for another 8–10 h at 25 °C. Cells were collected by centrifugation at 6000 ×g for 10 min, resuspended, and washed twice with 50 mM Tris-HCl buffer (pH 8.0). Biomass was monitored during cultivation by measuring the optical density of culture broth at 600 nm. Cells were disrupted by an ultrasonic cell disruptor (Ningbo Scientz, China) in an ice bath at a power controlled from 200 W to 400 W. Each circle worked for 4 s, intermittent for 5 s, and continued for 20 min. The disrupted cells were collected by centrifugation at 12,000 ×g for 10 min. The supernatants were used for SmL activity and SDS-PAGE analysis. 2.4. Optimization of recombinant SmL production in shake flask Shake flask culture experiments are usually performed in 250 mL flasks containing 50 mL of medium. In this study, LB medium was employed as the initial medium for optimization. After inoculation with 1% (v/v) seed culture, the flasks were shaken at 37 °C and 200 rpm for different times to attain different OD600. Different concentrations of lactose were then added. Induction continued for different times at varied temperatures. A systematic optimization of the culture medium compositions and conditions was conducted to enhance the recombinant SmL production in shake flask. 2.5. Batch fermentation in a 7.0 L fermenter The scale-up cultivation of recombinant E. coli BL21 (DE3)/pET32a-SmL was conducted in a 7.0 L
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fermenter with a working volume of 4.0 L. The fermentation medium was the same as the optimized culture medium in shake flask cultivation. Seed culture was performed in 500 mL flasks with a working volume of 100 mL. Cultivation in the fermenter was conducted at 37 °C until OD600 of recombinant E. coli cells reached 0.7. Lactose (15 g/L) was added to induce SmL expression at 20 °C. The pH was controlled at 7.0 using 2 M NaOH or H2SO4. The dissolved oxygen concentration was controlled above 20% by regulating the agitation speed. 2.6. SDS-PAGE and lipase activity analysis Protein expression was analyzed by SDS-PAGE as described by Laemmli [13]. Lipase activity was determined by alkali titration method using olive oil as a substrate [14]. Appropriately diluted enzyme solution (1 mL) was added to the substrate solution containing 5 mL of 10% emulsified olive oil in 10% gum acacia and 2.5 mL of 200 mM Tris-HCl buffer (pH 8.0). The enzyme-substrate solution was incubated in a water bath shaker at 30 °C and 150 rpm for 10 min. Ethanol (10 mL) was added to stop the reaction. Liberated fatty acids were titrated with 0.05 mol/L NaOH using phenolphthalein as an indicator. One unit (i.e., U) was defined as the amount of enzyme that released 1 μmol fatty acid per minute under standard assay conditions. 2.7. Chiral selectivity of the recombinant fusion SmL The chiral selectivity of the recombinant fusion SmL was investigated in a toluene: water two-phase system using (±)-MPGM as a substrate [10]. Approximately 5 mL of 200 mM Tris-HCl buffer (pH 8.0) containing 50 U recombinant fusion SmL was added to 5 mL of toluene solution containing 100 mM (±)-MPGM. The reaction was conducted at 30 °C and 150 rpm in a 50 mL flask equipped with a tight plug. The concentrations of optical isomers were determined by analyzing the enantiomers on a chiral OJ column (25 cm × 4.6 cm, Daicel Chemical Industries, Ltd., Japan) at a flow rate of 0.8 mL/min with an eluting solvent system of n-hexane: isopropanol (60:40, v/v). Absorbance was measured at 254 nm.
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3. Results and discussion 3.1. Cloning and analysis of S. marcescens H30 lipase gene The lipase gene from S. marcescens H30 was successfully cloned using specific primers, ligated with pMD19-T, and sequenced. A band of 1,848 bp length was amplified from the chromosomal DNA of S. marcescens H30. The sequencing results indicate a full-length lipase gene motif encoding a polypeptide of 615 amino acid residues with a predicted molecular mass of nearly 65 kDa. The amino acid sequence contains the lipase active-site consensus sequence Gly-X1-Ser-X2-Gly, which is found in the majority of bacterial lipases. After alignment by the programs of BLASTN and BLASTP in the NCBI database, the results reveal that the nucleotide sequence and deduced amino acid sequence of S. marcescens H30 lipase shared 95%–98% identity and 97%–99% identity with the lipases from S. marcescens Sr41, SM6, ES-2, and ECU1010 [7, 9, 15, 16]. 3.2. Recombinant expression of S. marcescens H30 lipase gene in E. coli The recombinant strains were constructed by transforming E. coli BL21 (DE3) with the three recombinant plasmids pET28a-SmL, pET32a-SmL, and pGEX-4T-1-SmL. The three recombinant E. coli strains were cultivated in LB medium at 25 °C for 10 h after induction by adding IPTG to a final concentration of 0.5 mM. The lipase expression results are shown in Fig. 1. E. coli BL21 (DE3)/pET28a-SmL expressed lipase mainly in the form of an inclusion body (Fig. 1(A), Lanes 4 and 5). Almost the same amount of soluble and insoluble lipase was expressed by E. coli BL21 (DE3)/pGEX-4T-1-SmL (Fig. 1(C), Lanes 4 and 5). E. coli BL21 (DE3)/pET32a-SmL expressed more soluble lipase than the aforementioned two recombinant strains. Most lipases were soluble (Fig. 1(B), Lanes 4 and 5). The soluble lipase activity in the cell lysate’s supernatant of different recombinant E. coli strains was also determined. The lipase activities of the plasmid pET28a-SmL system, plasmid pGEX-4T-1-SmL system, and plasmid pET32a-SmL system were 500, 2000, and 5000 U/L, respectively.
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Previous studies showed that several affinity tags can enhance the solubility of some of the partner proteins to which they are attached. Polyhistidine-tags are usually used for the affinity purification of recombinant proteins expressed in E. coli. In our previous work, we found that 6× His tag of pET28a can improve the solubility of D-amino acid oxidase expressed in E. coli [17]. However, it can not enhance the soluble expression of SmL in E. coli. Busso et al. [18] published a systematic study on the effect of His tag on the solubility of 24 proteins. Their results indicated that His tag affects the solubility of some target proteins, but these effects vary per target protein. Numerous solubility tags have been reported. The majority of recent studies have focused on a few major players, particularly glutathione-S-transferase (GST), maltose-binding protein (MBP), and thioredoxin (Trx). MBP and GST can function as affinity tags (MBP binds strongly to amylose resin, whereas GST binds to glutathione resin) [19]. However, increasing evidence has shown that GST is a poor solubility enhancer in E. coli [20]. In this work, the GST tag of pGEX-4T-1 could only improve the solubility of SmL expressed in E. coli to a certain extent, which was consistent with a previous report [20]. The pET Trx fusion system is designed for cloning and high-level expression of proteins fused with the 109-aa Trx tag protein. Numerous proteins that normally aggregate in an insoluble form in E. coli tend to become soluble when fused with the Trx tag sequence [21]. In this work, the Trx tag in pET32a obviously contributed to the soluble expression of SmL, and resulted in high lipase activity. The recombinant strain E. coli BL21 (DE3)/pET32a-SmL was selected for further study. 3.3. Effect of lactose as an inducer on lipase expression of E. coli BL21 (DE3)/pET32a-SmL The lac promoter and its derivatives are widely employed to express foreign genes. Considering its high cost and possible toxicity, inducer IPTG is often replaced with lactose for the induction of protein expression [22]. However, lactose can simultaneously serve as an inducer and carbon source, so using lactose is more complex than using IPTG as an inducer. Varied concentrations of lactose (2–20 g/L) were used to investigate
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their effects on the recombinant lipase expression. E. coli BL21 (DE3)/pET32a-SmL was cultured in LB medium until OD600 reached 0.6–0.8, and induced with different concentrations of lactose at 20 °C for 10 h. The lipase expression is shown in Fig. S1. Lactose could effectively substitute IPTG to induce lipase expression at different concentrations (Fig. S1, Lanes 1–5). Given the soluble lipase in the supernatants (Fig. S1, Lanes 6–10), 15 g/L lactose was the optimum inducer concentration (Fig. S1, Lane 9). This concentration was used in the following optimization. 3.4. Optimization of culture medium compositions Optimization of culture medium compositions is essential to enhance the accumulation of the recombinant protein and cell density (OD600), and elevate the level of recombinant protein expression in E. coli [23]. Therefore, the effects of culture medium compositions on the recombinant lipase expression by E. coli BL21 (DE3)/pET32a-SmL were investigated with LB medium as the initial fermentation medium. To reduce cost, cheap nitrogen and carbon sources available in the domestic market were chosen for optimization. 3.4.1. Effect of nitrogen sources Oxoid tryptone in LB medium constitutes the majority of the culture medium cost. Thus, Oxoid tryptone was first substituted with different concentrations of fish peptone. The results are shown in Fig. 2. The highest OD600 of recombinant E. coli strain was 8.7 at a fish peptone concentration of 20 g/L. However, this high OD600 did not lead to high lipase activity. The highest lipase activity of approximately 14,000 U/L appeared at a fish peptone concentration of 15 g/L. Compared with Oxoid tryptone, fish peptone may be more beneficial to the recombinant lipase expression because it produced higher lipase activity (12,000 U/L) than (10,000 U/L) Oxoid tryptone at the same concentration (10 g/L). In our previous work, the same phenomenon was found in the optimization of recombinant glutaryl-7-aminocephalosporanic acid acylase expression [24]. Fish peptone contains several essential amino acids and some other growth-promoting factors that may positively influence
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cell growth and protein expression [24]. Oxoid yeast extract is the other main component of LB medium. The use of a homemade yeast extract, such as Angel yeast extract, as a substitute will decrease the cost of culture medium. Different concentrations of Angel yeast extract were used to investigate their effects on the recombinant lipase expression. The results are shown in Fig. 3. The highest OD600 attained was 8.5 at an Angel yeast extract concentration of 15 g/L. However, the maximum lipase activity reached 16,000 U/L at an Angel yeast extract concentration of 10 g/L. Compared with Oxoid yeast extract, Angel yeast extract was inefficient because it resulted in lower OD600 and lipase activity (6.92 and 12,000 U/L, respectively) than those (8.22 and 14,000 U/L, respectively) of Oxoid yeast extract at the same concentration of 5 g/L. Nitrogen sources include organic and inorganic sources. Organic nitrogen sources are nutrient-rich. Besides being rich in protein, polypeptide, and free amino acids, they often contain small amounts of carbohydrates, fat, vitamins, and some inorganic growth factors. The microorganisms cultured on mediums containing organic nitrogen sources often thrive [25]. Inorganic nitrogen sources lack nutrients. However, the uptake and utilization of inorganic nitrogen sources are generally faster than those of organic nitrogen sources. They are also known as fast nitrogen sources [26]. Thus, an appropriate combination of organic and inorganic nitrogen sources may prompt cell growth and enhance the recombinant enzyme expression [27]. Different inorganic nitrogen sources, such as NH4Cl, (NH4)2SO4, and NH4NO3, were separately added to the culture medium to a final concentration of 2 g/L to investigate their synergistic effects with organic nitrogen sources. The results show that the effects of all inorganic nitrogen sources were not obvious (data not shown).
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3.4.2. Effect of phosphate Phosphate, which is one of the most abundant elements in E. coli in terms of cellular content, has a crucial function in cells [28]. This element is essential for E. coli cell growth. An appropriate concentration of phosphate can maintain the osmotic pressure of fermentation broth, and influence the recombinant plasmid stability and expression of target protein to a certain degree. Given the universal function of phosphate, it was added to the culture medium at different concentrations (20–200 mmol/L) to substitute NaCl. Their effects are shown in Fig. 4. Lipase activity was enhanced from 15,900 U/L to 17,600 U/L when the phosphate concentration increased from 0 mmol/L to 100 mmol/L. OD600 increased from 7.89 to 9.2 as the phosphate concentration increased from 0 mmol/L to 100 mmol/L. Thus, cell growth and lipase production were affected by phosphate. The optimum concentration of phosphate was 100 mmol/L in shake flask culture. 3.4.3. Effect of carbon sources During growth of recombinant E. coli strain, the nitrogen and carbon sources in the culture medium had important functions in the expression of the target protein. Although the initial LB medium did not contain a carbon source component, several types of carbon sources were still added to the fermentation medium to investigate their effects on the production of recombinant lipase. As indicated in Fig. 5(A), the highest OD600 of recombinant E. coli strain was more than 10 when sorbitol or mannitol was used as a carbon source. However, the high OD600 did not lead to high lipase activity. Glucose was the optimum carbon source for the production of recombinant lipase, and the level of lipase expression in E. coli was further improved to 19,500 U/L (Fig. 5(A)). When dextrin was used as a carbon source, both the lipase activity and OD600 were the lowest. This finding was not in accordance with our previous studies [17, 24]. This difference may be derived from the different induction times. In previous studies, the induction time was more than 30 h, and dextrin could satisfy the need of recombinant E. coli as a “slow” carbon source. However, in this study, the recombinant lipase was rapidly
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expressed, thereby shortening the induction time. “Fast” carbon sources, such as glucose, would be better for cell growth and lipase expression. The transcription of E. coli lac operon in a lactose-containing medium may be repressed by the presence of glucose [29]. Thus, the concentration of glucose was further optimized. Fig. 5(B) shows the obvious effects of glucose concentration on OD600 and lipase activity. Glucose (2 g/L) showed an inhibitory effect on cell growth and lipase expression. High glucose concentration (5 g/L or 10 g/L) showed stronger repression on lipase expression than that on cell growth. The highest lipase activity (21,000 U/L) and OD600 (11.29) were obtained at a glucose concentration of 1 g/L. 3.5. Optimization of induction conditions Aside from the culture medium compositions, the conditions used to induce recombinant E. coli strain also affected cell growth and lipase expression. Systematic optimization of the induction conditions, such as induction temperature, initial induction OD600, and induction time, was performed. The induction of lipase expression by E. coli BL21 (DE3)/pET32a-SmL was performed at different temperatures (15 °C, 20 °C, 25 °C, 30 °C, and 37 °C). The results are shown in Fig. S2. High or low induction temperature was unfavorable to lipase expression in the recombinant strain. Temperature higher than 20 °C would lead to the decrease in soluble lipase in the supernatant. Induction at 20 °C generated a good balance between lipase synthesis and post-translation in recombinant E. coli strain, and resulted in the most soluble lipase in the supernatant. Lipase expression by E. coli BL21 (DE3)/pET32a-SmL was induced at different initial OD600 values (0.5, 0.7, 0.9, 1.1, and 1.3). The total amount of lipase expression and amount of lipase expression in the supernatant were almost the same at an initial induction OD600 between 0.7 and 1.3 (Fig. S3). The effect of induction time on lipase expression by E. coli BL21 (DE3)/pET32a-SmL was also investigated. In the comparison of soluble lipase in the supernatants of cell lysates induced for different times, the amount of soluble lipase increased with the induction
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time, and the maximum value was obtained at 18 h (Fig. S4). Based on the aforementioned optimization, the optimum culture medium composition and induction conditions were 1 g/L glucose, 15 g/L fish peptone, 10 g/L Angel yeast extract, and 100 mmol/L phosphate with 15 g/L lactose induction at an initial OD600 of 0.7 and 20 °C for 18 h. Batch cultivation in shake flask was performed in the optimized medium under the optimized induction conditions, which resulted in the highest lipase activity and productivity of 23,000 U/L and 1278 U L−1 h−1, respectively. This lipase activity was 4.6 times higher than that (5000 U/L) before optimization. Compared with a reported SmL activity of 6,000 U/L [9], the current value is the highest SmL activity attained by heterogeneous recombinant expression in E. coli. 3.6. Scale-up cultivation in a 7.0 L fermenter To verify the feasibility of scaling up the recombinant lipase production under the optimized conditions and medium identified in shake flask, scale-up cultivation was conducted in a 7.0 L fermenter. The results are shown in Fig. 6. The highest OD600 and lipase activity reached 8.96 and 19,650 U/L, respectively. After 16 h, OD600 decreased because of the autolysis of E. coli cells. Lipase productivity was highest (1228 U L−1 h−1) at 16 h of induction, and then decreased. The lipase yield was insignificantly lower than that obtained at flask-scale. The differences in several variables, such as oxygen supply, shear stress, nutrients, and inducer distribution, were possibly due to the fermentation scale changing from shake flask to fermenter. Optimization in fermenter may improve lipase activity and yield. 3.7. Effect of fusion expression on the chiral selectivity of SmL Fusion expression of S. marcescens H30 lipase with the Trx tag of pET-32a obviously enhanced the active lipase level. SmL is generally used for the kinetic resolution of (±)-MPGM. Whether the Trx tag influences the chiral selectivity of SmL was investigated. This fusion lipase was used to enantioselectively hydrolyze (±)-MPGM in a biphasic system. The fusion lipase enantioselectively catalyzed the hydrolysis of (+)-MPGM
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(Fig. S5). The reaction proceeded at a substrate concentration of 100 mmol/L, which resulted in a highly pure product [i.e., (-)-MPGM] with high enantiomeric excess (99.2%). These results suggest that the fusion lipase possessed the same chiral selectivity toward (±)-MPGM similar to other wild-type SmL [5]. Therefore, the existence of the Trx tag did not influence the chiral selectivity of SmL. 4. Conclusions In this work, the effects of fusion tags on SmL recombinant expression were revealed. Trx-tagged SmL was found to be a suitable choice. To optimize SmL production by E. coli BL21 (DE3)/pET32a-SmL, systematic optimization of the culture medium compositions and induction conditions was conducted. The recombinant SmL activity and productivity reached approximately 23,000 U/L and 1278 U L−1 h−1 in shake flask, respectively. Scale-up cultivation in a 7.0 L fermenter resulted in lipase activity of 19,650 U/L and productivity of 1228 U L−1 h−1. These results are the highest levels that have been reported so far, and completely meet the requirements of industrial SmL production in a recombinant E. coli strain.
Acknowledgments This work was supported by “A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions，PAPD”, the “Scientific Research Fund of Nanjing Forestry University for the Introduction of High-level talents” and the “Program for the Top Young Talents of Nanjing Forestry University”. All the authors have no conflict of interest to declare.
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[10] S. X. Li, H. Y. Pang, K. Lin, J. H. Xu, J. Zhao, L. Q. Fan (2011) Refolding, purification and characterization of an organic solvent-tolerant lipase from Serratia marcescens ECU1010. J. Mol. Catal B-Enzym. 71, 171-176. [11] L. Y. Zhang, J. A. Sun, Y. L. Hao, J. W. Zhu, J. Chu, D. Z. Wei, Y. L. Shen (2010) Microbial production of 2,3-butanediol by a surfactant (serrawettin)-deficient mutant of Serratia marcescens H30. J. Ind. Microbiol. Biotechnol. 37, 857-862. [12] S. F. Altschul, W. Gish, W. Miller, E. W. Myers, D. J. Lipman (1990) Basic local alignment search tool. J. Mol. Biol. 215, 403-410. [13] U. K. Laemmli (1970) Cleavage of structure proteins during the assembly of the head of bacteriophage T4. Nature. 227, 680-685. [14] P. S. Borkar, R. G. Bodade (2009) Purification and characterization of extracellular lipase from a new strain-Pseudomonas Aeruginosa SRT 9. Braz. J. Microbiol. 40, 358-366. [15] H. Akatsuka, E. Kawai, K. Omori, S. Komatsubara, T. Shibatani, T. Tosa (1996) The lipA gene of Serratia marcescens which encodes an extracellular lipase having no N-terminal signal peptide. J. Bacteriol. 176, 1949-1956. [16] K. W. Lee, H. A. Bae, Y. H. Lee (2007) Molecular cloning and functional expression of esf gene encoding enantioselective lipase from Serratia marcescens ES-2 for kinetic resolution of optically active (S)-flurbiprofen. J. Microbiol. Biotechnol. 17, 74-80. [17] S. W. Deng, E. Z. Su, X. Q. Ma, S. L. Yang, D. Z. Wei (2014) High-level soluble and functional expression of Trigonopsis variabilis D-amino acid oxidase in Escherichia coli. Bioprocess. Biosyst. Eng. DOI 10.1007/s00449-013-1123-z. [18] D. Busso, R. Kim, S. H. Kim (2003) Expression of soluble recombinant proteins in a cell-free system using
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a 96-well format. J. Biochem. Biophys. Methods. 55, 233-240. [19] D. Esposito, D. K. Chatterjee (2006) Enhancement of soluble protein expression through the use of fusion tags. Curr. Opin. Biotechnol.17, 353-358. [20] M. R. Dyson, S. P. Shadbolt, K. J. Vincent, R. L. Perera, J. McCafferty (2004) Production of soluble mammalian proteins in Escherichia coli: identification of protein features that correlate with successful expression. BMC. Biotechnol. 4, 32. [21] A. Dummler, A. M. Lawrence, A. D. Marco (2005) Simplified screening for the detection of soluble fusion constructs expressed in E. coli using a modular set of vectors. Microb. Cell. Fact. 4, 34. [22] R. S. Donovan, C. W. Robinson, B. R. Glick (1996) Review: Optimizing inducer and culture conditions for expression of foreign proteins under the control of the lac promoter. J. Ind. Microbiol.16, 145-154. [23] S. Sahdev, S. K. Khattar, K. S. Saini (2008) Production of active eukaryotic proteins through bacterial expression systems: a review of the existing biotechnology strategies. Mol. Cell. Biochem. 307, 249-264. [24] X. Q. Ma, E. Z. Su, Y. Zhu, S. W. Deng, D. Z. Wei (2013) High-level expression of glutaryl-7-aminocephalosporanic acid acylase from Pseudomonas diminuta NK703 in Escherichia coli by combined optimization strategies. J. Biotechnol. 168, 607-615. [25] S. Mahmoudi, H. Abtahi, A. Bahador, G. Mosayebi, A. H. Salmanian, M. Teymuri (2012) Optimizing of nutrients for high level expression of recombinant streptokinase using pET32a expression system. Maedica (Buchar). 7, 241-246. [26] J. Zhang, R. Greasham (1999) Chemically defined media for commercial fermentations. Appl. Microbiol. Biotechnol. 51, 407-421. [27] J. Shiloach, R Fass (2005) Growing E. coli to high cell density-A historical perspective on method development. Biotechnol. Adv. 23, 345-357.
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[28] H. Robert, R. Simon, R. Natalie, B. Jochen (2011) Utilizing high-throughput experimentation to enhance specific productivity of an E. coli T7 expression system by phosphate limitation. BMC Biotechnol. 11, 22. [29] K. Kimata, H. Takahashi, T. Inada, P. Postma, H. Aiba (1997) cAMP receptor protein-cAMP plays a crucial role in glucose-lactose diauxie by activating the major glucose transporter gene in Escherichia coli. PNAS. 94, 12914-12919.
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Figure 1.
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Figure 2.
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Figure 3.
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Figure 4.
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Figure 5.
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Figure 6.
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Enzyme and Fermentation Technology Laboratory, College of Light Industry Science and Engineering, Nanjing Forestry University, Nanjing 210037, P. R. China.
b
State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, P.R .China.
* Corresponding author: Erzheng Su Tel. +86 25 85428906
Fax: +86 25 85428906
E-mail address: [email protected]
Running Title：High-level soluble expression of lipase
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/bab.1248. This article is protected by copyright. All rights reserved.
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Abstract Serratia marcescens lipase (SmL) is an important biocatalyst used to enantioselectively hydrolyze (±)-trans-3-(4-methoxyphynyl) glycidic acid methyl ester. However, the economically justified level recombinant soluble expression of SmL in Escherichia coli has not been established. Thus, fusion genes of lipase from S. marcescens H30 with different fusion tags were constructed and expressed in E. coli. The effects of fusion tags were revealed. A significant increase in recombinant lipase solubility showed that E. coli BL21 (DE3)/pET32a-SmL was a suitable choice for SmL production. To optimize the performance of recombinant SmL production, changes in culture medium compositions and induction conditions were systematically tested. Finally, the recombinant SmL activity and productivity reached approximately 23,000 U/L and 1278 U L−1 h−1 in shake flasks, respectively. This value is the highest SmL activity attained by heterogeneous recombinant expression in E. coli. Lipase activity and productivity reached 19,650 U/L and 1228 U L−1 h−1, respectively, by scaling up SmL production in a 7.0 L fermenter. The existence of the Trx tag did not influence the chiral selectivity of recombinant SmL. These findings indicate a possibility for soluble and economical SmL expression in E. coli to meet industrial needs. Keywords: Serratia marcescens H30; Lipase; Fusion tags; Solubility; Culturing medium; induction conditions.
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1. Introduction Lipases (triacylglycerol ester hydrolases, EC 3.1.1.3) are ubiquitous in animals, plants, and microorganisms. They can catalyze the hydrolysis of medium- and long-chain triglycerides into fatty acids and glycerol at the oil–water interface, as well as ester synthesis and transesterification reactions with high regioselectivity and stereoselectivity in non-aqueous medium systems [1]. As of this writing, lipases have become excellent biocatalysts in a broad range of industrial applications, including pharmaceutical, fine chemical, and other industrial areas, such as detergents, oil/fats, cheese making, hard-surface cleaning, and leather and paper processing [2]. Among many successful examples of lipase applications, the Serratia marcescens lipase (SmL) is well-known in the pharmaceutical industry for its excellent enantioselectivity in the biocatalytic hydrolysis of trans-3-(4-methoxyphynyl) glycidic acid methyl ester [(±)-MPGM] to produce (2R, 3S)-MPGM [(−)-MPGM], which is a key intermediate for the synthesis of diltiazem hydrochloride [3, 4]. Given the importance of SmL, a productive bioprocess for SmL production should be developed. To increase enzyme production, the effects of medium compositions and cultivation conditions on the accumulation of S. marcescens ECU1010 lipase were optimized in shake flasks, with lipase production only being 4,780 U/L [5]. The strain S. marcescens ECU1010 was further subjected to UV irradiation. A mutant strain (UV-01) has only given a 2.3-fold increase in lipase activity compared with a wild-type strain [6]. SmL genes from different S. marcescens have also been cloned and expressed in recent years. An SmL gene from S. marcescens SM6 was expressed in Escherichia coli, and the lipase activity only reached wild-type level. This gene was further subcloned into the expression vector pSE420, providing a strong trc promoter and containing a bacteriophage T7 gene 10 translational enhancer. However, the level of lipase activity has remained very low [7]. An SmL gene was cloned into pET11d and overexpressed in E. coli JM109/DE3. The overexpressed protein was not secreted but formed insoluble inclusion bodies inside the bacterial cytoplasm, which were refolded to produce
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enzymatically active lipase [8]. The SmL gene from S. marcescens ECU1010 has also been cloned and ligated to the plasmid pET-24a to create an overexpression plasmid. An overproducing strain has been constructed by transforming E. coli BL21 (DE3) with this recombinant plasmid. After the optimization of many physical and chemical factors, lipase production has only increased to 5000–6000 U/L [9]. The aforementioned review of previous studies revealed that an economically justified level expression of SmL in E. coli for industrial purposes has not been achieved. Most SmL genes have been expressed in the form of inclusion bodies; for example, the SmL gene from S. marcescens ECU1010 expressed in soluble fraction is estimated to be only 30% in cell-free extract after optimization [9]. Refolding of inclusion bodies is highly complicated that it wastes considerable money and time [10]. In the current study, a lipase gene from S. marcescens H30, which is a Gram-negative enteric organism previously used for the efficient production of 2,3-butanediol in our laboratory [11], was ligated to three plasmids with different fusion tags to overexpress active lipase in soluble form. Changes in culture medium compositions and conditions were investigated to optimize the performance of SmL production in E. coli. Scale-up cultivation was conducted in a 7.0 L fermenter to determine the feasibility of our strategy.
2. Materials and methods 2.1. Materials Restriction enzymes, T4 DNA ligase, and Taq polymerase premix were supplied by MBI Fermentas (Germany). DNA gel and plasmid extracts were purchased from Generay Biotech (Shanghai) Co., Ltd. Molecular markers (DNA and protein markers) were bought from Thermo Fisher Scientific Inc. (Shanghai, China). Tryptone and yeast extract were obtained from Oxoid Co., Ltd., UK. Carbon sources (glycerol, sucrose, glucose, sorbitol, mannitol, starch, and dextrin), nitrogen sources [fish tryptone, Angel yeast extract, NH4Cl, (NH4)2SO4, and NH4NO3], and inorganic salt (Na2HPO4) were employed for medium composition screening and This article is protected by copyright. All rights reserved.
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optimization. All these sources were of industrial grade and purchased from the Chinese market with the lowest possible price. All other chemicals were of reagent grade and obtained from commercial sources. 2.2. Bacterial strains, plasmids, and culture conditions S. marcescens H30 was deposited in our laboratory. Genomic DNA from S. marcescens H30 was used as the source of SmL gene for PCR amplification. E. coli DH5α and BL21(DE3) were employed as host strains in gene manipulation and protein expression, respectively. The plasmids used for cloning and protein expression were pMD19-T, pET-28a, pET-32a, and pGEX-4T-1. S. marcescens H30 was grown at 30 °C in a nutrient broth (3 g/L beef extract, 5 g/L peptone, and 5 g/L NaCl). Wild-type and recombinant E. coli cells were regularly cultured in Luria–Bertani (LB) medium (10 g/L tryptone, 5 g/L yeast extract, and 5 g/L NaCl) at 37 °C. An appropriate amount of ampicillin or kanamycin was added. 2.3. Cloning and expression of S. marcescens H30 lipase gene in E. coli To obtain the full-length lipase gene from S. marcescens H30, total genome was extracted. Two specific primers, namely, forward 5'-GGTTGAATTCGGCATCTTTAGCTATAAGGA-3' (EcoRI cutting site is underlined) and reverse 5'-CCTTGCGGCCGCTAGGCCAACACCACCTGATC-3' (NotI cutting site is underlined), were designed according to the reported SmL gene sequence [7]. PCR amplifications were performed at 94 °C for 4 min, followed by 30 cycles of 94 °C for 30 s, 58 °C for 1 min, and 72 °C for 1 min, with a final extension step of 8 min at 72 °C. The PCR product was separated, cloned into pMD19-T, and sequenced. The DNA and protein sequence similarities were assessed using BLASTN and BLASTP programs [12]. Three plasmids, namely, pET28a (6 × His tag), pET32a (Trx tag), and pGEX-4T-1 (GST tag), with different fusion tags were chosen as recombinant expression plasmids. The obtained SmL gene and plasmids were digested with EcoRI and NotI restriction enzymes simultaneously, and then ligated to construct the SmL
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expression vectors pET28a-SmL, pET32a-SmL, and pGEX-4T-1-SmL. These expression plasmids were transformed into E. coli BL21 (DE3). A single colony was inoculated into 3 mL of LB medium in a 10 mL test tube supplemented with kanamycin (50 μg/mL) or ampicillin (100 μg/mL) according to the different selection markers of plasmids and hosts, and cultivated at 37 °C and 200 rpm overnight as seed. Subsequently, 1% (v/v) seed was inoculated into 100 mL of LB medium in a 500 mL Erlenmeyer flask, and grown for 2.5–3.0 h at 37 °C and 200 rpm until OD600 reached 0.6–0.8. IPTG was added to a final concentration of 0.5 mM for inducing SmL expression. Cultivation continued for another 8–10 h at 25 °C. Cells were collected by centrifugation at 6000 ×g for 10 min, resuspended, and washed twice with 50 mM Tris-HCl buffer (pH 8.0). Biomass was monitored during cultivation by measuring the optical density of culture broth at 600 nm. Cells were disrupted by an ultrasonic cell disruptor (Ningbo Scientz, China) in an ice bath at a power controlled from 200 W to 400 W. Each circle worked for 4 s, intermittent for 5 s, and continued for 20 min. The disrupted cells were collected by centrifugation at 12,000 ×g for 10 min. The supernatants were used for SmL activity and SDS-PAGE analysis. 2.4. Optimization of recombinant SmL production in shake flask Shake flask culture experiments are usually performed in 250 mL flasks containing 50 mL of medium. In this study, LB medium was employed as the initial medium for optimization. After inoculation with 1% (v/v) seed culture, the flasks were shaken at 37 °C and 200 rpm for different times to attain different OD600. Different concentrations of lactose were then added. Induction continued for different times at varied temperatures. A systematic optimization of the culture medium compositions and conditions was conducted to enhance the recombinant SmL production in shake flask. 2.5. Batch fermentation in a 7.0 L fermenter The scale-up cultivation of recombinant E. coli BL21 (DE3)/pET32a-SmL was conducted in a 7.0 L
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fermenter with a working volume of 4.0 L. The fermentation medium was the same as the optimized culture medium in shake flask cultivation. Seed culture was performed in 500 mL flasks with a working volume of 100 mL. Cultivation in the fermenter was conducted at 37 °C until OD600 of recombinant E. coli cells reached 0.7. Lactose (15 g/L) was added to induce SmL expression at 20 °C. The pH was controlled at 7.0 using 2 M NaOH or H2SO4. The dissolved oxygen concentration was controlled above 20% by regulating the agitation speed. 2.6. SDS-PAGE and lipase activity analysis Protein expression was analyzed by SDS-PAGE as described by Laemmli [13]. Lipase activity was determined by alkali titration method using olive oil as a substrate [14]. Appropriately diluted enzyme solution (1 mL) was added to the substrate solution containing 5 mL of 10% emulsified olive oil in 10% gum acacia and 2.5 mL of 200 mM Tris-HCl buffer (pH 8.0). The enzyme-substrate solution was incubated in a water bath shaker at 30 °C and 150 rpm for 10 min. Ethanol (10 mL) was added to stop the reaction. Liberated fatty acids were titrated with 0.05 mol/L NaOH using phenolphthalein as an indicator. One unit (i.e., U) was defined as the amount of enzyme that released 1 μmol fatty acid per minute under standard assay conditions. 2.7. Chiral selectivity of the recombinant fusion SmL The chiral selectivity of the recombinant fusion SmL was investigated in a toluene: water two-phase system using (±)-MPGM as a substrate [10]. Approximately 5 mL of 200 mM Tris-HCl buffer (pH 8.0) containing 50 U recombinant fusion SmL was added to 5 mL of toluene solution containing 100 mM (±)-MPGM. The reaction was conducted at 30 °C and 150 rpm in a 50 mL flask equipped with a tight plug. The concentrations of optical isomers were determined by analyzing the enantiomers on a chiral OJ column (25 cm × 4.6 cm, Daicel Chemical Industries, Ltd., Japan) at a flow rate of 0.8 mL/min with an eluting solvent system of n-hexane: isopropanol (60:40, v/v). Absorbance was measured at 254 nm.
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3. Results and discussion 3.1. Cloning and analysis of S. marcescens H30 lipase gene The lipase gene from S. marcescens H30 was successfully cloned using specific primers, ligated with pMD19-T, and sequenced. A band of 1,848 bp length was amplified from the chromosomal DNA of S. marcescens H30. The sequencing results indicate a full-length lipase gene motif encoding a polypeptide of 615 amino acid residues with a predicted molecular mass of nearly 65 kDa. The amino acid sequence contains the lipase active-site consensus sequence Gly-X1-Ser-X2-Gly, which is found in the majority of bacterial lipases. After alignment by the programs of BLASTN and BLASTP in the NCBI database, the results reveal that the nucleotide sequence and deduced amino acid sequence of S. marcescens H30 lipase shared 95%–98% identity and 97%–99% identity with the lipases from S. marcescens Sr41, SM6, ES-2, and ECU1010 [7, 9, 15, 16]. 3.2. Recombinant expression of S. marcescens H30 lipase gene in E. coli The recombinant strains were constructed by transforming E. coli BL21 (DE3) with the three recombinant plasmids pET28a-SmL, pET32a-SmL, and pGEX-4T-1-SmL. The three recombinant E. coli strains were cultivated in LB medium at 25 °C for 10 h after induction by adding IPTG to a final concentration of 0.5 mM. The lipase expression results are shown in Fig. 1. E. coli BL21 (DE3)/pET28a-SmL expressed lipase mainly in the form of an inclusion body (Fig. 1(A), Lanes 4 and 5). Almost the same amount of soluble and insoluble lipase was expressed by E. coli BL21 (DE3)/pGEX-4T-1-SmL (Fig. 1(C), Lanes 4 and 5). E. coli BL21 (DE3)/pET32a-SmL expressed more soluble lipase than the aforementioned two recombinant strains. Most lipases were soluble (Fig. 1(B), Lanes 4 and 5). The soluble lipase activity in the cell lysate’s supernatant of different recombinant E. coli strains was also determined. The lipase activities of the plasmid pET28a-SmL system, plasmid pGEX-4T-1-SmL system, and plasmid pET32a-SmL system were 500, 2000, and 5000 U/L, respectively.
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Previous studies showed that several affinity tags can enhance the solubility of some of the partner proteins to which they are attached. Polyhistidine-tags are usually used for the affinity purification of recombinant proteins expressed in E. coli. In our previous work, we found that 6× His tag of pET28a can improve the solubility of D-amino acid oxidase expressed in E. coli [17]. However, it can not enhance the soluble expression of SmL in E. coli. Busso et al. [18] published a systematic study on the effect of His tag on the solubility of 24 proteins. Their results indicated that His tag affects the solubility of some target proteins, but these effects vary per target protein. Numerous solubility tags have been reported. The majority of recent studies have focused on a few major players, particularly glutathione-S-transferase (GST), maltose-binding protein (MBP), and thioredoxin (Trx). MBP and GST can function as affinity tags (MBP binds strongly to amylose resin, whereas GST binds to glutathione resin) [19]. However, increasing evidence has shown that GST is a poor solubility enhancer in E. coli [20]. In this work, the GST tag of pGEX-4T-1 could only improve the solubility of SmL expressed in E. coli to a certain extent, which was consistent with a previous report [20]. The pET Trx fusion system is designed for cloning and high-level expression of proteins fused with the 109-aa Trx tag protein. Numerous proteins that normally aggregate in an insoluble form in E. coli tend to become soluble when fused with the Trx tag sequence [21]. In this work, the Trx tag in pET32a obviously contributed to the soluble expression of SmL, and resulted in high lipase activity. The recombinant strain E. coli BL21 (DE3)/pET32a-SmL was selected for further study. 3.3. Effect of lactose as an inducer on lipase expression of E. coli BL21 (DE3)/pET32a-SmL The lac promoter and its derivatives are widely employed to express foreign genes. Considering its high cost and possible toxicity, inducer IPTG is often replaced with lactose for the induction of protein expression [22]. However, lactose can simultaneously serve as an inducer and carbon source, so using lactose is more complex than using IPTG as an inducer. Varied concentrations of lactose (2–20 g/L) were used to investigate
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their effects on the recombinant lipase expression. E. coli BL21 (DE3)/pET32a-SmL was cultured in LB medium until OD600 reached 0.6–0.8, and induced with different concentrations of lactose at 20 °C for 10 h. The lipase expression is shown in Fig. S1. Lactose could effectively substitute IPTG to induce lipase expression at different concentrations (Fig. S1, Lanes 1–5). Given the soluble lipase in the supernatants (Fig. S1, Lanes 6–10), 15 g/L lactose was the optimum inducer concentration (Fig. S1, Lane 9). This concentration was used in the following optimization. 3.4. Optimization of culture medium compositions Optimization of culture medium compositions is essential to enhance the accumulation of the recombinant protein and cell density (OD600), and elevate the level of recombinant protein expression in E. coli [23]. Therefore, the effects of culture medium compositions on the recombinant lipase expression by E. coli BL21 (DE3)/pET32a-SmL were investigated with LB medium as the initial fermentation medium. To reduce cost, cheap nitrogen and carbon sources available in the domestic market were chosen for optimization. 3.4.1. Effect of nitrogen sources Oxoid tryptone in LB medium constitutes the majority of the culture medium cost. Thus, Oxoid tryptone was first substituted with different concentrations of fish peptone. The results are shown in Fig. 2. The highest OD600 of recombinant E. coli strain was 8.7 at a fish peptone concentration of 20 g/L. However, this high OD600 did not lead to high lipase activity. The highest lipase activity of approximately 14,000 U/L appeared at a fish peptone concentration of 15 g/L. Compared with Oxoid tryptone, fish peptone may be more beneficial to the recombinant lipase expression because it produced higher lipase activity (12,000 U/L) than (10,000 U/L) Oxoid tryptone at the same concentration (10 g/L). In our previous work, the same phenomenon was found in the optimization of recombinant glutaryl-7-aminocephalosporanic acid acylase expression [24]. Fish peptone contains several essential amino acids and some other growth-promoting factors that may positively influence
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cell growth and protein expression [24]. Oxoid yeast extract is the other main component of LB medium. The use of a homemade yeast extract, such as Angel yeast extract, as a substitute will decrease the cost of culture medium. Different concentrations of Angel yeast extract were used to investigate their effects on the recombinant lipase expression. The results are shown in Fig. 3. The highest OD600 attained was 8.5 at an Angel yeast extract concentration of 15 g/L. However, the maximum lipase activity reached 16,000 U/L at an Angel yeast extract concentration of 10 g/L. Compared with Oxoid yeast extract, Angel yeast extract was inefficient because it resulted in lower OD600 and lipase activity (6.92 and 12,000 U/L, respectively) than those (8.22 and 14,000 U/L, respectively) of Oxoid yeast extract at the same concentration of 5 g/L. Nitrogen sources include organic and inorganic sources. Organic nitrogen sources are nutrient-rich. Besides being rich in protein, polypeptide, and free amino acids, they often contain small amounts of carbohydrates, fat, vitamins, and some inorganic growth factors. The microorganisms cultured on mediums containing organic nitrogen sources often thrive [25]. Inorganic nitrogen sources lack nutrients. However, the uptake and utilization of inorganic nitrogen sources are generally faster than those of organic nitrogen sources. They are also known as fast nitrogen sources [26]. Thus, an appropriate combination of organic and inorganic nitrogen sources may prompt cell growth and enhance the recombinant enzyme expression [27]. Different inorganic nitrogen sources, such as NH4Cl, (NH4)2SO4, and NH4NO3, were separately added to the culture medium to a final concentration of 2 g/L to investigate their synergistic effects with organic nitrogen sources. The results show that the effects of all inorganic nitrogen sources were not obvious (data not shown).
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3.4.2. Effect of phosphate Phosphate, which is one of the most abundant elements in E. coli in terms of cellular content, has a crucial function in cells [28]. This element is essential for E. coli cell growth. An appropriate concentration of phosphate can maintain the osmotic pressure of fermentation broth, and influence the recombinant plasmid stability and expression of target protein to a certain degree. Given the universal function of phosphate, it was added to the culture medium at different concentrations (20–200 mmol/L) to substitute NaCl. Their effects are shown in Fig. 4. Lipase activity was enhanced from 15,900 U/L to 17,600 U/L when the phosphate concentration increased from 0 mmol/L to 100 mmol/L. OD600 increased from 7.89 to 9.2 as the phosphate concentration increased from 0 mmol/L to 100 mmol/L. Thus, cell growth and lipase production were affected by phosphate. The optimum concentration of phosphate was 100 mmol/L in shake flask culture. 3.4.3. Effect of carbon sources During growth of recombinant E. coli strain, the nitrogen and carbon sources in the culture medium had important functions in the expression of the target protein. Although the initial LB medium did not contain a carbon source component, several types of carbon sources were still added to the fermentation medium to investigate their effects on the production of recombinant lipase. As indicated in Fig. 5(A), the highest OD600 of recombinant E. coli strain was more than 10 when sorbitol or mannitol was used as a carbon source. However, the high OD600 did not lead to high lipase activity. Glucose was the optimum carbon source for the production of recombinant lipase, and the level of lipase expression in E. coli was further improved to 19,500 U/L (Fig. 5(A)). When dextrin was used as a carbon source, both the lipase activity and OD600 were the lowest. This finding was not in accordance with our previous studies [17, 24]. This difference may be derived from the different induction times. In previous studies, the induction time was more than 30 h, and dextrin could satisfy the need of recombinant E. coli as a “slow” carbon source. However, in this study, the recombinant lipase was rapidly
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expressed, thereby shortening the induction time. “Fast” carbon sources, such as glucose, would be better for cell growth and lipase expression. The transcription of E. coli lac operon in a lactose-containing medium may be repressed by the presence of glucose [29]. Thus, the concentration of glucose was further optimized. Fig. 5(B) shows the obvious effects of glucose concentration on OD600 and lipase activity. Glucose (2 g/L) showed an inhibitory effect on cell growth and lipase expression. High glucose concentration (5 g/L or 10 g/L) showed stronger repression on lipase expression than that on cell growth. The highest lipase activity (21,000 U/L) and OD600 (11.29) were obtained at a glucose concentration of 1 g/L. 3.5. Optimization of induction conditions Aside from the culture medium compositions, the conditions used to induce recombinant E. coli strain also affected cell growth and lipase expression. Systematic optimization of the induction conditions, such as induction temperature, initial induction OD600, and induction time, was performed. The induction of lipase expression by E. coli BL21 (DE3)/pET32a-SmL was performed at different temperatures (15 °C, 20 °C, 25 °C, 30 °C, and 37 °C). The results are shown in Fig. S2. High or low induction temperature was unfavorable to lipase expression in the recombinant strain. Temperature higher than 20 °C would lead to the decrease in soluble lipase in the supernatant. Induction at 20 °C generated a good balance between lipase synthesis and post-translation in recombinant E. coli strain, and resulted in the most soluble lipase in the supernatant. Lipase expression by E. coli BL21 (DE3)/pET32a-SmL was induced at different initial OD600 values (0.5, 0.7, 0.9, 1.1, and 1.3). The total amount of lipase expression and amount of lipase expression in the supernatant were almost the same at an initial induction OD600 between 0.7 and 1.3 (Fig. S3). The effect of induction time on lipase expression by E. coli BL21 (DE3)/pET32a-SmL was also investigated. In the comparison of soluble lipase in the supernatants of cell lysates induced for different times, the amount of soluble lipase increased with the induction
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time, and the maximum value was obtained at 18 h (Fig. S4). Based on the aforementioned optimization, the optimum culture medium composition and induction conditions were 1 g/L glucose, 15 g/L fish peptone, 10 g/L Angel yeast extract, and 100 mmol/L phosphate with 15 g/L lactose induction at an initial OD600 of 0.7 and 20 °C for 18 h. Batch cultivation in shake flask was performed in the optimized medium under the optimized induction conditions, which resulted in the highest lipase activity and productivity of 23,000 U/L and 1278 U L−1 h−1, respectively. This lipase activity was 4.6 times higher than that (5000 U/L) before optimization. Compared with a reported SmL activity of 6,000 U/L [9], the current value is the highest SmL activity attained by heterogeneous recombinant expression in E. coli. 3.6. Scale-up cultivation in a 7.0 L fermenter To verify the feasibility of scaling up the recombinant lipase production under the optimized conditions and medium identified in shake flask, scale-up cultivation was conducted in a 7.0 L fermenter. The results are shown in Fig. 6. The highest OD600 and lipase activity reached 8.96 and 19,650 U/L, respectively. After 16 h, OD600 decreased because of the autolysis of E. coli cells. Lipase productivity was highest (1228 U L−1 h−1) at 16 h of induction, and then decreased. The lipase yield was insignificantly lower than that obtained at flask-scale. The differences in several variables, such as oxygen supply, shear stress, nutrients, and inducer distribution, were possibly due to the fermentation scale changing from shake flask to fermenter. Optimization in fermenter may improve lipase activity and yield. 3.7. Effect of fusion expression on the chiral selectivity of SmL Fusion expression of S. marcescens H30 lipase with the Trx tag of pET-32a obviously enhanced the active lipase level. SmL is generally used for the kinetic resolution of (±)-MPGM. Whether the Trx tag influences the chiral selectivity of SmL was investigated. This fusion lipase was used to enantioselectively hydrolyze (±)-MPGM in a biphasic system. The fusion lipase enantioselectively catalyzed the hydrolysis of (+)-MPGM
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(Fig. S5). The reaction proceeded at a substrate concentration of 100 mmol/L, which resulted in a highly pure product [i.e., (-)-MPGM] with high enantiomeric excess (99.2%). These results suggest that the fusion lipase possessed the same chiral selectivity toward (±)-MPGM similar to other wild-type SmL [5]. Therefore, the existence of the Trx tag did not influence the chiral selectivity of SmL. 4. Conclusions In this work, the effects of fusion tags on SmL recombinant expression were revealed. Trx-tagged SmL was found to be a suitable choice. To optimize SmL production by E. coli BL21 (DE3)/pET32a-SmL, systematic optimization of the culture medium compositions and induction conditions was conducted. The recombinant SmL activity and productivity reached approximately 23,000 U/L and 1278 U L−1 h−1 in shake flask, respectively. Scale-up cultivation in a 7.0 L fermenter resulted in lipase activity of 19,650 U/L and productivity of 1228 U L−1 h−1. These results are the highest levels that have been reported so far, and completely meet the requirements of industrial SmL production in a recombinant E. coli strain.
Acknowledgments This work was supported by “A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions，PAPD”, the “Scientific Research Fund of Nanjing Forestry University for the Introduction of High-level talents” and the “Program for the Top Young Talents of Nanjing Forestry University”. All the authors have no conflict of interest to declare.
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Figure 1.
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Figure 2.
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Figure 3.
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Figure 4.
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Figure 5.
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Figure 6.
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