CN109136207B

CN109136207B - Method for producing phospholipase D by recombinant escherichia coli

Info

Publication number: CN109136207B
Application number: CN201810843255.2A
Authority: CN
Inventors: 卢英华; 熊维德; 曾宪海; 姚传义; 沈亮; 陈翠雪
Original assignee: Xiamen University
Current assignee: Xiamen University
Priority date: 2018-07-27
Filing date: 2018-07-27
Publication date: 2020-12-04
Anticipated expiration: 2038-07-27
Also published as: CN109136207A

Abstract

The invention discloses a method for producing phospholipase D by recombinant escherichia coli, which is characterized in that a phospholipase D gene is placed under the control of a strict promoter, a par region of pSC101 is inserted into an expression plasmid, meanwhile, an escherichia coli recA mutant strain is used for keeping the genetic stability of the plasmid, cells containing the plasmid are enriched and cultured to high density in a growth period, the induction period is saturated and induced, the temperature is reduced, the alkali metal salt stress is applied, the cytotoxicity of PLD to a host is relieved, the cell cracking is inhibited, the synthesis time of PLD is prolonged, and the expression of PLD is improved.

Description

Method for producing phospholipase D by recombinant escherichia coli

Technical Field

The invention belongs to the technical field of genetic engineering and microbial fermentation, and particularly relates to a method for producing phospholipase D by recombinant escherichia coli.

Background

Phospholipase D (EC 3.1.4.4, PLD) acts on phosphodiester bonds using phospholipids as substrates, and hydrolysis and transphosphatidylation occur depending on the receptor (water and alcohol). Among them, various alcohol groups can be introduced into substrate phospholipid through transphosphatidylation reaction to generate phospholipid with various biological activities and medicinal values, and the phospholipid is widely used in food and pharmaceutical industries. For example, when the substrate is Phosphatidylcholine (PC) and the receptor isPLD converts PC to Phosphatidylserine (PS), Phosphatidylglycerol (PG), and Phosphatidylethanolamine (PE) ((II) (P

and Iwasaki 2013). Among them, PS has received continuous attention as a brain health nutritional supplement with the aging population, and has been successively approved by the FDA in the united states, HBM in japan, and the ministry of health in china. PS prepared by PLD bioenzyme method with soybean PC as substrate, compared to PS extracted from bovine brain and plants, avoids the problems of food safety and low content of plant sources (Mor et al 2014). Further, some novel structural and functional phospholipids, some of which have anticancer and antioxidant activities, have been synthesized by PLD transphosphatidylation reactions, such as phosphatidylbatyl alcohol (Arranz-Martinez et al 2017), phosphatidylglucose (Song et al 2012), cardiolipin analogs (Muller et al 2012), phosphatidyltyrosol (Yamamoto et al 2011; Casado et al 2013), phosphatidylterpenes (Yamamoto et al 2008a; Yamamoto et al 2008b; Gliszcy ń a et al 2016), and phosphatidylserinol (Dippe et al 2008), among others.

For industrial applications and laboratory studies, PLD used for transphosphatidylation reactions mainly comes from plants (cucumber, cabbage and peanut) and actinomycetes (mainly Streptomyces). They show higher transphosphatidylation activity than other sources, and the sources are easily available. Dippe et al (Dippe et al.2008) compared PLDs_cab(derived cabbage) and PLD_str(Streptomyces sp origin) catalyzing the synthesis of cephalins of different polarity in yield and purity; in all reactions, the comparison was with PLD_cab，PLD_strShows obviously higher transphosphatidylation reaction activity.

Limited by the shortage of phospholipase D sources and high price (Streptomyces PLD, 6516.9 mg/1000U, Sigma), the wide industrial application is restricted. The highest yield of PLD is 5.5X 10, which is obtained by Ogino et al (Ogino et al 2004) in 2004 by genetic engineering in recombinant Streptomyces tenebrionus secretion and expression⁴U·L^-1(118mg·L^-1) (ii) a In China, the method comprises the steps of,in 2013, Zhangying (Zhanging 2013) obtains equivalent expression quantity (58U/mL) in recombinant streptomyces plumbizicus by using similar technology; in the last 5 years, other researchers in China bred excellent strains and optimized culture media, and the yield of PLD was 10³U·L^-1Left and right. In E.coli and yeast expression systems, recombinant PLD exhibits severe cytotoxicity, causing problems of plasmid instability, cell lysis, short synthesis time, and enzyme leakage, and high-density fermentation expression is difficult to achieve (Iwasaki et al 1995; Mishima et al 1997; Zamboneli et al 2003).

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a method for producing phospholipase D by recombinant escherichia coli, wherein a stable region is inserted into an expression plasmid, the stable inheritance of the plasmid in an recA mutant strain of the escherichia coli is maintained, the plasmid is cultured to high density in a growth period, a strict arabinose promoter is adopted for saturation induction in an induction period, the temperature is reduced, and alkali metal salt stress is applied, so that the cytotoxicity of PLD on a host is relieved, the cell lysis is inhibited, the synthesis time of PLD is prolonged, the expression of PLD is greatly improved, and the 1.1 x 10 PLD is finally achieved⁶U·L^-1(748mg·L^-1) The enzyme activity yield is 20 times of the highest yield before.

The technical scheme adopted by the invention for solving the technical problems is as follows:

a method for producing phospholipase D by recombinant Escherichia coli, comprising:

1) amplifying the par sequence using plasmid pUC57-par containing the par region sequence of pSC101 shown in SEQ ID No.1 as a template; to contain the arabinose promoter P_BADThe plasmid pBADK is used as a template to amplify a plasmid pBADK framework; respectively taking the par sequence and the pBADK skeleton obtained by amplification as a primer and a template, amplifying and transforming to obtain a vector pBADKP; the vector pBADKP was ligated and transformed into E.coli recA by double digestion with Nco I and Xba I using plasmid pUC57-PLD containing codon-optimized Streptomyces antibioticus PLD gene sequence (GenBank accession No. MH237968) as shown in SEQ ID No.2 and the vector pBADKP^-Obtaining a recombinant strain containing pBADKP-PLD from the defective strain;

2) culturing the recombinant strain containing pBADKP-PLD obtained in step 1) to high density (high density refers to middle and late stage of logarithmic cell growth, and carbon source is consumed to reach OD ₆₀₀5 to 6); and then supplementing nutrition, performing saturation induction, adjusting the temperature to 16-22 ℃, adding alkali metal salt to the concentration of 0.05-0.75M, and performing induction expression to obtain phospholipase D.

In one embodiment: in the step 1), the par sequence is amplified by using a primer 1 shown as SEQ ID No.3 and a primer 2 shown as SEQ ID No. 4.

In one embodiment: in the step 1), a pBADK framework of the amplification plasmid adopts a primer 3 shown as SEQ ID No.5 and a primer 4 shown as SEQ ID No. 6.

In one embodiment: in the step 1), the par sequence and the pBADK skeleton are respectively used as a primer and a template, and POE-PCR amplification is adopted and the primers are transformed into E.coli DH5 alpha.

In one embodiment: in the step 1), Escherichia coli recA^-Coli TOP10 (recA)^-) The obtained recombinant strain is TOP 10/pBADKP-PLD.

In one embodiment: in the step 2), the alkali metal salt is at least one of sodium salt, potassium salt and lithium salt.

In one embodiment: in the step 2), the alkali metal salt is NaCl, and the concentration is 0.15-0.75M, preferably 0.35-0.65M.

In one embodiment: in the step 2), the induction temperature is 17-19 ℃, and is preferably 18 ℃.

In one embodiment: in the step 2), the nutrition is 1-1.25: 1 (for example, 1:1 or 1.25: 1) of glycerol and yeast powder by mass ratio, and the final concentration of the glycerol is 20-30 g.L^-1。

In one embodiment: in the step 2), saturation induction is to add the inducer arabinose to the concentration of 0.03-0.04% OD₆₀₀ ^-1。

Compared with the background technology, the technical scheme has the following advantages:

par region and recA of pSC101^-Arabinose promoter P for ensuring stable inheritance and strict regulation of plasmid_BADSuppression ofCell lysis and promotion of PLD expression; the cells containing plasmids are enriched to high density in the growth period, which is beneficial to improving the yield; supplementing nutrition and saturating induction in the induction period, so as to promote cells to keep the optimal physiological state and produce enzyme to the maximum extent; the addition of alkali metal salt stress and low-temperature stress in the induction period is favorable for relieving the toxicity of PLD on cells, inhibiting cell lysis, prolonging the synthesis time of PLD and promoting PLD expression. The composite optimization strategy determined by the invention ensures that the plasmid stability is 100 percent, the cell lysis is completely inhibited, more than 99 percent of recombinant PLD is kept in cells, the cell lysis and enzyme leakage are completely inhibited in the induction period, the synthesis time of the PLD is prolonged to 36 hours from 2-3 hours reported in the literature, and the yield is improved to 1.1 multiplied by 10⁶U·L^-1(748mg·L^-1) The enzyme activity yield is 90 times of the expression level of escherichia coli reported in the prior art and 20 times of the expression level of streptomycete reported previously.

Drawings

The invention is further illustrated by the following figures and examples.

FIG. 1 is a diagram for explaining the effect of a promoter on phospholipase D expression.

FIG. 2 is used to illustrate the effect of induction temperature on cell growth and phospholipase D expression.

FIG. 3 is a graph illustrating the effect of salt on cell growth and phospholipase expression. Potassium phosphate (A), NaCl (B), KCl (C) and LiCl (D).

FIG. 4 is used to illustrate production of phospholipase D by batch culture in 3L fermentors under salt stress. (A) The sequence diagram, (B) SDS-PAGE analysis of the induction process, lanes 1-6 represent induction for 0h, 6h, 12h, 24h, 36h, 70h, respectively. Lane 7 is purified PLD.

FIG. 5 is a map of the pBAD/gIIIC plasmid used in the examples of the present invention.

FIG. 6 is a map of the pBADK plasmid used in the examples of the present invention.

FIG. 7 is a map of the pBADKP plasmid used in the examples of the present invention.

FIG. 8 is a plasmid map of pBADKP-PLD used in the examples of the present invention.

FIG. 9 is a map of pET22KP-PLD plasmid used in the examples of the present invention.

FIG. 10 is a plasmid map of pLACKP-PLD used in the examples of the present invention.

FIG. 11 is a map of a pUC57-par plasmid used in the example of the present invention.

FIG. 12 is a map of a pUC57-PLD plasmid used in the examples of the present invention.

Detailed Description

The present invention will be described in detail with reference to the following examples:

remarking: the materials and the instruments used in the following examples are specifically described below, but not limited thereto.

The main experimental materials:

plasmid pBADK (original plasmid was derived from plasmid pBAD/gIIIC of Invitrogen corporation, Amp^RSubstitution of the resistance Gene for Kan as shown in SEQ ID No.7^RThen pBADK is obtained and contains a strict arabinose promoter P_BADGuzman et al 1995), plasmid pBADKP-PLD (containing a stringent arabinose promoter P)_BADAnd par region), plasmid pET22KP-PLD (containing T7 promoter P)_T7And par region), plasmid pLACKP-PLD (containing the stringent lactose promoter P)_lac/ara-1Lutz and Bujard 1997 and the par region), plasmid pUC57-par (containing the par region sequence of pSC101 shown in SEQ ID No.1, synthesized by Producer (Shanghai) Co., Ltd., resulting in plasmid pUC57-par containing the par sequence shown in SEQ ID No. 1) and plasmid pUC57-PLD (containing the codon-optimized Streptomyces antibioticus PLD gene sequence shown in SEQ ID No.2, synthesized by Producer (Shanghai) Co., Ltd., resulting in plasmid pUC57-PLD containing the PLD sequence shown in SEQ ID No. 2). Coli DH5 alpha from Biotechnology engineering (Shanghai) Ltd, E.coli TOP10 (recA)^-) Purchased from Saimer Feishale technologies (China) Co. The related plasmid map is shown in the figure, wherein the black filled symbol represents the promoter (P)_BAD、P_T7And P_lac/ara-1) Signal peptide sequences (pelB signal sequence and Gene III sequencing signal sequence) localized in the periplasmic space, par region, Kan^RResistance genes, insertion of PLD by Nco I and Xba I, myc epitope tag designed for West bonting experiments, and for Ni²⁺Affinity chromatography purified histidine tag of 6 × His, both of which are located at the C-terminus of PLD.

Example 1 construction of recombinant bacterium TOP10/pBADKP-PLD

PCR-amplified the par sequence using plasmid pUC57-par (containing the par region sequence of pSC101 shown in SEQ ID No. 1) as a template by primer 1 and primer 2 (Table 1); to contain the arabinose promoter P_BADThe plasmid pBADK of (1) is used as a template, and a plasmid pBADK framework is amplified by PCR through a primer 3 and a primer 4 (table 1); taking the par sequence and the pBADK skeleton obtained by the two sections of amplification as a primer and a template respectively, carrying out POE-PCR, directly transforming the product to E.coli DH5 alpha, carrying out colony PCR verification and sequencing to obtain a vector pBADKP; plasmid pUC57-PLD containing codon-optimized Streptomyces antibioticus PLD gene sequence shown in SEQ ID No.2 was ligated with vector pBADKP by double digestion with Nco I and Xba I, T4 ligase, and transformed into E.coli TOP10 (recA)^-) And carrying out colony PCR verification and sequencing to obtain the recombinant bacterium TOP 10/pBADKP-PLD. The reaction systems and thermal cycling conditions for PCR and POE-PCR are shown in tables 2, 3, 4 and 5, respectively.

TABLE 1 primer sequences

TABLE 2 PCR reaction System

TABLE 3 PCR thermocycling conditions

TABLE 4 POE-PCR reaction System

TABLE 5 POE-PCR thermocycling conditions

Example 2. Effect of promoters on phospholipase D expression.

Four promoters P_T7、P_lac/ara-1 ^a、P_lac/ara-1 ^bAnd P_BADWas used to examine the expression of phospholipase D, whose activation intensity decreased in turn. Wherein P is_lac/ara-1 ^aPartially initiated by IPTG induction, P_lac/ara-1 ^bCompletely started by IPTG and arabinose. The recombinant strain TOP10/pBADKP-PLD was induced at 25 ℃ for 16h, and the results are shown in FIG. 1. In the expression of PLD under saturation induction, P_lac/ara-1 ^bAnd P_BADIs better than P_T7And P_lac/ara-1 ^aIn which P is_BADThe maximum PLD enzyme activity and specific yield are produced. In the induction process, the activity of the PLD enzyme under all promoters almost reaches the maximum value after 1h of induction; after 16h of induction, the extracellular enzyme activity accounts for 100%, 25%, 34% and 26% respectively; throughout the process, the increase in extracellular enzyme activity was accompanied by a decrease in biomass, where BLR (DE3)/pET22KP-PLD (promoter P)_T7) The biomass is reduced most rapidly. These results indicate that the toxicity of PLD results in short PLD synthesis time and cell lysis, and that the stronger the promoter, the higher the extracellular PLD enzyme activity, probably due to accelerated cell lysis. Weaker stringent promoter (P)_lac/ara-1And P_BAD) Is more suitable for expressing PLD, and has higher enzyme activity and less extracellular quantity.

Example 3 Effect of Induction temperature on cell growth and expression of phospholipase D

In this experiment, TOP10/pBADKP-PLD was cultured in TB medium at 37 ℃ to OD₆₀₀5-6, then at 12 degrees, 18 degrees, 22 degrees, 25 degrees, 30 degrees and 37 degrees C, respectively, induced expression for 16 hours, the temperature of induced on TOP10/pBADKP-PLD expression PLD and biomass effect, the results are shown in figure 2. The induction temperature significantly affected the expression of PLD and cell growth. The expression level is higher at the temperature of 16-22 ℃,the highest expression level occurred at 18 ℃ with a specific yield of 3.8 times that of 30 ℃ and 92% of PLD in the cells. Meanwhile, when the temperature is more than 18 ℃, PLD expression decreases. Cell growth at moderately lower temperatures is significantly better than at higher temperatures, which reflects that certain lower temperatures reduce the toxicity of PLD to the cells, while the extent of cell lysis is reduced.

Example 4 Effect of salt stress on cell growth and phospholipase expression

TOP10/pBADKP-PLD was cultured to OD at 37 ℃ in TB medium₆₀₀5-6, salts (potassium phosphate, NaCl, KCl and LiCl) with different concentrations are respectively added, and the induction expression is carried out for 16h at 18 ℃, and the result is shown in figure 3. The salt concentration is increased in the induction stage, so that the expression of PLD is obviously increased, the toxicity of PLD on cells is relieved, and the biomass of the cells is increased. The NaCl is most obvious, compared with a control group, the enzyme activity is improved by 5 times to the maximum, the biomass is increased under 0.15-0.75M NaCl, and the optimal concentration is 0.35-0.65M.

Example 5 expression of phospholipase D in 3L fermentor under salt stress

A small amount of the cells were frozen in TOP10/pBADKP-PLD glycerol storage tubes, and inoculated into 10mL of LB medium at 37 ℃ and 200rpm for overnight culture. Transferring the strain into 200mL TB medium according to the inoculation amount of 1%, and culturing at 37 ℃ and 200rpm for 6-7 h. Then inoculating into 3L fermentation tank containing fermentation medium, controlling temperature at 37 deg.C, and culturing cell to OD₆₀₀50-60, and when the glycerol is consumed, adjusting the culture temperature to 18 ℃. Respectively adding sodium chloride, arabinose, glycerol and yeast powder into the fermentation tank, wherein the final concentration of the sodium chloride is 0.4M, and the final concentration of the arabinose is 0.035%. OD₆₀₀ ^-1And the mass ratio of the glycerol to the yeast powder is 1.25:1, final concentration of glycerol 25 g.L^-1. The induction process is continued until the biomass begins to decrease or the activity of the PLD enzyme is reduced. As a result, as shown in FIG. 4, PLD synthesis continued for 32h with glycerol consumption and cell growth. This indicates that cell growth is coupled to PLD expression, and that the toxicity of PLD is well overcome during expression. The final highest enzyme yield reaches 1.1 multiplied by 10⁶U·L^-1(748mg·L^-1) The extracellular enzyme production is 1.6X 10⁴U·L^-1Only 1.5% of the total enzyme activity, indicating that cell lysis did not occur.

The above description is only a preferred embodiment of the present invention, and therefore should not be taken as limiting the scope of the invention, which is defined by the appended claims and their equivalents.

Sequence listing

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Claims

1. A method for producing phospholipase D by recombinant Escherichia coli is characterized in that: the method comprises the following steps:

1) so as to contain pSC101 shown as SEQ ID No.1parThe plasmid pUC57-par of the region sequence was used as a template for amplificationparA sequence; to contain the arabinose promoter P_BADThe plasmid pBADK is used as a template to amplify the plasmid pBADK boneA frame; obtained by the above amplificationparThe sequence and pBADK skeleton are used as primer and template separately, and the vector pBADKP is obtained through amplification and conversion; optimized by using a codon containing a sequence as shown in SEQ ID No.2Streptomyces antibioticusPlasmid pUC57-PLD of PLD gene sequence and the vector pBADKP were synthesized byNcoI andXbai double digestion, ligation and transformation into E.colirecA ^-Obtaining a recombinant strain containing pBADKP-PLD from the defective strain;

2) culturing the recombinant strain containing pBADKP-PLD obtained in the step 1) to high density; then supplementing nutrition, performing saturation induction, adjusting the temperature to 16-22 ℃, adding alkali metal salt to the concentration of 0.15-0.75M, and performing induction expression to obtain phospholipase D; the saturation induction is to add an inducer arabinose to the concentration of 0.03-0.04%. OD₆₀₀ ^-1。

2. The method for producing phospholipase D using recombinant Escherichia coli according to claim 1, wherein: in the step 1), amplificationparThe sequence adopts a primer 1 shown as SEQ ID No.3 and a primer 2 shown as SEQ ID No. 4.

3. The method for producing phospholipase D using recombinant Escherichia coli according to claim 1, wherein: in the step 1), a pBADK framework of the amplification plasmid adopts a primer 3 shown as SEQ ID No.5 and a primer 4 shown as SEQ ID No. 6.

4. The method for producing phospholipase D using recombinant Escherichia coli according to claim 1, wherein: in the step 1), toparThe sequence and pBADK skeleton are respectively used as primer and template, and POE-PCR amplification and transformation are carried outE. coliDH5α。

5. The method for producing phospholipase D using recombinant Escherichia coli according to claim 1, wherein: in the step 1), Escherichia colirecA ^-The defective plant isE. coli TOP10 recA ^-Defective strain, the obtained recombinant bacterium is TOP10/pBADKP-PLD。

6. The method for producing phospholipase D using recombinant Escherichia coli according to claim 1, wherein: in the step 2), the alkali metal salt is at least one of sodium salt, potassium salt and lithium salt.

7. The method for producing phospholipase D using recombinant Escherichia coli according to claim 1, wherein: in the step 2), the alkali metal salt is NaCl, and the concentration is 0.15-0.75M.

8. The method for producing phospholipase D using recombinant Escherichia coli according to claim 1, wherein: in the step 2), the nutrition is 1-1.25: 1 of glycerol and yeast powder by mass ratio, and the final concentration of the glycerol is 20-30 g.L^-1。