CN119876216A - Bacillus subtilis expression system for producing feruloyl esterase by fermentation and application thereof - Google Patents
Bacillus subtilis expression system for producing feruloyl esterase by fermentation and application thereof Download PDFInfo
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Abstract
本发明公开了一种用于发酵生产阿魏酸酯酶的枯草芽孢杆菌表达系统及其应用。该枯草芽孢杆菌表达系统的构建方法包括以下步骤：1)合成序列如SEQ ID NO:1所示阿魏酸酯酶的编码基因：2)将所述阿魏酸酯酶的编码基因构建到一种穿梭表达质粒中，得到阿魏酸酯酶表达质粒；3)将所述阿魏酸酯酶表达质粒转入枯草芽孢杆菌底盘菌株中，即可得到一种用于发酵生产阿魏酸酯酶的枯草芽孢杆菌表达系统。本发明通过将一种经密码子优化的革兰氏阳性菌来源的阿魏酸酯酶基因构建至枯草芽孢杆菌的T7表达系统，只需补加木糖或廉价的麸皮，即可高效分泌表达阿魏酸酯酶。该酶可用于饲料或食品原料的加工以及阿魏酸的生产等领域，具有广阔的应用前景。
The invention discloses a Bacillus subtilis expression system for fermentation production of feruloyl esterase and its application. The construction method of the Bacillus subtilis expression system comprises the following steps: 1) synthesizing a feruloyl esterase encoding gene with a sequence as shown in SEQ ID NO: 1; 2) constructing the feruloyl esterase encoding gene into a shuttle expression plasmid to obtain a feruloyl esterase expression plasmid; 3) transferring the feruloyl esterase expression plasmid into a Bacillus subtilis chassis strain to obtain a Bacillus subtilis expression system for fermentation production of feruloyl esterase. The invention constructs a codon-optimized Gram-positive bacteria-derived feruloyl esterase gene into the T7 expression system of Bacillus subtilis, and only needs to add xylose or cheap bran to efficiently secrete and express feruloyl esterase. The enzyme can be used in the fields of processing feed or food raw materials and production of ferulic acid, and has broad application prospects.
Description
Technical Field
The invention belongs to the technical field of construction of bacillus subtilis engineering bacteria, and particularly relates to a bacillus subtilis expression system for producing feruloyl esterase by fermentation and application thereof.
Background
As GRAS (Generally Recognised As Safe) strain, bacillus subtilis chassis strain is widely applied to bioengineering, especially industrial enzymes used in the field of medical food processing. Nevertheless, the molecular genetic tools of bacillus subtilis, such as promoter elements, etc., are still limited compared to e.coli, and the most widely used promoter is P43. Several novel promoters have been reported in recent years, but their efficiency is still inferior to the T7 promoter in T7 expression systems. The T7 promoter was originally used in E.coli and was able to initiate transcription of DNA sequences downstream thereof very efficiently under the action of T7 RNA polymerase. In recent years, it has been reported that T7 expression system can function in Bacillus, but has not been reported in Bacillus subtilis ATCC 6051 a. The bacillus subtilis ATCC 6051a is also called as A164 strain, has better effect of secretion expression of heterologous protein, faster growth in a culture medium containing peptone and better potential of industrial application compared with bacillus subtilis model strain 168 and derivative strains thereof which are researched in a laboratory.
Feruloyl esterase (EC 3.1.1.73;Ferulic acid esterase) is a carboxylate hydrolase which hydrolyzes ester bonds formed by ferulic acid, xylan and arabinose in the plant cell wall, releasing free ferulic acid. Meanwhile, the cellulose and cellulose can be fully contacted, and the degradation of cellulose in the plant cell wall is improved. In addition, feruloyl esterase has wide application in medicine, food, feed, papermaking, textile and other industrial fields. Thus, more and more researchers are devoted to the study of feruloyl esterases. FAE is classified into A, B, C, D classes according to its primary protein structure and its action characteristics on four synthetic substrates (methyl coumarate, methyl ferulate, methyl sinapic acid and methyl caffeate).
FAE of type A can be specially applied to phenolic esters containing CH 3O-in benzene rings similar to FA structures, and can catalyze and hydrolyze methyl sinapic acid, methyl p-coumarate and methyl ferulate, but has no catalysis to methyl caffeate. Class a FAEs are capable of specifically cleaving FA and arabinose-linked 1, 5-ester linkages, which have amino acid sequences similar to those of lipases. FAE from aspergillus niger belongs to class a. Class B FAEs, except for having no catalytic effect on methyl sinapite, all three other substrates can catalyze the binding of 1, 6-ester linkages to galactose residues and 1, 2-ester linkages to arabinose residues, with a protein primary structure similar to that of acetylxylan esterase. Class C and class D FAEs hydrolyze all 4 hydroxycinnamate substrates, wherein class C FAEs are unable to hydrolyze FA dimers, their protein primary structure is similar to chlorogenic acid esterase, tannase, whereas class D FAEs show higher protein primary structure similarity than xylanases, releasing ferulic acid dimers. At present, bacterial feruloyl esterase has low expression level, usually uses escherichia coli for intracellular expression, uses expensive IPTG for induction expression, and needs to perform wall breaking treatment on host cells, so that the purification cost of the enzyme is too high, and the method has no commercial application prospect.
Disclosure of Invention
The invention aims to provide a bacillus subtilis expression system for producing ferulic acid esterase by fermentation and application thereof, so as to solve the problems that the yield of ferulic acid esterase in the prior art is low and commercial production cannot be realized.
In order to solve the problems, the invention adopts the following technical scheme:
According to a first aspect of the invention, a construction method of a bacillus subtilis expression system for producing ferulic acid esterase by fermentation is provided, which comprises the following steps of 1) synthesizing a ferulic acid esterase coding gene with a sequence shown as SEQ ID NO. 1, 2) constructing the ferulic acid esterase coding gene into a shuttle expression plasmid to obtain a ferulic acid esterase expression plasmid, and 3) transferring the ferulic acid esterase expression plasmid into a bacillus subtilis chassis strain to obtain the bacillus subtilis expression system for producing ferulic acid esterase by fermentation.
According to the present invention, the bacillus subtilis expression system transcribes the gene encoding feruloyl esterase through T7 RNA polymerase and its constitutive promoter pT 7.
Preferably, the shuttle expression plasmid is pMK4-T7. It should be understood that the invention is not limited to pMK4-T7 expression plasmids, but may be other shuttle expression plasmids.
Preferably, the bacillus subtilis chassis strain is bacillus subtilis 164T7P.
According to a preferred embodiment of the invention, the bacillus subtilis chassis strain is bacillus subtilis 164T7P capable of expressing exogenous proteins at high level (see Chinese patent application CN 112226451A), and the strain carries out high-level transcriptional expression on the exogenous proteins by inserting a xylose-induced T7 RNA polymerase expression system into an aprE site of a bacillus subtilis genome, and in addition, the strain integrates a comk gene expression frame at the nprE site, so that the genetic operation efficiency of the strain is improved. It should be understood, however, that the bacillus subtilis 164T7P is the most preferred embodiment of the present invention, and the method of the present invention is not limited to the bacillus subtilis 164T7P, and any bacillus subtilis may be used.
According to a preferred scheme of the invention, a construction method of a bacillus subtilis expression system is provided, which comprises the following steps of 1) constructing a feruloyl esterase encoding gene shown in SEQ ID NO.1 into a shuttle expression plasmid through a synthetic sequence shown in SEQ ID NO.1 after gene sequence optimization, 2) transferring pMK4-T7-fe4 into bacillus subtilis 164T7P to obtain a 164T7P-pMK4-T7-fe4 expression strain.
According to a second aspect of the present invention there is provided a bacillus subtilis expression system for fermentative production of feruloyl esterase according to the above construction method.
According to a third aspect of the present invention there is provided the use of a bacillus subtilis expression system in the fermentative production of feruloyl esterase.
And (3) fermenting and producing feruloyl esterase by using the bacillus subtilis expression system in an LB culture medium and under the induction of xylose or bran.
Preferably, 0.1-1w/V% xylose or 1.0-3.0w/V% bran is added to LB medium for efficient induction of feruloyl esterase expression and growth of host cell lines.
According to the invention, the bacillus subtilis expression system can be used for the production of feruloyl esterase and also for microbial bioconversion of feed or food materials.
It should be understood that the feruloyl esterase shown in SEQ ID NO. 1 used in the present invention is a series of mutation, codon optimized, gram positive bacteria derived feruloyl esterase gene of the inventor. Furthermore, the invention realizes secretion after the feruloyl esterase gene is expressed by bacillus subtilis, which is quite rare at present, and more surprisingly, the invention also realizes efficient secretion and expression of the feruloyl esterase. The inventors have previously screened a variety of enzymes, only this feruloyl esterase being the most excellent for expression. The selectivity between enzyme and expression plasmid and host cell is unknown before, but the invention constructs a bacillus subtilis expression system for the first time through the selection of feruloyl esterase gene, expression plasmid and host cell, and successfully realizes the efficient secretion expression of feruloyl esterase.
Secondly, the invention also provides a fermentation production method for realizing feruloyl esterase by using the bacillus subtilis expression system. The method can realize high expression amount by using xylose or cheap bran and the like as an expression inducer, and is easy to realize industrial production.
Compared with the prior art, the bacillus subtilis expression system for producing feruloyl esterase by fermentation and the application thereof have the following beneficial effects:
1) The feruloyl esterase expression strain constructed by the invention can realize fermentation production of feruloyl esterase by taking xylose or cheap bran as an inducer;
2) The feruloyl esterase expression strain constructed by the invention has the advantages of rapid growth, high enzyme protein expression quantity and specific enzyme activity, capability of autonomous secretion and expression, low purification and application cost and good industrial application prospect.
In conclusion, the feruloyl esterase gene derived from gram-positive bacteria subjected to codon optimization is constructed to a T7 expression system of bacillus subtilis, and the feruloyl esterase can be secreted and expressed efficiently only by supplementing xylose or cheap bran. The enzyme can be used in the fields of feed or food raw material processing, ferulic acid production and the like, and has wide application prospect.
Drawings
FIG. 1 is a schematic diagram of feruloyl esterase expression plasmid pMK4-T7-fe 4;
FIG. 2 is a photograph of SDS polyacrylamide gel electrophoresis (SDS-PAGE) of the Bacillus subtilis expression system after secretion of feruloyl esterase;
FIG. 3 shows the results of enzyme activity assay of feruloyl esterase.
Detailed Description
The following describes the technical scheme of the present invention in detail by referring to examples. The operations, for which specific conditions are not noted in the examples, were performed according to conventional conditions or conditions suggested by the manufacturer. The reagents and biological materials used hereinafter are commercial products unless otherwise specified.
EXAMPLE 1 construction of feruloyl esterase expression plasmid
FIG. 1 is a schematic diagram of the feruloyl esterase expression plasmid pMK4-T7-fe4. The construction is based on plasmid pMK4-T7, and linear DNA is amplified by taking the plasmid as a template, and linear pMK4-T7 plasmid fragments are amplified. The primer pair VecF (SEQ ID NO: 3)/VecR (SEQ ID NO: 4) is used, after PCR reaction, the purification kit of FastPure Gel DNA Extraction Mini Kit product from Novain company is used for cleaning and recovering DNA, then the chemically synthesized DNA coding for feruloyl esterase is used as a template to amplify fe4 gene fragment, the primer pair is fe4-F (SEQ ID NO: 5)/fe 4-R (SEQ ID NO: 6), the template is removed by restriction endonuclease Dpn I from the amplified PCR product, the purification kit of FastPure Gel DNA Extraction Mini Kit product from Novain company is used for cleaning and recovering DNA, then the construction of pMK4-T7-fe4 is completed by fusion PCR, and the fusion PCR is operated as a PCR reaction system comprising 1 mu L of fe4 fragment, 1 mu L of linearized pMK4-T7 1 mu L, 2X PHANTA MASTER Mix 25 mu L, and ddH 2 O is added to a reaction system of 50 mu L. The PCR reaction conditions were pre-denatured at 95℃for 5min, then denatured at 95℃for 15s, annealed at 55℃for 15s, extended at 72℃for 2min, cycle number 30. The 10 mu L PCR reaction products are used for transforming bacillus subtilis 164T7P, after incubation, the bacillus subtilis 164T7P is evenly coated on a resistance plate containing 10 mu g/mL chloramphenicol, the bacillus subtilis 164T7P-pMK4 is placed in a 37 ℃ incubator for overnight culture, the single colony which grows out is identified through colony PCR, positive transformants are successfully transformed strains, monoclonal extracted plasmids are randomly picked up, sequencing is carried out by Shanghai Jie Li biotechnology company, and the plasmid which is completely correct in identification is identified is the plasmid pMK4-T7-fe4, and the corresponding recombinant bacteria are named bacillus subtilis 164T7P-pMK4-T7-fe4.
EXAMPLE 2 secretion expression and Activity detection of feruloyl esterase
Unless otherwise specified, the following media contained 10. Mu.g/mL chloramphenicol. A shake flask fermentation experiment is carried out by using bacillus subtilis 164T7P-pMK4-T7-fe4, firstly, picking up a monoclonal into a test tube filled with 3mL of LB culture medium (10 g/L peptone, 10g/L NaCl and 5g/L yeast powder), culturing at 200rpm and 37 ℃ overnight in a constant temperature shaking table, transferring 500 mu L of culture into a 500mL shake flask filled with 50mL of LB culture medium, culturing at 200rpm and 37 ℃ in the constant temperature shaking table, adding xylose with a final concentration of 0.1-1.0% (w/V) into the culture medium for induction when the strain grows to about 4 hours or the strain reaches about 600 -1, taking a sample every 24 hours, and measuring OD 600 by using an enzyme marker instrument.
Various crop wastes (wheat bran, corn bran, beet pulp, brewer's grains, etc.) rich in lignocellulose and the like contain a large amount of ferulic acid. FAEs can be used as auxiliary enzyme to assist lignin degrading enzyme or pectase to enter into action sites of plant cell wall, promote biomass conversion in plants, and release ferulic acid or other phenolic acid compounds with biological activity. Bran is a byproduct of wheat processing and is rich in arabinoxylans. The method is basically the same as the fermentation method using xylose as an inducer, except that the inducer xylose is replaced by inexpensive bran, other method steps are the same, firstly, single clone is selected and cultured in a test tube filled with 3mL LB culture medium at 200rpm and 37 ℃ overnight in a constant temperature shaking table, then 500 mu L of culture is transferred into a 500mL shaking bottle filled with 50mL LB+1.0-3.0% bran (w/V, wheat bran or corn bran) culture medium, one sample is taken every 24 hours at 200rpm and 37 ℃ in the constant temperature shaking table culture, and FIG. 2 shows a chart of staining after SDS polyacrylamide gel electrophoresis (SDS-PAGE) after secretion of feruloyl esterase by using bacillus subtilis, and the feruloyl esterase can be expressed at high level under the induction of xylose or xylose/wheat bran.
The feruloyl esterase activity detection principle is that the feruloyl methyl ester has maximum light absorption at 340nm, the feruloyl esterase can degrade ester bonds in the feruloyl methyl ester to reduce absorbance, and the enzyme activities of different samples are calculated by measuring the absorbance difference (delta A340) at 340 nm. The preparation method comprises the steps of weighing 100mg of ferulic acid methyl ester, fixing the volume to 100mL by using absolute ethyl alcohol, sequentially taking 0.02, 0.04, 0.06, 0.08 and 0.1mL of ferulic acid methyl ester solution from the solution, and fixing the volume to 10mL by using absolute ethyl alcohol to obtain ferulic acid methyl ester standard solutions with the concentrations of 9.6, 19.2, 28.8, 38.4 and 48 mu mol/L respectively. Adding diluted 0.6mL of crude enzyme solution into a 2mL centrifuge tube, preheating for 15min in a water bath at 30-70 ℃, sequentially adding 0.2mL of ferulic acid methyl ester solution with pH of 3.0-8.0 respectively, reacting for 30min in constant temperature water baths at different temperatures, and detecting absorbance of a reaction system at the reaction termination time of 340nm by using a microplate reader by taking the boiling inactivated enzyme solution as a reference. Defining 1 mu mol of ferulic acid methyl ester hydrolyzed per min as an enzyme activity unit, and obtaining an enzyme activity calculation formula, wherein the enzyme activity is = [ x (inactivation) -x (normal) ]multipliedby V sample total multiplied by N/(1000 multiplied by T multiplied by V sample). Wherein x is the value calculated after the absorbance value of the sample delta A340 is substituted into the standard curve, the total volume of the extracting solution is 0.5mL, N is the dilution factor, the sample volume of the reaction system is added, and T is the reaction time for 30min. The obtained feruloyl esterase has the highest enzyme activity of 228.56U/L at a pH=7 and a temperature of 40 ℃ as shown in the enzyme activity data of FIG. 3.
The foregoing description is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention, and various modifications can be made to the above-described embodiment of the present invention. All simple, equivalent changes and modifications made in accordance with the claims and the specification of the present application fall within the scope of the patent claims. The present invention is not described in detail in the conventional art.
Claims (9)
1. A method for constructing a bacillus subtilis expression system for producing feruloyl esterase by fermentation, which is characterized by comprising the following steps:
1) The coding gene of feruloyl esterase with the sequence shown in SEQ ID NO.1 is synthesized:
2) Constructing the coding gene of the feruloyl esterase into a shuttle expression plasmid to obtain the feruloyl esterase expression plasmid;
3) Transferring the feruloyl esterase expression plasmid into a bacillus subtilis chassis strain to obtain the bacillus subtilis expression system for producing feruloyl esterase by fermentation.
2. The method of claim 1, wherein the bacillus subtilis expression system transcribes the gene encoding feruloyl esterase by T7 RNA polymerase and its constitutive promoter pT 7.
3. The method of claim 1, wherein the shuttle expression plasmid is pMK4-T7.
4. The method of claim 1, wherein the bacillus subtilis chassis strain is bacillus subtilis 164T7P.
5. A Bacillus subtilis expression system for fermentative production of feruloyl esterase according to the construction method of any one of claims 1 to 4.
6. Use of the bacillus subtilis expression system according to claim 5 for the fermentative production of feruloyl esterase.
7. The use according to claim 6, wherein the fermentation production of feruloyl esterase is carried out in LB medium and under induction of xylose or bran by the expression system of Bacillus subtilis.
8. The use according to claim 7, characterized in that 0.1-1w/v% xylose or 1.0-3.0w/v% bran is added to LB medium for efficient induction of feruloyl esterase expression and growth of host cell lines.
9. The use according to claim 6, wherein the bacillus subtilis expression system is useful for microbial bioconversion of feed or food materials.
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