Bacillus subtilis expression system for fermentation production of feruloyl esterase and application thereof

Abstract

The invention discloses a bacillus subtilis expression system for fermentation production of feruloyl esterase and application of the bacillus subtilis expression system. The construction method of the bacillus subtilis expression system comprises the following steps: 1) synthesizing a feruloyl esterase coding gene with a sequence as shown in SEQ ID NO: 1; 2) constructing the feruloyl esterase coding gene into a shuttle expression plasmid to obtain a feruloyl esterase expression plasmid; and 3) transferring the feruloyl esterase expression plasmid into a bacillus subtilis chassis strain to obtain the bacillus subtilis expression system for fermentation production of feruloyl esterase. According to the invention, the feruloyl esterase gene optimized by a codon and derived from gram-positive bacteria is constructed to a T7 expression system of bacillus subtilis, and the feruloyl esterase can be efficiently secreted and expressed 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 a wide application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of Bacillus subtilis engineering bacteria construction, and particularly relates to a Bacillus subtilis expression system for fermenting and producing ferulic acid esterase and an application thereof. Background Art

[0002] As a GRAS (Generally Recognised As Safe) strain, Bacillus subtilis chassis strains are widely used in bioengineering, especially for industrial enzymes in the field of pharmaceutical and food processing. Despite this, the molecular genetic tools of Bacillus subtilis, such as promoter elements, are still limited compared to Escherichia coli, and the most widely used promoter is P43. In recent years, several new promoters have been reported, but their efficiency is still not as good as the T7 promoter in the T7 expression system. The T7 promoter was first used in Escherichia coli. Under the action of T7 RNA polymerase, it can very efficiently initiate transcription of its downstream DNA sequence. In recent years, there have been literature reports that the T7 expression system can work in Bacillus, but it has not been reported in Bacillus subtilis ATCC 6051a. Bacillus subtilis ATCC 6051a, also known as A164 strain, has a better effect on secreting and expressing heterologous proteins than the Bacillus subtilis model strain 168 and its derivative strains used in laboratory research, and grows faster in a medium containing peptone, and has better potential for industrial application.

[0003] Ferulic acid esterase (EC 3.1.1.73; Ferulic acid esterase) is a carboxylic acid ester hydrolase, which can hydrolyze the ester bond formed by ferulic acid, xylan and arabinose in plant cell walls to release free ferulic acid. At the same time, cellulase and cellulose can be fully contacted to improve the degradation of cellulose in plant cell walls. In addition, ferulic acid esterase has a wide range of applications in industrial fields such as medicine, food, feed, papermaking, and textiles. Therefore, more and more researchers are committed to the research of ferulic acid esterase. According to the primary structure of FAE protein and the action characteristics on four synthetic substrates (methyl coumarate, methyl ferulate, methyl sinapinic acid and methyl caffeate), FAE is divided into four categories: A, B, C, and D.

[0004] Class A FAE can specifically act on phenolic esters containing CH3O- in the benzene ring that are similar to FA in structure. It can catalyze the hydrolysis of methyl sinapate, methyl p-coumarate and methyl ferulate, but has no catalytic effect on methyl caffeate. Class A FAE can specifically cut the 1,5-ester bond connecting FA and arabinose, and its amino acid sequence is similar to that of lipase. FAE from Aspergillus niger belongs to Class A. Class B FAE has no catalytic effect on methyl sinapate, but can catalyze the other three substrates. It has a catalytic effect on FA bound to galactose residues with 1,6-ester bonds and arabinose residues with 1,2-ester bonds. Its primary protein structure is similar to that of acetyl xylan esterase. Class C and D FAEs can hydrolyze all four hydroxycinnamic acid methyl ester substrates, of which Class C FAEs cannot hydrolyze FA dimers, and their protein primary structure is similar to chlorogenic acid esterase and tannase, while Class D FAEs show a higher protein primary structure similarity to xylanase and can release ferulic acid dimers. Currently, the expression level of bacterial ferulic acid esterase is very low, usually expressed intracellularly in Escherichia coli, induced by expensive IPTG, and the host cells need to be broken, resulting in high enzyme purification costs and no commercial application prospects. Summary of the invention

[0005] The purpose of the present invention is to provide a Bacillus subtilis expression system for fermenting and producing ferulic acid esterase and application thereof, so as to solve the problem that the yield of ferulic acid esterase in the prior art is low and commercial production cannot be achieved.

[0006] In order to solve the above problems, the technical solution adopted by the present invention is as follows:

[0007] According to a first aspect of the present invention, a method for constructing a Bacillus subtilis expression system for fermentative production of feruloyl esterase is provided, comprising the following steps: 1) synthesizing a gene encoding a feruloyl esterase whose sequence is shown in SEQ ID NO: 1; 2) constructing the gene encoding the feruloyl esterase into a shuttle expression plasmid to obtain a feruloyl esterase expression plasmid; and 3) transferring the feruloyl esterase expression plasmid into a Bacillus subtilis chassis strain to obtain a Bacillus subtilis expression system for fermentative production of feruloyl esterase.

[0008] According to the present invention, the Bacillus subtilis expression system transcribes the gene encoding ferulic acid esterase via T7 RNA polymerase and its constitutive promoter pT7.

[0009] Preferably, the shuttle expression plasmid is pMK4-T7. However, it should be understood that the present invention is not limited to the pMK4-T7 expression plasmid, and other shuttle expression plasmids may also be used.

[0010] Preferably, the Bacillus subtilis chassis strain is Bacillus subtilis 164T7P.

[0011] According to a preferred embodiment of the present invention, the Bacillus subtilis chassis strain used is a strain of Bacillus subtilis 164T7P (see Chinese invention patent application CN112226451A) that can express exogenous proteins at a high level. The strain inserts a xylose-induced T7 RNA polymerase expression system into the aprE site of the Bacillus subtilis genome to perform high-level transcriptional expression of exogenous proteins; in addition, the strain integrates a comk gene expression cassette at the nprE site to improve its genetic manipulation efficiency. However, it should be understood 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 can be used.

[0012] According to a preferred embodiment of the present invention, a method for constructing a Bacillus subtilis expression system is provided, comprising the following steps: 1) synthesizing a gene encoding a ferulic acid esterase as shown in SEQ ID NO:1 after gene sequence optimization; 2) constructing the gene encoding a ferulic acid esterase as shown in SEQ ID NO:1 into a shuttle expression plasmid to obtain an expression plasmid pMK4-T7-fe4 as shown in SEQ ID NO:2; 3) transferring pMK4-T7-fe4 into Bacillus subtilis 164T7P to obtain a 164T7P-pMK4-T7-fe4 expression strain.

[0013] According to a second aspect of the present invention, there is provided a Bacillus subtilis expression system for fermentative production of ferulic acid esterase obtained according to the above construction method.

[0014] According to a third aspect of the present invention, there is provided a use of a Bacillus subtilis expression system in the fermentation production of ferulic acid esterase.

[0015] The Bacillus subtilis expression system is placed in LB medium and induced by xylose or bran to produce ferulic acid esterase by fermentation.

[0016] Preferably, 0.1-1 w / V% xylose or 1.0-3.0 w / V% bran is added to the LB medium to efficiently induce the expression of ferulic acid esterase and the growth of the host cell strain.

[0017] According to the present invention, the Bacillus subtilis expression system can be used for both the production of ferulic acid esterase and the microbial bioconversion of feed or food raw materials.

[0018] It should be understood that the feruloyl esterase as shown in SEQ ID NO:1 used in the present invention is a feruloyl esterase gene from Gram-positive bacteria that has been optimized by the inventor through a series of mutations and codons. Further, the present invention realizes secretion after the feruloyl esterase gene is expressed by Bacillus subtilis, which is quite rare at present, and what is more surprising is that the efficient secretory expression of feruloyl esterase is also realized. The inventor has screened a variety of enzymes before, and only this kind of feruloyl esterase has the most outstanding expression effect. The selectivity between enzymes and expression plasmids and host cells was unknown before, and the present invention, through the selection of feruloyl esterase genes, expression plasmids and host cells, constructs a Bacillus subtilis expression system for the first time, and successfully realizes the efficient secretory expression of feruloyl esterase.

[0019] Secondly, the present invention also provides a fermentation production method of ferulic acid esterase using a Bacillus subtilis expression system. The method only needs to use xylose or cheap bran as an expression inducer to achieve high expression levels and is easy to implement industrial production.

[0020] Compared with the prior art, the Bacillus subtilis expression system for fermentation production of ferulic acid esterase and its application provided by the present invention have the following beneficial effects:

[0021] 1) The ferulic acid esterase expression strain constructed by the present invention can realize the fermentation production of ferulic acid esterase using xylose or cheap bran as an inducer;

[0022] 2) The ferulic acid esterase expression strain constructed by the present invention grows rapidly, has high enzyme protein expression and specific enzyme activity, can be secreted and expressed autonomously, has low purification and application costs, and has good industrial application prospects.

[0023] In summary, the present invention constructs a codon-optimized ferulic acid esterase gene from Gram-positive bacteria into the T7 expression system of Bacillus subtilis, and only needs to add xylose or cheap bran to efficiently secrete and express ferulic acid 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is a schematic diagram of the ferulic acid esterase expression plasmid pMK4-T7-fe4;

[0025] Figure 2 This is the SDS-polyacrylamide gel electrophoresis photo (SDS-PAGE) of ferulic acid esterase secreted and expressed in the Bacillus subtilis expression system;

[0026] Figure 3 This is the result of ferulic acid esterase activity determination. DETAILED DESCRIPTION

[0027] The technical scheme of the present invention is described in detail below in conjunction with the examples. The operations without specific conditions in the examples are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents and biological materials used below are all commercial products unless otherwise specified.

[0028] Example 1: Construction of ferulic acid esterase expression plasmid

[0029] Figure 1 The schematic diagram of ferulic acid esterase expression plasmid pMK4-T7-fe4 is shown in FIG. The construction of the plasmid pMK4-T7 is based on the plasmid pMK4-T7. First, the linear DNA of the plasmid pMK4-T7 is amplified using the plasmid as a template to amplify the linear pMK4-T7 plasmid fragment. Using the primer pair VecF (SEQ ID NO: 3) / VecR (SEQ ID NO: 4), after the PCR reaction, the DNA was cleaned and recovered using the FastPure Gel DNA Extraction Mini Kit product purification kit from Novagen. Then, the chemically synthesized DNA encoding ferulic acid esterase was used as a template to amplify the fe4 gene fragment, and the primer pair was fe4-F (SEQ ID NO: 5) / fe4-R (SEQ ID NO: 6). The template of the amplified PCR product was removed by restriction endonuclease Dpn I, and then the DNA was cleaned and recovered using the FastPure Gel DNA Extraction Mini Kit product purification kit from Novagen. Then, the construction of pMK4-T7-fe4 was completed by fusion PCR. The fusion PCR operation was as follows: PCR reaction system: 1 μL of fe4 fragment, 1 μL of linearized plasmid pMK4-T7, 25 μL of 2×Phanta Master Mix, and ddH2O was added to the reaction system to make 50 μL. The PCR reaction conditions were pre-denaturation at 95°C for 5 min, then denaturation at 95°C for 15 s, annealing at 55°C for 15 s, extension at 72°C for 2 min, and a cycle number of 30. 10 μL of the PCR reaction product was used to transform Bacillus subtilis 164T7P. After incubation, it was evenly spread on a resistant plate containing 10 μg / mL chloramphenicol and placed in a 37°C incubator for overnight culture. For the single colonies grown, they were identified by colony PCR. The positive transformants were the successfully transformed strains. Single clones were randomly selected to extract plasmids, which were sequenced by Shanghai Jie Li Biotechnology Co., Ltd. The completely correct plasmid was identified as plasmid pMK4-T7-fe4, and the corresponding recombinant bacteria were named Bacillus subtilis 164T7P-pMK4-T7-fe4.

[0030] Example 2 Secretory Expression and Activity Detection of Ferulic Acid Esterase

[0031] Unless otherwise specified, the following culture media contain 10 μg / mL chloramphenicol. Use Bacillus subtilis 164T7P-pMK4-T7-fe4 for shake flask fermentation experiments: First, pick a single clone into a test tube containing 3 mL LB medium (formula: 10 g / L peptone, 10 g / L NaCl, 5 g / L yeast powder), and culture it in a constant temperature shaker at 200 rpm and 37 °C overnight; then take 500 μL of the culture and transfer it to a 500 mL shake flask containing 50 mL LB medium, and culture it in a constant temperature shaker at 200 rpm and 37 °C; wait for growth for ~4 h or OD 600 When the concentration of xylose reaches about 1, a final concentration of 0.1-1.0% (w / V) of xylose is added to the culture medium for induction; samples are taken every 24 hours and the OD is measured using an enzyme marker. 600 .

[0032] Various agricultural wastes rich in lignocellulose (wheat bran, corn bran, beet pulp and brewer's grains, etc.) contain a large amount of ferulic acid. As auxiliary enzymes, FAEs can assist lignin degrading enzymes or pectinases to enter the action sites of plant cell walls, promote the conversion of biomass in plants, and release biologically active ferulic acid or other phenolic acid compounds. Bran is a by-product of wheat processing and is rich in arabinoxylan. This part is basically the same as the fermentation method using xylose as an inducer mentioned above. The only difference is that the inducer xylose is replaced with cheap bran. The other steps are the same: first, pick a single clone into a test tube containing 3mL LB medium, and culture it in a constant temperature shaker at 200rpm and 37℃ overnight; then take 500μL of the culture and transfer it to a 500mL shake flask containing 50mL LB+1.0-3.0% bran (w / V, wheat bran or corn bran) medium, and culture it in a constant temperature shaker at 200rpm and 37℃. Samples are taken every 24h. Figure 2 The figure shows the SDS-PAGE staining after feruloyl esterase was secreted and expressed by Bacillus subtilis. It can be seen that feruloyl esterase can be expressed at a high level under the induction of xylose or xylose / wheat bran.

[0033] The principle of ferulic acid esterase activity detection is that methyl ferulic acid has maximum light absorption at 340nm. Ferulic acid esterase can degrade the ester bond in methyl ferulic acid to reduce the absorbance. The enzyme activity of different samples is calculated by measuring the absorbance difference (ΔA340) at 340nm. The specific operation is as follows: weigh 100mg of methyl ferulic acid and dilute it to 100mL with anhydrous ethanol; take 0.02, 0.04, 0.06, 0.08 and 0.1mL of methyl ferulic acid solution in turn, and dilute it to 10mL with anhydrous ethanol respectively, to obtain methyl ferulic acid standard solutions with concentrations of 9.6, 19.2, 28.8, 38.4 and 48μmol / L respectively. Take 0.6 mL of the diluted crude enzyme solution and add it to a 2 mL centrifuge tube, preheat it in a 30-70°C water bath for 15 min, then add 0.2 mL of methyl ferulate solution with a pH of 3.0-8.0, and react in a constant temperature water bath at different temperatures for 30 min; use the boiled inactivated enzyme solution as a control, and use an enzyme marker to detect the absorbance of the reaction system at 340 nm at the end of the reaction. Define the hydrolysis of 1 μmol of methyl ferulate per min as one enzyme activity unit, and the enzyme activity calculation formula is: Enzyme activity = [x (inactivated) - x (normal)] × V total sample × N / (1000 × T × V sample). Among them, x is the value calculated by substituting the absorbance value of the sample ΔA340 into the standard curve; V total sample is the total volume of the extract, 0.5 mL; N is the dilution multiple; V sample is the sample volume added to the reaction system; T is the reaction time, 30 min. The enzyme activity data of ferulic acid esterase obtained are as follows: Figure 3 As shown, it can be seen that when pH = 7 and temperature is 40°C, the enzyme activity of ferulic acid esterase is the highest, which is 228.56 U / L.

[0034] The above is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. The above embodiments of the present invention can also be modified in various ways. All simple, equivalent changes and modifications made according to the claims and the description of the present invention fall within the scope of protection of the claims of the present invention. The contents not described in detail in the present invention are all conventional technical contents.

Claims

1. A method for constructing a Bacillus subtilis expression system for fermentation production of ferulic acid esterase, characterized in that: The following steps are involved: 1) Synthesize the gene encoding ferulic acid esterase as shown in SEQ ID NO: 1: 2) constructing the encoding gene of the feruloyl esterase into a shuttle expression plasmid to obtain a feruloyl esterase expression plasmid; 3) The ferulic acid esterase expression plasmid is transferred into a Bacillus subtilis chassis strain to obtain a Bacillus subtilis expression system for fermentation production of ferulic acid esterase.

2. The construction method according to claim 1, characterized in that: The Bacillus subtilis expression system transcribes the gene encoding ferulic acid esterase through T7 RNA polymerase and its constitutive promoter pT7.

3. The construction method according to claim 1, characterized in that: The shuttle expression plasmid is pMK4-T7.

4. The construction method according to claim 1, characterized in that: The Bacillus subtilis chassis strain is Bacillus subtilis 164T7P.

5. A Bacillus subtilis expression system for fermentative production of ferulic acid esterase obtained 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 in the fermentation production of ferulic acid esterase.

7. The use according to claim 6, characterized in that: The Bacillus subtilis expression system is placed in LB medium and induced by xylose or bran to produce ferulic acid esterase by fermentation.

8. The use according to claim 7, characterized in that: 0.1-1 w / V% xylose or 1.0-3.0 w / V% bran is added to the LB medium to efficiently induce the expression of ferulic acid esterase and the growth of the host cell strain.

9. The use according to claim 6, characterized in that: The Bacillus subtilis expression system can be used for microbial bioconversion of feed or food raw materials.

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