CN111826377B

CN111826377B - Signal peptide for promoting extracellular expression of pullulanase

Info

Publication number: CN111826377B
Application number: CN202010749154.6A
Authority: CN
Inventors: 佟毅; 李义; 陶进; 刘松; 徐奎栋; 李江华; 陈坚
Original assignee: Jiangnan University; Cofco Jilin Bio Chemical Technology Co Ltd
Current assignee: Jiangnan University; Cofco Jilin Bio Chemical Technology Co Ltd
Priority date: 2020-07-30
Filing date: 2020-07-30
Publication date: 2023-09-01
Anticipated expiration: 2040-07-30
Also published as: CN111826377A

Abstract

The invention discloses a signal peptide for promoting the extracellular expression of pullulanase, belonging to the technical fields of genetic engineering and enzyme engineering. The invention uses bacillus subtilis (Bacillus subtilis) to express host, and carries out synonymous mutation to bacillus subtilis endogenous signal peptide AprE, sacB, epr to obtain synonymous mutation sequence capable of obviously improving extracellular protein quantity, thereby ensuring normal translation of protein and improving protein expression quantity. The synonymous mutant sequences of the Apre, epr, sacB signal peptide can respectively improve the extracellular enzyme activity by 2.92,1.08,1.37 times compared with the wild type, thereby providing a strategy for the extracellular efficient expression of the pullulanase.

Description

Signal peptide for promoting extracellular expression of pullulanase

Technical Field

The invention relates to a signal peptide for promoting the extracellular expression of pullulanase, belonging to the technical fields of genetic engineering and enzyme engineering.

Background

Pullulanase (EC 3.2.1.41) belongs to the family of alpha-amylases, which are capable of specifically hydrolyzing branched alpha-1, 6-glycosidic bonds of amylopectin, also known as starch debranching enzymes. However, when wild bacteria are used as the production bacteria to produce pullulanase, various problems exist, including difficult culture, low yield and the like, so that the industrial mass production is difficult to realize. With the rapid development of genetic engineering, the pullulanase encoding gene realizes heterologous expression in different genetic engineering strains.

Soluble expression of pullulanase in bacillus subtilis has been successfully achieved in our earlier studies, and the expression of pullulanase has been driven by the strong promoter PlytR (Yang, S et al Characterization and application of endogenous phase-dependent promoters in Bacillus subtilis. Appl Microbiol Biotechnol, 2017). However, by means of these current approaches, although the extracellular enzymatic activity of pullulanase has been significantly improved, it is still being sought to meet the demands of large-scale industrial production. Therefore, how to further increase the extracellular expression of pullulanase is still a urgent problem to be solved.

Disclosure of Invention

In order to solve the problems, the extracellular expression quantity of the pullulanase is further improved, and the extracellular expression of the pullulanase can be improved by synonymous mutation means. Synonymous mutations in genes can leave the amino acid sequence unchanged, but can produce significant changes in gene transcript levels. The invention modifies the coding region of the N end of the gene by synonymous mutation means, which not only can realize the remarkable improvement of the gene expression quantity, but also has almost negligible influence on the enzyme activity. The signal peptide screened by synonymous mutation can obviously improve the extracellular enzyme activity of pullulanase.

The invention provides a kind of signal peptide, the nucleotide sequence of which is shown as any one of SEQ ID NO. 1-3.

In one embodiment of the invention, the nucleotide sequences of the signal peptides are synonymous mutant sequences of the signal peptides AprE, sacB, epr, respectively.

In one embodiment of the invention, the synonymous mutant sequence of the signal peptide AprE is shown in SEQ ID No. 1; the synonymous mutation sequence of the signal peptide SacB is shown in SEQ ID NO. 2; the synonymous mutant sequence of the signal peptide Epr is shown in SEQ ID NO. 3.

The invention provides a recombinant plasmid containing signal peptide with nucleotide sequence shown as SEQ ID NO. 1-3.

In one embodiment of the invention, the heavy plasmid is based on any vector that can be expressed in bacillus subtilis.

In one embodiment of the invention, the departure vector comprises P43NMK.

In one embodiment of the invention, the recombinant plasmid further comprises a constitutive promoter.

In one embodiment of the invention, the constitutive promoter comprises a LytR promoter, the LytR promoter nucleotide sequence of which is shown in SEQ ID NO. 4.

The invention provides recombinant bacteria containing signal peptides with nucleotide sequences shown as SEQ ID NO. 1-3.

In one embodiment of the invention, the recombinant bacterium is a bacillus subtilis host.

In one embodiment of the present invention, the recombinant bacterium is hosted by bacillus subtilis WB600, or bacillus subtilis 168.

The invention provides a method for improving the expression quantity of protein, which takes recombinant bacteria containing signal peptide with nucleotide sequences shown as SEQ ID NO. 1-3 as fermentation strain to produce protein.

In one embodiment of the present invention, the recombinant bacterium is cultured to OD ₆₀₀ Bacterial liquid not lower than 3.0, and inoculating the bacterial liquid into a reaction system according to the proportion of 1-10 mL/100 mL.

In one embodiment of the present invention, the medium of the culture system is TB medium, and is cultured at 30 to 40℃and 200 to 250rpm for 25 to 35 hours.

The invention provides a method for improving the expression quantity of extracellular proteins of bacillus subtilis, which adds signal peptide with nucleotide sequences shown as SEQ ID NO. 1-3 at the N end of a nucleotide sequence of a coded protein.

In one embodiment of the invention, a signal peptide with a nucleotide sequence shown as SEQ ID NO. 1-3 is added at the N-terminal of a target protein, and a recombinant plasmid is constructed and introduced into bacillus subtilis.

In one embodiment of the present invention, the signal peptide having the nucleotide sequence shown in SEQ ID NO.1 to 3 is inserted after the initiation codon ATG of the nucleotide sequence encoding the target protein.

In one embodiment of the invention, the protein of interest is any protein capable of extracellular expression in bacillus subtilis.

In one embodiment of the invention, the protein of interest comprises pullulanase and/or sfGFP.

In one embodiment of the present invention, the NCBI accession number of the pullulanase amino acid sequence is AMQ67157, and the amino acid sequence is shown in SEQ ID NO. 5.

In one embodiment of the invention, the nucleotide sequence encoding said sfGFP is shown in SEQ ID No. 7.

The invention also protects the application of the signal peptide with the nucleotide sequence shown as SEQ ID NO. 1-3, or the recombinant plasmid containing the signal peptide with the nucleotide sequence shown as SEQ ID NO. 1-3, or the recombinant bacteria containing the signal peptide with the nucleotide sequence shown as SEQ ID NO. 1-3 in improving the expression quantity of extracellular proteins.

The invention also protects the application of the method for improving the protein expression quantity in improving the extracellular protein expression quantity.

The invention also provides application of the method for improving the extracellular protein expression quantity of the bacillus subtilis in improving the extracellular protein expression quantity.

The invention also protects the application of the recombinant plasmid, the recombinant bacterium or the signal peptide AprE, sacB and Epr synonymous mutant sequences in improving the expression quantity of extracellular proteins.

The invention has the beneficial effects that:

the invention screens synonymous mutant sequences capable of improving the extracellular expression quantity of protein through synonymous mutation of signal peptides AprE, sacB and Epr, fuses the obtained mutant sequences at the N end of pullulanase, can obviously improve and reduce the extracellular enzyme activity yield of the pullulanase, and can respectively improve the extracellular enzyme activity of the pullulanase by 2.92,1.08,1.37 times in bacillus subtilis transformation production.

Drawings

FIG. 1 shows the expression plasmid map (for example AprE) of signal peptide and sfGFP fused to N-and C-termini of pullulanase, respectively.

FIG. 2 is a map of an expression plasmid fused to the N-terminus of pullulanase (AprE, for example) with a signal peptide.

FIG. 3 shows the extracellular enzyme activities of pullulanase secreted by wild-type and NCS engineered strains.

Detailed Description

Seed culture medium: peptone 10g/L, yeast extract 5g/L, sodium chloride 5g/L.

The fermentation medium is TB medium: peptone 12g/L, yeast extract 24g/L, glycerol 4mL/L, KH ₂ PO ₄ 0.017mol/L，K ₂ HPO ₄ 0.072mol/L。

Seed culture: and (3) picking a single colony of engineering bacteria, inoculating the single colony of engineering bacteria into a triangular flask (250 mL) with the liquid loading amount of 25mL, and culturing at the culture temperature of 37 ℃ and the rotation speed of a shaking table of 200r/min for 12h.

Fermentation culture: the culture was carried out at 37℃for 48 hours by inoculating the culture medium to a flask (250 mL) having a liquid loading amount of 25mL at an inoculum size of 4% (v/v).

Green fluorescent protein expression amount and biomass measurement: in a 96-well plate, if 200 μl of diluted broth was used, using a Cystation 3 cell imaging microplate detector (Bertoni instruments Co., U.S.) green fluorescence excitation wavelength: 480nm, green fluorescence emission wavelength: 520nm, cell growth OD absorbance wavelength: 600nm.

The enzyme activity determination method of the pullulanase comprises the following steps: 1mL of pullulan substrate and 0.9mL 100mM pH 4.5 acetic acid-sodium acetate buffer solution with 1mg/100mL are uniformly mixed, placed in a water bath kettle with the temperature of 60 ℃ for preheating for 10min, 0.1mL of pullulanase solution is added for reaction for 10min, 3mL of DNS color development solution is added, then the mixture is boiled in a boiling water bath for 7min, placed in ice water for stopping the color development reaction, 10mL of deionized water is added, uniformly mixed, and the light absorption value is measured at 540 nm.

Definition of enzyme activity: the amount of enzyme that produces 1. Mu. Mol of reducing sugar per unit time is one enzyme activity unit.

Example 1: construction of Apre Signal peptide NCS synonymous mutation library

Connecting an LytR promoter (the sequence information of which is shown as SEQ ID NO. 4) to a P43NMK plasmid through a one-step cloning kit (purchased from Nanjinouzan biotechnology Co., ltd.) to construct a recombinant plasmid, transferring the recombinant plasmid into E.coli JM109, coating bacterial liquid on an LB plate containing 100 mug/L ampicillin resistance, culturing at 37 ℃ until a monoclonal grows, and picking up the monoclonal to verify by sequencing to obtain the plasmid P43NMK-LytR; by using the same one-step cloning method, a pullulanase gene (the nucleotide sequence of which is shown as SEQ ID NO. 6) is fused to the downstream of LytR through a one-step cloning kit, and a recombinant plasmid P43NMK-LytR-Pul is constructed; by using the same one-step cloning method, sfGFP fluorescent protein and Apre signal peptide (the nucleotide sequences are shown as SEQ ID NO.7 and 8) are respectively connected to the C end and the N end of pullulanase, and recombinant plasmid P43NMK-Apre-Pul is constructed.

The P43NMK-Apre-Pul is used as a template, and an upstream and downstream degenerate primer is used to obtain a synonymous mutation library (synonymous mutation recombinant plasmid) of the front 30 bases of the N end of Apre through PCR, namely, the front 30 amino acid sequence is unchanged, and the nucleotide sequence is changed.

Upstream degenerate primer:

ATGAGRAGYAARAARTTRTGGATHAGYTTRTTRtttgcgttaacgttaatctttacgatgg(SEQ ID NO.12)；

downstream degenerate primers:

GTGTACATTTCACCTCCTTTAAATTACTTTCATTAT(SEQ ID NO.11)。

example 2: construction of SacB Signal peptide NCS synonymous mutation library

Specific embodiments are the same as in example 1, except that after P43NMK-LytR-Pul is obtained, a recombinant plasmid P43NMK-SacB-Pul is constructed by connecting sfGFP fluorescent protein and SacB signal peptide (nucleotide sequence shown as SEQ ID NO. 9) to the C-terminus (sfGFP fluorescent protein) and N-terminus (SacB signal peptide) of pullulanase, respectively, by a one-step cloning method.

The P43NMK-SacB-Pul is used as a template, and a synonymous mutation library (synonymous mutation recombinant plasmid) of the front 30 bases of the N end of the SacB is obtained by PCR (polymerase chain reaction) by using upstream and downstream degenerate primers, namely, the front 30 amino acid sequence is unchanged, and the nucleotide sequence is changed.

Upstream degenerate primer:

ATGCTNAARAGRACNTCNTTYGTNTCNTCNTTRttcatcagttcagctgttttactatcaatct(SEQ ID NO.13)；

downstream degenerate primers:

GTGTACATTTCACCTCCTTTAAATTACTTTCATTAT(SEQ ID NO.11)。

example 3: construction of Epr Signal peptide NCS synonymous mutation library

Specific embodiments are the same as in example 1, except that after P43NMK-LytR-Pul was obtained, a recombinant plasmid P43NMK-SacB-Pul was constructed by connecting sfGFP fluorescent protein and Epr signal peptide (nucleotide sequence shown in SEQ ID NO. 10) to the C-terminus (sfGFP fluorescent protein) and N-terminus (SacB signal peptide) of pullulanase, respectively, using a one-step cloning method.

And (3) taking P43NMK-Epr-Pul as a template, using upstream and downstream degenerate primers, and obtaining a synonymous mutation library (synonymous mutation recombinant plasmid) of the front 30 bases of the N end of Epr by PCR, wherein the front 30 amino acid sequence is unchanged, and the nucleotide sequence is changed.

Upstream degenerate primer:

ATGTTRAARAARGTNATHTTRGCNGCNTTYATHttagtaggaagtactttgggagctttta(SEQ ID NO.14)；

downstream degenerate primers:

GTGTACATTTCACCTCCTTTAAATTACTTTCATTAT(SEQ ID NO.11)。

example 4: screening of NCS synonymous mutant library Signal peptide nucleotide sequence

(1) The synonymous mutant recombinant plasmids constructed in examples 1-3 are respectively transformed into an expression host bacillus subtilis WB600, the transformation solution is coated on an LB plate containing 50 mug/mL of the resistance of the calicheamicin for challenging growth, the culture is carried out at 37 ℃ until monoclonal grows, the monoclonal is picked up to a 96 shallow pore plate containing 200 mug/mL of LB culture medium containing 50 mug/mL of the resistance of the calicheamicin, and the culture is carried out for 8 hours, so as to obtain seed solution;

(2) Inoculating the seed solution into a 96-deep well plate containing 50 mug/mL of a calicheamicin resistant 800 mug TB culture medium according to an inoculum size of 4mL/100mL, and culturing for 24 hours to obtain a fermentation broth;

(3) The fermentation broth was rapidly frozen on ice, centrifuged, the supernatant removed, diluted to a suitable multiple with 100mM PBS buffer (pH 7.2), and the fluorescence (excitation light 480, absorption light 520) and OD were measured by a microplate reader ₆₀₀ 。

Defining the relative fluorescence intensity RFI=fluorescence value/OD, and selecting the cell with the highest relative fluorescence intensity according to the RFI value to send to the Shanghai engineering company for sequencing.

After sequencing and identification, the AprE signal peptide successfully realizes synonymous mutation at the N end, and the nucleotide sequence of a mutant part of the AprE signal peptide is shown as SEQ ID NO. 15; the SacB signal peptide successfully realizes synonymous mutation at the N end, and the nucleotide sequence of a mutant part of the SacB signal peptide is shown as SEQ ID NO. 16; the Epr signal peptide successfully realizes synonymous mutation at the N end, and the nucleotide sequence of the mutant part is shown as SEQ ID NO.17.

Example 5: application of synonymous mutant signal peptide in improving extracellular expression quantity of pullulanase

Extracting plasmids from sequenced cells having the highest fluorescence intensity obtained in example 4, removing sfGFP fluorescent protein by PCR using primers to obtain recombinant plasmids from which fluorescent protein was removed, transforming recombinant plasmids each containing AprE signal peptide, sacB signal peptide, and Epr signal peptide synonymous mutant sequences into Bacillus subtilis WB600 to obtain recombinant bacteria, inoculating the recombinant bacteria into 250mL shake flask containing 50. Mu.g/mL of calicheamicin-resistant 20mL LB medium, fermenting at 37℃and 220rpm for 8 hours to obtain OD ₆₀₀ Above 4, the cells were inoculated in a proportion of 4mL/100mL into 250mL shake flasks containing 50. Mu.g/mL of a calicheamicin-resistant 25mL TB medium, and after fermentation at 37℃and 250rpm for 30 hours, the extracellular enzyme activities of pullulanase were measured, and the results are shown in FIG. 3.

Removal of sfGFP upstream primer:

CGGTAAAAAATAAtgagattatcaaaaaggatcttcacctagat(SEQ ID NO.18)；

removal of sfGFP downstream primer:

gataatctcaTTATTTTTTACCGTGATCTGGAGAAACttc(SEQ ID NO.19)。

by measuring the extracellular enzyme activity of pullulanase, the synonymous mutant sequences of the Apre, epr, sacB signal peptide are found to respectively increase the extracellular enzyme activity 2.92,1.08,1.37 times compared with the wild type.

TABLE 1 pullulanase extracellular enzyme Activity (U/mL)

While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

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Jiangnan University

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<213> artificial sequence

<400> 12

atgagragya araarttrtg gathagyttr ttrtttgcgt taacgttaat ctttacgatg 60

g 61

<210> 13

<211> 64

<212> DNA

<213> artificial sequence

<220>

<221> misc_feature

<222> (6)..(6)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (15)..(15)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (18)..(18)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (24)..(24)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (27)..(27)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (30)..(30)

<223> n is a, c, g, or t

<400> 13

atgctnaara gracntcntt ygtntcntcn ttrttcatca gttcagctgt tttactatca 60

atct 64

<210> 14

<211> 61

<212> DNA

<213> artificial sequence

<220>

<221> misc_feature

<222> (15)..(15)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (24)..(24)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (27)..(27)

<223> n is a, c, g, or t

<400> 14

atgttraara argtnathtt rgcngcntty athttagtag gaagtacttt gggagctttt 60

a 61

<210> 15

<211> 30

<212> DNA

<213> artificial sequence

<400> 15

agaagtaaaa aattatggat aagtttatta 30

<210> 16

<211> 30

<212> DNA

<213> artificial sequence

<400> 16

aatataaaaa aatttgcaaa acaagctaca 30

<210> 17

<211> 30

<212> DNA

<213> artificial sequence

<400> 17

aaaaatatgt cttgtaaact agttgtatct 30

<210> 18

<211> 44

<212> DNA

<213> artificial sequence

<400> 18

cggtaaaaaa taatgagatt atcaaaaagg atcttcacct agat 44

<210> 19

<211> 40

<212> DNA

<213> artificial sequence

<400> 19

gataatctca ttatttttta ccgtgatctg gagaaacttc 40

Claims

1. A method for improving the expression quantity of extracellular proteins of bacillus subtilis is characterized in that a signal peptide is added at the N end of a nucleotide sequence of an encoded protein; the nucleotide sequences of the signal peptide are respectively shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, the protein comprises pullulanase and/or sfGFP, and the nucleotide sequence of the pullulanase is shown as NCBI accession number: AMQ 67157; the nucleotide sequence for encoding the sfGFP is shown as SEQ ID NO. 7.

2. The method of claim 1, wherein the signal peptide is added to the N-terminal of the target protein to construct a recombinant plasmid; and then the recombinant plasmid is guided into bacillus subtilis to construct recombinant bacteria.

3. The method according to claim 2, wherein the recombinant bacterium is cultured and OD is obtained ₆₀₀ At least 3 bacterial liquid, and inoculating the bacterial liquid into the reaction system in an amount of 1-10 mL/100 mL.