Method for efficiently expressing pullulanase in bacillus subtilis and recombinant bacillus subtilis

Abstract

The present invention relates to the field of genetic engineering, particularly to a method for efficiently expressing pullulanase in Bacillus subtilis and recombinant Bacillus subtilis. said method includes steps of constructing modified Bacillus subtilis strain with deletion of alkaline protease gene and neutral protease gene, constructing expression vector including an optimized combination of promoter and signal peptide and pullulanase gene, and transforming said modified Bacillus subtilis strain with by said expression vector. A series of combinations of promoter and signal peptide are optimized to obtain the combination for efficiently expressing pullulanase, provide an industrial application basis.

Claims

1. A method for efficiently expressing pullulanase in Bacillus subtilis comprising the steps of constructing Bacillus subtilis strain with deletion of alkaline protease gene and neutral protease gene, constructing expression vector including a combination of promoter and signal peptide and pullulanase gene, and transforming said Bacillus subtilis strain with deletion of alkaline protease gene and neutral protease gene by said expression vector, wherein said promoter is a hybrid promoter having the nucleotide sequence of SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16 or SEQ ID NO.17, respectively, and said signal peptide has the nucleotide sequence of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 or SEQ ID NO:13.

2. A recombinant Bacillus subtilis efficiently expressing pullulanase, comprising a combination of promoter and signal peptide for expressing pullulanase, and pullulanase gene, and having deletion of alkaline protease gene and neutral protease gene, wherein said promoter is a hybrid promoter having the nucleotide sequence of SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16 or SEQ ID NO.17, respectively; and said signal peptide has the nucleotide sequence of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 or SEQ ID NO:13.

Description

BRIEF DESCRIPTIONS OF THE DRAWINGS

(1) FIG. 1 is a schematic diagram of construct of knock-out vector Puc-Ats.

(2) FIG. 2 is a schematic diagram of construct of expression vector Puc-vtr.

EMBODIMENT

(3) The present invention is further illustrated with reference to the following Examples and the appended drawings, which should by no means be construed as limitations of the present invention.

(4) Suitable biology laboratory methods not particularly mentioned in the examples as below can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and other kit laboratory manuals.

(5) Test Materials and Reagents:

(6) 1. Strains and Vectors:

(7) Escherichia coli. Top10, Bacillus subtilis 10033, Bacillus licheniformis 10037, Bacillus amylolytic 10079, Bacillus thuringiensis 20557 were purchased from China Center of Industrial Culture Collection (CICC). The maps of structures of the knockout vector puc-ats and expression vector puc-vtr are shown in FIG. 1 and FIG. 2.

(8) 2. Enzymes and Other Biochemical Reagents:

(9) Q5 High-Fidelity DNA Polymerase (NEB), Plasmid extraction Kit, Agarose Gel DNA Purification Kit, and restriction enzyme were commercially available.

(10) 3. Medium:

(11) E. coli. LB medium: 1% of peptone, 0.5% of yeast extract, and 1% of NaCl, natural pH.

Example 1 Constructing a Bacillus subtilis Strain with Deletion of Alkaline Protease Gene

(12) Bacillus subtilis 10033 was inoculated into LB medium and cultured at 37° C. for 24 h for being extracted genomic DNA. The upper and lower homologous arms of the alkaline protease gene were amplified respectively, and then were fused by overlapping PCR. Finally, the PCR products were digested by the restriction enzymes BamHI and XbaI, purified, recovered, and then inserted into the vector Puc-Ats, followed by being transformed into Escherichia coli top10, in order to obtain an alkaline protease gene Knockout vector Puc-Ats-AprE.

(13) Bacillus subtilis 10033 was eletro-transformed by Knockout vector Puc-Ats-AprE, and screened on tetracycline-resistant plates. The transformants growing on the tetracycline-resistant plates were induced at 45° C. to inactivate the replicator of the vector Puc-Ats-AprE, and promote the homologous recombination between the vector Puc-Ats-Apre and Bacillus subtilis 10033. 5′-terminal homologous arm and 3′-terminal homologous arm were amplified by PCR to determine the location of homologous arm single exchange.

(14) The transformants for which the location of homologous single exchange has been determined were antibiotic-freely cultured and passaged for 10 times continuously, to screen the positive transformants with deletion of alkaline protease gene which growed on the antibiotic-free plates, but not on the antibiotic plates. The obtained positive transformants were identified by PCR, so as to obtain Bacillus subtilis strain with deletion of alkaline protease gene, named as Bs-1.

Example 2 Constructing Bacillus subtilis Strain with Deletion of Neutral Protease Gene

(15) The neutral protease gene was knocked out with the same method as in Example 1 taking Bs-1 as the starting stain. 5′-terminal homologous arm and 3′-terminal homologous arm were amplified by PCR to construct Knockout vector of neutral protease gene, Puc-Ats-NprE, which was electro-transformed into Bs-1. The transformants were induced at 45° C. to obtain single-exchange Bacillus subtilis, followed by antibiotic-freely passaging to obtain double-exchange Bacillus subtilis which was identified by PCR to obtain Bacillus subtilis with deletion of alkaline protease gene and neutral protease gene, named as Bs-vtr.

Example 3 Optimizing Combination of Promoter and Signal Peptide for Expressing Pullulanase

(16) 1. Cloning Promoter and Signal Peptide Gene

(17) Bacillus subtilis 10033, Bacillus licheniformis 10037, Bacillus amylolysis 10079 and Bacillus thuringiensis 20557 were respectively inoculated into LB medium for expanding culture, followed by collecting the culture by centrifuging for extracting genomic DNA. The promoters and signal peptides are amplified by PCR to obtain the sequence of the promoter of thermophilic α-amylase gene in Bacillus licheniformis (P.sub.bl), the promoter of alkaline protease gene in Bacillus subtilis (P.sub.ap), promoter of neutral protease gene in Bacillus subtilis(P.sub.np), the promoter of medium temperature α-amylase gene in Bacillus subtilis (P .sub.ma), the promoter of medium temperature α-Amylase gene in Bacillus amyloliquefaciens(P .sub.ba), and the promoter from Bacillus thuringiensis(P.sub.bt), having the nucleotide sequence as set in forth in SEQ ID NO.2 to SEQ ID NO.7 respectively, and sequence of the signal peptide of thermophilic α-amylase in Bacillus licheniformis (S.sub.bl), the signal peptide of alkaline protease in Bacillus subtilis(S.sub.ap), the signal peptide of neutral protease in Bacillus subtilis (S .sub.np), the signal peptide of medium temperature α-amylase in Bacillus subtilis (S.sub.ma), the signal peptide of the medium temperature α-amylase in Bacillus amyloliquefaciens (S .sub.ba), and the signal peptide of chitinase in Bacillus licheniformis. (S.sub.c), having the amino sequence as set in forth in SEQ ID NO.8 to SEQ ID NO.13 respectively.

(18) 2. Combination of Promoter and Signal Peptide

(19) The different promoters, the signal peptides and pullulanase Bdp gene were fused together by overlapping PCR, followed by being inserted into the expression vector Puc-vtr to obtain the desired expression vector of pullulanase gene, for electro-transforming the host, Bs-vtr. The transformants were cultured in the enzyme-producing medium for 24 h, followed by detecting the enzyme activity according to national standard GB 1886.174-2016.

(20) The efficient combinations of the promoter and the signal peptide were selected according to the relative activity of pullulanase with respect to a series of combinations of the different promoters and the different signal peptides as list in Table 1. As showed in Table 1, the relative activity of pullulanase for all of the optimized combinations of the promoter and the signal peptides was improved compared to that of the control combination, wherein the highest relative enzyme activity, being 300%, was detected in the combination of the promoter of alkaline protease gene in Bacillus subtilis and the signal peptide of medium temperature α-amylase in Bacillus subtilis, and the second highest relative enzyme activity, being 210%, was detected in the combination of the promoter of medium temperature α-Amylase gene in Bacillus subtilis and the signal peptide of neutral protease in Bacillus subtilis.

(21) TABLE-US-00001 TABLE 1 Combination of promoter The relative activity of the and signal peptide expressed pullulanase (%) Control: P43 + SacB 100 P.sub.ap + S.sub.ma 300 P.sub.ap + S.sub.bl 225 P.sub.ap + S.sub.np 178 P.sub.ma + S.sub.np 210 P.sub.ba + S.sub.c 198 P.sub.ma + S.sub.ap 168 P.sub.bl + S.sub.ba 190 P.sub.bl + S.sub.c 180 P.sub.np + S.sub.bl 160 P.sub.np + S.sub.ma 189 P.sub.ma + S.sub.ap 175 P.sub.ma + S.sub.bl 180 P.sub.bt + S.sub.bl 169 P.sub.bt + S.sub.ap 186 In Table 1, “P.sub.bl” represents promoter of thermophilic α-amylase gene in bacillus licheniformis, “P.sub.ap” represents promoter of alkaline protease gene in bacillus subtilis, “P.sub.np” represents promoter of neutral protease gene in bacillus subtilis, “P.sub.ma” represents promoter of medium temperature α-amylase gene in bacillus subtilis, “P.sub.ba” represents promoter of medium temperature α-Amylase gene in bacillus amyloliquefaciens, “P.sub.bt” represents promoter from bacillus thuringiensis, “S.sub.bl”represents signal peptide of thermophilic α-amylase in bacillus licheniformis, “S.sub.ap,” represents the signal peptide of alkaline protease in bacillus subtilis, “S.sub.np” represents the signal peptide of neutral protease in bacillus subtilis, “S.sub.ma” represents the signal peptide of medium temperature α-amylase in bacillus subtilis, “S.sub.ba” represents the signal peptide of the medium temperature α-amylase in bacillus amyloliquefaciens, and “S.sub.c” presents signal peptide of chitinase in bacillus licheniformis.

Example 4 Constructing Hybrid Promoter

(22) One or more promoter selected from the group of P.sub.bl, P.sub.ap, P.sub.np, P .sub.ma, P .sub.ba and P.sub.bt were combined, and were further combined with different signal peptides selected from the group of S.sub.bl, S.sub.ap, S .sub.np, S .sub.ma, S .sub.ba or S.sub.c. Four hybrid promoters efficiently expressing pullulanase were selected, named as promoter P .sub.1, P 2, P 3 and P 4, with the nucleotides sequence as set in forth in SEQ ID NO.14 to SEQ ID NO.17 respectively, wherein the promoter P1 consists of P.sub.bl, P.sub.ap and P .sub.ba, the promoter P2 consists of P.sub.bl, P .sub.ba and P.sub.bt, the promoter P3 consists of P.sub.bl, P.sub.np and P.sub.bt, and the promoter P 4 consists of P.sub.ap, P .sub.ba and P.sub.np. The table 2 lists the relative activity of expressed pullulanase for the combinations of the above four hybrid promoters and the signal peptides selected from S.sub.bl, S.sub.ap, S .sub.np, S .sub.ma, S .sub.ba or S.sub.c.

(23) TABLE-US-00002 TABLE 2 Combination of promoter The relative activity of the and singal peptide expressed pullulanase (%) Control: P43 + SacB 100 P.sub.1 + S.sub.ma 400 P.sub.1 + S.sub.bl 620 P.sub.1 + S.sub.np 580 P.sub.1 + S.sub.ba 450 P.sub.1 + S.sub.c 620 P.sub.2 + S.sub.ap 480 P.sub.2 + S.sub.ba 450 P.sub.2 + S.sub.c 470 P.sub.2 + S.sub.bl 490 P.sub.2 + S.sub.ma 430 P.sub.3 + S.sub.ap 560 P.sub.3 + S.sub.bl 550 P.sub.3 + S.sub.c 630 P.sub.3 + S.sub.ba 650 P.sub.3 + S.sub.ma 590 P.sub.4 + S.sub.c 600 P.sub.4 + S.sub.ap 580 P.sub.4 + S.sub.ba 610 P.sub.4 + S.sub.ma 570 P.sub.4 + S.sub.bl 620

Example 5 Constructing Pullulanase-Integrated Bacillus subtilis

(24) The combinations of promoters and signal peptides obtained in the examples 3 and 4, pullulanase gene, and the part of the medium temperature α-amylase gene as the homologous arm were integrated into the Knock-out integrating vector Puc-Ats, taking the medium temperatures-amylase gene as the target integration site, to obtain the corresponding Knock-out integrating vector, followed by constructing pullulanase-integrated Bacillus subtilis by the same method as described in Example 1, as so to obtain the corresponding pullulanase-integrated Bacillus subtilis.