Recombinant bacillus subtilis strain expressing and secreting beta-mannanase efficiently and method for obtaining strain

A technology of Bacillus subtilis and mannanase, applied in the field of molecular biology, can solve the problem of predicting the most effective signal peptide sequence without downstream protein sequence, affecting the correct folding of β-mannanase, and β-mannanase Degradation and other issues

Inactive Publication Date: 2019-07-12
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no software that can predict the most effective signal peptide sequence based on the downstream protein sequence, so it is necessary to determine the most efficient signal peptide sequence through experiments
In addition, because β-mannanase may have bottleneck factors in the process of protein transport and secretion, when the regulatory elements involved in protein transport in cells cannot meet the demand, or when the limiting factors involved in protein secretion are lacking, β-mannan may be affected. Correct folding of glycanase will cause β-mannanase to be degraded by protease in the cell wall

Method used

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  • Recombinant bacillus subtilis strain expressing and secreting beta-mannanase efficiently and method for obtaining strain
  • Recombinant bacillus subtilis strain expressing and secreting beta-mannanase efficiently and method for obtaining strain
  • Recombinant bacillus subtilis strain expressing and secreting beta-mannanase efficiently and method for obtaining strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] The β-mannanase gene in the expression vector pMA5 used in this example is derived from Bacillus licheniformis DSM13 and does not contain the original signal peptide DNA sequence. After codon optimization, its nucleotide sequence is the sequence SEQ ID NO: 1. The signal peptide DNA fragments used were derived from amyL (KEGG BSU03040), lipA (KEGG BSU32330), nprB (KEGGBSU11100), nprE (KEGG BSU14700) and phoD (KEGG BSU02620) of the Sec secretion pathway of Bacillus subtilis (Bacillus subtilis 168) and Tat secretion pathway. ), ywbN(KEGGBSU38260).

[0042] 1. Construction of expression vectors containing different signal peptide sequences.

[0043] First, the codon-optimized β-mannanase gene sequence (SEQ ID NO: 1) was cloned into the pMA5 vector (derived from BGSC) by overlapping extension PCR (POE-PCR). On this basis, the Overlap extension PCR (POE-PCR) method, the carrier pMA5 containing the codon-optimized β-mannanase gene sequence and different signal peptide DNA fr...

Embodiment 2

[0069] The expression vector and signal peptide sequence of the β-mannanase in this example are pMA5-2Man and lipA, respectively. The different Bacillus subtilis host bacteria used in this example overexpressed the relevant elements in the Sec secretion pathway in a single copy form on the genome, as well as genes such as signal peptidase and molecular chaperones that may affect the secretion efficiency of the pathway. The Bacillus subtilis host bacteria used to express the β-mannanase gene and the corresponding overexpressed genes are: B. subtilis 1A751S1 (secY), 1A751S2 (secE), 1A751S3 (secG), 1A751S4 ( secYEG), 1A751S5(secDF), 1A751S6(secA), 1A751S7(ffh), 1A751S8(scr), 1A751S9(hbs), 1A751S10(SRP), 1A751S11(ftsY), 1A751P1(sipS), 1A751P 2(sipT), 1A (sipU), 1A751P4(sipV), 1A751P5(sipW), 1A751P6(dnaK / J), 1A751P7(groESL) and 1A751P8(prsA). The overexpression of the above-mentioned genes was integrated into the amyE site of the Bacillus subtilis genome in a single copy form thro...

Embodiment 3

[0077] The expression vector of the β-mannanase used in this example is pMA5-2Man, the promoter it contains is the constitutively expressed hpaII, and the signal peptide is lipA. Using the method described in Example 1, the promoter hpaII Replaced with constitutive promoter fragments aprE and P43 derived from Bacillus subtilis strain 168. The other three inducible promoters used were xylose-inducible promoter xyl (derived from gene BSU17600), IPTG-inducible promoter grac (derived from gene BSU06020) and maltose promoter mglv (derived from gene BSU08180).

[0078] The primer sequences used in this example are listed in Table 4.

[0079] Primer sequences used in table 4 embodiment 3

[0080]

[0081]

[0082] The plasmids and bacterial strains constructed in this example and their characteristics are shown in Table 5.

[0083] Plasmids and bacterial strains constructed in table 5 embodiment 3 and their characteristics

[0084]

[0085] 1. Determination of the concent...

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Abstract

The invention belongs to the technical field of molecular biology, and in particular, relates to a recombinant bacillus subtilis strain expressing and secreting beta-mannanase efficiently and a methodfor obtaining the strain. Specifically, the method comprises the steps: a codon-optimized beta-mannanase gene is cloned to the downstreams of different signal peptide sequences in a pMA5 vector to secrete to extracellular sites; then restriction factors involved in the process of secretion are overexpressed in a host bacteria genome; and finally promoters on the expression vector are replaced, and the production of beta-mannanase is increased at the level of protein synthesis by enhancing transcription and translation processes. The recombinant bacillus subtilis expressing and secreting beta-mannanase efficiently utilizes a 2*SR culture medium for fermentation production of mannanase under conditions of the temperature of 37 DEG C and the rotating speed of 220 rpm, the highest enzyme activity of beta-mannanase in 72 h fermentation broth can reach 2207 U/mL, and the recombinant bacillus subtilis strain is a strain with good application prospect.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and specifically relates to one or more recombinant Bacillus subtilis strains highly expressing and secreting β-mannanase and an obtaining method thereof. Background technique [0002] Bacillus subtilis (Bacillus subtilis) is a Gram-positive bacterium and an important non-pathogenic industrial microorganism. Its whole genome sequencing work has been completed, and its genetic background and physiological characteristics are clear. The use of Bacillus subtilis to express foreign proteins has many advantages: (1) high safety, and can be used in industrial production such as food and medicine; (2) can directly release secreted proteins into the medium, which is beneficial for separation and purification; (3) has The ability to efficiently secrete the target protein has a variety of secretory pathways, and the relevant secretory components in the pathway have been analyzed; the secreted re...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/56C12N15/75C12N15/67C12N9/24C12N1/21C12R1/125
CPCC12N9/2494C12N15/75C12N15/67C12Y302/01078
Inventor 张大伟付刚宋亚凤
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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