CN104818291A - Construction and application of streptomycete recombinant expression vector - Google Patents

Abstract

The invention discloses construction and application of a streptomycete recombinant expression vector, and belongs to the field of gene engineering. A streptomycete expression vector disclosed by the invention can be used for facilitating the insertion of exogenous genes and preventing from adding multiple cloning sites and terminators on the vector by virtue of transcription and reading-through, and can also be used for massively secreting and expressing TGase in three streptomycetes.

Description

Construction and application of a streptomyces recombinant expression vector

technical field

The invention relates to the construction and application of a Streptomyces recombinant expression vector, which belongs to the field of genetic engineering.

Background technique

Streptomyces is a Gram-positive bacterium, which has its own advantages as a genetically engineered recipient: First, the use of Streptomyces for the industrial-scale production of antibiotics has a long history, and considerable experience has been accumulated in industrial-scale fermentation technology. Therefore, existing technology and equipment can be used to produce exogenous gene products expressed by Streptomyces: secondly, Streptomyces produces less extracellular enzymes than Bacillus subtilis, which can prevent the degradation of exogenous proteins. The extracellular secretion system is developed, which can be used for the secretion and expression of exogenous genes, and simplifies the isolation and purification of exogenous gene products; at the same time, great progress has been made in the study of molecular genetics of Streptomyces, especially the molecular biology of Streptomyces plasmids, chain Comprehensive progress in research on fungal promoters and signal sequences for protein secretion in Streptomyces. It provides a theoretical basis for the expression of foreign genes in Streptomyces. Proteins expressed in Streptomyces are often soluble, so there is no need to re-dissolve and fold expressed proteins into the correct configuration in order to obtain biologically active proteins; Sickness is small. Streptomyces has become another valuable host for gene expression after Escherichia coli and Bacillus subtilis.

There have been a large number of reports on the expression of eukaryotic genes and some prokaryotic genes of non-Streptomyces origin in Streptomyces. Such as human insulin Glucagon (Qi. et al., J. Microbiol. Biotechnol, 2008, 1076–1080.), Aspergillus xylanase Xylanase (Díaz et al., 2004, Appl. Microbiol. Biotechnol., 401–406. ), streptokinase (Pimienta et al., 2007, Microb. Cell Fact.6, 20.).

Although the cloning system of Streptomyces has been established relatively well, the promoters and vectors used for gene expression that can be compared with those in Escherichia coli are still very limited. Therefore, it is of great significance to explore and discover new promoters and construct expression vectors for expanding the expression system of Streptomyces.

The Streptomyces expression vector provided by the invention facilitates the insertion of foreign genes and prevents transcription read-through. Multiple cloning sites and terminators are added to the vector, and can secrete and express TGase in large quantities in three Streptomyces species.

Contents of the invention

In the present invention, the carrier pISG86 (SEQ ID NO.3) is used as the starting carrier, and the promoter and signal peptide whose sequence is shown in SEQ ID NO.1 are connected to it, and the polyclonal sequence shown in SEQ ID NO.2 is designed. sites and terminators to construct a high-efficiency secretion expression vector for Streptomyces.

The present invention also provides a method for constructing the secretory expression vector, which mainly includes the following steps:

(1) After amplifying the promoter and signal peptide sequence, it was treated with KpnI and BglII double enzyme digestion, and connected to the vector pISG86 after the same enzyme digestion treatment to obtain pISG86-1;

(2) Using the primers whose sequences are shown in SEQ ID NO.6 and 7, and using pISG86-1 as a template, connect the multiple cloning site sequence and terminator sequence to the downstream of the signal peptide of pISG86-1 by overlapping PCR to obtain the sequence The correct vector pSG01; using pSG01 as a template, using the primer pair whose sequence is shown in SEQ ID NO.8 and 9, and the primer pair whose sequence is shown in SEQ ID NO10 and 11, respectively, carry out PCR, and pSG01 is obtained by codon optimization Streptomyces high-efficiency secretion expression vector pSG02.

The expression vector provided by the present invention, when expressing the exogenous protein TGase, has a higher expression level than the commonly used strong promoter erythromycin promoter _PermE* in Streptomyces, indicating that the promoter can be used as a strong promoter expression of other foreign proteins in Streptomyces. In addition, the vector constructed in the present invention can successfully express TGase in S. lividans TK24, S. lividans 1326 and S. griseus, indicating that the expression vector has the potential to express other foreign proteins in these three Streptomyces.

Description of drawings

Figure 1. Protein electrophoresis pattern of pSG02-TGase expressed in Streptomyces S.lividans 1326, (a) protein electrophoresis pattern of pSG02-TGase expressed in Streptomyces S.lividans 1326, (b) pSG03-TGase expressed in Streptomyces S.lividans 1326 Electrophoretic pattern of protein expressed in 1326.

Fig. 2 Expression of pSG02-TGase in three kinds of Streptomyces.

Fig. 3 Protein electrophoresis patterns of phenylalanine deaminase and aminopeptidase expressed in S. lividans TK24 by pSG02; (a) aminopeptidase, (b) phenylalanine deaminase.

Detailed ways

Relevant methods involved in the following examples:

TGase enzyme activity assay method: colorimetric assay enzyme activity. α-N-CBZ-Gln-Gly was used as the substrate, and L-glutamic acid-γ-monohydroxamic acid was used as the standard curve. One unit of TGase enzyme activity is defined as: the amount of enzyme (U/mL) that catalyzes the substrate to form 1 μmol L-glutamic acid-γ-monohydroxylamine per minute at 37°C. Enzyme activity assay conditions: react at 37°C for 10 minutes.

Phenylalanine deaminase (PAL) enzyme activity assay method: Take an appropriate amount of fermentation supernatant and mix with 225μL Tris-HCL buffer (25mM, pH 8.0), 250μL substrate L-phenylalanine, the total reaction volume is 500μL . React at 40°C for 30 min, then add 500 μL of methanol to terminate the reaction. Definition of enzyme activity unit: The amount of enzyme required to generate 1mM trans-cinnamic acid per minute at 40°C is one enzyme activity unit.

Aminopeptidase (BSAP) enzyme activity assay method: take an appropriate amount of fermentation supernatant and mix it with 4mmol/L substrate aminoacyl-p-nitroanilines, react at 37°C for 10min, measure the absorbance value at 405nm, and define the enzyme activity unit: 37 degrees per minute The amount of enzyme needed to form 1 μmol p-nitroaniline is one unit of enzyme activity.

The construction of embodiment 1 vector pSG02-TGase and pSG03-TGase

Design primers (P1/P2) to amplify the promoter signal peptide sequence and then digest (KpnI/BglII) and connect to the vector pISG86; on this basis, insert MCS and fd-ter into Downstream of the signal peptide. Then use site-directed mutagenesis (P5/P6) to mutate the rare codon TTA of leucine at position 7 in the signal peptide sequence to CTG, and the gene AGC encoding the amino acid serine at position 21 to GCC (P7/P8) to obtain the vector pSG02, 6.6 kb in size. Using the S. hygroscopicus WSH03-13 genome as a template, primers were designed to obtain the TGase gene fragment, which was ligated to pSG02 by enzyme digestion to obtain the TGase expression vector pSG02-TGase. Using the vector pISG86 as a template, the _PermE* promoter fragment was obtained by PCR method (P9/P10), the promoter of pSG02 was replaced by the _PermE* promoter by the method of whole plasmid PCR, and the TGase gene was connected to obtain the vector pSG03-TGase.

P1: 5'-CGGGGTACCGCCAGGAGCAGGGGAACGC-3'

P2: 5'-GAAGATCTGGCATGGCTGACCGACGGC-3'

P3:

5’-GGAGTCATGCCGTCGGTCAGCCATGCCATCGATTTTAAAGGATCCGTTAACAAGCTTCCCGGGTAAACCGATACAATTAAAGGCTCCTTTTGGAGCCTTTTTTTTTGGAGTCACGCGCCCAACGGCGGCG-3’

P4:

5'-GGGCCCACGCCGCCGTTGGGCGCGTGACTCCAAAAAAAAAGGCTCCAAAAGGAGCCTTTAATTGTATCGGTTTAACCCGGGAAGCTTGTTAACGGATCCTTTAAAATCGATGGCATGGCTGACCGACGGC-3'

P5: 5'-GGAGTCATGCCGTCGGTCGCCCATGCCATCGATTTTAAAGGATCC-3'

P6: 5'-CCTTTAAAATCGATGGCATGGGCGACCGACGGCATGACTCCAGCGGTG-3'

P7: 5'-ATGTACAAGCGTCGGAGTCTGCTCGCCTTCGCCACTGTGAG-3'

P8: 5'-CAGTGGCGAAGGCGAGCAGACTCCGACGCTTGTACATGAAAATCG-3'

P9: 5'-CCACAACAAGGGAGTCACCGATTTTCGGTACCAGCCCGACCCGAGC-3'

P10: 5'-CGAGTAAACTCCGACGCTTGTACATAGATCTGCAGCCAAGCTTGC-3'

The expression of embodiment 2TGase

Transfer pSG02-TGase and pSG03-TGase into S.lividans 1326 respectively to obtain recombinant bacteria S.lividans1326/pSG02-TGase and S.lividans 1326/pSG03-TGase. The recombinant bacteria were cultured in the seed medium at 37°C and 200rpm for 2 days, then transferred to the fermentation medium with 2% inoculum size at 37°C and 200rpm for 6 days, and samples were taken every 6h to obtain the maximum enzyme activity. In addition, pSG02-TGase was transferred to S.lividans TK24 and S.griseus and cultivated under the same conditions.

The recombinant strain S. lividans 1326/pSG02-TGase began to express the zymogen proTGase at about 24 hours. With the prolongation of the culture time, proTGase was gradually activated into TGase. The amount of TGase obtained at 84 hours was the largest, corresponding to an enzyme activity of 9.62U/ ml (Fig. 1a); while S.lividans 1326/pSG03-TGase obtained the maximum expression level at 84h, corresponding to an enzyme activity of 4.6U/ml (Fig. 1b). By comparing the expression levels of TGase in the two recombinant bacteria, it can be seen that the promoter provided by the invention is better than the erythromycin promoter _PermE* commonly used in Streptomyces when expressing TGase, and can be used as a strong promoter for the expression of TGase. Mold as host for recombinant expression. In addition, in order to evaluate the host versatility of the expression vector pSG02, pSG02-TGase was transformed into Streptomyces hosts S. lividans TK24 and S. griseus respectively. TGase was expressed in the above two hosts, and the corresponding enzyme activity was 7.6 U/ml and 6.84U/ml. This result shows that the recombinant vector pSG02-TGase can be applied to different Streptomyces host bacteria ( FIG. 2 ).

The expression of embodiment 3PAL and AP

The gene PAL encoding phenylalanine deaminase derived from Rhodotorula viscosus and the gene AP encoding aminopeptidase derived from Bacillus subtilis were respectively connected to the vector pSG02 by enzyme digestion and ligation to construct the vector pSG02-PAL , pSG02-BSAP were transferred into S.lividans TK24 for expression. The culture conditions were consistent with those of the recombinant strain S.lividans1326/pSG02-TGase. After culturing for 84 hours, 2.82U/mL aminopeptidase and 20.6U/mL phenylalanine deaminase were obtained.

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore The scope of protection of the present invention should be defined by the claims.

Claims (7)

1. a streptomycete recombinant secretor expression vector, it is characterized in that, with the carrier pISG86 of nucleotide sequence as shown in SEQ ID NO.3 for the carrier that sets out, design is thereon connected to multiple clone site as shown in SEQ ID NO.2 of the promotor of sequence as shown in SEQ ID NO.1 and signal peptide, sequence and terminator, constructs streptomycete efficient secretory expression carrier.

2. build a method for recombinant secretor expression vector described in claim 1, it is characterized in that, mainly comprise the following steps:

(1) increase by the process of KpnI, BglII double digestion after promotor and signal peptide sequence, and be connected to same enzyme cut process after carrier pISG86, obtain pISG86-1;

(2) take pISG86-1 as template, with the primer pair of sequence as shown in SEQ ID NO.6,7, by over-lap PCR, multiple clone site sequence and terminator sequence are connected to the signal peptide downstream of pISG86-1, obtain carrier pSG01; Take pSG01 as template, respectively with the primer pair of sequence as shown in SEQ ID NO.8,9, the primer pair of sequence as shown in SEQ ID NO10,11, carries out PCR, carries out codon optimizedly obtaining streptomycete efficient secretory expression carrier pSG02 to pSG01.

3. recombinant secretor expression vector described in claim 1 with streptomycete be host carry out recombinant expressed in application.

4. application according to claim 3, is characterized in that, described streptomycete is Streptomyces lividans TK24, Streptomyces lividans 1326 or Streptomyces griseus.

5. application according to claim 3, is characterized in that, recombinant expressed Transglutaminase EC2.3.2.13.

6. application according to claim 3, is characterized in that, recombinant expressed phenylalanine deaminase.

7. application according to claim 3, is characterized in that, recombinant expressed aminopeptidase.