A genetically engineered bacterium for improving polygalacturonase secretion and its construction method and application

Abstract

The present invention discloses a genetically engineered bacterium for improving the secretion of polygalacturonase, a construction method and an application thereof, and belongs to the field of genetic engineering technology. The present invention provides a gene Km_JEN1 related to the secretion of polygalacturonase, and the nucleotide sequence is shown in SEQ ID NO.1. Through functional verification of the gene, the Km_JEN1 gene has the effect of promoting the secretion of polygalacturonase. The present invention genetically engineered Kluyveromyces marxianus to obtain a genetically engineered bacterium with overexpression of Km_JEN1, and the amount of polygalacturonase secreted by the strain is 1.32 times that of the control. The present invention provides a theoretical basis and related genes for optimizing the protein secretion system.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to the construction of a genetic engineering bacterium for improving the secretion of polygalacturonase and the application of the strain. Background Art

[0002] Pectin is a kind of heteropolysaccharide widely present in the primary wall and the middle layer of the cell wall of plants. It exists with cellulose and constitutes the middle layer adhesive of adjacent cells, which makes the plant tissue cells stick together tightly. Pectin exists in large quantities in the peels of citrus, lemon, grapefruit, etc. The pectin content is high in the waste and discharged water of fruit processing. The COD content in pectin is high and has high viscosity. Therefore, it is necessary to treat these wastes and discharged water to meet the discharge requirements. At present, the method of pectin treatment can achieve good results by using pectinase treatment, which not only plays an important role in energy consumption and waste discharge, but also can bring huge economic benefits to society.

[0003] Various hosts from bacteria to higher eukaryotes can produce heterologous proteins. Using model organisms such as bacteria, yeast, animal cells or plant cells to express foreign gene proteins is a molecular biology technology for protein expression, which occupies a core position in genetic engineering technology. Compared with eukaryotic organisms, the prokaryotic protein expression system is crude in terms of post-translational modification and folding of protein molecules. Yeast is a eukaryotic unicellular fungus. The yeast protein expression system has the advantages of high expression, inducibility, glycosylation mechanism close to higher eukaryotes, easy purification of secreted proteins, and easy high-density fermentation. However, the secretory expression of heterologous proteins in yeast is usually limited to a relatively low level, so it is particularly difficult and crucial to perform molecular engineering of secreted proteins to optimize their secretion pathways.

[0004] Kluyveromyces marxianus is a food safety yeast with the advantages of short growth cycle and high temperature resistance. Kluyveromyces marxianus has endogenous polygalacturonase. Polygalacturonase (PG) is a helicase belonging to the 28th family of glycoside hydrolases (GH28). It is a type of pectin or pectinase that can decompose pectin or pectinase. It can hydrolyze polygalacturonic acid, the main component of pectin, to generate oligogalacturonic acid. Kluyveromyces marxianus is an ideal host for the production of polygalacturonic acid. However, Kluyveromyces marxianus also has the problem of low endogenous pectinase secretion level. It is necessary to carry out molecular engineering transformation of Kluyveromyces marxianus to optimize its secretion pathway and increase the secretion of pectinase.

[0005] At present, there are two main ways to improve the expression of heterologous proteins. The first is to increase the expression of heterologous proteins, which can be achieved by increasing the number of gene copies and optimizing codons. The second is to enhance the secretion pathway of heterologous proteins, which can be achieved by regulating key genes related to the secretion pathway to improve the secretion efficiency of proteins. Yeast uses a highly conserved transport mechanism to deliver newly synthesized secretory proteins. After being synthesized from ribosomes, secretory proteins are first transferred to the endoplasmic reticulum for folding and modification, and then these proteins are transported to the Golgi apparatus for further modification, and then secreted outside the cell through the plasma membrane.

[0006] Therefore, it is of great significance for the development of proteases to analyze the transcription level of Kluyveromyces marxianus, explore the genes related to the secretion of polygalacturonan hydrolase, explore the mechanism of yeast protein secretion, and construct genetically engineered bacteria that improve the secretion of polygalacturonan hydrolase. Summary of the invention

[0007] The purpose of the present invention is to screen out genes related to transmembrane transport by analyzing the transcription level of Kluyveromyces marxianus, regulate key genes and screen out genes that can improve the secretion of polygalacturonase. A genetically engineered bacterium that improves the secretion of polygalacturonase is constructed through genetic engineering and applied to the development of enzyme preparations.

[0008] To achieve the above object, the present invention adopts the following technical solution:

[0009] The present invention screened a key gene Km_JEN1 for protein secretion by sequencing transcriptomes of Kluyveromyces marxianus and knockout polygalacturonase strains, and its nucleotide sequence is shown in SEQ ID NO.1. The relative expression level of the gene in the wild-type strain is 3.8 times that of the knockout strain. The gene was cloned by molecular biological means, a vector was constructed and transferred into the Kluyveromyces marxianus strain, and the results showed that the amount of polygalacturonase secreted by the strain into which the gene was transferred was significantly increased, indicating that the Km_JEN1 gene has the effect of promoting enzyme secretion.

[0010] Therefore, the present invention provides the use of Km_JEN1 gene in promoting the secretion of polygalacturonase in yeast. The nucleotide sequence of the Km_JEN1 gene is shown as SEQ ID NO.1, and the amino acid sequence of the protein encoded by the Km_JEN1 gene is shown as SEQ ID NO.2.

[0011] Furthermore, the application includes: using yeast as a starting bacterium, inserting an overexpression module containing the Km_JEN1 gene into the genome by biological technology means, or introducing a recombinant expression plasmid containing the Km_JEN1 gene into the host bacterium to obtain functional genetically engineered bacteria.

[0012] The yeast has the function of expressing endogenous polygalacturonase. Further, the yeast is Kluyveromyces marxianus.

[0013] Furthermore, the original plasmid of the recombinant expression plasmid is the pIW1146 vector. The pIW1146 vector is a publicly available material, see the literature (CRISPR-mediated multigene integration enables Shikimate pathway refactoring for enhanced 2-phenylethanol biosynthesis in Kluyveromyces marxianus [J]. Biotechnology for Biofuels, 2021, 14 (1).).

[0014] Furthermore, the genetically engineered bacteria are fermented and cultured using SD medium. Studies have found that Kluyveromyces marxianus can secrete polygalacturonic acid hydrolase to the extracellular space when cultured in SD medium. The composition of SD medium includes: yeast basic nitrogen source (without amino acids and ammonium sulfate), amino acid nucleotide mixture, and glucose.

[0015] The present invention also provides a genetically engineered bacterium for improving the secretion of polygalacturonase. The genetically engineered bacterium uses Kluyveromyces marxianus as a starting bacterium, and the genetically engineered bacterium has an overexpression module containing a Km_JEN1 gene with a nucleotide sequence as shown in SEQ ID NO.1.

[0016] In the overexpression module, there is an overexpression promoter upstream of the Km_JEN1 gene, which can be expressed by P GPD , P TEF etc. promoter.

[0017] Furthermore, the genetic engineering contains a recombinant expression plasmid containing the Km_JEN1 gene, the original vector of the recombinant expression plasmid is the pIW1146 vector, the Km_JEN1 gene replaces the GFP gene in the original vector, and the pIW1146 vector contains the GPD promoter and the CYC1 terminator.

[0018] The present invention also provides a method for constructing the genetic engineering, which comprises: using Kluyveromyces marxianus as a starting bacterium, inserting an overexpression module containing the Km_JEN1 gene into the genome by biological technology means, or introducing a recombinant expression plasmid containing the Km_JEN1 gene into the host bacterium.

[0019] Furthermore, the present invention provides a method for removing the GFP gene in the pIW1146 vector by double enzyme digestion, connecting the Km_JEN1 gene fragment with the vector by seamless cloning, constructing a recombinant expression vector pIW1146-Km_JEN1, and then transferring it into Kluyveromyces marxianus to construct a genetically engineered bacterium.

[0020] The invention also provides the use of the genetically engineered bacteria in the preparation of polygalacturonase preparations.

[0021] Further, the application includes: the application includes: inoculating the genetically engineered bacteria after the expanded culture into SD liquid culture medium for fermentation culture, collecting the fermentation broth, and isolating and obtaining polygalacturonase from the fermentation broth. For the genetically engineered bacteria containing the recombinant expression vector pIW1146-Km_JEN1, the fermentation culture adopts SD culture medium lacking histidine.

[0022] The fermentation culture comprises: the genetically engineered bacteria are inoculated into the SD medium at an initial OD 600 The value is about 0.05 to 1, and the culture is carried out at 30 to 37 ° C and 200 to 220 rpm until the OD 600 The value is 6-7.

[0023] The present invention has the following beneficial effects:

[0024] (1) The present invention provides a gene Km_JEN1 related to polygalacturonase secretion. Functional verification of the gene shows that the gene has the effect of promoting the secretion of polygalacturonase, providing a theoretical basis and related genes for the optimization of the protein secretion system.

[0025] (2) The present invention genetically engineered Kluyveromyces marxianus to obtain a genetically engineered bacterium with overexpression of Km_JEN1. The amount of polygalacturonase secreted by the strain was 1.32 times that of the control, which is of great significance for the development of protease preparations. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The relative expression levels of Km_JEN1 in the control strain (Km) and the polygalacturonase knockout strain (Km_ΔPG) shown in the transcriptome sequencing results.

[0027] Figure 2 Prediction of the transmembrane region in the protein structure of Km_JEN1.

[0028] Figure 3 The cloning results of Km_JEN1, M is DNA marker, and lane 1 is the PCR product.

[0029] Figure 4The results of SDS-PAGE, where control is the wild-type strain, Km_JEN1 is the engineered strain into which the Km_JEN1 gene plasmid was transferred, and Km_ΔPG is the polygalacturonase knockout strain, the same below.

[0030] Figure 5 Relative gray value analysis for imageJ.

[0031] Figure 6 is the relative enzyme activity of PG. DETAILED DESCRIPTION

[0032] The present invention will be further described below in conjunction with specific embodiments. The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of application of the present invention. Without departing from the spirit and essence of the present invention, modifications or replacements made to the inventive method, steps or conditions all belong to the scope of the present invention.

[0033] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0034] The strain Kluyveromyces marxianus CBS6556 was purchased from Ningbo Mingzhou Biotechnology Co., Ltd.

[0035] Construction of polygalacturonase knockout strain (Km_ΔPG): The polygalacturonase gene was completely knocked out from the genome of Kluyveromyces marxianus CBS6556 using CRISPR-Cas9 technology. The nucleotide sequence of the polygalacturonase gene is shown in SEQ ID NO.3.

[0036] Strain culture medium: YPD medium: 10 g / L yeast extract (Oxoid), 20 g / L peptone (Oxoid), 20 g / L glucose (Coolaber).

[0037] SD medium: 1.7 g / L BD Difco TM Yeast nitrogen base without amino acids and ammonium sulfate (BD Company), amino acid mixture (including nucleotides) (Coolaber Company), 20g / L glucose (Coolaber Company).

[0038] SD-His medium is the above SD medium without histidine.

[0039] Example 1

[0040] Transcriptome sequencing samples: Kluyveromyces marxianus CBS6556 (control strain) and polygalacturonase knockout strain Km_ΔPG were cultured in SD medium, and yeasts at 8 h were selected as logarithmic phase samples, and yeasts at 14 h were selected as stationary phase samples.

[0041] Transcriptome sequencing was performed on the logarithmic phase samples and the stationary phase samples, respectively, and genes related to transmembrane transport with significant differences were screened out by analyzing the transcriptome data.

[0042] Transcriptome sequencing results showed that compared with the control strain, in the logarithmic phase, a total of 449 genes were differentially expressed genes, of which 313 genes were down-regulated in Km_ΔPG and 136 genes were up-regulated. At the same time, in the plateau phase, a total of 177 genes were differentially expressed genes, of which 63 genes were down-regulated genes and 114 genes were up-regulated genes. Polygalacturonase is a secreted protein, so the differentially expressed genes in the plateau phase were mainly screened and analyzed.

[0043] According to the GO enrichment analysis of differentially expressed genes, they are divided into three parts: molecular function, biological process, and cellular component. We mainly screen the key genes we need from the 30 most significantly enriched GO terms (each part has 10 terms). Because we are studying secretory proteins, we mainly analyze proteins related to the transport process, including membrane component proteins, proteins related to transport, and proteins related to endocytosis and exocytosis. Among them, in biological process, 13 differentially expressed genes were enriched in transmembrane transport, and 16 differentially expressed genes were enriched in transport. In cellular component, 13 differentially expressed genes were annotated as membrane-related genes, 2 genes were related to cell periphery, 6 genes were related to integral component of membrane, 6 genes were related to intrinsic component of membrane, 1 gene was related to nuclear envelope, 7 genes were related to membrane part, 1 gene was related to plasma membrane, 1 gene was related to cytoplasmic vesicle, 1 gene was related to vesicle, and 1 gene was related to cytoplasmic vesicle part. In molecular function, 1 differentially expressed genes were annotated as membrane-related genes, 2 genes were related to cell periphery, 6 genes were related to integral component of membrane, 6 genes were related to intrinsic component of membrane, 1 gene was related to nuclear envelope, 7 genes were related to membrane part, 1 gene was related to plasma membrane, 1 gene was related to cytoplasmic vesicle, 1 gene was related to vesicle, and 1 gene was related to cytoplasmic vesicle part. Among the differentially expressed genes, 8 were related to transmembrane transporter activity, 8 were related to transporter activity, and 2 were related to metal ion transmembrane transporter activity.

[0044] Combined with the GO enrichment results, it was found that the Km_JEN1 gene is not only a membrane component-related protein, but also related to the transmembrane transport process, so the Km_JEN1 gene was experimentally verified. The nucleotide sequence of the Km_JEN1 gene is shown in SEQ ID NO.1, and the encoded amino acid sequence is shown in SEQ ID NO.2.

[0045] Transcriptome sequencing results showed that the relative expression level of Km_JEN1 in the control strain was 3.8 times that of Km_ΔPG in the treatment group. Figure 1 .

[0046] The functional domains of Km_JEN1 protein were predicted and analyzed by hmmscan (https: / / www.ebi.ac.uk / Tools / hmmer / search / hmmscan) on the HMMER website. The results showed that Km_JEN1 protein has 12 transmembrane regions, which are located at the following amino acid positions: 139-157, 177-199, 206-225, 231-252, 264-286, 298-317, 365-384, 404-422, 434-452, 458-479, 491-511, 537-556, see Figure 2 .

[0047] Example 2

[0048] 1. Based on the key genes screened out from the transcriptome data, we extracted the genome of Kluyveromyces marxianus CBS6556 (using the Tiangen yeast genomic DNA extraction kit) and cloned the Km_JEN1 gene.

[0049] PCR primers were designed with the following sequences (5'-3'): Km_JEN1_F: agaactagtggatcccccgggATGTCAGAAGGAAACGAAGAAAAAG; Km_JEN1_R: taactaattacatgactcgagCTAAGCGTTCTCGACTTGTTCTTCC.

[0050] PCR reaction system (50 μL): 2×Phanta Max Buffer 25 μL, dNTP Mix (10 mM) 1 μL, upstream and downstream primers (10 μM) 2 μL, Phanta Max Super-Fidelity DNA Polymerase 1 μL, template DNA 50-400 ng, ddH 2 OFill in.

[0051] PCR reaction program: 95°C for 3 min, 95°C for 15 sec, 56-72°C for 15 sec, 72°C for 1 min / kb, 35 cycles, 72°C for 5 min.

[0052] The PCR products were tested by gel electrophoresis. Figure 3 shown.

[0053] 2. Construct a recombinant expression plasmid. The plasmid vector uses the pIW1146 vector framework (Li M, Lang X, Cabrera MM, et al. CRISPR-mediated multigene integration enables Shikimate pathway refactoring for enhanced 2-phenylethanol biosynthesis in Kluyveromyces marxianus[J]. Biotechnology for Biofuels, 2021, 14(1).), which is modified from pIW 578 and contains the GPD promoter sequence, GFP encoding gene, and CYC1 terminator sequence.

[0054] First, the GFP gene was removed from the vector by enzyme digestion, and then Km_JEN1 was constructed into the pIW1146 vector by seamless cloning. The plasmid was then transfected into yeast to construct a new strain. The construction of the empty plasmid removed GFP.

[0055] The transfection method is as follows: the original strain was placed in 2 mL YPD medium and shaken in a 30°C incubator for 16 h, 1 mL of the bacteria was centrifuged at 5000 × g for 1 min, the supernatant was discarded, and 1 mL of ddH 2 After washing twice, add 500μL transfection buffer (40% 3350PEG, 0.1M LiAc, 10mM Tris-HCl at pH7.5, 1mM EDTA, 10mM DTT), 10μL salmon sperm DNA, 1μg plasmid, place at room temperature for 15min, heat shock at 47℃ for 15min, centrifuge at 5000×g for 1min, resuspend with 500μL selection medium (SD-His medium), then aspirate 50μL in 2mL selection medium and shake culture for 24-48h, finally spread on the selection medium solid culture medium plate to obtain positive clones. The positive clones are sent to the company for sequencing, and the single clones with correct sequencing are expanded and the bacterial liquid is preserved.

[0056] Example 3

[0057] The engineered bacteria transformed with the Km_JEN1 gene plasmid constructed in Example 2 were activated and inoculated into 2 ml of SD-His liquid culture medium. The OD was measured after culturing at 30°C and 220 rpm for 17 h. 600 , and then inoculated into a 25 mL SD-His liquid medium shake flask (initial OD 600All were 0.05), cultured at 30°C, 220rpm for 20h, centrifuged 2mL of bacterial solution, took the supernatant into a concentrator, centrifuged and finally concentrated to 180μL (washed twice with PBS buffer during this period). Protein analysis was performed using SDS-PAGE, and its gray value was analyzed using imageJ.

[0058] The results are as follows Figure 4 and Figure 5 As shown, the knockout polygalacturonase strain Km_ΔPG has no target protein band, indicating that the protein secreted into the extracellular space by the wild-type strain and the recombinant strain is polygalacturonase. Compared with the control (Kluyveromyces marxianus CBS6556), the amount of polygalacturonase secreted by the recombinant strain is 1.32 times that of the control.

[0059] Determination of enzyme activity: Take 100 μL of the protein solution in the above-mentioned concentrator tube, mix it with 900 μL of pectin solution (2 g / L), incubate it at 50°C for 5 min, add 4 mL of DNS solution (100 mL of DNS solution contains 80 mL of 0.5 M NaOH, 1 g of DNS, and 30 g of sodium potassium tartrate), react at 100°C for 10 min, immediately put it on ice, and measure the absorbance at 540 nm. The results are as follows: Figure 6 As shown, the relative enzyme activity of the recombinant strain group was 1.4 times that of the control, which was consistent with the enzyme amount, proving that overexpression of Km_JEN1 promoted the secretion of polygalacturonase.

[0060] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any form, and any technical solution obtained by equivalent replacement or equivalent transformation falls within the protection scope of the present invention.

Claims

1. Application of Km_JEN1 gene in promoting the secretion of polygalacturonase in yeast, characterized in that: The application includes: using yeast as a starting bacterium, introducing a recombinant expression plasmid containing the Km_JEN1 gene into a host bacterium by biological technology means, and obtaining a genetically engineered bacterium overexpressing Km_JEN1, wherein the nucleotide sequence of the Km_JEN1 gene is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that The amino acid sequence of the protein encoded by the Km_JEN1 gene is shown in SEQ ID NO.

2.

3. The use according to claim 1, characterized in that The yeast is Kluyveromyces marxianus.

4. The application according to claim 3, characterized in that: The original plasmid of the recombinant expression plasmid is pIW1146 vector.

5. The use according to claim 4, characterized in that The Km_JEN1 gene replaced the GFP gene in the original vector.

6. The use according to claim 3, characterized in that The application comprises: inoculating the expanded cultured genetically engineered bacteria into a SD liquid culture medium for fermentation, collecting the fermentation liquid, and separating and obtaining polygalacturonase from the fermentation liquid.

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