CN116445465A - Method for efficiently expressing glucose isomerase in Streptomyces corset and recombinant mutant - Google Patents

Method for efficiently expressing glucose isomerase in Streptomyces corset and recombinant mutant Download PDF

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CN116445465A
CN116445465A CN202210816219.3A CN202210816219A CN116445465A CN 116445465 A CN116445465 A CN 116445465A CN 202210816219 A CN202210816219 A CN 202210816219A CN 116445465 A CN116445465 A CN 116445465A
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汪小杰
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Shanghai Jikaixing Biotechnology Co ltd
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Abstract

The invention relates to the technical field of genetic engineering, and discloses a method for efficiently expressing glucose isomerase in Streptomyces corset and a recombinant mutant. The Streptomyces corset engineering bacteria constructed by the method can produce glucose isomerase with high yield, is environment-friendly because of not carrying antibiotic screening marks, and can ensure the production stability of the glucose isomerase. The invention also provides a mutant strain Streptomyces rust brown JKX, the preservation number is CCTCC NO: M20221030, the fermentation enzyme activity is up to 925.3U/ml, which is improved by about 58.52% compared with that of the parent strain, the mutant strain Streptomyces rust brown is widely applicable to the production of glucose isomerase, the production cost is reduced, and the application prospect is wide.

Description

Method for efficiently expressing glucose isomerase in Streptomyces corset and recombinant mutant
Technical Field
The invention relates to the technical fields of genetic engineering and protein engineering, in particular to a glucose isomerase mutant and application thereof.
Background
Glucose isomerase (Glucose isomerase, GI) is also known as xylose isomerase (ec 5.3.1.5), which catalyzes the isomerisation of glucose to fructose, and also the isomerisation of xylose to xylulose. Glucose isomerase is one of the most important industrial enzymes since fructose is the main sweetener for beverages and foods. In addition, pentoses and hexoses are also good substrates for glucose isomerase, including D-ribose, L-arabinose, L-rhamnose and D-allose. GI is also used to produce rare sugars that have important pharmaceutical applications, for example, GI can efficiently catalyze the conversion of L-arabinose to L-ribose, an important component of nucleosides, glycosyl complexes, and oligonucleotides antiviral and anticancer drugs.
In industry, the most important use of GI is to use its activity in catalyzing the conversion of glucose to fructose for the production of high fructose syrup (HFCS). Fructose is a natural ketohexose and is the highest known sugar, which is abundant in honey and fruit. Fructose is predicted to be a new source of functional sugars that globally replaces sucrose and glucose in the 21 st century. Fructose bypasses the rate-limiting enzyme in glycolysis (phosphofructokinase), and the rate of decomposition of fructose in the liver is faster than glucose. The metabolic intensity of fructose depends on the concentration of fructose and is not affected by insulin. Therefore, the intake of fructose by human body can not cause serious postprandial blood sugar peak and hypoglycemia symptoms which are easily caused by the intake of glucose and sucrose, and the potential factors for inducing diabetes are eliminated. Thus being a healthy sugar which can be eaten by diabetics. Fructose is used in many countries to produce low-energy foods, infant foods, and food for weak people, as well as nutritional and therapeutic foods, because of its low calorific value, water-retaining properties, freezing resistance, corrosion resistance, flavor enhancement, and affinity. The European and American countries use fructose in sweets and beverages without sucrose. As specified by canadian law, all beverages must use high fructose syrup as a sweetener. Statistics prove that the high fructose syrup in 2013 is eliminatedThe consumption is 10 7 Ton/year is the most produced product of industrial enzymes, and thus the amount of glucose isomerase used is also huge.
The current global production of glucose isomerase is mainly monopolized by two companies, dupont and novelin in denmark, usa. The most of the production strains are Streptomyces corsetStreptomyces rubiginosus) And bacillus coagulansBacillus coagulans）。
Glucose isomerase genes are ubiquitous in prokaryotes, and are found in a wide variety of microorganisms such as E.coli, streptomyces, and lactic acid bacteria. In order to increase the expression of glucose isomerase in the early stage, a method of optimizing a medium is mainly used. The former findings that the addition of D-xylose to the fermentation medium can increase the expression of glucose isomerase, but the addition of D-xylose increases the industrial production cost, and later findings that the addition of starch, glucose, sorbitol, glycerol, etc. can achieve the effect of 75% of the addition of D-xylose. Later, bejar S et al found that the strong promoter could increase the expression level of glucose isomerase, and that the strong promoter (P1) was used to replace the original promoter of glucose isomerase gene, and found that the strain increased the expression level of glucose isomerase by 7 times compared with the xylose-induced wild strain. However, the expression level of glucose isomerase is still low and cannot meet the requirements of industrial production. Therefore, the glucose isomerase high-yield strain with independent intellectual property is constructed, the production cost is reduced, the industrial production is realized, and the need is felt.
Disclosure of Invention
The invention aims to construct a Streptomyces corset strain with high glucose isomerase yield, so as to be applied to the industrial production of glucose isomerase. The applicant firstly constructs and obtains a streptomyces rusticans strain for recombining and expressing glucose isomerase genes, then carries out ultraviolet mutagenesis on the streptomyces rusticans strain, and screens and obtains mutant strains with obviously improved glucose isomerase yield, thereby being beneficial to reducing the production cost of the enzyme and promoting the wide application of glucose isomerase.
In one aspect, the invention provides an engineering strain of a strain of Streptomyces corset with high glucose isomerase yield, which carries genes for expressing glucose isomerase.
The gene sequence of the glucose isomerase is SEQ ID NO. 1, and the encoded amino acid sequence is SEQ ID NO. 2.
One aspect of the invention provides a mutant strain Streptomyces rust brown JKX%Streptomyces rubiginosusJKX 102) has been preserved in China center for type culture collection (CCTCC NO: M20221030) of university of Wuhan, china, at 7.5 of 2022.
The invention also provides a method for producing glucose isomerase, which takes the streptomyces rustoffii as a fermentation strain.
The invention also provides glucose isomerase which is obtained by fermenting the streptomyces rustoffii.
Advantageous effects
The invention firstly expresses glucose isomerase gene in a Streptomyces corset host, and constructs engineering strain Streptomyces corset JKX101 for recombinant expression of the enzyme. The glucose isomerase activity in the supernatant of the shake flask fermentation and the 15L tank fermentation of the strain reaches 62.5U/mL and 583.7U/mL respectively.
In order to increase the yield of glucose isomerase, the applicant uses Streptomyces rust brown JKX101 as an initial strain, and screens and obtains a mutant strain Streptomyces rust brown JKX102 by an ultraviolet mutagenesis method. The glucose isomerase in the shake-flask fermentation supernatant of the mutant strain is as high as 94.6U/mL, which is 51.28% higher than that of the parent strain; the glucose isomerase in 15L tank fermentation crude enzyme liquid has the enzyme activity as high as 925.3U/mL, which is improved by 58.52% compared with the starting strain, and unexpected technical effects are obtained. The mutant strain can be widely applied to the production of glucose isomerase, is beneficial to reducing the production cost of the glucose isomerase and promotes the application of the glucose isomerase.
Drawings
FIG. 1 is a pXW403 plasmid map;
FIG. 2 is a diagram of SDS-PAGE electrophoretic analysis of fermentation supernatant of Streptomyces carborundum JKX 102;
biological material preservation information
Mutant strain rust palmStreptomyces chromogenes JKX [ ]Streptomyces rubiginosusJKX 102), the preservation number is CCTCC NO: M20221030, and the preservation is carried out in China Center for Type Culture Collection (CCTCC), address: chinese, wuhan, university of Wuhan, post code: 430072 and the preservation time is 2022, 7, 5 days.
Detailed Description
The method of the present invention will be further described with reference to examples, in which the experimental method without specifying the specific conditions in the examples may be performed under conventional conditions, such as those described in Streptomyces genetic manipulation laboratory Manual written in Hopwood (D.A.), etc., or under conditions recommended by the manufacturer. The present invention may be better understood and appreciated by those skilled in the art by reference to examples. However, the method of implementing the present invention should not be limited to the specific method steps described in the embodiments of the present invention.
The formula of the culture medium related in the embodiment of the invention is as follows:
LB plate: 1% of tryptone, 0.5% of yeast powder, 1% of NaCl and 1.5% of agar powder;
MS medium: mannitol 20 g, soybean cake powder 20 g, agar powder 20 g, pH value adjusted to 7.2-7.4, and tap water was used to fix volume to 1,000, 000 ml.
SS medium: oxoid Tryptic Soy Broth (TSB) 30 g,15g tryptone, 10 g K 2 HPO 4 ， pH 7.2。
SPP medium: glucose 30 g, yeast extract 2 g, sodium chloride 2 g, citric acid 2 g, ammonium sulfate 2 g, magnesium sulfate heptahydrate 2 g, dipotassium phosphate 4 g, ferric sulfate heptahydrate 0.05 g, zinc sulfate heptahydrate 0.05 g, manganese sulfate tetrahydrate 0.05 g, pH7.2.
GI Activity was determined using spectrophotometric analysis, and GI in one enzyme activity unit was defined as the amount of enzyme required to produce 1. Mu. Mol fructose per minute. The method comprises the following steps: 100. mu.l of the fermentation supernatant (containing GI) was added to the mixed solution (5 ml of 70% glucose, 4 ml of 0.2M phosphate buffer pH 7.5,0.5 ml of 0.5M magnesium sulfate, 0.5 ml of 0.01M cobalt chloride), and the mixture was left to stand at 70 o C,150 rpm, for 30 min. The reaction was then quenched by the addition of 5 ml of 0.5M perchloric acid. Taking the solution 0.1ml dilution to a suitable multiple (final fructose concentration of 10-50. Mu.g/ml), 1ml of the above solution was taken and added with 0.2ml of L-cysteine hydrochloride solution (0.12%, w/v) and 6 ml of 70% sulfuric acid at 60 o C, standing for 10min, then standing on ice for 1.5 min, and then allowing to reach room temperature. The absorbance was then measured at an absorption wavelength of 560 nm. The fructose concentration can be calculated through a standard curve.
EXAMPLE 1 construction of glucose isomerase-integrating expression plasmid pXW403
Through literature studies, xylR genes have negative regulation effect on Glucose Isomerase (GI) gene expression. The present patent thus employs insertion of a glucose isomerase gene expression cassette at the xylR position. On one hand, the negative regulation effect of the expression of the xylR gene glucose isomerase can be eliminated, and on the other hand, the glucose isomerase gene can be inserted, so that the genetic operation steps are simplified. The glucose isomerase integrated expression plasmid pXW403 is constructed by taking pSP72 as a framework, and the xylR genes of 500 bp are respectively connected to the upstream and downstream of the glucose isomerase expression cassette, and the constructed pXW403 vector map is shown in FIG. 1. Meanwhile, the streptomycete promoter PrrnA (SEQ ID NO: 3) is used as a promoter for expressing glucose isomerase, so that the expression of the glucose isomerase is promoted. The primers used were as follows:
assxylR left F：tcgactctagaggatcccagcaccgtgacgcggtgga
assxylR left Rv：ggtcgtcggcggagaggacccggcgggccgcgggagc
ass403-GI-F： ccatgattacgaattgtacgtaaagcttggatccatggcagcaccgtgacgcggtggaagttgcacc
ass403-GI-Rv：gtatctagagctagcgggcccatggcgcggtgtccaccctggtcgacgagctcatccgct
assxylR right F：gcggcgcgtatgcgcttgcgaagttggcctcgttggc
assxylR right Rv：gagctcggtacccggggatccgcggtgtccaccctgg
assrrnA-F： gccgacgaagtctagagacccgcctcctgcgcagggtggagc
assrrnA-Rv： ctcgctgaggctatttcacaccacgagagcggaacagtcggag
ass255-pSP72-F： gaatcatggcgcgggtgccctgagttgctggcgtttttccataggctccgccc
ass255-pSP72-Rv：attgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcc
the DNA sequence of the synthesized glucose isomerase gene, SEQ ID NO:1 (which encodes the amino acid sequence of SEQ ID NO: 2), was cloned into the pUC57 plasmid and designated pUC57-gi.
The newly activated Streptomyces corset spores were inoculated into 25 mL of SS liquid medium, shake-cultured overnight at 30℃with shaking at 200 rpm, and the genome of Streptomyces corset was extracted according to the instructions of TIANGEN bacterial genomic DNA extraction kit. PCR amplification was performed using the assxylR left F, assxylR left Rv primer, assxylR right F, assxylR right Rv primer, respectively, using the Phusion fidelity enzyme of NEB company, and the PCR amplified product was recovered by OMEGA gel recovery kit to obtain xylR left and xylR right homology arm fragments with a size of about 0.5kb.
PCR amplification was performed using the pSP72 plasmid as a template, using ass255-pSP72-F, ass255-pSP72-Rv primer, and using Phusion fidelity enzyme from NEB company. The OMEGA gel recovery kit recovered the PCR amplification product to give the backbond sequence of pSP72, approximately 2.5. 2.5 kb.
The synthesized glucose isomerase gene and rrnA DNA sequence were PCR amplified using ass403-GI-F, ass403-GI-Rv and assrrnA-F, assrrnA-Rv primers, respectively, with Phusion fidelity enzyme from NEB company, and the sizes of the products were 1.2kb and 0.5kb, respectively.
The PCR products obtained by the above extension were taken out and ligated using the GeneArt ™ Gibson Assembly and EX Cloning Kit. Ligation product conversionE. coliDH 5. Alpha. Competent cells were plated on LB plates containing 25. Mu.g/mL of apramycin, colony PCR was performed on the transformants, plasmids were extracted from positive transformants, and plasmids were sent to Suzhou gold and other intelligent biotechnology Co., ltd for sequencing, and the plasmid sequenced correctly was designated pXW403, and the map is shown in FIG. 1. The glucose isomerase integrated expression plasmid is constructed.
EXAMPLE 2 construction and fermentation verification of genetically engineered Strain recombinantly expressing glucose isomerase
Coli ET12567/pUZ8002 is used as donor bacteria for conjugal transfer, wherein plasmid pUZ8002 contains oriT conjugal transfer initiation site, which can assist transfer of target plasmid.
Firstly, the target plasmid pXW403 is transferred into the Escherichia coli ET12567/pUZ8002 by electrotransformation or chemical transformation method, and the Escherichia coli is cultivated to OD by LB containing the corresponding antibiotics 600 0.4-0.6, collecting thallus, washing twice with LB medium without antibiotic, and removing antibiotic from supernatant. Meanwhile, freshly harvested Streptomyces corset spores are suspended in a volume of 2 XYT medium (if spores stored in 20% glycerol, the glycerol is removed by centrifugation, washed twice with sterile water, and suspended in a volume of 2 XYT medium), heat-shocked at 50℃for 10min, and cooled sufficiently. Mixing the treated Streptomyces corset spores with Escherichia coli, coating on MS culture medium without antibiotics, culturing at 30deg.C for 12-16 hr, covering the plate with antibiotics corresponding to the target plasmid and naphthalenedone acid or trimethoprim An; the growth of the conjugative transfer was seen 2-3 days after the covering.
10 zygotes were streaked onto MS plates containing 25. Mu.g/mL of apramycin, and then each plate was inoculated with a single colony of seed medium 20 mL, and cultured at 28℃under shaking at 200 rpm for 48 h. Then respectively inoculating 2.5. 2.5 mL seed cultures into 50 mL fermentation medium, and shake culturing at 28deg.C and 200 rpm for 96 h;12000 Centrifuging at rpm for 10min to obtain supernatant; the glucose isomerase enzyme activities of the strain fermentation supernatants are respectively measured by a method for measuring the glucose isomerase activity of national standards of the people's republic of China (GB 23533-2009).
The results show that: the enzyme activity of glucose isomerase in the fermentation supernatant of one recombinant strain is up to 62.5U/mL, which is obviously higher than that of the other 9 recombinant strains. The applicant named the strain as Streptomyces corset JKX @ 101Streptomyces rubiginosusJKX 101). The fermentation supernatant of the strain was subjected to SDS-PAGE electrophoresis. The results are shown in FIG. 2, and the arrow points to the glucose isomerase expressed by the recombinant strain.
EXAMPLE 3 mutagenesis screening of glucose isomerase-producing high-yield strains
At present, two main methods for breeding industrial microorganisms exist: one is the traditional mutation breeding technology, and the other is the modern genetic engineering technology, namely, the production performance of the strain is improved by directionally modifying specific genes of the strain, but due to the protein expression of streptomyces, the expected aim is hardly achieved by changing single genes or regulatory factors. The traditional mutation breeding technology can obtain the strain with excellent production performance under the condition of not clarifying the genetic background of microorganisms only by repeatedly carrying out mutation breeding, fermentation and analysis. Therefore, the traditional mutation breeding technology is still an important means for streptomycete breeding.
Mutation caused by ultraviolet mutagenesis is very random, and the effect of mutation is also random and difficult to predict. Therefore, in order to obtain effective positive mutation, the skilled person is usually required to perform multiple rounds of ultraviolet mutagenesis, the screening effort is large, and there is a possibility that effective positive mutation cannot be obtained. However, since the equipment required for ultraviolet mutagenesis is simple and low in cost, and a large number of mutants can be obtained in a short time, it is still a commonly used mutagenesis breeding method.
The applicant uses the Streptomyces rust brown JKX101 constructed in example 2 as an initial strain, and genetically modifies the Streptomyces rust brown JKX by an ultraviolet mutagenesis method to further improve the yield of glucose isomerase.
3.1 preparation of monospore suspension
Taking a spore plate for culturing mature Streptomyces rust brown JKX101, adding sterile water, gently scraping spores with an inoculating loop, inoculating spore suspension containing spores into a 50 mL triangular flask with a plug, adding 5-10 glass beads, scattering spores on a 250 r/min shaking table, and filtering with filter paper to obtain monospore suspension with monospore content of more than 98%.
3.2 ultraviolet mutagenesis method
And (3) selecting a 30W ultraviolet lamp for irradiation, starting the ultraviolet lamp for preheating for 20 min before mutagenesis, and then placing an empty plate containing 200 mu L of bacterial suspension at a position which is about 20 cm away from the ultraviolet lamp for irradiation, wherein the irradiation time is respectively 10, 20, 30, 60, 90 and 120 s. After the irradiation is finished, 100 mu L of bacterial liquid is respectively taken from each culture dish, a flat plate is coated by dilution according to a certain concentration, and the culture is carried out in the dark at 28 ℃ for 3 to 5 dThe number of colonies of the single spore suspension coated plate without mutagenesis was used as a blank control, and the mortality M was calculated to determine the optimal UV irradiation time. The calculated formula of the mortality rate M is M= (N) 0 －N）/N 0 X 100%. Wherein: n (N) 0 For biomass density before mutagenesis, individual/mL; biomass density, in/mL, remained after N.
TABLE 1 ultraviolet mutagenesis mortality of Streptomyces carborundum JKX101
Time/s 10 20 30 60 90 120
Mortality/% 20.2 34.6 59.7 80.1 96.2 99.9
Too high or too low mortality is unfavorable for the occurrence of positive mutation, and the mortality rate is 70-80% and the effect is good. As can be seen from table 1: the mortality rate of the strain increases along with the increase of the irradiation time, and when the irradiation time is 60 s, the mortality rate of the strain reaches 80.1 percent; continuing to increase the irradiation time, the mortality rate of the strain had reached 96.2% at an irradiation time of 90 s. 60 s was therefore selected as the uv irradiation treatment time.
3.3 mutant Strain screening
And (3) taking spores which survive ultraviolet mutagenesis, properly diluting and coating on an MS plate, properly diluting spore suspension which is not subjected to mutagenesis treatment, coating the plate as a control, picking single colonies after spores on the plate grow to maturity, inoculating seed liquid into a fermentation medium for culturing for 48 hours, inoculating the seed liquid into the fermentation medium at an inoculum size of 5%, culturing for 4 d at a temperature of 28 ℃ at a speed of 200 r/min, and measuring the glucose isomerase activity of the supernatant of the fermentation liquid. Meanwhile, the original strain is used as a control, and 30 single colonies with glucose isomerase activity more than 30% of that of the original strain are selected for shake flask rescreening.
3.4 shaking bottle re-screening
And respectively inoculating the 30 mutant strains obtained by screening into a 50 mL shake flask fermentation medium, fermenting and culturing at 28 ℃ and 200 rpm for 96 h, centrifuging to obtain supernatant, respectively measuring the glucose isomerase enzyme activity in the fermentation supernatant, simultaneously taking the starting strain as a control, and selecting a mutant strain with the shake flask fermentation enzyme activity improved by more than 15% compared with the starting strain for carrying out second round of ultraviolet mutation screening.
The applicant continuously carries out 7 rounds of ultraviolet mutagenesis screening according to the method to finally obtain 1 mutant strain with the glucose isomerase yield obviously higher than that of the parent strain, and the mutant strain is named as Streptomyces corymbose JKX and JKX #Streptomyces rubiginosusJKX 102). After the strain is fermented and cultured in a 50 mL shake flask fermentation medium at 28 ℃ and 200 rpm for 96 h, supernatant is centrifugally taken, the activity of glucose isomerase in the supernatant is up to 96.4U/mL, which is 54.7% higher than that of the starting strain, and the activity of glucose isomerase in a 15L tank fermentation crude enzyme liquid is up to 925.3U/mL, which is 63.9% higher than that of the starting strain, so that unexpected technical effects are achieved.
Example 4 stability verification of Streptomyces rust brown JKX102
Streptomyces rust-brown JKX is inoculated in a seed culture medium, after 10 times of continuous subculture, 2.5 ml seed cultures are inoculated in a 50 mL fermentation culture medium for each passage, 96 h is cultivated at 28 ℃ under 200 rpm shaking, after fermentation is finished, supernatant is obtained by centrifugation at 12000 rpm for 10min, and glucose isomerase enzyme activities of the supernatant are respectively measured.
The results show that after 10 times of subculture, the fermentation enzyme activity of the Streptomyces corymbose JKX102 still exceeds 96.4U/mL, and the enzyme activity is not obviously reduced compared with that before the subculture. Therefore, the mutant strain of the high-yield glucose isomerase obtained by further screening by the ultraviolet mutagenesis method can still effectively keep the consistency of fermentation enzyme activities of different production batches, and has stable passage and remarkable effect.
The applicant has carried out the mutation of the strain Streptomyces carborundum JKX @ 5.2022Streptomyces rubiginosusJKX 102) is preserved in China center for type culture collection (CCTCC NO: M20221030) of university of Wuhan in Wuhan, china.

Claims (6)

1. The glucose isomerase is characterized in that the amino acid sequence of the glucose isomerase is SEQ ID NO. 1, and the encoding nucleotide sequence of the glucose isomerase is SEQ ID NO. 2.
2. A recombinant expression vector, characterized in that the recombinant expression vector carries a sequence encoded by SEQ ID NO:2, a glucose isomerase gene.
3. Streptomyces corymboseStreptomyces rubiginosus) The Streptomyces rust brown strain comprises the recombinant expression vector of claim 1 or 2.
4. The Streptomyces corset mutant strain is characterized in that the Streptomyces corset mutant strain is obtained by ultraviolet mutagenesis by taking the Streptomyces corset engineering strain as set forth in claim 3 as a starting strain.
5. The Streptomyces rust brown mutant strain according to claim 4, wherein the Streptomyces rust brown mutant strain has a preservation number of CCTCC NO: M20221030.
6. Use of the mutant Streptomyces rust brown bacteria according to claim 4 or 5 for the production of high fructose syrups.
CN202210816219.3A 2022-07-10 2022-07-10 Method for efficiently expressing glucose isomerase in Streptomyces corset and recombinant mutant Pending CN116445465A (en)

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