1. Corynebacterium glutamicum strain (A)Corynebacterium glutamicum) The microbial inoculum is characterized by being preserved in China general microbiological culture Collection center (CGMCC) at 6 months and 9 days in 2021 with the preservation number of CGMCC No. 22679.

2. Use of a strain of Corynebacterium glutamicum as claimed in claim 1, for the metabolism of mannose.

3. The use according to claim 2, wherein mannose is added to the medium in which the strain of Corynebacterium glutamicum is cultured.

4. The use according to claim 3, wherein the mannose concentration in the medium is 0.1 g/L to 200 g/L and the temperature of the culture is 25 ℃ to 35 ℃.

5. A recombinant strain of Corynebacterium glutamicum, which is obtained by introducing a mannose 6-phosphoglucomutase gene and a mannose 1-phosphoglucomutase gene into the starting strain of the Corynebacterium glutamicum strain of claim 1.

6. Use of the recombinant strain of Corynebacterium glutamicum of claim 5 for the synthesis of GDP-mannose.

7. The use of claim 6, wherein the GDP-mannose is obtained by fermentation from glucose, or mannose.

8. Use according to claim 7, wherein the fermentation conditions are: the concentration of mannose or glucose is 5-50 g/L, and the culture temperature is 25-35 deg.C.

9. A recombinant strain of Corynebacterium glutamicum, which is obtained by introducing a mannose 6-phosphoglucomutase gene, a mannose 1-phosphoglucomutase gene, a GDP-mannose 4, 6-dehydratase gene and a GDP-fucose synthetase gene into the Corynebacterium glutamicum strain of claim 1.

10. Use of a recombinant strain of corynebacterium glutamicum as claimed in claim 9 in the synthesis of GDP-fucose.

11. The use of claim 10, wherein the GDP-fucose is obtained by fermentation using glucose and mannose as raw materials.

12. Use according to claim 11, wherein the fermentation conditions are: the concentration of mannose or glucose is 5-50 g/L, and the culture temperature is 25-35 deg.C.

Background

Corynebacterium glutamicum is a facultative anaerobic, fast-growing gram-positive bacterium, which was first isolated from nature in 1957. The strain was named Corynebacterium glutamicum because of its ability to synthesize glutamic acid in excess. At present, corynebacterium glutamicum is considered to be a GRAS safe strain, the research on the physiological characteristics of the strain is clear, a genetic operation and exogenous gene expression system is established, the corynebacterium glutamicum recombinant strain which is modified by metabolic engineering is widely applied to the industrial production of amino acid, nucleotide, bioplastic and other fine chemicals, and about 2/3 of the current amino acid productivity is obtained by fermenting and producing corynebacterium glutamicum serving as a chassis cell and plays an important role in the biological manufacturing industry.

D-mannose is a aldohexose, is only used for clinical carbohydrate nutrients, has various physiological functions, promotes insulin secretion, induces anti-inflammatory cytokine expression, prevents urinary tract infection and promotes the growth of intestinal probiotics. Mannose is widely present in a plurality of biomass resources such as konjak, yeast cell walls and the like, is also a main six-carbon sugar unit in hemicellulose, and has the content second to glucose, xylose and arabinose. Until now, researchers have developed industrial strains for efficiently utilizing xylose and arabinose, but reports on the utilization of mannose metabolism are few, so that the efficient metabolism of mannose and the promotion of the conversion and utilization of hemicellulose have important significance in improving the additional value of hemicellulose and even natural lignocellulose.

Mannose can be metabolized and utilized by yeast strains, but bacteria capable of fermenting and utilizing mannose are reported less frequently, and research on improving the mannose metabolizing capacity of corynebacterium glutamicum by improving the activity of mannose 6-phosphate isomerase in cells is available, however, the strains still need higher thallus concentration for metabolizing and utilizing mannose and the mannose metabolizing rate is lower. GDP-mannose and GDP-fucose are key precursors for the in vivo synthesis of mannoglycans and fucoidans, the efficient preparation of which is essential for the synthesis of making mannooligosaccharides and fucosyllactose. Mannose metabolism greatly contributes to the improvement of intracellular content of GDP-mannose and GDP-fucose, so that bacterial strains capable of efficiently metabolizing and utilizing mannose are urgently developed to synthesize more various chemicals.

Disclosure of Invention

The present inventors have completed the present invention by screening a strain capable of utilizing mannose and a Corynebacterium glutamicum strain exhibiting a relatively excellent biomass.

The invention aims to provide a corynebacterium glutamicum strain named as corynebacterium glutamicum (C.) (Corynebacterium glutamicum) The Cgl-M19 CGMCC No.22679 strain has been deposited in China general microbiological culture Collection center (accession number: west road No.1 hospital No.3, north morning area, beijing), the preservation number is CGMCC No. 22679.

The invention also aims to apply the corynebacterium glutamicum Cgl-M19 in the mannose metabolism. It is characterized in that the Cgl-M19 of Corynebacterium glutamicum can be subjected to growth metabolism in a mannose-rich medium.

The culture medium not only comprises rich culture medium such as LB culture medium, brain heart soaking powder, corn steep liquor, corn flour and the like, but also comprises basic salt culture medium such as M9 basic salt culture medium, CGXII basic salt culture medium and the like.

The mannose-rich medium may include not only a medium to which mannose is added but also a medium in which mannose is obtained after hydrolysis of mannan.

The concentration of mannose in the culture medium is 0.1 g/L-200 g/L, and the culture temperature is 25-35 ℃.

The third purpose of the invention is to provide a corynebacterium glutamicum recombinant strain Cgl-GM, which is characterized in that the corynebacterium glutamicum Cgl-M19 is used as an initial strain, and a mannose 6-phosphoglucomutase gene (ManB) and a mannose 1-phosphoglucomutase gene (ManC) are introduced to obtain the corynebacterium glutamicum recombinant strain Cgl-GM. The specific construction method comprises the following steps:

(1) construction of recombinant vector pEC-ManB-ManC

Designing primers according to nucleotide sequences (SEQ ID NO. 2) of mannose 6-phosphate mutase (ManB) (SEQ ID NO.1) and mannose 1-phosphate glycosyltransferase (ManC) derived from Corynebacterium glutamicum, and performing PCR amplification by using Corynebacterium glutamicum genome as templatemanBAndmanCthe gene is connected to a recombinant expression vector pEC-XK99E through a restriction enzyme digestion connection method to obtain a recombinant plasmid pEC-ManB-ManC.

(2) A recombinant vector pEC-ManB-ManC is introduced into a corynebacterium glutamicum recombinant strain Cgl-M19 through an electrotransformation method to obtain a corynebacterium glutamicum recombinant strain named as strain Cgl-GM.

The fourth purpose of the invention is to provide the application of the recombinant strain Cgl-GM of the corynebacterium glutamicum in the synthesis of GDP-mannose, which is characterized in that the recombinant strain Cgl-GM of the corynebacterium glutamicum can ferment glucose and mannose to obtain GDP-mannose, wherein the content of the GDP-mannose is higher than that of the glucose when the recombinant strain Cgl-GM of the corynebacterium glutamicum ferments the mannose.

The fermentation condition is that the concentration of mannose or glucose is 5-50 g/L, and the culture temperature is 25-35 ℃.

The fifth purpose of the invention is to provide a corynebacterium glutamicum recombinant strain Cgl-GF, which is characterized in that corynebacterium glutamicum Cgl-M19 is used as an initial strain, and a mannose 6-phosphoglucomutase gene (ManB), a mannose 1-phosphoglucomutase gene (ManC), a GDP-mannose 4, 6-dehydratase gene (Gmd) and a GDP-fucose synthetase gene (Wcag) are introduced to obtain the corynebacterium glutamicum recombinant strain Cgl-GF. The specific construction method comprises the following steps:

(1) construction of recombinant vector pEC-ManB-ManC

Designing primers according to nucleotide sequences of mannose-6-phosphate mutase (ManB) and mannose-1-phosphate glycosyltransferase (ManC) derived from Corynebacterium glutamicum, and performing PCR amplification by using Corynebacterium glutamicum genome as templatemanBAndmanCthe gene is ligated by restriction enzyme ligation,and connecting the plasmid into a recombinant expression vector pEC-XK99E to obtain a recombinant plasmid pEC-ManB-ManC.

(2) Construction of recombinant vector pXMJ19-Gmd-Wcag

Designing primers according to nucleotide sequences of GDP-mannose 4, 6-dehydratase (Gmd) (SEQ ID NO. 3) and GDP-fucose synthetase (Wcag) (SEQ ID NO. 4) derived from Escherichia coli, and performing PCR amplification by using Escherichia coli genome as templategmdAndwcaGthe gene was ligated to the vector pXMJ19 by restriction ligation to obtain the recombinant plasmid pXMJ 19-Gmd-Wcag.

(3) Recombinant vectors pEC-ManB-ManC and pXMJ19-Gmd-WcaG are introduced into the recombinant strain Cgl-M19 of Corynebacterium glutamicum by an electrotransformation method to obtain the recombinant strain of Corynebacterium glutamicum, which is named as the strain Cgl-GF.

The invention aims to provide the application of the recombinant strain Cgl-GF of the corynebacterium glutamicum in GDP-fucose synthesis, which is characterized in that the recombinant strain Cgl-GF of the corynebacterium glutamicum can ferment glucose and mannose to obtain GDP-fucose, wherein the content of the GDP-fucose is higher than that of the glucose when the recombinant strain Cgl-GF of the corynebacterium glutamicum ferments the mannose.

The fermentation conditions are that the concentration of mannose or glucose is 5-50 g/L, and the culture temperature is 25-35 ℃.

The invention screens the glutamic acid corynebacterium strain which can utilize mannose and shows relatively excellent thallus biomass. Further, the invention provides application of the strain in the aspect of producing mannose metabolism. The recombinant protein can efficiently produce GDP-mannose and GDP-fucose synthesis after gene modification. The invention has proved the above effect through the experiment, therefore has greater application value.

Drawings

FIG. 1 Cgl-M19 fermentation of Corynebacterium glutamicum metabolizes mannose to plot growth curves and mannose consumption curves.

Detailed Description

The present invention will be described in further detail with reference to examples.

The percentage concentrations mentioned in the present invention and examples are mass/mass (W/W, unit g/100g) percentage concentrations, mass/volume (W/V, unit g/100mL) percentage concentrations or volume/volume (V/V, unit mL/100mL) percentage concentrations unless otherwise specified.

The methods used in the following examples are conventional unless otherwise specified, and specific procedures can be found in: molecular Cloning: A Laboratory Manual (Sambrook, J., Russell, David W., Molecular Cloning: A Laboratory Manual, 3rd edition, 2001, NY, Cold Spring Harbor).

Materials or reagents having the same names used in the respective examples are the same unless otherwise specified. The various biological material access approaches described in the examples are provided for the purpose of specific disclosure, and should not be construed as limiting the source of biological material in practicing the invention. In fact, the sources of the biological materials used are wide and any biological material that can be obtained without violating the law and ethics can be used instead as suggested in the examples.

The primers used in the present invention were synthesized by Jiangsu Jinzhi Biotechnology GmbH.

The embodiments are provided in order to provide detailed embodiments and specific procedures, which will help understanding of the present invention, but the scope of the present invention is not limited to the following embodiments.

Example 1: screening and identification of C.glutamicum Cgl-M19

The screening sample is taken from a Tianjin Hangu rhizosphere soil sample.

And (3) a separation process: weighing 2g of soil sample, placing the soil sample into a triangular flask containing 30 mL of sterile water, oscillating the soil sample for 30min at 30 ℃ under 180rmp, and taking 1mL of soil suspension to perform gradient dilution for 10^-1-10^-7Then take 10 out^-5，10^-6，10^-7The three dilutions were plated on enrichment medium M containing mannose (mannose: 5g/L; agar: 15g/L; (NH)₄)₂SO₄(5g/L), urea (5g/L), KH₂PO₄ (1g/L), K₂HPO₄ (1g/L), MgSO₄∙7H₂O (0.25g/L), CaCl₂ (10mg/L), FeSO₄∙7H₂O (10mg/L), MnSO₄∙H₂O (0.1mg/L), ZnSO₄∙7H₂O (1mg/L), CuSO₄∙5H₂O (0.2mg/L), NiCl₂∙6H₂O (20. mu.g/L), biotin (0.4mg/L), MOPS (42g/L), pH adjusted to 7.4), and cultured at 28 ℃ for 1 to 3 days with inversion.

And (3) purification process: colonies grown on enrichment medium M plates were streaked in another enrichment medium containing mannose. And after continuously enriching for 5 times, coating a plate and drawing a line.

Strain comparison: 12 strains capable of utilizing mannose are obtained through continuous enrichment, and the 12 strains are inoculated to a basic salt culture medium (mannose: 10g/L; (NH) taking mannose as a unique carbon source₄)₂SO₄(5g/L), urea (5g/L), KH₂PO₄ (1g/L), K₂HPO₄ (1g/L), MgSO₄∙7H₂O (0.25g/L), CaCl₂ (10mg/L), FeSO₄∙7H₂O (10mg/L), MnSO₄∙H₂O (0.1mg/L), ZnSO₄∙7H₂O (1mg/L), CuSO₄∙5H₂O (0.2mg/L), NiCl₂∙6H₂O (20. mu.g/L), biotin (0.4mg/L), MOPS (42g/L), pH adjusted to 7.4), fermented for 48h, comparing the final biomass of the different strains.

Finally, a strain M19 was obtained by screening, which exhibited a relatively excellent biomass, and 16S rDNA sequencing was performed on the strain, and the strain was identified as Corynebacterium glutamicum based on gene sequence analysis (C.) (Corynebacterium glutamicum) And was named as Corynebacterium glutamicum Cgl-M19.

The strain of Corynebacterium glutamicum Cgl-M19 has been deposited in China general microbiological culture Collection center (address: No.3, Xilu 1, Kyoto, the rising area, Beijing) at 9.6.1.1 with the deposit number of CGMCC No. 22679.

Example 2: fermentation of metabolic mannose by Cgl-M19 Corynebacterium glutamicum

(1) Cgl-M19 activation

The strain Cgl-M19 preserved in glycerin pipe is streaked on the self-made solid culture medium plate and cultured for 48h for activation. The self-made solid culture medium is prepared from the following components: CGXII medium, mannose 10g/L, agar 15g/L, pH =7.0, sterilized at 121 ℃ under high pressure steam for 20 min.

(2) Cgl-M19 seed liquid

Selecting Cgl-M19 single colony on self-made solid culture medium, inoculating in 100mL triangular flask containing 10-30 mL fermentation culture medium, culturing at 30 deg.C, and culturing on a shaker at rotation speed of 150-. The fermentation medium is prepared from the following components: CGXII medium, mannose 10g/L, pH =7.0, sterilized at 121 ℃ under high pressure steam for 20 min.

(3) Cgl-M19 fermentation metabolizes mannose

Inoculating 0.5-3% seed solution into a 250 mL triangular flask containing 50-100 mL fermentation medium M, culturing at 30 deg.C, placing on a shaking table with rotation speed of 150-₆₀₀And a growth curve and a mannose consumption curve are plotted.

As a result, the Cgl-M19 fermented mannose has higher utilization efficiency.

Example 3: construction of recombinant Strain Cgl-GM of Corynebacterium glutamicum

The method specifically comprises the following steps:

(1) construction of recombinant vector pEC-ManB-ManC

Designing primers according to nucleotide sequences of mannose-6-phosphate mutase (ManB) and mannose-1-phosphate glycosyltransferase (ManC) derived from Corynebacterium glutamicum, and performing PCR amplification by using Corynebacterium glutamicum genome as templatemanBAndmanCthe gene is connected to a vector pEC-XK99E through a restriction enzyme digestion connection method to obtain a recombinant plasmid pEC-ManB-ManC.

(2) A recombinant vector pEC-ManB-ManC is introduced into a corynebacterium glutamicum recombinant strain Cgl-M19 through an electrotransformation method to obtain a corynebacterium glutamicum recombinant strain named as strain Cgl-GM.

Example 4: synthesis of GDP-mannose by fermentation of mannose by Corynebacterium glutamicum Cgl-GM

Selecting a culture medium (CGXII), adding 20g/L mannose into the culture medium, culturing a corynebacterium glutamicum recombinant strain Cgl-20 at 30 ℃ under 200rmp, fermenting for 24h, collecting a sample, and detecting the content of GDP-mannose.

HPLC detection method: adopting chromatographic column Inertsil ODS-SP and ultraviolet detector, the mobile phase is A (20 mM triethylamine acetic acid, pH 6.0); b: acetonitrile; adopting a gradient elution mode: firstly, adopting 100% of mobile phase A for 10 min; secondly, the mobile phase B adopts a linear gradient of 0-4% for 10 min; thirdly, the linear gradient of the mobile phase B is 4-0 percent; finally, 100% mobile phase A, 25 min; the flow rate of the mobile phase was 0.6 ml/min; the ultraviolet detection wavelength is 254 nm.

The content of GDP-mannose reaches up to 10.4 +/-1.9 mg per gram of dry cell weight by HPLC detection.

Example 5: construction of recombinant Strain of Corynebacterium glutamicum Cgl-GF

The method specifically comprises the following steps:

(1) construction of recombinant vector pXMJ19-Gmd-Wcag

Designing primers according to nucleotide sequences of GDP-mannose 4, 6-dehydratase (Gmd) and GDP-fucose synthetase (Wcag) derived from Escherichia coli, and performing PCR amplification by using Escherichia coli genome as templategmdAndwcaGthe gene was ligated to the vector pXMJ19 by restriction ligation to obtain the recombinant plasmid pXMJ 19-Gmd-Wcag.

(2) Recombinant vectors pEC-ManB-ManC and pXMJ19-Gmd-WcaG are introduced into the recombinant strain Cgl-M19 of Corynebacterium glutamicum by an electrotransformation method to obtain the recombinant strain of Corynebacterium glutamicum, which is named as the strain Cgl-GF.

Example 6: synthesis of GDP-fucose by fermentation of mannose by Corynebacterium glutamicum Cgl-GF

Selecting a culture medium (CGXII), adding 20g/L mannose into the culture medium, culturing the recombinant strain Cgl-GF of the corynebacterium glutamicum at 30 ℃ under the condition of 200rmp, fermenting for 24 hours, collecting samples, and detecting the content of GDP-fucose.

HPLC detection method: adopting chromatographic column Inertsil ODS-SP and ultraviolet detector, the mobile phase is A (20 mM triethylamine acetic acid, pH 6.0); b: acetonitrile; adopting a gradient elution mode: firstly, adopting 100% of mobile phase A for 10 min; secondly, the mobile phase B adopts a linear gradient of 0-4% for 10 min; thirdly, the linear gradient of the mobile phase B is 4-0 percent; finally, 100% mobile phase A, 25 min; the flow rate of the mobile phase was 0.6 ml/min; the ultraviolet detection wavelength is 254 nm.

The content of GDP-fucose reaches 6.3 +/-1.2 mg per gram of dry cell weight by HPLC detection.

Sequence listing

<110> institute of biotechnology for Tianjin industry of Chinese academy of sciences

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