CN108795969B

CN108795969B - Engineering bacterium NXdP and construction method and application thereof

Info

Publication number: CN108795969B
Application number: CN201810737183.3A
Authority: CN
Inventors: 马挺; 史忠; 李国强; 吴萌萌; 田学峰; 沈雅琪
Original assignee: Nankai University
Current assignee: Nankai University
Priority date: 2018-07-06
Filing date: 2018-07-06
Publication date: 2020-08-07
Anticipated expiration: 2038-07-06
Also published as: CN108795969A

Abstract

The invention provides an engineering bacterium NXdP and a construction method and application thereof, belonging to the technical field of genetic engineering and biological materials.A preservation number of the engineering bacterium NXdP is CGMCC15406, a preservation place is a China general microbiological culture Collection center, and the preservation time is 2018, 03 and 01 days.A hydrogel (Xuan Fu Gu) is only generated during fermentation by using the engineering bacterium NXdP constructed by the invention, the yield of a finally obtained pure product is 15-22 g/L, the total yield is 30-35 g/L, the conversion rate is more than 48%, the yield of the hydrogel can be effectively improved, and the strain has a very high sugar-gum conversion rate, and has a wide industrial application prospect.

Description

Engineering bacterium NXdP and construction method and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering and biological materials, and particularly relates to an engineering bacterium NXdP and a construction method and application thereof.

Background

The Sphingomonas (Sphingomonas sp.) is a new genus proposed according to the characteristics of 16s rRNA sequence, respiratory quinone species, cell polar lipid pattern and the like, the important characteristic of the Sphingomonas sp is that the cell membrane of other gram-negative bacteria contains sphingoglycolipids, but no lipopolysaccharide, the Sphingomonas sp is widely distributed in water, soil and air, at present, the research on the Sphingomonas is mainly focused on the ability of degrading refractory organic pollutants in the genus, for example, diphenyl furan can be used as a unique carbon source substance by the Sphingomonas sp.RW 1, the ability of synthesizing β carotene, and the ability of synthesizing a class of acidic capsular polysaccharides with similar but different structures, which are collectively called as sphingol glue.

Currently, sphingosine gums that have been produced in large quantities and widely used mainly include: gellan gum synthesized by Sphingomonas elodea ATCC3161, welan gum synthesized by Sphingomonas sp.atcc31555, diutan gum synthesized by Sphingomonas sp.atcc53159, rhamsan gum synthesized by Sphingomonas sp.atcc 31961, and the like. The sphingosine glue has a relatively conservative main chain structure, and the types and positions of side chain groups have great diversity, so that the structure and the functions of the sphingosine are more abundant, and the unique physical properties of each sphingosine glue are endowed. For example, gellan gum without glycosyl side chain can form gel, thereby being widely applied to food, daily chemicals and medicine; the welan gum with rhamnose or mannose side chains can form a high-viscosity solution which is resistant to acid, alkali and high temperature, the diutan gum with one or two rhamnose side chains can form a high-viscosity solution under the condition of low concentration, and the welan gum is widely applied to the high-tech fields of buildings, well drilling, oil extraction and the like; with the development of biotechnology, more and more strains capable of producing sphingosine gum are identified, and these newly discovered natural polymer resources will certainly play more important roles in future application of biological gum.

Sphingomonas sp.T-3(CGMCC No.10150) is a production strain capable of producing transparent hydrogel (Sphingomonas T-3 and a method for producing biological polysaccharide and poly β hydroxybutyric acid by co-fermentation thereof, CN201510110078.3 is authorized).

Disclosure of Invention

In view of the above, the present invention aims to provide a hydrogel-producing engineering bacterium NXdP, which does not produce poly β hydroxybutyric acid and realizes a high conversion rate of the carbohydrate gum.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides an engineering bacterium NXdP (Sphingomonas sanxanigens) for producing hydrogel, wherein the preservation number of the engineering bacterium NXdP is CGMCC15406, the preservation place is the China general microbiological culture preservation management center, the concrete address is the microbial research institute of China academy of sciences No.3 of the West Lu No.1 Hospital in the sunward area of Beijing, and the preservation time is 03 months and 01 days in 2018.

The invention provides a method for constructing the engineering bacteria NXdP, which comprises the following steps:

1) knocking out phbB gene of Sphingomonas (Sphingomonas sp.T-3) by using a double-exchange homologous recombination method to obtain engineering bacteria T-3-delta PHB which does not produce poly β hydroxybutyric acid;

2) culturing the engineering bacteria T-3-delta PHB obtained in the step 1) in a TPG liquid culture medium, and performing plasma mutagenesis after culturing to obtain mutagenic engineering bacteria T-3-delta PHB, wherein the TPG liquid culture medium comprises 8-12 g/L of glucose, 3-7 g/L of peptone, 1-5 g/L of yeast powder and 1-5 g/L of beef extract powder;

3) culturing the mutagenic engineering bacteria T-3-delta PHB obtained in the step 2) on a TPG solid culture medium, and screening a strain with the highest hydrogel yield, wherein the strain with the highest hydrogel yield is engineering bacteria NXdP for producing hydrogel, and the TPG solid culture medium comprises 8-12 g/L of glucose, 3-7 g/L of peptone, 1-5 g/L of yeast powder, 1-5 g/L of beef extract powder and 15 g/L of agar powder.

Preferably, the double crossover homologous recombination method in step 1) comprises the following steps:

a) using sphingomonas T-3 genomic DNA as a template, and using the nucleotide sequence shown in SEQ ID NO: 1-4, performing overlapping PCR, purifying and recovering to obtain a recombinant fragment;

b) respectively carrying out double digestion on the recombinant fragment and a p L O3 plasmid by utilizing restriction enzymes SacI and XbaI or SacI and PacI, and connecting the digestion products to obtain a recombinant plasmid p L O3-delta gene;

c) transforming the recombinant plasmid p L O3-delta gene obtained in the step b) into E.Coli S17 competent cells to obtain E.Coli S17/p L O3-delta gene;

d) the single colony of the E.coli S17/p L O3-delta gene obtained in the step c) is arranged in a tet-containing manner^rCulturing in L B liquid culture medium for 6-10 h, and centrifuging to collect E.coli S17/p L O3-delta gene thallus;

e) selecting sphingomonas T-3 single colonyInoculating to a culture medium containing Cm^rCulturing the strain in the seed culture medium for 20-28 h, and centrifugally collecting sphingomonas T-3 bacteria;

f) respectively using the E.coli S17/p L O3-delta gene thallus obtained in the step d) and the sphingomonas T-3 thallus obtained in the step e) with MgSO₄After resuspension of the solution, the ratio of 1: (1.5-2.5), mixing and filtering in a volume ratio, and performing joint transfer on a TPG solid culture medium for 10-15 h to obtain a single-exchange recombinant;

g) inoculating the single-exchange recombinant obtained in the step F) into a nonresistant seed culture medium, and carrying out passage twice to obtain an F2 generation single-exchange recombinant, wherein the seed culture medium comprises 8-12 g/L of sucrose, 1.8-3 g/L of peptone, 1-2.5 g/L of yeast powder and K₂HPO₄1.8-3 g/L and MgSO₄0.06～0.15 g/L；

h) Inoculating the F2 generation single-exchange recombinant obtained in the step g) to a TPG solid culture medium containing cane sugar, and culturing for 65-78 h to obtain a double-exchange recombinant with single copy of phbB gene knocked out;

i) taking the double-exchange recon with the single copy of the phbB gene knocked out obtained in the step h) as a template, and repeating the steps a) to h) by using a primer with a sequence shown in SEQ ID NO. 9-12 to obtain the double-exchange recon with the double copy of the phbB gene knocked out;

j) and (3) taking the double-exchange recon with the phbB gene double copies knocked out as a template, and sequentially using primers of SEQ ID NO. 17-20, SEQ ID NO. 23-26, SEQ ID NO. 27-30 and SEQ ID NO. 33-36 to repeat the steps a) -h), so as to obtain the engineering bacterium T-3-delta PHB without producing β hydroxybutyric acid.

Preferably, the procedure of overlapping PCR of step a) comprises: 1-10 cycles, denaturation at 98 ℃ for 10s, annealing at 55 ℃ to 72 ℃ within 15s, and extension at 72 ℃ for 1 min; performing 11-20 cycles, performing denaturation at 98 ℃ for 10s, cooling to 55 ℃ within 15s at 72 ℃, annealing, and extending for 2min at 72 ℃; performing 21-31 cycles, performing denaturation at 98 ℃ for 10s, annealing at 60 ℃ for 15s, and extending at 72 ℃ for 2 min; 32 nd cycle, extension at 72 ℃ for 10 min.

Preferably, the mass concentration of the sucrose in the TPG solid medium in the step h) is 8-12%.

The invention also provides application of the engineering bacteria NXdP or the engineering bacteria NXdP constructed by the method in hydrogel production.

Preferably, the method comprises the following steps:

①, inoculating the engineering bacteria NXdP single colony to a TPG liquid culture medium, and performing shake culture for 20-26 h to obtain a culture solution;

②, inoculating the culture solution obtained in the step ① to a seed culture medium, and performing shake culture for 20-26 hours to obtain a seed solution;

③, inoculating the seed solution obtained in the step ② into a fermentation medium, and fermenting for 68-75 hours to obtain a fermentation liquid;

④ diluting the fermentation liquid obtained in step ③ with distilled water, heating and centrifuging, and separating to obtain supernatant liquid and thallus precipitate;

⑤ adjusting the pH of the supernatant from step ④ to 3.0, and collecting the precipitate to obtain hydrogel named as Xuefang Gum.

Preferably, the temperature of the shaking culture in the steps ① and ② is 28-35 ℃ independently.

Preferably, the fermentation medium of step ③ includes glucose 30-70 g/L, bean cake powder 0.5-2 g/L, and K₂HPO₄1～2g/L，MgSO₄0.1-1 g/L and NaNO ₃1～2g/L。

Preferably, the fermentation temperature in the step ③ is 28-35 ℃.

The invention provides an engineering bacterium NXdP, which does not produce poly β hydroxybutyric acid but only produces the rice gum, realizes high conversion rate of the rice gum, thereby improving the purity of the hydrogel (rice gum). As can be seen from the test results and data, when the engineering bacterium NXdP is applied to fermentation, the yield of a pure product is 21.20 +/-0.38 g/L, the total yield is 32.03 +/-2.21 g/L, the content of the residual glucose in fermentation liquor in different fermentation periods is different from 40 g/L to 0 g/L, the conversion rate is 80%, the viscosity of the fermentation liquor reaches the maximum value of about 6.4pa.s, and the shear rate range of the obtained hydrogel (rice gum) is 0.001s^-1～1000s^-1The presence of poly β hydroxybutyrate was not detected.

Drawings

FIG. 1 is a flow chart of the construction of a knock-out vector;

FIG. 2 is a diagram showing the results of knocking out and verifying a key gene for synthesizing poly β hydroxybutyrate;

FIG. 3 is a graph of the fermentation yield of the NXdP strain;

figure 4 is a graph of rheological results for the gum.

Detailed Description

When the engineering bacteria NXdP is constructed, firstly, the phbB gene of Sphingomonas (Sphingomonas sp.T-3) is knocked out by using a double-exchange homologous recombination method to obtain the engineering bacteria T-3-delta PHB which does not produce poly β hydroxybutyric acid, the double-exchange homologous recombination method preferably comprises the following steps:

b) respectively carrying out double enzyme digestion on the recombinant fragment and a p L O3 plasmid by utilizing restriction enzymes SacI and XbaI or acI and PacI, and connecting enzyme digestion products to obtain a recombinant plasmid p L O3-delta gene;

e) selecting sphingomonas T-3 single colony to inoculate Cm-containing^rCulturing the strain in the seed culture medium for 20-28 h, and centrifugally collecting sphingomonas T-3 bacteria;

j) and (3) repeating the steps a) to h) by using the double-exchange recon with the phbB gene double copies knocked out as a template and sequentially using primers with sequences shown as SEQ ID NO. 17-20, SEQ ID NO. 23-26, SEQ ID NO. 27-30 and SEQ ID NO. 33-36 to obtain the engineering bacterium T-3-delta PHB which does not produce poly β hydroxybutyrate.

The invention takes sphingomonas T-3 genome DNA as a template, and utilizes SEQ ID NO: 1-4, performing overlapping PCR, purifying and recovering to obtain a recombinant fragment. In the present invention, the system of the overlap PCR preferably comprises 1ul of template, 0.4ul of upstream and downstream primers, 12.5ul of premixed high fidelity DNA polymerase Prime star, and 25ul of water. The procedure for overlapping PCR according to the present invention preferably comprises: 1-10 cycles, denaturation at 98 ℃ for 10s, annealing at 55 ℃ to 72 ℃ within 15s, and extension at 72 ℃ for 1 min; performing 11-20 cycles, performing denaturation at 98 ℃ for 10s, cooling to 55 ℃ within 15s at 72 ℃, annealing, and extending for 2min at 72 ℃; performing 21-31 cycles, performing denaturation at 98 ℃ for 10s, annealing at 60 ℃ for 15s, and extending at 72 ℃ for 2 min; 32 nd cycle, extension at 72 ℃ for 10 min.

After a recombinant fragment is obtained, the recombinant fragment and a p L O3 plasmid are respectively subjected to double enzyme digestion by utilizing restriction enzymes SacI and XbaI or SacI and PacI, the enzyme digestion product is connected to obtain a recombinant plasmid p L O3-delta gene, the temperature of double enzyme digestion is preferably 32-40 ℃, more preferably 35-38 ℃, and most preferably 37 ℃, the time of double enzyme digestion is preferably 60-120 min, more preferably 80-100 min, and most preferably 90 min.

The linked enzyme of the present invention preferably comprises a ligase, preferably a T4 ligase. The temperature of the connection is preferably 12-20 ℃, more preferably 14-18 ℃, and most preferably 16 ℃. The connection time is preferably 8-16 h, more preferably 10-14 h, and most preferably 12 h. The present invention is not particularly limited to the system of the above-mentioned linkage, and may be carried out by a conventional linkage method in the art.

After obtaining the recombinant plasmid p L O3-delta gene, the invention transforms the recombinant plasmid p L O3-delta gene into E.ColiS17 competent cells to obtain E.Coli S17/p L O3-delta gene, when the transformation is carried out, the volume ratio of the recombinant plasmid p L O3-delta gene to the E.Coli S17 competent cells is preferably 1:10, the transformation can amplify the recombinant plasmid p L O3-delta gene.

After obtaining the E.coli S17/p L O3-delta gene, the invention puts the single colony of the E.coli S17/p L O3-delta gene into a medium containing tet^rThe culture is carried out for 6-10 h in L B liquid culture medium, E.coli S17/p L O3-delta gene thallus is collected by centrifugation, the amount of the single colony and L B liquid culture medium is 1/5 m L, and tet in L B liquid culture medium^rThe mass concentration of (b) is preferably 50-150 [ mu ] g/m L, more preferably 80-120 [ mu ] g/m L, and most preferably 100 [ mu ] g/m L. the culture of the invention is preferably performed by shaking table culture, the temperature of the culture is preferably 35-40 ℃, more preferably 36-39 ℃, and most preferably 37 ℃, the rotation speed of the shaking table is preferably 180-220 rpm, more preferably 190-210 rpm, and most preferably 200 rpm.

The invention selects sphingomonas T-3 single colony to be inoculated to the colony containing Cm^rThe method comprises the steps of culturing the sphingomonas T-3 in a seed culture medium for 20-28 h, and centrifugally collecting sphingomonas T-3 thallus, wherein the seed culture medium comprises 8-12 g/L of sucrose, 1.8-3 g/L of peptone, 1-2.5 g/L of yeast powder and K₂HPO₄1.8-3 g/L and MgSO₄0.06-0.15 g/L, wherein the amount of the single colony and the seed culture medium is 1/5 m L, and Cm in the seed culture medium^rThe mass concentration of (b) is preferably 50 to 150. mu.g/m L, more preferably 80 to 120. mu.g/m L, and most preferably 100. mu.g/m L.The culture of the invention is preferably shaking culture, the temperature of the culture is preferably 25-35 ℃, more preferably 28-32 ℃, and most preferably 30 ℃, and the rotation speed of the shaking table is preferably 180-220 rpm, more preferably 190-210 rpm, and most preferably 200 rpm. The culture time is preferably 22-26 h, more preferably 23-25 h, and most preferably 24 h. The rotating speed of the centrifugation is preferably 4000-8000 rpm, more preferably 5000-7000 rpm, and most preferably 6000rpm, and the time of the centrifugation is preferably 3-8 min, more preferably 4-6 min, and most preferably 5 min.

After obtaining E.coli S17/p L O3-delta gene thallus and sphingomonas T-3 thallus, the invention uses MgSO respectively₄After resuspension of the solution, the ratio of 1: (1.5-2.5), and carrying out vacuum filtration, and carrying out joint transfer on the TPG solid culture medium for 10-15 h to obtain the single-exchange recombinant. Before resuspension, MgSO₄Procedure for washing of solution, said washing being with MgSO₄The concentration of the solution is preferably 8-12 mmol/L, more preferably 9-11 mmol/L, and most preferably 10 mmol/L. the washing frequency is preferably 2 times, each washing preferably comprises a slight centrifugation process, the rotation speed of the slight centrifugation is preferably 5000-7000 rpm, and the time of the slight centrifugation is preferably 5-7 min₄The solution dosage is preferably 150-250 mu L, more preferably 180-220 mu L, and most preferably 200 mu L, the volume ratio of the E.Coli S17/p L O3-delta gene cells to the Sphingomonas T-3 cells is preferably 1 (1.8-2.2), more preferably 1:2, the suction filtration is preferably performed on a filter membrane, the pore diameter of the filter membrane is preferably 0.22 mu m, the binding transfer is preferably performed on a TPG solid culture medium, the filter membrane preferably has a cell surface facing upwards, the temperature of the binding transfer is preferably 25-35 ℃, more preferably 28-32 ℃, most preferably 30 ℃, the time of the binding transfer is preferably 11-13 h, and more preferably 12 h.

After obtaining the single crossover recombinants, the invention inoculates the single crossover recombinants to the non-resistant speciesThe invention also preferably comprises colony PCR verification before the inoculation, and the colony PCR verification is preferably performed by using 200u L MgSO₄The cells on the filter membrane are washed with the solution and then coated with the solution containing Cm^rAnd tet^rThe method comprises the steps of culturing the double-antibody solid TPG in a 30 ℃ constant temperature incubator for 72 hours, because p L O3 cannot be copied in Sphingomonas T-3, and exchanging and integrating the p L O3-delta gene into a genome through an upstream homology arm or a downstream homology arm after the p L O3-delta gene is transferred to the T-3 in a conjugal manner, picking a single colony on a double-antibody plate to 5ml of a seed liquid culture medium containing the double antibody, and culturing the double-antibody solid TPG for 36 hours at 200rpm in a 30 ℃ constant temperature shaker, wherein the primer sequence of the PCR is shown in SEQ ID NO. 5-6, the colony PCR is carried out to verify the single-exchange recombinant, the PCR product is subjected to agarose gel electrophoresis, and the result is shown in figure 2, whether two target bands with different lengths are provided or not, the single-exchange recombinant is inoculated into a non-resistant seed culture medium for two times after the colony PCR is completed, the passage is preferably carried out in a shaker, the temperature of the shaker is preferably 25-35 ℃, the most preferably 28-32 ℃, the most preferably 30 ℃ is 30 rpm, and the most preferably the passage is preferably carried out at 250-24 rpm, and the most preferably at the passage time of the passage is preferably carried out at the passage of the shaker is preferably at the passage of the.

After the single-exchange recombinant of the F2 generation is obtained, the single-exchange recombinant of the F2 generation is inoculated to a TPG solid culture medium containing cane sugar and cultured for 65-78 h, and the double-exchange recombinant with the single copy of the phbB gene knocked out is obtained. The mass concentration of the sucrose in the TPG solid culture medium is preferably 8-12%, more preferably 9-11%, and most preferably 10%. The culture time is preferably 66-75 h, more preferably 70-73 h, and most preferably 72 h.

After obtaining the double-exchange recombinants with single copies of the phbB gene knocked out, preferably, the method further comprises colony PCR verification, wherein the colony PCR preferably comprises the steps of selecting single colonies of the double-exchange recombinants with single copies of the phbB gene knocked out to 5m L seed culture medium without antibodies, carrying out shake cultivation for 24 hours at the temperature of preferably 25-35 ℃, more preferably 28-32 ℃, and most preferably 30 ℃, and carrying out shake cultivation at the rotating speed of preferably 150-250 rpm, more preferably 180-220 rpm, and most preferably 200 rpm.

After obtaining the double-exchange recon with the single copy of the phbB gene knocked out, the invention takes the double-exchange recon with the single copy of the phbB gene knocked out as a template, and repeats the steps a) -h) by using the primers of SEQ ID NO. 9-12 to obtain the double-exchange recon with the double copies of the phbB gene knocked out. In the present invention, when the above steps are repeated, the steps g) and h) are preferably repeated, and the colony PCR verification is also preferably included, wherein the primer sequences for colony PCR verification are respectively shown in SEQ ID NO 13-14 and SEQ ID NO 15-16, and a short target band is observed by agarose gel electrophoresis of each obtained PCR product, and the result is expected.

The double-exchange recombinants obtained by the technical scheme are used as templates, and the primers of SEQ ID NO. 17-20, SEQ ID NO. 23-26, SEQ ID NO. 27-30 and SEQ ID NO. 33-36 are sequentially utilized to repeat the steps a) -h) and the steps a) -h) for 4 cycles to obtain the engineering bacteria T-3-delta PHB which does not produce poly β hydroxybutyric acid, wherein the information of the primers is shown in Table 1:

TABLE 1 primer information

After the engineering bacterium T-3-delta PHB is obtained, the engineering bacterium T-3-delta PHB is cultured in a TPG liquid culture medium and subjected to plasma mutagenesis to obtain the mutagenized engineering bacterium T-3-delta PHB. The TPG liquid culture medium comprises glucose, peptone, yeast powder and beef extract powderThe content of glucose in the TPG liquid culture medium is preferably 8-12 g/L, more preferably 9-11 g/L, and most preferably 10 g/L0, the content of peptone in the TPG liquid culture medium is preferably 3-7 g/L1, more preferably 4-6 g/L, and most preferably 5 g/L, the content of yeast powder in the TPG liquid culture medium is preferably 1-5 g/L, more preferably 2-4 g/L, and most preferably 3 g/L, the content of beef extract powder in the TPG liquid culture medium is preferably 1-5 g/L, more preferably 2-4 g/L, and most preferably 3 g/L, the sources of the components in the TPG liquid culture medium are not particularly limited, and conventional commercial products in the field can be utilized to culture the engineering bacteria T-3-0.5L 210, and the cell density of the engineering bacteria T-3-355 is 0.210.3568⁵～2×10⁵A unit/m L, more preferably 0.8 × 10⁵～1.2×10⁵A unit/m L, most preferably 1 × 10⁵And each m L.

The plasma mutagenesis process preferably comprises the steps of dripping bacteria to the bottom of a sample injector and carrying out mutagenesis by using an MPMS instrument to obtain mutagenesis engineering bacteria, wherein the dripping is preferably carried out under an aseptic condition, more preferably in an ultra-clean workbench, the sample injector is preferably a sample injector of a multifunctional plasma mutagenesis instrument, and the method for sample injection is not particularly limited in the invention₂The air flow is preferably 8 to 15slm, more preferably 10 to 13slm, and most preferably 12 slm. The time for mutagenesis is preferably 30-180 s, more preferably 60-150 s, and most preferably 90 s.

After obtaining the mutagenic engineering bacteria T-3-delta PHB, culturing the mutagenic engineering bacteria T-3-delta PHB on a TPG solid culture medium, screening the strains with the highest hydrogel yield, wherein the strains with the highest hydrogel yield are engineering bacteria NXdP producing hydrogel, before culturing on the TPG solid culture medium, preferably, resuspending the mutagenic engineering bacteria T-3-delta PHB, preferably, mixing the obtained mutagenic engineering bacteria and a fresh TPG liquid culture medium according to a volume ratio of 1:50 to obtain a TPG suspension of the mutagenic engineering bacteria, and preferably, coating 100 mu L suspension on the TPG solid culture medium, wherein the culturing temperature is preferably 25-35 ℃, more preferably 28-32 ℃, most preferably 30 ℃, the culturing time is preferably 1-5 d, more preferably 2-4 d, most preferably 3d, the larger the agar powder content in the TPG liquid culture medium is, and the colony formation time is preferably the larger the strain is the strain yield is L g of the screened agar powder in the TPG solid culture medium, and the colony formation time is preferably the same as the strain obtained after culturing.

The invention also provides a method for producing hydrogel by the engineering bacteria NXdP or the engineering bacteria NXdP constructed by the method, which comprises the following steps:

④ diluting the fermentation liquid obtained in step ③ with distilled water, heating and centrifuging, and separating supernatant liquid and thallus precipitate;

When the hydrogel is produced by using the engineering bacteria NXdP, the single bacterial colony of the engineering bacteria NXdP is inoculated into a TPG liquid culture medium and shake culture is carried out for 20-26 hours to obtain a culture solution, the inoculation relation between the single bacterial colony of the engineering bacteria NXdP and the TPG liquid culture medium is preferably 1/3-8 m L, more preferably 1/4-6 m L, and most preferably 1/5 m L, the shake culture temperature is preferably 28-35 ℃, more preferably 29-32 ℃, and most preferably 30 ℃, and the shake culture time is preferably 22-25 hours, more preferably 23-24.5 hours, and most preferably 24 hours.

After the culture solution is obtained, the culture solution is inoculated into a seed culture medium, and shake culture is carried out for 20-26 h to obtain the seed solution. The volume ratio of the culture solution to the seed culture medium is preferably 1: (20-60), when the shake flask method is used for fermentation, the volume ratio is 1: 20; when fermentation is performed using a batch fermentation process, the volume ratio is 1: 60. The temperature of the shake culture is preferably 28-35 ℃, more preferably 29-32 ℃, and most preferably 30 ℃. The shake culture time is preferably 22-25 h, more preferably 23-24.5 h, and most preferably 24 h.

After the seed solution is obtained, the seed solution is inoculated in a fermentation culture medium and fermented for 68-75 hours to obtain the fermentation liquor. The inoculation volume of the seed liquid is preferably 3-10%, more preferably 4-6%, and most preferably 5% of the volume of the fermentation medium. The fermentation medium preferably comprises glucose, soybean meal, K₂HPO₄，MgSO₄And NaNO₃In the fermentation medium, when the shake flask method is used for fermentation, the concentration of the glucose is preferably 30-70 g/L, more preferably 40-50 g/L, and most preferably 40 g/L, the concentration of the bean cake powder is preferably 0.5-2 g/L, more preferably 1-1.8 g/L, and most preferably 1.54 g/L, and the K is₂HPO₄The concentration of (b) is preferably 0.5-2 g/L, more preferably 0.8-1.2 g/L, most preferably 0.9 g/L, and the concentration of (b) is MgSO₄The concentration of (b) is preferably 0.1-1 g/L, more preferably 0.3-0.5 g/L, most preferably 0.4 g/L, and the NaNO is₃The concentration of (b) is preferably 1 to 2 g/L, more preferably 1.2 to 1.8 g/L, and most preferably 1.54 g/L, the concentration of glucose is preferably 30 to 50 g/L, more preferably 40 to 50 g/L, and most preferably 40 g/L, and the concentration of soybean cake powder is preferably 0.5 to 2 g/L, more preferably 40 g/L, when the fermentation is carried out by a batch fermentation methodIs 1-1.8 g/L, most preferably 1.54 g/L, and the K₂HPO₄The concentration of (b) is preferably 0.5-2 g/L, more preferably 0.8-1.2 g/L, most preferably 0.9 g/L, and the concentration of (b) is MgSO₄The concentration of (b) is preferably 0.1-1 g/L, more preferably 0.3-0.5 g/L, most preferably 0.4 g/L, and the NaNO is₃The concentration of (b) is preferably 1-2 g/L, more preferably 1.2-1.8 g/L, and most preferably 1.54 g/L. the sources of the components in the fermentation medium are not particularly limited, and conventional commercial products in the field can be used, the fermentation temperature in the invention is preferably 28-35 ℃, more preferably 29-32 ℃, and most preferably 30 ℃.

After the fermentation liquor is obtained, the invention dilutes the fermentation liquor with distilled water, heats and centrifuges the fermentation liquor, and separates upper-layer liquid and thallus sediment. The dilution is preferably 5 to 10 times, more preferably 6 to 8 times, and most preferably 7 times. The heating temperature is preferably 95-121 ℃, more preferably 99-110 ℃, and most preferably 105 ℃; the heating time is preferably 15 to 25min, more preferably 18 to 22min, and most preferably 20 min. The rotation speed of the centrifugation is preferably 10000-15000 rpm, more preferably 11000-13000 rpm, most preferably 12000rpm, and the time of the centrifugation is preferably 10-50 min, more preferably 20-40 min, most preferably 30 min.

After obtaining the upper liquid, adjusting the pH of the upper liquid to 3.0 by the invention, collecting the generated precipitate to obtain hydrogel, namely Xuefang gel, adjusting the pH of the upper liquid by using dilute hydrochloric acid preferably with the concentration of 1 mol/L, adjusting the pH of the precipitate by using NaOH solution preferably with the concentration of 1 mol/L, and drying to obtain the hydrogel, wherein the concentration of the NaOH solution is preferably 1 mol/L, and the drying condition is preferably 60 ℃ overnight.

The engineering bacteria NXdP provided by the present invention, the construction method and the application thereof are described in detail below with reference to the examples, but they should not be construed as limiting the scope of the present invention.

Example 1

Construction of a key gene knockout vector for the synthetic pathway of poly β hydroxybutyrate:

according to the flow chart shown in figure 1, an extraction kit is used for extracting a T-3 genome, the upstream and downstream homologous arms of a target gene take the T-3 genome as a template, primers SEQ ID NO: 1-4 and PrimeSTARDNA polymerase are respectively used for amplification, the upstream and downstream DNA fragments are connected through overlap PCR, a product is detected through electrophoresis, a target gene strip is purified and recovered through a gel recovery kit to obtain a recombinant fragment, the recombinant fragment and a p L O3 plasmid are simultaneously subjected to enzyme digestion through restriction enzymes SacI and XbaI, the temperature is 37 ℃ and the time is 90min, the fragment subjected to the enzyme digestion is subjected to PCR purification and recovery through the kit, the recovered products of the two are connected through T4DNA ligase at the temperature of 16 ℃ overnight to obtain a recombinant plasmid p L O3-delta gene, the recombinant plasmid is transferred into E.coli S17 competent cells for recombinant amplification, and a single colony which is correctly sequenced is selected for storage of the glycerol.

Example 2

according to the flow chart shown in figure 1, an extraction kit is used for extracting a T-3 genome, the upstream and downstream homologous arms of a target gene take the T-3 genome as a template, primers SEQ ID NO: 1-4 and PrimeSTARDNA polymerase are respectively used for amplification, the upstream and downstream DNA fragments are connected through overlap PCR, a product is detected through electrophoresis, a target gene strip is purified and recovered through a glue recovery kit to obtain a recombinant fragment, the recombinant fragment and a p L O3 plasmid are simultaneously subjected to enzyme digestion by using restriction enzymes SacI and PacI, the temperature is 37 ℃ and 90min, the fragment subjected to enzyme digestion is subjected to PCR purification and recovery by using the kit, the recovered products of the two are connected through a night at the temperature of 16 ℃ by using T4DNA ligase to obtain a recombinant plasmid p L O3-delta gene, the recombinant plasmid is transferred into E.coli S17 competent cells for recombinant amplification, and a single colony which is correctly sequenced is selected for glycerol storage.

Example 3

Construction of a gene engineering strain which does not produce poly β hydroxybutyrate:

selecting a T-3 single colony in a TPG solid culture medium and inoculating the single colony to the culture medium containing Cm^rCulturing in a 30 ℃ constant temperature shaker at 200rpm for 24h, picking single colony of E.coli s17/p L O3-delta gene to tet^rCulturing in L B liquid culture medium at 37 deg.C under 200rpm shaking table for 8 hr, centrifuging two strains at 5ml and 6000rpm for 5min, collecting thallus, and culturing with 10 mmol/L MgSO 2₄The solution was washed twice, centrifuged, and 200ul of MgSO₄Resuspending the thallus in the solution, mixing T-3 and E.coli s17/p L O3-delta gene at a ratio of 2:1, vacuum filtering to obtain a mixture with an aperture of 0.22um, placing the mixture on a filter membrane with the thallus facing upwards, culturing in a 30 ℃ constant temperature incubator for 12h, performing conjugation transfer, and culturing with 200ul MgSO 2₄The bacteria on the filter membrane are washed by the solution, and the solution is coated on the membrane containing Cm after being diluted in a gradient manner^rAnd tet^rThe double-resistant plate is cultured for 72h in a constant-temperature incubator at 30 ℃, a single colony is picked from the double-resistant plate to 5ml of a seed liquid culture medium containing the double-resistant, the double-resistant plate is cultured for 36h at 200rpm in a constant-temperature shaking table at 30 ℃, a colony PCR (polymerase chain reaction) is carried out by using primers SEQ ID NO. 5-6 to verify a single-exchange recombinant, a PCR product is subjected to agarose gel electrophoresis, whether a target strip exists or not is observed, the single-exchange recombinant is inoculated into 5ml of a seed culture medium without resistance, the single-exchange recombinant is cultured for 24h at 200rpm in a constant-temperature shaking table at 30 ℃, passaged for two times, then the single-exchange recombinant is coated on a 10% sucrose plate and cultured for 72h in a constant-temperature incubator at 30 ℃, the single colony is picked into a 5ml test tube, the single colony is cultured for 24h at 30 ℃, the 200rpm, the primers SEQ ID NO. 7-8 are used to verify the colony PCR, the correct double-exchange recombinant is determined, the double-exchange recombinant is stored and continuously knocked out in the next step until an engineering strain which no poly β hydroxybutyrate is obtained.

Example 4

Plasma mutagenesis was performed using a Multifunctional Plasma Mutagenesis System (MPMS):

prior to plasma mutagenesis, T-3-delta PHB strains were precultured to about 10 in TPG medium⁵Cell density of/m L, dropping 20 μ L bacterial suspension on the bottom of the sample applicator of the multifunctional plasma mutagen in a clean bench, and standing at room temperatureStanding for 10min to form a bacterial film on the bottom of the sample injector. Bacteria were mutagenized using an MPMS instrument that had been previously uv sterilized for 30 minutes. When subjected to mutagenesis, N₂The air flow was fixed at 12slm and the cells were impacted with air flow and subjected to gradient mutagenesis for various lengths of time (30-180 s.) the cells were resuspended in 1m L fresh TPG broth and each mixed suspension (100. mu. L) was spread onto two TPG plates and then incubated at 30 ℃ for 3 days.

Example 5

The genetic engineering bacteria NXdP shake flask method is used for producing hydrogel (Xuefu gum) by fermentation:

① inoculating the single colony of the genetically engineered bacterium NXdP into 5ml of TPG liquid culture medium, and performing shaking culture at 30 ℃ for 24 hours;

② inoculating the culture solution prepared in step (1) into 100ml of seed culture medium, and culturing for 24 hours at 30 ℃ with shaking;

③ inoculating the seed solution prepared in the step (2) into a glass shake flask with the inoculation amount of 6-10%, culturing at 30 ℃ and constant pH of 6.8-7.2 for 72 hours;

④ diluting the fermentation liquid of step (3) with distilled water by at least 5 times of volume, heating at 105 deg.C for 20min, and centrifuging at high speed to separate the fermentation liquid and thallus precipitate;

⑤, adjusting the pH of the fermentation liquid in the step (4) to about 3.0 by using dilute hydrochloric acid, adjusting the pH of the precipitate to be neutral by using NaOH, and drying to obtain the biological polysaccharide, wherein the yield of the pure product is 15.03 +/-2.35 g/L, the total yield is 32.51 +/-3.18 g/L, and the conversion rate is 48.8%.

Example 6

The gene engineering bacterium NXdP batch fermentation method is used for producing hydrogel (Xuefu gelatin):

② inoculating the culture solution prepared in step (1) into 300ml of seed culture medium, and culturing for 24 hours at 30 ℃ with shaking;

③ inoculating the seed solution prepared in the step (2) into a 5L fermentation tank with the inoculation amount of 6-10%, culturing for 72 hours at the constant pH of 6.8-7.2 at the temperature of 30 ℃;

⑤ adjusting pH of the fermentation liquid in step (4) to about 3.0 with dilute hydrochloric acid, adjusting pH of the precipitate to neutral with NaOH, and oven drying to obtain biological polysaccharide with pure product yield of 21.20 + -0.38 g/L, total yield of 32.03 + -2.21 g/L, and conversion rate of 80%.

Experimental example 1

And (3) measuring the glucose content in different periods of batch fermentation:

the method comprises the steps of adopting a biosensor analyzer SBA-40C (important laboratory of biosensor in Shandong province) provided with a glucose enzyme membrane ring to measure the content of glucose in fermentation liquor, diluting the fermentation liquor by 5 times of volume with distilled water after fermentation is finished, heating for 10 minutes at 100 ℃, centrifuging and separating supernatant and precipitate, wherein the supernatant is used for measuring the content of glucose in the fermentation liquor, 100mg/dl of glucose is used as standard liquid, and the measurement result is shown in figure 3.

Experimental example 2

Measuring the viscosity of the fermentation liquor in different periods of batch fermentation:

viscosity measurements were performed using a Brookfield viscometer DV _ II +, using a number 64 spindle at 60 rpm. As shown in FIG. 3b, the viscosity of the fermentation broth increased when the medium was cultured for 40 hours, and reached a maximum value of about 6.4pa.s when the medium was cultured for 72 hours, thus confirming that the gum was highly pure.

Experimental example 3

Determination of rheological Properties of Xuefu rubber:

the rheological property of the rice gum is determined by TA rheometer, dissolving rice gum in ultrapure water (10mg/ml), with the detection condition of 25 deg.C, the detection result is shown in FIG. 4, and the shear rate range is 0.001s^-1-1000s^-1。

Experimental example 4

The fermentation production of the Xuefan gum by the genetic engineering strain is compared with the wild strain: the fermentation is carried out by using a fermentation tank, except that the degerming strains are different, the other conditions are the same, and the detection results are shown in the table 2:

TABLE 2

In conclusion, the genetic engineering strain NXdP constructed by the invention can effectively improve the yield of transparent hydrogel (rice glue), has high carbohydrate-gum conversion rate, and has wide industrial application prospect.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

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Claims

1. The application of the engineering bacterium NXdP for producing hydrogel in producing hydrogel is characterized by comprising the following steps:

⑤ adjusting pH of the upper layer liquid obtained in step ④ to 3.0, collecting the generated precipitate to obtain hydrogel named as Xuanfu gel;

the shake culture temperature in the step ① and the shake culture temperature in the step ② are both 28-35 ℃;

the fermentation medium of step ③ comprises glucose 30-70 g/L, bean cake powder 0.5-2 g/L, and K₂HPO₄1～2g/L，MgSO₄0.1-1 g/L and NaNO₃1～2g/L；

The fermentation temperature in the step ③ is 28-35 ℃;

the preservation number of the engineering bacteria NXdP is CGMCC15406, the preservation place is the China general microbiological culture Collection center, and the preservation time is 03 and 01 days in 2018.