Sphingomonas engineering bacteria for producing welan gum and fermentation method

Abstract

The invention relates to the technical field of bioengineering, in particular to sphingomonas engineering bacteria for producing welan gum and a fermentation method. According to the engineering bacterium disclosed by the invention, the gene for coding Hsp60 is transferred, so that the robustness of the sphingomonas engineering bacterium is improved, renaturation and folding of protein in the bacterium are promoted, especially the normal function of the protein is maintained in a high-viscosity, high-permeability and low-oxygen adverse environment in the later stage of fermentation, the synthesis efficiency of a product is improved, the growth of the bacterium is promoted, and the yield of the bacterium is increased. Therefore, the yield and the quality of welan gum production are improved.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, and in particular to a Sphingomonas engineering bacterium for producing Welan gum and a fermentation method. Background Art

[0002] Welan Gum is an extracellular polysaccharide secreted by Sphingomonas, and its main chain is composed of D-glucose, D-glucuronic acid, D-glucose and L-rhamnose tetrasaccharide repeating units. Due to its physical and chemical properties such as high viscosity, high thermal stability and excellent shear thinning, Welan Gum is used as a suspending agent, emulsifier, thickener, lubricant and stabilizer in the fields of petroleum, biomedicine, chemical industry, building materials, etc. Welan Gum's unique high temperature resistance and thermal stability are often used in oil production under extreme conditions to increase oil recovery.

[0003] The low yield of Wellan gum fermentation and high production cost are the main factors restricting large-scale production. At present, most studies focus on genetic modification of the original strain of Wellan gum production, Sphingomonas, so as to obtain Wellan gum with higher yield or improved product performance. For example, the introduction of polysaccharide transport proteins and multi-enzyme complex carriers into Sphingomonas can increase the yield of gum (CN112725372A). The construction of pigment-deficient and encapsulated Sphingomonas genetically engineered bacteria can produce pigment-free low molecular weight colloids (CN110144318A).

[0004] Welan gum fermentation is a growth-coupled fermentation, that is, product production and cell growth are positively correlated. In the early stage of fermentation, the viscosity of the fermentation liquid increases with the production of products, which limits the increase in bacterial biomass. In the middle and late stages of fermentation, the high-concentration products form a hypertonic and high-viscosity environment, and the production of metabolites causes the pH of the system to decrease, leading to denaturation of key enzyme proteins in anabolism, reduced enzyme activity, and weakened mass transfer and oxygen transfer capacity, which in turn causes the growth performance of the bacteria to decline or even die, thereby reducing the production efficiency of Welan gum. Summary of the invention

[0005] In view of the problem of low production efficiency of Welan gum, the present invention provides a Sphingomonas engineered bacterium and a fermentation method for producing Welan gum. The engineered bacterium of the present invention improves the robustness of the Sphingomonas engineered bacterium by transferring the gene encoding Hsp60, promotes the renaturation and folding of proteins in the bacteria, especially maintains the normal function of proteins in the unfavorable environment of high viscosity, hypertonicity and hypoxia in the late fermentation period, improves the synthesis efficiency of the product, promotes the growth of the bacteria, and thus improves the yield and quality of Welan gum production.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides the use of Hsp60 in improving the production of welan gum by Sphingomonas, and the gene sequence encoding Hsp60 is shown in SEQ ID NO: 1.

[0008] In a second aspect, the present invention provides a method for constructing an engineered bacterium of Sphingomonas, the method comprising the following steps: constructing an expression vector containing a gene sequence encoding Hsp60, and expressing the obtained expression vector in Sphingomonas; the gene sequence encoding Hsp60 is shown in SEQ ID NO: 1.

[0009] Furthermore, the backbone of the expression vector used is pBBR1MCS-5.

[0010] The third aspect of the present invention provides an engineered Sphingomonas bacterium constructed by the method described in the second aspect.

[0011] In a fourth aspect, the present invention provides a fermentation method for producing welan gum, wherein the Sphingomonas engineered bacteria described in the third aspect are used for fermentation, and the fermentation process includes: primary seed culture, secondary seed liquid culture and fermentation culture;

[0012] The fermentation medium comprises the following components: 30-50 g / L sucrose, 20-30 g / L glucose, 1-10 g / L soybean powder, 1-10 g / L NaNO3, 1-3 g / L K2HPO4, 0.1-1.0 g / L MgSO4·7H2O, and 1-5 mL / L soybean oil.

[0013] Furthermore, NaCl is added during the fermentation culture process, preferably, the amount of NaCl added is 0.3%-1.0% of the volume of the fermentation liquid.

[0014] Furthermore, the rotation speed is controlled at 200-400 rpm during the fermentation culture process.

[0015] Compared with the prior art, the present invention has the following technical advantages:

[0016] The engineered strain HSP60 has better salt tolerance than the original strain. By adding NaCl, inorganic nitrogen source NaNO3 and other process optimizations, the yield of Welland gum produced by the engineered strain HSP60 was significantly improved. The 0.66% Welland gum aqueous solution of this strain has good high temperature resistance. When heated at 85°C, the colloidal viscosity of the 0.66% Welland gum aqueous solution did not decrease but increased, and its viscosity was as high as 3551mPa·s at 85°C. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Construction map for recombinant plasmids;

[0018] Figure 2PCR agarose gel electrophoresis diagram: 1: PCR amplification electrophoresis diagram, 2: PCR amplification gel recovery electrophoresis diagram, 3: PCR identification diagram of DH5α bacterial solution;

[0019] Figure 3 This is the observation picture of colony morphology after NaCl treatment. DETAILED DESCRIPTION

[0020] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0021] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations and / or combinations thereof.

[0022] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0023] Example 1 Construction of high-yield Weilan gum fermentation engineering strain HSP60

[0024] (1) Gene sequence selection: Extract molecular chaperone protein gene sequences from extreme microorganisms, download the sequences from the NCBI website, and submit them to professional biological companies for gene synthesis.

[0025] (2) Vector construction: Design primer sequences SEQ ID NO.2 and SEQ ID NO.3, amplify the HSP60 sequence with T7 promoter, recover the target fragment by gel, double digest the pBBR1MCS5 plasmid with BamHI and HindIII, connect the linearized vector with the target fragment, 10μL reaction system: 4.5μL linearized vector, 0.5μL target gene fragment, 5μL 2×ClonExpress Mix, mix the reaction mixture and place it at 50℃ for 5min, then cool to 4℃. Add 10μL of ligation product to 100μL E. coli DH5α competent cells, mix gently, place on ice for 25-30min; heat shock at 42℃ for 90s, place on ice for 2min, and add 1mL LB culture medium. Then place in a shaker at 180rpm and culture at 37℃ for 1h; centrifuge at 5000rpm for 4min, collect the bacteria, and spread on a resistant plate containing 50μg / mL gentamicin for overnight culture. When a single colony grows, pick a single colony for liquid culture, take the bacterial solution for PCR verification and send it for sequencing. The single colony with the correct sequencing sequence is the successfully constructed HSP60 recombinant bacteria Ecoli DH5α. The construction process of the vector is as follows Figure 1 As shown, the nucleic acid gel electrophoresis diagram is as follows Figure 2 shown.

[0026] (3) Transfer to Sphingomonas sp. by triparental conjugation: Inoculate Sphingomonas sp. into seed culture medium (glucose 20 g / L, yeast extract 1 g / L, peptone 3 g / L, K2HPO4·3H2O 2 g / L, MgSO4 0.1 g / L, pH 7.2-7.4) and culture at 28°C for about 20 h. Dilute the Sphingomonas sp. culture medium to 10% using dd H2O. -6 , 10 -7 , 10 -8After the growth rate was doubled, 50 μL, 100 μL, and 150 μL were respectively applied to the seed solid culture medium, and cultured in a 28°C incubator for 3-4 days. After the single colony grew, the single colony was picked and cultured in the seed culture medium for 24 hours. The E. colipBBR1MCS5-HSP60 and auxiliary bacteria E. colipRK2013 bacterial liquids were streaked with an inoculation loop on solid plates containing 50 μg / mL gentamicin and 50 μg / mL kanamycin sulfate, respectively, and cultured in a 37°C incubator for 16 hours. After the single colony grew, the single colony was picked and cultured in the corresponding resistance LB culture medium for 16 hours. Take 2 mL of Sphingomonas sp. bacterial liquid and transfer it to 50 mL of seed culture medium for 16 hours. Take 2 mL of E. colipBBR1MCS5-HSP60 and auxiliary bacteria E. colipRK2013 bacterial liquid to 50 mL of corresponding resistance LB culture medium for 16 hours. Take appropriate amount of the above bacterial solution, centrifuge at 5000rpm for 10min to collect the bacteria, resuspend the precipitate with 1mL LB culture medium, and wash it twice. Resuspend the washed bacterial sludge with 50-100μL LB culture medium and mix them in the ratio of donor bacteria: receptor bacteria: auxiliary bacteria = 1:3:1. Add the mixed bacterial solution dropwise to the filter paper sheet spread on the binding culture medium (glucose 10g / L, peptone 10g / L, beef extract powder 3g / L, NaCl5g / L, agar 20g / L, adjust pH to 7.2-7.4) plate, place it for a period of time, and then invert it in a 28℃ incubator for 48h of bonding culture. Wash the cultured binding bacteria from the solid culture medium with sterile water, apply it to the double-resistant screening culture medium containing gentamicin and streptomycin sulfate, and culture it for 5-7 days until the yellow sphingomonas grows.

[0027] (4) Detection of HSP60-transfected Sphingomonas: Pick a single yellow colony and culture it in a liquid culture medium containing gentamicin resistance at 28°C and 220rpm for 16h. Take 0.8mL of bacterial solution, centrifuge at 12000rpm for 1min, wash twice with sterile water, collect the bacteria, add 50μL of sterile water to the bacteria, boil in a water bath for 3min, centrifuge at 12000rpm for 5min, and take the supernatant as a PCR template. Use the M13F / M13R primer pair for detection. The PCR reaction system is as follows: 7.5μL 2×Taq Plus MasterMix II, 1μL M13F (10μmol / L), 1μL M13R (10μmol / L), 4.5μL ddH2O, 1μL PCR template. PCR amplification program: 95℃ pre-denaturation for 3min; 95℃ denaturation for 15s, 56℃ annealing for 20s, 72℃ extension for 1.5min, 30 cycles; 72℃ hold for 10min. The bacteria with the same band amplified with the positive plasmid as template are the engineered strain HSP60.

[0028] Example 2 Salt tolerance test of engineered strain HSP60

[0029] Pick the bacteria from the test tube into the seed culture medium, place it in a shaker at 28°C, 220 rpm and culture for 16-24 hours until the OD 600 At 1.6-2.0, 1 mL of bacterial solution was diluted to 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 . Dilute to 10 -6 The bacterial solution was spread on a solid culture medium plate containing 0.5% and 2.5% NaCl, and an original bacterial control group was set up. The colony morphology on the plate was observed and the number of live bacteria was counted after 4-10 days of culture. The colony growth was as follows: Figure 3 . Under 2.5% NaCl treatment, bacterial growth was inhibited, among which the growth of the control strain was most obviously inhibited. The survival rate of the engineered strain HSP60 was 84.9%, and the survival rate of the control strain was 62.4% (Table 1). This shows that the engineered strain HSP60 is more salt-tolerant than the original strain. Under 2.5% NaCl treatment, on the 3rd day, the colonies of the engineered strain HSP60 and the original bacteria were smaller than those of the culture medium containing 0.5% NaCl (normal growth concentration); but on the 10th day of culture, the colony morphology of the engineered strain HSP60 and the original bacteria was larger than the bacterial circle cultured on the culture medium containing 0.5% NaCl, and the colonies of the HSP60 strain were larger than those of the original bacteria, and there was more viscous material.

[0030] Table 1 Effect of 2.5% NaCl on the growth of engineered strain HSP60

[0031]

[0032]

[0033] Example 3 Test of different strains producing Weilan gum

[0034] First-level seed liquid culture: Pick bacteria from the fresh test tube to a 250mL culture bottle containing 50mL seed culture solution, and culture at 28°C, 220rpm for 16-24h; Second-level seed liquid culture: 2mL seed liquid (2% inoculum) is inoculated into a 500mL shake flask containing 100mL seed culture solution, and culture at 28°C, 220rpm for about 10h until OD 600At 1.3-1.8; Fermentation culture: transfer 10% inoculum to a bottle of 100mL fermentation culture medium, 30℃, 250rpm shaking culture for 72h. The seed culture medium contains 20g / L sucrose, 1g / L yeast extract, 2g / L K2HPO4·3H2O, 0.1g / L MgSO4, 0.5g / L (NH4)2HPO4, pH 7.2-7.4, sterilized at 121℃ for 20min. Fermentation culture medium (g / L): sucrose 40.0, soybean powder 5.0, K2HPO4 2.0, MgSO4·7H2O 0.3, soybean oil 1.5mL, pH 7.5, sterilized at 121℃ for 20min.

[0035] The control group strain produced 14.1 g / L of glue, and the engineered strain HSP60 produced 16.0 g / L of glue. The engineered strain HSP60 produced slightly more glue than the original strain, but the bacterial mass was less. When the bacterial liquid was cultured, the engineered strain HSP60 tended to produce glue in advance, and began to produce glue when the bacterial mass was extremely low. The culture liquid tended to become viscous, and the viscosity of the bacterial liquid led to a decrease in bacterial mass. It was necessary to improve the culture medium formula to increase the growth of bacterial cells.

[0036] Table 2 Test of fermentation production of Weilan gum by engineering strain HSP60 and control strain

[0037] Group Rubber production (g / L) <![CDATA[Cell mass (OD 600 )]]> Control group 14.1 15.64 HSP60 group 16.0 14.48

[0038] Example 4 Production of Welan Gum Using Fermentation Medium with Quick-Acting Nitrogen Sources of Corn Steep Powder, (NH4)2HPO4, NaNO3, and KNO3

[0039] The primary and secondary seed liquid culture methods are the same as those in Example 3. Fermentation culture: 10% inoculation amount is transferred to 500mL shaking flasks with 100mL fermentation culture medium, 30℃, 250rpm shaking culture for 80h. Fermentation culture medium (g / L): sucrose 40.0, glucose 20.0, soybean powder 5.0, K2HPO4 2.0, MgSO4·7H2O 0.3, soybean oil 1.5mL, pH 7.5, sterilized at 115℃ for 30min. In addition to the slow-acting nitrogen source soybean powder, appropriate amounts of fast-acting nitrogen sources corn syrup powder, (NH4)2HPO4, NaNO3, and KNO3 are added respectively. When NaNO3 and KNO3 are used as nitrogen sources, the production of glue and the amount of bacteria of HSP60 engineering bacteria are higher, followed by corn syrup powder and (NH4)2HPO4. Yeast extract powder has the worst effect. NaNO3 as a fast-acting nitrogen source can significantly increase the production of glue (Table 3).

[0040] Table 3 Effects of different available nitrogen sources on the production of Weilan gum by engineered strain HSP60

[0041] Group Rubber production (g / L) <![CDATA[Cell mass (OD 600 )]]> Corn steep powder as nitrogen source 18.3 18.20 Yeast extract powder as nitrogen source 14.3 15.70 <![CDATA[(NH4)2HPO4 is used as the nitrogen source]]> 17.0 16.02 <![CDATA[NaNO3 is used as the nitrogen source]]> 21.1 28.00 <![CDATA[KNO3 is used as the nitrogen source]]> 19.6 26.40

[0042] Example 5 Effect of different concentrations of NaCl on the yield of welan gum from different strains

[0043] The primary and secondary seed liquid culture methods are the same as those in Example 3. Fermentation culture: transfer the inoculation amount to 100 mL of fermentation culture medium in a 500 mL shake flask, and culture at 30°C and 250 rpm for 80 h. Fermentation culture medium (g / L): sucrose 40.0, glucose 20.0, soybean powder 5.0, NaNO3 5.0, K2HPO4 2.0, MgSO4·7H2O 0.3, soybean oil 1.5 mL, sterilize at 115°C for 30 min. After adding NaCl, the engineered strain HSP60 and the control strain both increased the rubber production compared to those without adding NaCl. The highest rubber production rate of the engineered strain HSP60 can reach 23.2 g / L, and the highest rubber production rate of the control strain can reach 19.2 g / L. After adding 0.3%-0.7% NaCl for fermentation, the bacterial mass of the engineered strain HSP60 showed an increasing trend after adding NaCl, and the bacterial mass OD 600 The maximum value increased from 28.21 to 31.52, while the control group strain showed a decreasing trend when NaCl was added, and the bacterial volume decreased from 29.76 to 27.00 (Table 4). When NaCl was added to the fermentation broth, the bacterial volume of the salt-tolerant engineered strain HSP60 increased significantly. The tolerance range of Sphingomonas to NaCl is 0-0.3%. When NaCl is added beyond the tolerance concentration, the bacterial cell may enter the stress stage prematurely, change the secondary metabolites of the microorganism, and increase the synthesis of Weilan gum.

[0044] Table 4 Effects of different concentrations of NaCl on the fermentation production of welan gum by engineered strain HSP60 and control strain

[0045]

[0046] Example 6 Fermentation of engineered strain HSP60 to produce Weilan gum

[0047] The primary and secondary seed liquid culture methods are the same as those in Example 3. Fermentation culture: 10% inoculum is transferred to a bottle of 100 mL fermentation culture liquid, 30°C, 350 rpm shaking culture for 60 h, 30°C 300 rpm shaking culture for 20 h. Fermentation culture liquid (g / L): sucrose 40.0, glucose 25.0, soybean powder 5.0, NaNO3 5.0, K2HPO4 2.0, MgSO4·7H2O 0.3, NaCl 5.0, soybean oil 1.5 mL, pH 7.0, sterilized at 115°C for 30 min. The amount of glue produced by the engineered strain HSP60 in the fermentation liquid containing NaCl can reach 30.0 g / L (Table 5).

[0048] Table 5 Fermentation production of Weilan gum by engineered strain HSP60

[0049] Group Rubber yield (g / L) <![CDATA[Cell mass (OD 600 )]]> 0 24.2 36.0 0.5% NaCl 30.0 40.2

[0050] Example 7 Fermentation of engineered strain HSP60 in a fermenter (10 L) to produce welan gum

[0051] 600mL seed solution was prepared, and the seed solution was transferred to a 10L fermentation tank containing 6L fermentation solution at a 10% inoculation amount, and the fermentation culture solution (g / L) was as follows: sucrose 40.0, glucose 25.0, soybean powder 5.0, NaNO3 5.0, K2HPO4 2.0, MgSO4·7H2O 0.3, NaCl 5.0, soybean oil 1.5mL, 1mL bubble enemy, pH 7.0, 115°C sterilization for 30min. The ventilation rate was maintained at 1.0vvm, the stirring speed was 600rpm, and the highest production of welan gum by the engineered strain HSP60 fermentation reached 35.0g / L.

[0052] Example 8 0.66% colloid heat resistance test

[0053] Weigh 0.66 g of the welan gum obtained in Example 5, dissolve it in 100 mL of distilled water, stir and mix, place it in a sealed container, place it in a water bath at 20°C and 85°C for 30 minutes, and immediately use an NDJ-4S digital viscometer to test the viscosity. At room temperature of 20°C, the 0.66% colloid viscosity obtained without adding NaCl to the fermentation broth is higher than the 0.66% colloid viscosity of the fermentation broth with 0.5% NaCl added, but after heating at 85°C for 30 minutes, the colloid viscosity does not decrease but increases, and the 0.66% colloid viscosity of the fermentation broth with or without adding NaCl increases, and can reach up to 3551 mPa·s at 85°C, which has good high temperature resistance (Table 6).

[0054] Table 6 High temperature resistance test of 0.66% colloid obtained by fermentation of engineering strain HSP60

[0055]

[0056] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. The application of Hsp60 in improving the production of welan gum by Sphingomonas, characterized in that: The gene sequence encoding Hsp60 is shown in SEQ ID NO:

1.

2. A method for constructing an engineered bacterium of Sphingomonas, characterized in that: The following steps are involved: An expression vector containing a gene sequence encoding Hsp60 is constructed, and the obtained expression vector is expressed in Sphingomonas; the gene sequence encoding Hsp60 is shown in SEQ ID NO:

1.

3. The construction method according to claim 2, characterized in that: Hsp60 is introduced into the T7 promoter; preferably, the backbone of the expression vector used is pBBR1MCS-5.

4. The engineered Sphingomonas bacteria constructed by the method of claim 2 or 3.

5. A fermentation method for producing welan gum, characterized in that: The engineered bacteria of Sphingomonas described in claim 4 are used for fermentation, and the fermentation process includes: primary seed culture, secondary seed liquid culture and fermentation culture; The fermentation medium comprises the following components: 30-50 g / L sucrose, 20-30 g / L glucose, 1-10 g / L soybean powder, 1-10 g / L NaNO3, 1-10 g / L K2HPO4, 1-3 g / L MgSO4·7H2O, 0.1-1.0 g / L, and 1-5 mL / L soybean oil.

6. The fermentation method according to claim 5, characterized in that NaCl was added during the fermentation process.

7. The fermentation method according to claim 6, characterized in that: The amount of NaCl added is 0.3%-1.0% of the volume of the fermentation liquid.

8. The fermentation method according to claim 5, characterized in that During the fermentation process, the rotation speed was controlled at 200-400 rpm.

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