CN112725372B - Multi-enzyme complex vector for improving expression of welan gum and recombinant bacterium for high yield of welan gum - Google Patents

Abstract

The invention discloses a multienzyme complex carrier for improving the expression of welan gum and a recombinant bacterium for high yield of welan gum, belonging to the technical field of molecular biology. The multienzyme complex vector for improving the expression of welan gum is obtained by constructing a DNA scaffold sequence, TALE1-ugpG and TALE2-welB into a pBBR1SMCS-BB plasmid; the multienzyme complex carrier improves the expression of welan gum by simulating the substrate channel transfer effect. The multienzyme complex expression vector successfully constructed by the invention is integrated into the welan gum strain, the yield of the welan gum fermented by the obtained recombinant strain is obviously improved compared with that of a wild strain, and a foundation is laid for industrial production of the welan gum.

Description

Multi-enzyme complex carrier for improving expression of welan gum and recombinant bacterium for high-yield welan gum

Technical Field

The invention belongs to the technical field of molecular biology, and particularly relates to a multienzyme complex carrier for improving the expression of welan gum and a recombinant bacterium for high yield of welan gum.

Background

Welan gum (Welan gum, with the number S-130) also known as Welan gum, Welan gum or valencia gum, is an extracellular heteropolysaccharide synthesized from gram-negative sphingomonas sp, and has a molecular weight of up to millions, which is one of the most promising microbial polysaccharides currently available in the market after xanthan gum and gellan gum developed by KELCO corporation of america.

The main chain of the welan gum mainly comprises tetrasaccharide repeating units consisting of D-glucose, D-glucuronic acid, D-glucose and L-rhamnose in sequence, and the synthetic process is multienzyme cascade reaction. At present, the domestic research work on welan gum mainly comprises the aspects of welan gum strain breeding, shaking culture medium optimization, welan gum rheological property, influence factors thereof and the like, the production method is that sphingomonas utilizes natural carbohydrate to ferment in cells and then is secreted to the outside of the cells, the fermentation yield of the production of natural microorganisms is low, and the requirement of people on novel microbial polysaccharide is difficult to meet.

The process of biosynthesis of extracellular polysaccharide needs the participation of various enzymes, and the action mechanism is very complex. Specific synthetic routes to welan gum are still being explored. According to previous research reports, the synthetic genes of the sphingosine glue have higher homology, which indicates that the synthetic genes have very similar synthetic mechanisms. Speculation of the specific biosynthetic pathway of welan gum from the synthetic pathway of gellan gum it was found that, in the synthetic pathway of welan gum, after converting glucose to UDP-glucose by UDP-glucose pyrophosphorylase (UgpG), the glycosyltransferase glucose phosphate prenyltransferase WelB then transfers and integrates glucose-1-phosphate from UDP-glucose to the lipid carrier Isoprene Phosphate (IP), synthesizing glucosyl-prenylphosphate. The product formed by catalysis of UgpG is a substrate acted by WelB, and the reactions catalyzed by the two enzymes are natural cascade enzymatic reactions which have important significance in the synthesis process of welan gum.

With the continuous exploration of the synthesis path and key enzymes of microbial polysaccharides, the method for improving the yield of microbial polysaccharides through the metabolic engineering of microbial polysaccharides has made great progress. However, the method of merely changing the activity of the metabolic enzyme and increasing the content of the metabolic enzyme or knocking out the competitive pathway does not necessarily achieve the purpose of increasing the yield of the target product, and sometimes causes a stress effect, but reduces the yield of the target product. In recent years, many natural multienzyme complexes have been found in intracellular metabolic pathways, and the catalytic efficiency of metabolic enzymes is greatly improved by substrate channeling effects. Inspired by the substrate channel transfer effect, people try to adopt a mode of constructing an artificial bracket to spatially draw close to the distance of the cascade enzyme, construct a multienzyme complex, simulate the substrate channel transfer effect and improve the catalytic efficiency of the cascade enzyme. However, conventional artificial scaffolds such as DNA scaffolds, RNA scaffolds and protein scaffolds have limited their in vivo applications due to their own defects.

Disclosure of Invention

Aiming at the problems in the prior art, the invention aims to provide a multienzyme complex vector for improving the expression of welan gum and a recombinant bacterium for high-yield welan gum. According to the invention, an artificial scaffold is constructed by utilizing the specific interaction of TALE and a DNA scaffold, metabolic enzymes in the welan gum synthesis process are spatially assembled, a multienzyme complex is constructed, a substrate channel transfer and processing mechanism is simulated, a metabolic enzyme spatial tissue engineering method is established, a welan gum high-yield strain is constructed, and a foundation is laid for the subsequent fermentation production of welan gum by recombinant bacteria.

In order to achieve the purpose, the invention adopts the following technical scheme:

a multienzyme complex scaffold for improving expression of welan gum comprises a DNA scaffold sequence, TALE1-UgpG and TALE 2-WelB;

wherein the DNA scaffold sequence comprises 11 repeat units, each repeat unit comprising one binding motif BM1 and one binding motif BM 2; a 6bp spacer sequence is arranged between the binding motif BM1 and the binding motif BM 2;

the nucleic acid sequence of the binding motif BM1 is: 5'-CTGCCCTCCTGGTT-3', respectively;

the nucleic acid sequence of the binding motif BM2 is: 5'-CCAGAAACAACACT-3', respectively;

the coding sequence of the TALE1-UgpG is shown as SEQ ID NO. 12;

the coding sequence of TALE2-WelB is shown in SEQ ID NO. 13.

A multienzyme complex vector for improving the expression of welan gum is disclosed, wherein pBBR1SMCS-BB-T1U-T2B-DS obtained by constructing a DNA scaffold sequence, TALE1-ugpG and TALE2-welB into a pBBR1SMCS-BB plasmid is the multienzyme complex vector for improving the expression of welan gum;

wherein the DNA scaffold sequence comprises 11 repeat units, each repeat unit comprising one binding motif BM1 and one binding motif BM 2; a 6bp spacer sequence is arranged between the binding motif BM1 and the binding motif BM 2;

the nucleic acid sequence of the binding motif BM1 is: 5'-CTGCCCTCCTGGTT-3', respectively;

the nucleic acid sequence of the binding motif BM2 is: 5'-CCAGAAACAACACT-3' the flow of the air in the air conditioner,

the nucleic acid sequence of the TALE1-ugpG is shown as SEQ ID NO 12;

the nucleic acid sequence of the TALE2-welB is shown as SEQ ID NO. 13;

the pBBR1SMCS-BB plasmid is a plasmid which is modified by eliminating enzyme cutting sites Spe I and Xba I carried by a pBBR1MCS-3 plasmid and respectively introducing two groups of enzyme cutting sites of Afl II, Spe I, Xba I and Bgl II into the upstream of a Lac promoter and the downstream of a terminator.

On the basis of the scheme, the construction method of the pBBR1SMCS-BB plasmid comprises the following steps:

carrying out double enzyme digestion on a plasmid pBBR1MCS-3 by using restriction enzymes Spe I and Xba I, and carrying out self-ligation on the plasmids subjected to double enzyme digestion to obtain a plasmid pBBR1SMCS forming a new scar sequence;

secondly, taking the pBBR1SMCS plasmid as a template, respectively amplifying a pBBR1SMCS plasmid gene expression region and a main body part of the plasmid except the expression region by utilizing PCR, and respectively introducing two groups of enzyme cutting sites including Afl II, Spe I, Xba I and Bgl II at the upstream of a promoter and the downstream of a terminator of the pBBR1SMCS plasmid Lac through primers in the PCR process;

and thirdly, connecting the two fragments obtained In the PCR step II by using an In-fusion cloning method to obtain the modified plasmid pBBR1 SMCS-BB.

On the basis of the scheme, the amplification primer pair of the pBBR1SMCS plasmid gene expression region is as follows:

SFFor：5’-CTTAAGCCTAGGACTAGTGCGCAACGCAATTAATGTGA-3’；

SFRev：5’-AGATCTACGCGTTCTAGATCGGCCTATTGGTTAAAAAATGAGC-3’；

the amplification primer pair of the main body part of the pBBR1SMCS plasmid except the expression region is as follows:

LFFor：5’-TCTAGAACGCGTAGATCTCTGCGATGAGTGGCAGGGCGG-3’；

LFRev：5’-ACTAGTCCTAGGCTTAAGTCACTGCCCGCTTTCCA-3’。

the use of the above multienzyme complex scaffold and the above multienzyme complex vector in welan gum expression.

A recombinant bacterium for producing welan gum at high yield integrates the multienzyme complex vector into welan gum production bacterium sphingomonas.

The construction method of the high-yield welan gum recombinant bacteria adopts a triparental hybridization method to integrate the multienzyme complex vector into welan gum production bacteria sphingomonas, and specifically adopts sphingomonas as a receptor bacteria; coli carrying the above-mentioned multienzyme complex vector as a donor bacterium; e.coli carrying pRK2013 as an auxiliary bacterium; the three are mixed and cultured in proportion to integrate pBBR1SMCS-BB-T1U-T2B-DS into Sphingomonas sp.

On the basis of the scheme, the recipient bacterium is Sphingomonas sp.wg with the deposit number: CCTCC No. M2013161.

The method for producing the welan gum by fermenting the recombinant bacterium of the high-yield welan gum comprises the following steps:

(1) inoculating the activated recombinant bacteria into a seed culture medium, and culturing at 28 ℃ and 150rpm for 16h to logarithmic phase to obtain a seed solution;

(2) inoculating the seed solution into fermentation culture medium at 5% (v/v), fermenting at 32.5 deg.C and 200rpm for 72 hr.

On the basis of the scheme, the seed culture medium is 10g of glucose, 1g of yeast powder, 5g of peptone, 2g of monopotassium phosphate, 0.1g of magnesium sulfate and 1L of deionized water in constant volume;

the fermentation culture medium comprises 67.37g of glucose, 3.42g of yeast powder and K ₂ HPO ₄ 3.89g、MgSO ₄ 0.1g、ZnSO ₄ 0.1g、CaSO ₄ 0.2g, 1M NaOH to adjust the pH to 7, and deionized water to 1L.

The technical scheme of the invention has the advantages that:

the invention takes enzymes UgpG and WelB which catalyze cascade reaction in the welan gum synthesis process as target objects, constructs a multienzyme complex thereof by means of a TALE-DNA bracket, and simulates the substrate channel transfer effect; improving the expression of welan gum. The constructed multienzyme complex expression vector is successfully integrated into the welan gum strain, the yield of the welan gum fermented by the obtained recombinant strain is obviously improved compared with that of a wild strain, and a foundation is laid for industrial production of the welan gum.

In addition, the invention also successfully constructs a new biological brick vector pBBR1SMCS-BB, the biological brick is constructed by utilizing the characteristics that cohesive ends generated after enzyme digestion by isocaudarner Spe I and Xba I can be complemented, and a sequence formed after connection is not recognized by the two enzymes any more; the method overcomes the defects that each unit is connected with a traditional vector, a unique PCR primer and a specific enzyme cutting site need to be designed, and the enzyme cutting site is not suitable for assembling the next unit after the connection is finished, and solves the problems that a large amount of intermediate products generated in the traditional cloning process cannot be reused, the operation is complex, and materials and time are wasted.

Drawings

FIG. 1 is a construction method of pBBR1SMCS-BB plasmid;

FIG. 2 is a DNA scaffold structure;

FIG. 3 shows the construction of a vector containing a DNA scaffold sequence;

FIG. 4 illustrates a three-segment ligation method;

FIG. 5 shows the bacterial liquid forms of three bacteria cultured for 24h, 36h and 60h in a conjugation manner;

fig. 6 colony morphology of sphingans sp.wg recombinant bacteria (right side, left side, right side);

fig. 7 yields and broth viscosities of whiskery gum fermented by sphingans sp.

Detailed Description

Terms used in the present invention have generally meanings as commonly understood by one of ordinary skill in the art, unless otherwise specified.

The present invention will be described in further detail with reference to the following data in conjunction with specific examples. The following examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way.

Example 1

A multienzyme complex vector for improving the expression of welan gum is disclosed, wherein pBBR1SMCS-BB-T1U-T2B-DS obtained by constructing a DNA scaffold sequence, TALE1-ugpG and TALE2-welB into a pBBR1SMCS-BB plasmid is the multienzyme complex vector for improving the expression of welan gum;

(1) construction of pBBR1SMCS-BB plasmid

The construction method of the pBBR1SMCS-BB plasmid is shown in figure 1, and comprises the following specific steps:

extracting pBBR1MCS-3 plasmid, and simultaneously performing double enzyme digestion by using two restriction enzymes Spe I and Xba I; spe I and Xba I are a group of homocercla enzymes, cohesive ends are complementary after double enzyme digestion, and pBBR1SMCS plasmids are formed after self-ligation; the plasmid eliminates the enzyme cutting sites Spe I and Xba I of pBBR1MCS-3, and is not recognized by any enzyme of Spe I and Xba I any more.

Taking pBBR1SMCS plasmid as a template, respectively amplifying by using primer pairs SFFor/SFRev and LFFor/LFRev to obtain two different fragments, amplifying by using primers SFFor and SFRev to obtain a small fragment of an expression region including a promoter, an MCS region and a terminator on the pBBR1SMCS plasmid, and marking as SF; the primers LFFor and LFRev are amplified to obtain a skeleton large fragment of other regions of the pBBR1SMCS plasmid vector, and the fragment is marked as LF. The four cleavage sites Afl II, Spe I, Xba I and Bgl II (underlined in the sequence) introduced by the primers are respectively at the upstream position of the promoter and the downstream position of the terminator.

The amplification primer pair of the pBBR1SMCS plasmid gene expression region is as follows:

SFFor：5’-CTTAAGCCTAGGACTAGTGCGCAACGCAATTAATGTGA-3’(SEQ ID NO:1)；

SFRev：5’-AGATCTACGCGTTCTAGATCGGCCTATTGGTTAAAAAATGAGC-3’(SEQ ID NO:2)；

the amplification primer pair of the main body part of the pBBR1SMCS plasmid except the expression region is as follows:

LFFor：5’-TCTAGAACGCGTAGATCTCTGCGATGAGTGGCAGGGCGG-3’(SEQ ID NO:3)；

LFRev：5’-ACTAGTCCTAGGCTTAAGTCACTGCCCGCTTTCCA-3’(SEQ ID NO:4)。

purification of amplified fragments SF and LF pBBR1SMCS-BB plasmid was obtained using In-Fusion HD Cloning System construction.

(2) Design of DNA scaffold sequences

The DNA scaffold design contained two TALE binding motifs BMs (BM1 and BM 2). The principles of BMs design are as follows: firstly, there is no homology with sphingomonas genome to avoid off-target phenomenon; secondly, this step was done with the help of the TALE-NT website (https:// TALE-nt.cac.corn.edu /) established by the earliest cooperation of Bogdannove and Voytas laboratories, in line with the characteristics of the DNA sequences recognized by TALE. Finally, BM length is designed according to the structural characteristics of the DNA double helix so as to keep metabolic enzymes on the same side of the DNA, and the metabolic enzymes and the DNA double helix can be assembled into a multienzyme complex smoothly.

The DNA scaffold sequence designed by the invention comprises 2 binding motifs BM1 and BM2 with the size of 14bp, BM 1: 5'-CTGCCCTCCTGGTT-3' (SEQ ID NO: 5); BM 2: 5'-CCAGAAACAACACT-3' (SEQ ID NO:6), wherein the two motifs are separated by 6bp, the spacer sequence being 5 '-TCTAGT-3' (SEQ ID NO: 7); repeat 11 times (shown in FIG. 2).

Because the DNA scaffold contains 11 repetitive sequences, the synthesis difficulty is high, and then a segmented synthesis method is adopted, two DNA scaffold fragments S1-1 and S1-2 respectively containing 6 repetitive sequences and 5 repetitive sequences are firstly synthesized, and the S1-1 and S1-2 sequences are connected with a pNW33N plasmid vector by using a three-fragment connection method to construct a complete DNA scaffold sequence vector pDNA-scaffold plasmid, as shown in FIG. 3, wherein one end of the S1-1 and S1-2 sequences respectively contains a isocaudarner, one end of the S1-1 and S1-2 sequences contains the enzyme cutting site of the plasmid pNW33N, and the enzyme cutting sites of the two ends of S1-1 are EcoR I and Xba I; enzyme cutting sites at two ends of S1-2 are Spe I and BamH I.

(3) TALE scaffold construction

Selecting corresponding RVDs according to sequence base compositions of BM1 and BM2 respectively, determining TALE sequences capable of specifically recognizing the RVDs, and assembling corresponding repeated sequence coding segments by using a Golden Gate TALEN and TAL Effect Kit 2.0 Kit to obtain coding gene sequences of TALE1 and TALE 2;

WG (CCTCC No: M2013161) whole genome is used as a template, and primer pairs of ugpGbioFor/ugpGbioRev and welBbioFor/welBbioRev are used for respectively amplifying ugpG and welB; the primers are designed with Sac I restriction enzyme sites (underlined), and the specific sequences are as follows:

ugpGbioFor：5’-ATAGAGCTCATGACGATCAAGCCGCTGCGCA-3’(SEQ ID NO:8)；

ugpGbioRev：5’-TATGAGCTCTCAATGATGATGATGATGGTGACCGAGCGCACGCTCGGCAAAATC-3’(SEQ ID NO:9)；

welBbioFor：5’-TATGAGCTCATGTTGCGCAAGTCCGCGCTTCGGTT-3’(SEQ ID NO:10)；

welBbioRev：5’-TATGAGCTCTCACTTTTCGAACTGCGGGTGGCTCCAGAAGGCGTTGGAGTGGACGAT-3’(SEQ ID NO:11)。

the cloned ugpG sequence is connected with the coding sequence of TALE1 to obtain a nucleic acid sequence 5 '→ 3' of TALE1-ugpG (SEQ ID NO: 12):

the cloned welB sequence is connected with the coding sequence of TALE2, and the nucleic acid sequence 5 '→ 3' of the TALE2-welB is (SEQ ID NO: 13):

(4) assembly of multienzyme complex vector for improving expression of welan gum

Firstly, the nucleic acid sequences of TALE1-ugpG and TALE2-welB are respectively constructed into a pBBR1SMCS-BB vector through kpn I and Sac I enzyme cutting sites, and plasmids of pBBR1SMCS-BB-T1-ugpG and pBBR1SMCS-BB-T2-welB are obtained.

Cutting the pBBR1SMCS-BB-T1-ugpG plasmid by using Afl II, Xba I and EcoRI to obtain a cutting fragment of TALE 1-ugpG;

using Spe I, Bgl II and EcoRI to cut pBBR1SMCS-BB-T2-welB plasmid to obtain a TALE2-welB enzyme cutting fragment;

the Afl II and Bgl II are used for double digestion of pBBR1SMCS-BB plasmid, and alkaline phosphatase is used for removing phosphate group at 5' end of the digested pBBR1SMCS-BB vector segment, so that the carrier self-ligation phenomenon is reduced.

And (3) carrying out overnight connection on the dephosphorylated pBBR1SMCS-BB vector fragment, the enzyme digestion fragment of TALE1-ugpG and the enzyme digestion fragment of TALE2-welB by using T4 DNA ligase at 16 ℃ to obtain a vector pBBR1SMCS-BB-T1U-T2B, wherein the specific method is shown in figure 4.

Extracting pDNA-scaffold plasmid containing DNA scaffold sequence, using said plasmid as template, using LDFor and LDrev as primer to make amplification, recovering and purifying the obtained DNA scaffold sequence, using restriction enzyme Bgl II to make enzyme digestion at 37 deg.C overnight; meanwhile, the plasmid pBBR1SMCS-BB-T1U-T2B is digested by the same enzyme; alkaline phosphatase removes phosphate groups at the 5' end of the pBBR1SMCS-BB-T1U-T2B fragment, and the dephosphorylated pBBR1SMCS-BB-T1U-T2B vector and the DNA scaffold fragment recovered by enzyme digestion are connected overnight at 16 ℃ by using T4 DNA ligase to construct a plasmid pBBR1SMCS-BB-T1U-T2B-DS, namely the multienzyme complex vector for improving welan gum expression.

LDFor：5’-ATAAGATCTGTTGCTGAAAGGTGCGTTGAAG-3’(SEQ ID NO:14)；

LDRev：5’-TATAGATCTAATTGAATTCGCGGCCGC-3’(SEQ ID NO:15)。

Example 2

A construction method of a recombinant bacterium for producing welan gum at high yield is characterized in that a triparental hybridization method is adopted to integrate the multienzyme complex vector into a welan gum production bacterium sphingomonas, and specifically sphingomonas is used as a receptor bacterium; coli carrying the above-mentioned multienzyme complex vector as a donor bacterium; e.coli carrying pRK2013 as an auxiliary bacterium; the three are mixed and cultured according to a certain proportion to integrate pBBR1SMCS-BB-T1U-T2B-DS into Sphingomonas sp.WG (the preservation number is CCTCC No: M2013161). The method comprises the following specific steps:

1) the strain Sphingomonas sp.WG was inoculated in LB medium and cultured at 28 ℃ for about 20 hours.

2) Using ddH ₂ O diluting Sphingomonas sp.WG bacterial liquid to 10 ⁵ 、10 ⁶ 、10 ⁷ After doubling, respectively taking 50 mu L, 100 mu L and 150 mu L to coat on an LB solid culture medium, culturing in an incubator at 28 ℃ for 3-4d, and picking single colonies to culture in the LB solid culture medium for 36h after the single colonies grow out. Meanwhile, E.coli pBBR1SMCS-BB-T-UB-DS and helper E.coli pRK2013 were inoculated into LB medium with final concentration of tetracycline 50. mu.g/mL and kanamycin 50. mu.g/mL, respectively, and cultured overnight at 37 ℃.

3) The bacterial solution of E.coli pBBR1SMCS-BB-T-UB-DS and helper E.coli pRK2013 was treated with ddH ₂ And O, respectively coating the diluted products on LB solid culture media after the same steps are carried out, culturing for 16h in an incubator at 37 ℃, and picking single colonies to culture for 16h in the LB solid culture media after the single colonies grow out.

4) Transferring 2mL of Sphingomonas sp.WG bacterial liquid in the step II to 50mL of LB culture medium for culturing for 36 h.

5) Culturing the E.coli pBBR1SMCS-BB-T1U-T2B-DS and the auxiliary bacteria E.coli pRK2013 in LB culture medium of 2-50 mL for 16 h.

6) And respectively taking an appropriate amount of the bacterial liquid, centrifuging at 5000rpm for 10min, collecting thalli, resuspending the precipitate by using 1mL of LB culture medium, and repeatedly washing twice.

7) Adding 50-100 mul of conjugation culture medium into each washed bacterial liquid for re-suspension, and providing the donor bacteria according to the bacterial mass: recipient bacterium: mixing the auxiliary bacteria at a ratio of 1:3: 1.

8) The mixed bacterial liquid is dripped on a filter paper sheet paved on an LB flat plate, placed for a period of time and then inverted to be placed in an incubator at 28 ℃ for culture and jointing for 48 hours.

9) After the filter paper sheet has obvious mycoderm, scraping all the mycoderm into 1mL LB culture medium, fully mixing uniformly to prepare bacterial suspension, coating the bacterial suspension on LB solid culture medium containing 50 mu g/mL tetracycline and 50 mu g/mL streptomycin according to gradient, placing the bacterial suspension right side for 30min, and inversely placing the bacterial suspension in an incubator at 28 ℃ for culturing for 3-7 d.

Wg in the seed medium containing 10g/L glucose showed the fastest growth rate, the best state and the thick bacterial liquid. Therefore, the culture medium used in the conjugation process is a seed culture medium containing 10g/L glucose, and the specific components are as follows: 10g of glucose, 1g of yeast powder, 5g of peptone, 2g of monopotassium phosphate, 0.1g of magnesium sulfate and 1L of deionized water in constant volume;

the combination of three kinds of bacteria through mixed culture is the most critical step in the triparental hybridization experiment. This step is to transfer the plasmid from the donor strain to the recipient strain by virtue of the sexual pili produced by the helper strain during conjugation. Too short conjugation time is detrimental to plasmid transport. However, after the conjugation time is prolonged, the culture medium has a certain contamination condition, the viscosity of the mixed bacteria liquid is reduced, the conjugation area is concave and light in color, a white transparent ring is arranged on the outer edge of the mixed bacteria liquid, and the bacteria liquid is separated, so that the conjugation time is 48 hours.

The plasmid to be transferred is large, and donor bacteria are adopted: recipient bacterium: the ratio of helper bacteria to 1:3:1 favors conjugation.

WG adopts strain preserved in refrigerator at-80 deg.C; the conjugation effect showed that the conditions of the recipient strains stored in a refrigerator at-80 ℃ were superior to those of the strains stored at-20 ℃. The recipient bacterium cultured by using the high-nutrition seed culture medium has high bacterium liquid concentration and high growth speed, and after the recipient bacterium is cultured for 24 hours, 36 hours and 60 hours respectively, the joint culture state of the three kinds of bacterium is observed and is shown in figure 5: the bacterial contamination phenomenon is avoided, the color of the thallus subjected to joint culture is bright, the thallus is full, the whole body is in an arc shape, the central part is slightly convex, and the growth of the thallus can be shown. The mixed bacteria after the joint culture are inoculated into an LB culture medium, are gently blown and beaten until the suspension is uniform, are all coated on an LB solid culture medium containing 10 mu g/mL streptomycin and 10 mu g/mL tetracycline, and are subjected to inverted culture at 28 ℃. After culturing, a certain number of microcolonies appear on the culture medium. The colony is round, convex and opaque, and has a smooth and viscous surface. The culture morphology of the strain is consistent with that of a wild strain Sphingomonas sp.WG in LB solid medium (FIG. 6).

Example 3

The method for producing welan gum by fermenting the recombinant bacterium of the high-yield welan gum obtained in the embodiment 2 comprises the following steps:

(1) activating the recombinant strain in the embodiment 2 on an LB (Langmuir-Blodgett) plate, selecting a single colony, inoculating the single colony into a seed culture medium, and culturing at 28 ℃ and 150rpm for 16h to logarithmic phase to obtain liquid fermentation seed liquid;

(2) the seed solution was inoculated into a 250mL Erlenmeyer flask containing 50mL of fermentation medium at an inoculum size of 5% (v/v), and fermented at 32.5 ℃ and 200rpm for 72 hours.

The seed culture medium is 10g of glucose, 1g of yeast powder, 5g of peptone, 2g of monopotassium phosphate, 0.1g of magnesium sulfate and 1L of deionized water in constant volume;

the fermentation culture medium comprises 67.37g of glucose, 3.42g of yeast powder and K ₂ HPO ₄ 3.89g、MgSO ₄ 0.1g、ZnSO ₄ 0.1g、CaSO ₄ 0.2g, 1M NaOH to adjust the pH to 7, and deionized water to 1L.

Using Sphingomonas sp.WG wild bacteria which are not transferred into pBBR1SMCS-BB-T1U-T2B-DS plasmid as a reference, and fermenting the wild bacteria in the same way as the recombinant bacteria; the yield of welan gum in the fermentation broth and the viscosity of the fermentation broth are measured after fermentation is carried out for 72 hours, and the result is shown in figure 7, the yield of welan gum of the recombinant strain reaches 43.89g/L and is improved by 21 percent compared with the wild strain.

The foregoing is directed to preferred embodiments of the present invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow. However, any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present invention are within the protection scope of the technical solution of the present invention.

Sequence listing

<110> China university of Petroleum (east China)

Fujian Normal University

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agtcagcagc agcaagagaa gatcaaaccg aaggtgcgtt cgacagtggc gcagcaccac 540

gaggcactgg tgggccatgg gtttacacac gcgcacatcg ttgcgctcag ccaacacccg 600

gcagcgttag ggaccgtcgc tgtcacgtat cagcacataa tcacggcgtt gccagaggcg 660

acacacgaag acatcgttgg cgtcggcaaa cagtggtccg gcgcacgcgc cctggaggcc 720

ttgctcacgg atgcggggga gttgagaggt ccgccgttac agttggacac aggccaactt 780

gtgaagattg caaaacgtgg cggcgtgacc gcaatggagg cagtgcatgc atcgcgcaat 840

gcactgacgg gtgcccccct gaacctgacc ccggaccaag tggtggctat cgccagccac 900

gatggcggca agcaagcgct cgaaacggtg cagcggctgt tgccggtgct gtgccaggac 960

catggcctga ccccggacca agtggtggct atcgccagca acggtggcgg caagcaagcg 1020

ctcgaaacgg tgcagcggct gttgccggtg ctgtgccagg accatggcct gaccccggac 1080

caagtggtgg ctatcgccag caacaatggc ggcaagcaag cgctcgaaac ggtgcagcgg 1140

ctgttgccgg tgctgtgcca ggaccatggc ctgactccgg accaagtggt ggctatcgcc 1200

agccacgatg gcggcaagca agcgctcgaa acggtgcagc ggctgttgcc ggtgctgtgc 1260

caggaccatg gcctgactcc ggaccaagtg gtggctatcg ccagccacga tggcggcaag 1320

caagcgctcg aaacggtgca gcggctgttg ccggtgctgt gccaggacca tggcctgact 1380

ccggaccaag tggtggctat cgccagccac gatggcggca agcaagcgct cgaaacggtg 1440

cagcggctgt tgccggtgct gtgccaggac catggcctga ccccggacca agtggtggct 1500

atcgccagca acggtggcgg caagcaagcg ctcgaaacgg tgcagcggct gttgccggtg 1560

ctgtgccagg accatggcct gactccggac caagtggtgg ctatcgccag ccacgatggc 1620

ggcaagcaag cgctcgaaac ggtgcagcgg ctgttgccgg tgctgtgcca ggaccatggc 1680

ctgactccgg accaagtggt ggctatcgcc agccacgatg gcggcaagca agcgctcgaa 1740

acggtgcagc ggctgttgcc ggtgctgtgc caggaccatg gcctgacccc ggaccaagtg 1800

gtggctatcg ccagcaacgg tggcggcaag caagcgctcg aaacggtgca gcggctgttg 1860

ccggtgctgt gccaggacca tggcctgacc ccggaccaag tggtggctat cgccagcaac 1920

aatggcggca agcaagcgct cgaaacggtg cagcggctgt tgccggtgct gtgccaggac 1980

catggcctga ccccggacca agtggtggct atcgccagca acaatggcgg caagcaagcg 2040

ctcgaaacgg tgcagcggct gttgccggtg ctgtgccagg accatggcct gaccccggac 2100

caagtggtgg ctatcgccag caacggtggc ggcaagcaag cgctcgaaac ggtgcagcgg 2160

ctgttgccgg tgctgtgcca ggaccatggc ctgaccccgg accaagtggt ggctatcgcc 2220

agcaacggtg gcggcaagca agcgctcgaa agcattgtgg cccagctgag ccggcctgat 2280

ccggcgttgg ccgcgttgac caacgaccac ctcgtcgcct tggcctgcct cggcggacgt 2340

cctgccatgg atgcagtgaa aaagggattg ccgcacgcgc cggaattgat cagaagagtc 2400

aatcgccgta ttggcgaacg cacgtcccat cgcgttgccg actacgcgca agtggttcgc 2460

gtgctggagt ttttccagtg ccactcccac ccagcgtacg catttgatga ggccatgacg 2520

cagttcggga tgagcaggaa cgggttggta cagctctttc gcagagtggg cgtcaccgaa 2580

ctcgaagccc gcggtggaac gctcccccca gcctcgcagc gttgggaccg tatcctccag 2640

gcatcaggga tgaaaagggc caaaccgtcc cctacttcag ctcaaacacc ggatcaggcg 2700

tctttgcatg cattcgccga ttcgctggag cgtgaccttg atgcgcccag cccaatgcac 2760

gagggagatc agacgcgggc aagcagccgt aaacggtccc gatcggatcg tgctgtcacc 2820

ggcccctccg cacagcaggc tgtcgaggtg cgcgttcccg aacagcgcga tgcgctgcat 2880

ttgcccctca gctggagggt aaaacgcccg cgtaccagga tctggggcgg cctcccggat 2940

cctggtacgc ccatggctgc cgacctggca gcgtccagca ccgtgatgtg ggaacaagat 3000

gcggacccct tcgcaggggc agcggatgat ttcccggcat tcaacgaaga ggaactcgca 3060

tggttgatgg agctattgcc tcaggagctc atgacgatca agccgctgcg caatgacgat 3120

caagccgctg cgcaaggctg ttttcccggt tgcgggactc ggcacccgat ttctgcccgc 3180

caccaaggcg atgccgaagg agatgctgcc cgtcgtcgat cgaccgctga tccaatatgc 3240

ggtggacgag gcgatcgagg ccgggatcga gcagatgatc ttcgtcaccg gtcgcggcaa 3300

gtccgcgctc gaggaccatt tcgacatcgc gtacgaactg gaagcgacga tgaccgcgcg 3360

cggaaagtcg cttgaggtgc tcgacggcac ccggttgaag cccggcaaca tcgcctatgt 3420

tcgccagcag gagccgatgg gcctgggcca tgccgtatgg tgcgcgcgcg acatcgtggg 3480

ggacgagccg ttcgcggtgc tgctgccgga cgatttcatg ttcggcaagc cgggctgcct 3540

gaagcagatg gtggatgctt ataaccgcgt cggcggcaac ctgatctgcg ccgagatcgt 3600

gccggacgac cagacgcatc gctatggcat catcaccccg ggcacgcagg aaggcacgct 3660

cacggaagtg aaggggctgg tggaaaagcc ggcgccgggc accgcgccgt cgaacctgtc 3720

ggtgatcgga cgctacatcc tgcagccgga agtcatgcgg atcctggaga accagggcaa 3780

gggcgccggc ggggagatcc agctgaccga cgcgatgcag cagatgatcg gcgaccagcc 3840

gttccacggc gtgaccttcg aaggcacgcg ctatgactgt ggcgacaagg ccggcttcat 3900

caccgccaac atcgccgtcg cgctttcgcg gccggatctg gcgccagcgg tgcgcgattt 3960

tgccgagcgt gcgctcggtt ga 3982

<210> 13

<211> 4425

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

atggatccca ttcgtccgcg caggccaagt cctgcccgcg agcttctgcc cggaccccaa 60

ccggataggg ttcagccgac tgcagatcgt ggggtgtctg cgcctgctgg cagccctctg 120

gatggcttgc ccgctcggcg gacggtgtcc cggacccggc tgccatctcc ccctgcgccc 180

tcacctgcgt tctcggcggg cagcttcagc gatctgctcc gtccgttcga tccgtcgctt 240

cttgatacat cgcttcttga ttcgatgcct gccgtcggca cgccgcatac agcggctgcc 300

ccagcagagt gggatgaggc gcaatcggct ctgcgtgcag ccgatgaccc gccacccacc 360

gtgcgtgtcg ctgtcactgc cgcgcggccg ccgcgcgcca agccggcccc gcgacggcgt 420

gctgcgcaac cctccgacgc ttcgccggcc gcgcaggtgg atctacgcac gctcggctac 480

agtcagcagc agcaagagaa gatcaaaccg aaggtgcgtt cgacagtggc gcagcaccac 540

gaggcactgg tgggccatgg gtttacacac gcgcacatcg ttgcgctcag ccaacacccg 600

gcagcgttag ggaccgtcgc tgtcacgtat cagcacataa tcacggcgtt gccagaggcg 660

acacacgaag acatcgttgg cgtcggcaaa cagtggtccg gcgcacgcgc cctggaggcc 720

ttgctcacgg atgcggggga gttgagaggt ccgccgttac agttggacac aggccaactt 780

gtgaagattg caaaacgtgg cggcgtgacc gcaatggagg cagtgcatgc atcgcgcaat 840

gcactgacgg gtgcccccct gaacctgacc ccggaccaag tggtggctat cgccagccac 900

gatggcggca agcaagcgct cgaaacggtg cagcggctgt tgccggtgct gtgccaggac 960

catggcctga ctccggacca agtggtggct atcgccagcc acgatggcgg caagcaagcg 1020

ctcgaaacgg tgcagcggct gttgccggtg ctgtgccagg accatggcct gaccccggac 1080

caagtggtgg ctatcgccag caacattggc ggcaagcaag cgctcgaaac ggtgcagcgg 1140

ctgttgccgg tgctgtgcca ggaccatggc ctgaccccgg accaagtggt ggctatcgcc 1200

agcaacaatg gcggcaagca agcgctcgaa acggtgcagc ggctgttgcc ggtgctgtgc 1260

caggaccatg gcctgacccc ggaccaagtg gtggctatcg ccagcaacat tggcggcaag 1320

caagcgctcg aaacggtgca gcggctgttg ccggtgctgt gccaggacca tggcctgacc 1380

ccggaccaag tggtggctat cgccagcaac attggcggca agcaagcgct cgaaacggtg 1440

cagcggctgt tgccggtgct gtgccaggac catggcctga ccccggacca agtggtggct 1500

atcgccagca acattggcgg caagcaagcg ctcgaaacgg tgcagcggct gttgccggtg 1560

ctgtgccagg accatggcct gactccggac caagtggtgg ctatcgccag ccacgatggc 1620

ggcaagcaag cgctcgaaac ggtgcagcgg ctgttgccgg tgctgtgcca ggaccatggc 1680

ctgaccccgg accaagtggt ggctatcgcc agcaacattg gcggcaagca agcgctcgaa 1740

acggtgcagc ggctgttgcc ggtgctgtgc caggaccatg gcctgacccc ggaccaagtg 1800

gtggctatcg ccagcaacat tggcggcaag caagcgctcg aaacggtgca gcggctgttg 1860

ccggtgctgt gccaggacca tggcctgacc ccggaccaag tggtggctat cgccagccac 1920

gatggcggca agcaagcgct cgaaacggtg cagcggctgt tgccggtgct gtgccaggac 1980

catggcctga ccccggacca agtggtggct atcgccagca acattggcgg caagcaagcg 2040

ctcgaaacgg tgcagcggct gttgccggtg ctgtgccagg accatggcct gactccggac 2100

caagtggtgg ctatcgccag ccacgatggc ggcaagcaag cgctcgaaac ggtgcagcgg 2160

ctgttgccgg tgctgtgcca ggaccatggc ctgaccccgg accaagtggt ggctatcgcc 2220

agcaacggtg gcggcaagca agcgctcgaa agcattgtgg cccagctgag ccggcctgat 2280

ccggcgttgg ccgcgttgac caacgaccac ctcgtcgcct tggcctgcct cggcggacgt 2340

cctgccatgg atgcagtgaa aaagggattg ccgcacgcgc cggaattgat cagaagagtc 2400

aatcgccgta ttggcgaacg cacgtcccat cgcgttgccg actacgcgca agtggttcgc 2460

gtgctggagt ttttccagtg ccactcccac ccagcgtacg catttgatga ggccatgacg 2520

cagttcggga tgagcaggaa cgggttggta cagctctttc gcagagtggg cgtcaccgaa 2580

ctcgaagccc gcggtggaac gctcccccca gcctcgcagc gttgggaccg tatcctccag 2640

gcatcaggga tgaaaagggc caaaccgtcc cctacttcag ctcaaacacc ggatcaggcg 2700

tctttgcatg cattcgccga ttcgctggag cgtgaccttg atgcgcccag cccaatgcac 2760

gagggagatc agacgcgggc aagcagccgt aaacggtccc gatcggatcg tgctgtcacc 2820

ggcccctccg cacagcaggc tgtcgaggtg cgcgttcccg aacagcgcga tgcgctgcat 2880

ttgcccctca gctggagggt aaaacgcccg cgtaccagga tctggggcgg cctcccggat 2940

cctggtacgc ccatggctgc cgacctggca gcgtccagca ccgtgatgtg ggaacaagat 3000

gcggacccct tcgcaggggc agcggatgat ttcccggcat tcaacgaaga ggaactcgca 3060

tggttgatgg agctattgcc tcaggagctc atgttgcgca agtccgcgct tcggttgctc 3120

ttgtacaccg agcttgtcct gttcgacagc attgcgatcc tgatcgcgtt ctatctcgcg 3180

gcgtgcgttc gggacagcaa ctggctgtcg ctggccggcg tgaacgtcgg catattcctg 3240

ctgccggtga cgctcggtac ggccgtcgcc agcggcgcct atgggctctc cgccctgcgg 3300

catcccgtga gcggtgtgaa aaacatcttc tccgccttct tcttctcggt atttatcgtg 3360

ctgctcggta gctatttgtt gacagccgag ctgccgctgt cgcgcgtgca gctcgcgctt 3420

ggtgtcgttc tcgcattgac cttcatcatg gccgcccgtc tgggctttcg tcgccatgtg 3480

cgccgcatga ccggcggcaa gctgctggac gagttggtaa ttgtcgacgg cgtgtcgctg 3540

gacgtaaccg acggtgcggt ggcgctcgat gcgcgcatcc tcaacctgac gcccgatccc 3600

cacaatccgc agatgttgca tcggctcggc tcgatggtcg ccggcttcga tcgcgtggtg 3660

gtggcatgca cctcggaaca acgcgcggtc tgggcgttgc tgctcaaggg gatgaacgtg 3720

aagggcgaga tcctcgtccc gcagttcaat gcgctcggcg caatcggcgt cgacgcctat 3780

gacgggcagg atacgctggt cgtgtcgcag ggcccgctca acatgcccaa tcgggcgaag 3840

aagcgcattc tcgacctggc aatcaccgtg ccggtgctga ttgcgctcgc gccgctgatg 3900

atcctggtgg ccatcgccat aaaggtcgag agcccggggc cggtgctgtt cgcgcaggat 3960

cgcgtcggtc gcggcaaccg cttgttcaag atcctgaaat tccggtcgat gcgccaggca 4020

ttgtgcgacg cggacggcaa tgtctcggcg agccgagatg acgatcgcat caccaaggtc 4080

ggcggtttca tccgcaagac cagcatcgac gaattgccgc aactcctgaa cgtgctgcgc 4140

ggcgacatga gcgtggtcgg gccgcggccg cacgcactcg gatccagagc ggccgatcat 4200

tacttctggg aaatcgacga acgctactgg catcgtcaca cgctgaagcc cggcatgacc 4260

ggcctggcac aggtgcgcgg gttccgcggc gccacggatc gccgggtcga ccttaccaac 4320

cggctgcagg cggacatgga atatatcgac ggctgggaca tctggcgcga tatcaccatc 4380

ctgttcaaga cactgcgcgt gatcgtccac tccaacgcct tctga 4425

<210> 14

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

ataagatctg ttgctgaaag gtgcgttgaa g 31

<210> 15

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

tatagatcta attgaattcg cggccgc 27

Claims (8)

1. A multienzyme complex vector for improving the expression of welan gum is characterized in that a pBBR1SMCS-BB-T1U-T2B-DS obtained by constructing a DNA scaffold sequence, TALE1-ugpG and TALE2-welB into a pBBR1SMCS-BB plasmid is the multienzyme complex vector for improving the expression of welan gum;

wherein the DNA scaffold sequence comprises 11 repeat units, each repeat unit comprising one binding motif BM1 and one binding motif BM 2; a 6bp spacer sequence is arranged between the binding motif BM1 and the binding motif BM 2;

the nucleic acid sequence of the binding motif BM1 is: 5'-CTGCCCTCCTGGTT-3', respectively;

the nucleic acid sequence of the binding motif BM2 is: 5'-CCAGAAACAACACT-3' the flow of the air in the air conditioner,

the nucleic acid sequence of the TALE1-ugpG is shown as SEQ ID NO 12;

the nucleic acid sequence of the TALE2-welB is shown as SEQ ID NO. 13;

the pBBR1SMCS-BB plasmid is a plasmid which is modified by eliminating enzyme cutting sites Spe I and Xba I carried by a pBBR1MCS-3 plasmid and respectively introducing two groups of enzyme cutting sites of Afl II, Spe I, Xba I and Bgl II into the upstream of a Lac promoter and the downstream of a terminator;

the construction method of the pBBR1SMCS-BB plasmid comprises the following steps:

carrying out double enzyme digestion on a plasmid pBBR1MCS-3 by using restriction enzymes Spe I and Xba I, and carrying out self-ligation on the plasmid subjected to double enzyme digestion to obtain a plasmid pBBR1SMCS forming a new scar sequence;

secondly, taking the pBBR1SMCS plasmid as a template, respectively amplifying a pBBR1SMCS plasmid gene expression region and a main body part of the plasmid except the expression region by utilizing PCR, and respectively introducing two groups of enzyme cutting sites including Afl II, Spe I, Xba I and Bgl II at the upstream of a promoter and the downstream of a terminator of the pBBR1SMCS plasmid Lac through primers in the PCR process;

and thirdly, connecting the two fragments obtained In the PCR step II by using an In-fusion cloning method to obtain the modified plasmid pBBR1 SMCS-BB.

2. The multi-enzyme complex vector for improving the expression of welan gum according to claim 1,

the amplification primer pair of the pBBR1SMCS plasmid gene expression region is as follows:

SFFor：5’-CTTAAGCCTAGGACTAGTGCGCAACGCAATTAATGTGA-3’；

SFRev：5’-AGATCTACGCGTTCTAGATCGGCCTATTGGTTAAAAAATGAGC-3’；

the amplification primer pair of the main body part of the pBBR1SMCS plasmid except the expression region is as follows:

LFFor：5’-TCTAGAACGCGTAGATCTCTGCGATGAGTGGCAGGGCGG-3’；

LFRev：5’-ACTAGTCCTAGGCTTAAGTCACTGCCCGCTTTCCA-3’。

3. use of a multi-enzyme complex vector according to claim 2 for expression of welan gum.

4. A recombinant bacterium capable of producing welan gum at a high yield, which is characterized in that the multienzyme complex vector of claim 1 is incorporated into the welan gum-producing bacterium Sphingomonas sp.

5. The construction method of the high-yield welan gum recombinant bacteria of claim 4, characterized in that the multienzyme complex vector of claim 1 is integrated into welan gum producing bacteria sphingomonas by a triparental hybridization method, and specifically sphingomonas is used as a recipient bacterium; coli as a donor bacterium carrying the multi-enzyme complex vector of claim 1; e.coli carrying pRK2013 as an auxiliary bacterium; the three are mixed and cultured in proportion to integrate pBBR1SMCS-BB-T1U-T2B-DS into Sphingomonas sp.

6. The construction method of the high-yield welan gum recombinant strain as claimed in claim 5, wherein the recipient strain is Sphingomonas sp.WG with the accession number: CCTCC No. M2013161.

7. A method for producing welan gum by fermenting recombinant bacteria for high yield of welan gum as claimed in claim 6, comprising the steps of:

(1) inoculating the activated recombinant bacteria into a seed culture medium, and culturing at 28 ℃ and 150rpm for 16h to logarithmic phase to obtain a seed solution;

(2) inoculating the seed solution into fermentation medium at 5% (v/v), fermenting at 32.5 deg.C and 200rpm for 72 h.

8. The method for producing welan gum by fermenting the recombinant bacterium of high welan gum according to claim 7,

the seed culture medium is 10g of glucose, 1g of yeast powder, 5g of peptone, 2g of monopotassium phosphate, 0.1g of magnesium sulfate and deionized water to a constant volume of 1L;

the fermentation culture medium comprises 67.37g of glucose, 3.42g of yeast powder and K ₂ HPO ₄ 3.89g、MgSO ₄ 0.1g、ZnSO ₄ 0.1g、CaSO ₄ 0.2g, 1M NaOH to adjust the pH to 7, and deionized water to 1L.

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