CN109706110A

CN109706110A - One plant of engineering strain for producing gel state xanthan gum and its construction method and application

Info

Publication number: CN109706110A
Application number: CN201910089794.6A
Authority: CN
Inventors: 马挺; 吴萌萌; 李国强; 史忠
Original assignee: Nankai University
Current assignee: Nankai University
Priority date: 2019-01-31
Filing date: 2019-01-31
Publication date: 2019-05-03

Abstract

The present invention provides the genetic engineering bacteriums that one plant produces gel state xanthan gum; belong to building and the applied technical field of engineering bacteria; the genetic engineering bacterium is using Xanthomonas campestris NK-01 as starting strain; I gene gumF of acetyltransferase and II gene gumG of acetyltransferase is knocked out in the starting strain, and is overexpressed transketolases gene gumL；The culture presevation number of the Xanthomonas campestris NK-01 is CGMCC No.15155.Gel state is presented in the xanthan gum of the genetic engineering bacterium production in aqueous solution or salting liquid, and gel strength increases with the increase of xanthan gum concentration and ion concentration；There is more superior thickening and gel characteristic than spontaneous xanthan gum, and do not limited by fermentation condition, can stability and high efficiency production, there is extensive prospects for commercial application.

Description

One plant of engineering strain for producing gel state xanthan gum and its construction method and application

Technical field

The invention belongs to the building of engineering bacteria and applied technical fields more particularly to one plant to produce gel state xanthan gum Genetic engineering bacterium and its construction method and application.

Background technique

Xanthan gum is a kind of extracellular anion generated by xanthomonas campestris (Xanthomonas campestris) Polysaccharide is that collection is thickened, suspended, emulsifying, being stable at one in the world, and the biogum that best performance is got over is widely used to eat The industries such as product, agricultural, petroleum, printing and dyeing and daily use chemicals.China is the important production base of xanthan gum, and yield of xanthan gum accounts for about the whole world The 67% of total output.Demand of the whole world to xanthan gum went up year by year in recent years, and China's yield of xanthan gum, which also presents always to increase, to become Gesture.With the promotion that the continuous extension of xanthan gum application field and downstream industry require product quality, exploitation, which has, stablizes The xanthan gum product of good characteristic is a main trend of industrial development.

Xanthan gum has the skeleton structure of like fibrous element, and contains α-on second glucose_D-Man-(2→1)- β-_D-GlcA-(4→1)-β-_DThe side-chain structure of-Man.According to different xanthan gum production bacterial strains and fermentation condition, close to 90% Inside mannose occur acetylation, and 30~50% outside mannose group occur acetone acidification.Acetyl group on side chain Group can stablize intramolecular helical conformation, and pyruvic acid group can promote the crosslinking between xanthan molecules, therefore, third Ketone acid content is higher, and the viscosity of xanthan gum solution is bigger.According to the monomer type of xanthan gum, acetone acid content is in xanthan gum point Shared maximum ratio is 8.69% in son, and acetone acid content is generally below 5.0% in the xanthan gum of naturally occurring.

The difference of pyruvic acid and acetyl content can significantly affect the rheological properties of xanthan gum in xanthan molecules.? During traditional zymotic, due to fermentation condition, starting strain, batch fermentation difference, pyruvic acid and acetyl in xanthan molecules Base content has differences, and then affects the rheological properties of xanthan gum.This status to the quality analysis of xanthan gum product, Sorting causes puzzlement, and increases the production cost and unstability of xanthan gum.

Summary of the invention

In view of this, the purpose of the present invention is to provide genetic engineering bacteriums and its structure that one plant produces gel state xanthan gum Construction method and application；The xanthan gum that the genetic engineering bacterium generates has the acetone acid content of saturation, can be formed under low concentration Weak Gels, product quality and rheological properties are stablized.

In order to achieve the above-mentioned object of the invention, the present invention provides following technical schemes:

The genetic engineering bacterium of one plant of production gel state xanthan gum, the genetic engineering bacterium is with Xanthomonas Campestris NK-01 is starting strain, and I gene gumF of acetyltransferase and acetyl group are knocked out in the starting strain II gene gumG of transferase, and it is overexpressed transketolases gene gumL；

The culture presevation number of the Xanthomonas campestris NK-01 is CGMCC No.15155.

Preferably, the overexpression transketolases gene gumL is by being transferred into the starting strain comprising ketone The multicopy expression plasmid pBBRL of based transferase gene gumL is realized.

The present invention provides the preparation methods of the genetic engineering bacterium, comprising the following steps:

1) the gumF gene and gumG gene in the starting strain are knocked out, gumF gene and gumG gene delection are obtained Strain X C (△ FG)；

2) ketone group turn is overexpressed in the gumF gene described in step 1) and the strain X C (△ FG) of gumG gene delection It moves enzyme gene gumL and obtains genetic engineering bacterium.

Preferably, gumF gene in the starting strain and gumG gene are knocked out in step 1) the following steps are included:

1.1) using the genome of the starting strain as template, the upstream homology arm that PCR amplification obtains gene gumF is carried out The downstream homology arm segment of segment and gene gumG；The nucleotide sequence such as SEQ of the upstream homology arm segment of the gene gumF Shown in ID No:1；The nucleotide sequence of the downstream homology arm segment of the gene gumG is as shown in SEQ ID No:2；

1.2) the downstream homology arm of the upstream homology arm segment for the gene gumF for obtaining step 1) and gene gumG It after segment is recombinated by overlap PCR, is connected in suicide plasmid pLO3, obtains seamless knockout plasmid plO3-FG；

1.3) plO3-FG of seamless knockout plasmid described in step 2) is transferred to progress gumF and gumG base in starting strain The knockout of cause obtains the strain X C (△ FG) of gumF and gumG gene delection.

Preferably, described in step 2) overexpression transketolases gene gumL the following steps are included:

2.1) transketolases gene gumL is connect with carrier pBBR1mcs-2 and obtains multicopy expression plasmid pBBRL；

2.2) pBBRL of multicopy expression plasmid described in step 2.1) is transferred to gumF the and gumG gene delection Strain X C (△ FG) in obtain genetic engineering bacterium XC (△ FG::pBBRL).

Preferably, it is transferred to described in step 1.3) as engagement transfer, the starting strain and seamless knockout plasmid The quantity ratio of plO3-FG is 1:(1.5~2.5)；It is described engagement transfer temperature be 28~32 DEG C, it is described engagement transfer when Between be 8~12h.

The present invention provides a kind of methods using genetic engineering bacterium production xanthan gum, comprising the following steps:

S1) genetic engineering bacterium is inoculated into seed culture medium and carries out activation acquisition activation bacterium solution；

S2) the activation bacterium solution is inoculated into fermentation medium and carries out fermented and cultured acquisition fermentation liquid；The fermentation training Feeding temperature is 28~32 DEG C, and the time of the fermented and cultured is 60~84h；

S3) fermentation liquid described in alcohol precipitation, collection solid phase components are xanthan gum.

Preferably, step S1) described in activate bacterium solution bacteria concentration OD₆₀₀It is 0.4~0.6.

Preferably, step S2) described in activation bacterium solution inoculum concentration be 8~12% (V/V).

Preferably, the culture medium of the fermented and cultured is in terms of 1L, including following components: cornstarch, 40~60g；Fish egg White peptone, 3.5~4.5g；The water of calcium carbonate, 3.5~4.5g and surplus.

Beneficial effects of the present invention: the genetic engineering bacterium of production gel state xanthan gum of the present invention, with Xanthomonas campestris NK-01 is starting strain, has knocked out I base of acetyltransferase in the starting strain Because of II gene gumG of gumF and acetyltransferase, and transketolases gene gumL has been overexpressed it；The genetic engineering bacterium is raw The xanthan gum of production has higher acetone acid content, close to saturation；It is Weak Gels under low consistency conditions, with concentration Increase, zero-shear viscosity and storage modulus gradually increase, and after reaching 2% concentration, stronger gel state is presented in xanthan gum, With certain self-maintaining.Gel is presented in aqueous solution or salting liquid in the xanthan gum of the genetic engineering bacterium production State, gel strength increase with the increase of xanthan gum concentration and ion concentration；Have than spontaneous xanthan gum more excellent Thickening and gel characteristic more, and do not limited by fermentation condition, it can stability and high efficiency production.Therefore, gene work of the present invention Journey bacterial strain has extensive prospects for commercial application.

Detailed description of the invention

Fig. 1 is the knockout flow chart of gumF and gumG gene in the embodiment of the present invention 1；

Fig. 2 is the PCR proof diagram of deletion mycopremna, and wherein M is DNA Marker DL2000, and swimming lane W is starting strain PCR is as a result, swimming lane Δ is the PCR result of the strain X C (△ FG) of gumF and gumG gene delection；

Fig. 3 is the viscosity of the different xanthan gum products of embodiment 3 with shearing force change curve；

Fig. 4 is the viscosity (a) and modulus (b) change curve of the xanthan gum XC-L of various concentration in embodiment 3；

Fig. 5 is viscosity (a) of the xanthan gum XC-L under different NaCl concentrations and modulus (b) change curve；

The gel state that Fig. 6 is xanthan gum XC-L is observed, and a refers to that the gel state of the XC-L of various concentration, b refer to 1.0% XC-L In various concentration Na⁺In gel state；Photo is shot after being inverted 12 hours；

Fig. 7 is xanthan gum XC-L (a) and microstructure and height line chart of the XC-5 (b) in NaCl solution, 1 He in a figure 2 represent more coiled strands, and 1-3 represents double helix chain in b figure；

Fig. 8 is the plasmid map of plasmid pLO3.

Biological deposits explanation

Xanthomonas campestris Xanthomonas campestris NK-01 is preserved in China General Microbiological strain guarantor Hiding center, preservation address are Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica；Preservation day Phase is on 01 03rd, 2018, and deposit number is CGMCC No.15155.

Specific embodiment

The present invention provides one plant produce gel state xanthan gum genetic engineering bacterium, the genetic engineering bacterium with Xanthomonas campestris NK-01 is starting strain, and I gene of acetyltransferase is knocked out in the starting strain II gene gumG of gumF and acetyltransferase, and it is overexpressed transketolases gene gumL；The Xanthomonas The culture presevation number of campestris NK-01 is CGMCC No.15155.

In the present invention, preferably through the downstream for upstream the homology arm sequence and gene gumG that will be connected with gene gumF The seamless knockout plasmid of homology arm sequence is transferred in starting strain Xanthomonas campestris NK-01 and carries out gene The knockout of gumF and gene gumG.In the present invention, the overexpression transketolases gene gumL is preferably by described The multicopy expression plasmid pBBRL comprising transketolases gene gumL is transferred in starting strain to realize.

Heretofore described genetic engineering bacterium has knocked out I gene gumF of acetyltransferase and II base of acetyltransferase Because of gumG；And transketolases gene gumL has been overexpressed it；So that process of the genetic engineering bacterium in synthesis xanthan gum In, the content of acetyl group in xanthan gum is reduced, the content of pyruvic acid in xanthan gum is improved；So that the genetic engineering bacterium It is able to produce gel state xanthan gum.The xanthan gum of genetic engineering bacterium synthesis of the present invention has the acetone acid content of saturation, low Weak Gels can be formed under concentration, product quality and rheological properties are stablized, and the orientation for being conducive to xanthan gum product stablizes life It produces.

The present invention also provides the preparation methods of genetic engineering bacterium, comprising the following steps: 1) knocks out in the starting strain GumF gene and gumG gene, obtain gumF gene and gumG gene delection strain X C (△ FG)；2) institute in step 1) The gumF gene stated and the middle transketolases gene gumL that is overexpressed of the strain X C (△ FG) of gumG gene delection obtain gene work Journey bacterium.

In the present invention, the gumF gene in the starting strain and gumG gene are preferred the following steps are included: 1.1) Using the genome of the starting strain as template, upstream homology arm segment and gene that PCR amplification obtains gene gumF are carried out The downstream homology arm segment of gumG；The nucleotide sequence of the upstream homology arm segment of the gene gumF such as SEQ ID No:1 institute Show；The nucleotide sequence of the downstream homology arm segment of the gene gumG is as shown in SEQ ID No:2；1.2) step 1) is obtained The upstream homology arm segment of the gene gumF obtained and the downstream homology arm segment of gene gumG pass through overlap PCR weight It after group, is connected in suicide plasmid pLO3, obtains seamless knockout plasmid plO3-FG；1.3) by seamless knockout described in step 2) Plasmid plO3-FG is transferred to the bacterium that the knockout acquisition gumF and gumG gene delection of gumF and gumG gene is carried out in starting strain Strain XC (△ FG).

For the present invention using the genome of the starting strain as template, the upstream for carrying out PCR amplification acquisition gene gumF is homologous The downstream homology arm segment of arm pieces section and gene gumG.The present invention does not have the extracting method of the genome of the starting strain Particular determination, using the Extraction Methods of Genome of this field routine, in specific implementation process of the present invention, using gene Group reagent box extracts.In the present invention, expand the upstream homology arm segment of the gene gumF primer be preferably fgsu and fgsl；The nucleotide sequence of the fgsu is as shown in SEQ ID No:3；The nucleotide sequence of the fgsl such as SEQ ID No:4 It is shown；The primer for expanding the upstream homology arm segment of the gene gumG is preferably fgxu and fgxl；The nucleosides of the fgxu Acid sequence is as shown in SEQ ID No:5；The nucleotide sequence of the fgxl is as shown in SEQ ID No:6.Heretofore described PCR Amplification enzyme used is preferably PrimeSTAR archaeal dna polymerase；In the present invention, the system of the PCR amplification is excellent in terms of 25 μ l Choosing includes following components:

The genomic DNA of starting strain, 1 μ l；

Primer fgsu and fgsl (or primer fgxu and fgxl), each 0.4 μ l；

PrimeSTAR archaeal dna polymerase, 12.5 μ l；

The water of surplus.

The program of the PCR amplification preferably includes: 98 DEG C, 15s；55 DEG C, 15s；72 DEG C, 40s；30 circulations；16 DEG C of guarantors It holds.

Downstream homology arm segment of the present invention in the upstream homology arm segment and gene gumG for obtaining the gene gumF Afterwards, above-mentioned segment is recombinated by overlap PCR, then the segment of recombination is connected in suicide plasmid pLO3, obtained seamless Knock out plasmid plO3-FG.

In the present invention, the overlap PCR amplification system is in terms of 25 μ l, including following components:

Upstream and downstream amplified fragments, each 0.5 μ l；

Primer fgsu, fgxl, each 0.3 μ l；

PrimeSTAR archaeal dna polymerase, 12.5 μ l；

The water of surplus.

After the present invention obtains the segment of recombination, preferably by the segment of the recombination and suicide plasmid with identical enzyme into Row digestion, then connection obtains seamless knockout plasmid plO3-FG.In the present invention, the digestion is preferably double digestion；It is described double Digestion is preferably restriction enzyme Pac I and Xba I with enzyme；The temperature of the double digestion is preferably 35~39 DEG C, more preferably 37 DEG C, the time of the double digestion is preferably 1.5~2.5h, more preferably 2h.

The digestion system is in terms of 10 μ l, including following components:

Recombinant fragment or plasmid, 7 μ l；

Restriction enzyme Pac I and Xba I, each 1 μ l；

Digestion buffer, 1 μ l.

In the present invention, after the digestion, the recombinant fragment after preferred collection digestion and the plasmid after digestion are carried out Connection；The temperature of the connection is preferably 16 DEG C, and the time of the connection is preferably 10~14h.The present invention is in the connection Afterwards, preferably the connection product is transferred to and obtains the recombination for having seamless knockout plasmid plO3-FG in E. coli competent Bacterium；It is currently preferred to be verified using bacterium colony PCR, screen correct transformant.The tool that the present invention verifies the bacterium colony PCR Gymnastics is not particularly limited the bacterium colony PCR verification method using this field routine；In specific implementation process of the present invention In, the primer of the bacterium colony PCR verifying is fg1 and fg2；The nucleotide sequence of the fg1 is as shown in SEQ ID No:7；Institute The nucleotide sequence of fg2 is stated as shown in SEQ ID No:8.In the present invention, plasmid map such as Fig. 8 of the suicide plasmid pLO3 It is shown.

The seamless knockout plasmid plO3-FG is transferred to by the present invention after obtaining the seamless knockout plasmid plO3-FG The knockout that gumF and gumG gene is carried out in starting strain obtains the strain X C (△ FG) of gumF and gumG gene delection.At this In invention, the preferably engagement that is transferred to is shifted, and the quantity of the starting strain and seamless knockout plasmid plO3-FG are than excellent It is selected as 1:(1.5~2.5), more preferably 1:2；The temperature of the engagement transfer is preferably 28~32 DEG C, and more preferably 30 DEG C； The time of the engagement transfer is preferably 8~12h, more preferably 9~11h.In the present invention, seamless knockout will be preferably had The recombinant bacterium and starting strain of plasmid plO3-FG carries out engagement transfer.

In specific implementation process of the present invention, preferably by the starting strain and with seamless knockout plasmid plO3-FG Recombinant bacterium be respectively placed in and cultivated in the fluid nutrient medium of resistance containing Cm and the resistance containing tet after, collect thallus；It will be described After the cell washing being collected into is resuspended, mixing obtains Mixed Microbes, and the Mixed Microbes are placed in non-resistant solid medium and are carried out Engagement transfer.In the present invention, the solution of the washing resuspension is preferably MgSO₄Solution, the MgSO₄The concentration of solution is preferred For 8~12mmol/L.In the present invention, filter membrane is preferably contained in the non-resistant solid medium, the filter membrane is conducive to bacterium The separation of body.The present invention, preferably from washing thalline on the filter membrane, then will wash after engagement transfer from filter membrane It washs and is screened and verified on dual anti-culture medium of the thallus coating containing Cm and tet to get off.It is currently preferred to use bacterium PCR verifying is fallen, correct transformant is screened.The present invention is not particularly limited use to the bacterium colony PCR concrete operations verified The bacterium colony PCR verification method of this field routine；In specific implementation process of the present invention, the primer of the bacterium colony PCR verifying For fg1 and fg2.

In the present invention, ketone group is overexpressed in the strain X C (△ FG) of the gumF gene and gumG gene delection and is turned It moves enzyme gene gumL and obtains genetic engineering bacterium.The overexpression transketolases gene gumL it is preferred the following steps are included: 2.1) transketolases gene gumL is connect with carrier pBBR1mcs-2 and obtains multicopy expression plasmid pBBRL；It 2.2) will step It is rapid 2.1) described in multicopy expression plasmid pBBRL be transferred in the strain X C (△ FG) of the gumF and gumG gene delection It obtains genetic engineering bacterium XC (△ FG::pBBRL).

In the present invention, PCR amplification is preferably carried out as template using the genome of starting strain and obtains the transketolases Gene gumL.The present invention is not particularly limited the extracting method of the genome of the starting strain, conventional using this field Extraction Methods of Genome is extracted in specific implementation process of the present invention using genomic kit.In the present invention, amplification The primer of the gene gumL is preferably l-br1 and l-br2；The nucleotide sequence of the l-br1 is as shown in SEQ ID No:9； The nucleotide sequence of the l-br2 is as shown in SEQ ID No:10.In the present invention, the system of the PCR amplification in terms of 25 μ l, Preferably include following components:

The genomic DNA of starting strain, 1 μ l；

Primer l-br1 and l-br2, each 0.4 μ l；

PrimeSTAR archaeal dna polymerase, 12.5 μ l；

The water of surplus.

The present invention is after obtaining the transketolases gene gumL, by transketolases gene gumL and carrier PBBR1mcs-2 connection obtains multicopy expression plasmid pBBRL.In the present invention, the digestion is preferably double digestion；Double enzymes Cutting with enzyme is preferably restriction enzyme Kpn I and Hind III；The temperature of the double digestion is preferably 35~39 DEG C, more preferably 37 DEG C, the time of the double digestion is preferably 1.5~2.5h, more preferably 2h.The digestion system in terms of 10 μ l, including with Lower component:

Recombinant fragment or plasmid, 7 μ l；

Restriction enzyme Kpn I and Hind III, each 1 μ l；

Digestion buffer, 1 μ l.

In the present invention, after the digestion, the gene gumL after preferred collection digestion and the plasmid after digestion are carried out Connection；The temperature of the connection is preferably 16 DEG C, and the time of the connection is preferably 10~14h.The present invention is in the connection Afterwards, preferably the connection product is transferred to and obtains the recombination for having multicopy expression plasmid pBBRL in E. coli competent Bacterium；It is currently preferred to be verified using bacterium colony PCR, screen correct transformant.

The multicopy expression plasmid pBBRL is transferred to by the present invention after obtaining the multicopy expression plasmid pBBRL Genetic engineering bacterium XC (△ FG::pBBRL) is obtained into the strain X C (△ FG) of the gumF and gumG gene delection.The present invention Described in be transferred to consistent with the above-mentioned operation that seamless knockout plasmid plO3-FG is transferred in starting strain, details are not described herein.

The present invention provides a kind of methods using genetic engineering bacterium production xanthan gum, comprising the following steps: S1) The genetic engineering bacterium is inoculated into seed culture medium and carries out activation acquisition activation bacterium solution；S2) the activation bacterium solution is inoculated with Fermented and cultured is carried out into fermentation medium obtains fermentation liquid；The temperature of the fermented and cultured is 28~32 DEG C, the fermentation training The feeding time is 60~84h；S3) fermentation liquid described in alcohol precipitation, collection solid phase components are xanthan gum.

In the present invention, the genetic engineering bacterium is inoculated into seed culture medium and carries out activation acquisition activation bacterium solution.? In the present invention, it more preferably includes activating in 2 generations that the activation, which preferably includes activating in 1~3 generation,.Heretofore described activation bacterium The bacteria concentration OD of liquid₆₀₀Preferably 0.4~0.6, more preferably 0.5.In the present invention, the seed culture medium is preferred in terms of 1L Including following components: sucrose, 15~25g；Peptone, 2.5~3.5g；Yeast powder, 0.8~1.2g；Beef extract, 4~6g；More Preferably comprise following components: sucrose, 20g；Peptone, 3g；Yeast powder, 1g；Beef extract, 5g；The seed culture medium PH value is preferably 6.98~7.02.

The activation bacterium solution is inoculated into fermentation medium after obtaining activation bacterium solution and carries out fermented and cultured by the present invention Obtain fermentation liquid.The inoculum concentration of the activation bacterium solution is preferably 8~12% (V/V), more preferably 10% (V/V).The present invention In, the culture medium of the fermented and cultured preferably includes following components: cornstarch, 40~60g in terms of 1L；Fish peptone, 3.5~4.5g；The water of calcium carbonate, 3.5~4.5g and surplus；It more preferably include following components: cornstarch, 50g；Fish egg White peptone, 4g；The water of calcium carbonate, 4g and surplus.The temperature of the fermented and cultured be 28~32 DEG C, preferably 30 DEG C, the hair The time of ferment culture is preferably 60~84h, more preferably 65~80h, most preferably 72h.In the present invention, the activation and training In feeding process, preferred with stirring, the revolving speed of the stirring is preferably 150~250rpm, more preferably 200rpm.

The present invention it is described after fermentation, fermentation liquid described in alcohol precipitation, collection solid phase components be xanthan gum.In the present invention, Alcohol used in the alcohol precipitation is preferably ethyl alcohol, and the volume ratio of the ethyl alcohol and fermentation liquid is preferably (2~3): 1；The alcohol precipitation mistake Preferred with stirring in journey, the present invention is not particularly limited the revolving speed of the stirring.The present invention terminates in the alcohol precipitation Afterwards, the preferred method using filtering collects solid phase components and obtains xanthan gum.The present invention is preferred after obtaining the xanthan gum Further including the steps that dry and crushes, the temperature of the drying is preferably 85~95 DEG C, and more preferably 90 DEG C；The drying Time is preferably 4~8h, more preferably 6h；The granularity of the crushing is preferably 60~100 mesh, more preferably 80 mesh.

Technical solution provided by the invention is described in detail below with reference to embodiment, but they cannot be managed Solution is limiting the scope of the present invention.

Primer sequence used in the embodiment of the present invention is shown in Table 1.

Table 1, primer sequence information table

Embodiment 1

The building of seamless knockout plasmid plO3-FG

Drawn using the genome that genomic kit extracts XC using it as template with fgsu/fgsl and fgxu/fgxl Object expands gumF upstream region of gene homology arm by PrimeSTAR archaeal dna polymerase (Takara Bio, Tokyo, Japan) respectively The downstream homology arm segment of segment and gumG gene；Overlap PCR recombination upstream and downstream segment, agarose gel electrophoresis separation, Plastic recovery kit recycles target fragment.With restriction enzyme Pac I and Xba I, 37 DEG C, 2h distinguishes digestion and recombinates purpose piece Section and plasmid pLO3 (deriving from Oliver doctor Lenz, plasmid map is as shown in Figure 8), overnight, conversion is extremely for 16 DEG C of connections In E.coli S17 competent cell；Bacterium colony PCR verifying is carried out to screen correct transformant using primer fg1/fg2, and is protected It is spare to deposit glycerol tube.

The PCR and amplification system of gene gumF and gene gumG are as follows:

25 μ l of PCR system: 1 μ l of template,

Each 0.4 μ l of primer,

12.5 μ l of enzyme,

Water polishing.

The overlap PCR amplification system is as follows:

Upstream and downstream amplified fragments, each 0.5 μ l；

Primer fgsu, fgxl, each 0.3 μ l；

PrimeSTAR archaeal dna polymerase, 12.5 μ l；

The water of surplus.

PCR and two gene overlap PCR amplification program of gene gumF and gene gumG is as follows:

98℃15s

55℃15s

72 DEG C of 40s 30 are recycled

16℃。

The knockout process of gene gumF and gumG are as shown in Figure 1.

The building of broad host range expression vector pBBRL

It is primer with l-br 1/l-br 2, passes through using it as template using the genome that genomic kit extracts XC PrimeSTAR archaeal dna polymerase (Takara Bio, Tokyo, Japan) expands gumL gene；With I He of restriction enzyme Kpn Hind III, 37 DEG C, 2h distinguishes digestion gumL and plasmid pBBR1mcs-2, and overnight, conversion to E.coli S17 is experienced for 16 DEG C of connections In state cell；Bacterium colony PCR verifying is carried out to screen correct transformant using primer l-br 1/l-br 2, and saves glycerol tube It is spare.

The PCR and amplification system of gene gumL is as follows:

25 μ l of PCR system: 1 μ l of template,

Each 0.4 μ l of primer,

12.5 μ l of enzyme,

Water polishing.

Amplification program:

98℃15s

55℃15s

72 DEG C of 40s 30 are recycled

16℃

Bacterium colony PCR program:

95℃45s

55℃45s

72 DEG C of 2min 30 are recycled

16℃。

The building of the engineering strain XC (△ FG::pBBRL) of gel state xanthan gum

It chooses XC single colonie and is seeded to the 5ml seed culture medium containing Cm, 200rpm cultivates 15h, picking in 30 DEG C of constant-temperature tables The single bacterium of E.coli s17/pLO3-FG drops down onto the LB liquid medium containing tet, and 200rpm is trained in 37 DEG C of constant-temperature tables 8h is supported, 6000rpm is centrifuged the thallus that 5min collects two plants of bacterium, the MgSO of 10 mmol/L₄Solution washing thalline twice, 200 μ l's MgSO₄Thallus is resuspended in solution, is placed on after XC and E.coli s17 (pLO3-FG) is mixed according to the ratio of 1:2 containing 0.22 μm On the non-resistant solid medium of filter membrane, 8~12h is cultivated under the conditions of 30 DEG C.With 200 μ l MgSO₄Solution washs filter membrane, ladder Coating contains Cm after degree dilution^rAnd tet^rDual anti-plate on, 30 DEG C, 48~72h culture.PLO3 can not be replicated in XC, when After pLO3-FG engagement is transferred to XC, genome can be entered by upstream homology arm or downstream homology arm recombination and integration.Dual anti- Picking single bacterium is dropped down onto containing in dual anti-seed culture medium on plate, 30 DEG C, 200rpm culture 36h obtain single-swap recon.With Afterwards, single-swap recon is seeded in the seed culture medium of non-resistant, 30 DEG C, 200rpm culture for 24 hours, passage twice, then It is applied on the plate containing 10% sucrose, cultivates 72h under the conditions of 30 DEG C.Picking single bacterium drops down onto 5ml test tube, and 30 DEG C, 200rpm is cultivated for 24 hours, is carried out bacterium colony PCR verifying using primer fg 1/fg 2, is determined correct deletion mycopremna, be named as XC(△FG).Using identical method, with XC (△ FG) for F-strain, with E.coli s17 (pBBRL) be F+strain into Row engagement transfer, building can produce the engineering strain of gel state xanthan gum, be named as XC (△ FG::pBBRL), ferment The xanthan gum of generation is named as XC-L.

Wherein non-resistant solid medium group is divided into (g/L):

Glucose: 15g, beef extract: 3g, peptone: 5g, yeast powder: 3g, agar: 15g, pH=7.

The program for the bacterium colony PCR being related in above-mentioned steps is as follows:

95℃45s

55℃45s

72 DEG C of 2min 30 are recycled

16℃。

The PCR verification result of deletion mycopremna enters shown in Fig. 2, and wherein M is DNAMarker DL2000, and swimming lane W is bacterium germination The PCR of strain is as a result, swimming lane Δ is the strain X C (△ FG) of gumF and gumG gene delection.

Embodiment 2

The engineering bacteria fermentation production gel state xanthan gum constructed in embodiment 1

It chooses genetic engineering bacterium single colonie and is inoculated into 5ml seed culture medium, 200rpm, 30 DEG C of culture 20h, then with 1% Inoculum concentration, which is inoculated into the seed culture medium of 100ml, makes the dense OD of its bacterium₆₀₀Reach 0.5, in 200rpm, 30 DEG C of culture 18h, then It is inoculated into the 500ml shaking flask of the culture medium containing 100ml with 10% inoculum concentration, 30 DEG C, cultivate 72h under the conditions of 200rpm.

Wherein, seed culture medium (g/L): sucrose, 20g；Peptone, 3g；Yeast powder, 1g；Beef extract, 5g；pH 7.0± 0.02；Fermentation medium (g/L): cornstarch, 50g；Fish peptone, 4g；Calcium carbonate, 4g.

The ethyl alcohol (v/v) of 2~3 times of volumes is added into fermentation liquid, stirring obtains xanthan gum sediment, and filtering will sink Shallow lake is put into 90 DEG C of baking oven dry 6h, and being crushed to partial size is 80 mesh, saves backup.

Embodiment 3

The performance study of the xanthan gum of genetic engineering bacterium production

The measurement of acetyl group and acetone acid content in xanthan gum

The content of acetyl group and pyruvic acid is measured by high pressure liquid chromatograph (HPLC, Aglient 1100, USA).It will Xanthan gum is dissolved in the xanthan gum solution that water is configured to 5mg/mL, and solution saves standby after 90 DEG C of heating stirrings 1h, ultrasonic 2min With.Pyruvic acid content assaying method is as follows: taking 1mL xanthan gum solution and the phosphoric acid solution of 1mL 0.1mol/L to mix, at 90 DEG C Constant temperature 90min adds water to supply 3mL, obtains acetone acidity test liquid；Acetyl content measuring method is as follows: taking 1mL xanthan peptization Liquid is mixed with the potassium hydroxide solution of 1mL 0.2mol/L, inflated with nitrogen, sealing, after 45 DEG C of constant temperature 6h, with 0.1 mol/L's of 1mL Phosphoric acid solution supplies 3mL, obtains acetyl based assays liquid.Isometric 0.7g/L is added in pyruvic acid and acetyl based assays liquid Propionic acid be internal standard, filtering, take 20 μ L sample introductions carry out HPLC analysis.Pyruvic acid used in pyruvic acid content standard curve determination is dense Degree are as follows: 15,30,45,75,100mg/mL；Acetic acid concentration used in acetyl content standard curve are as follows: 15,30,45,75, 150mg/mL.The measurement result of separate sources xanthan gum is shown in Table 2.

The acetyl group and acetone acid content of 2 separate sources xanthan gum product of table

Wherein a indicates the different fermentations condition of XC bacterial strain.The fermentation condition of XC-L, XC-2 and XC-3 are 28 DEG C, revolving speed 200rpm, ferment 72h；XC-4 fermentation condition is 33 DEG C, revolving speed 100rpm, and ferment 72h；XC-4 fermentation condition is 37 DEG C, revolving speed 100rpm, ferment 72h.

The rheological behavior of xanthan gum solution is assessed

Rheometer (the Discovery HR-2hybrid that the ftheoloqical measurements of xanthan gum utilize stress to adjust Rheometer, TA Instruments), the aluminium sheet (1000 μm of gap) of diameter 40mm is installed.Viscosimetric analysis parameter are as follows: turn Fast 0.01-101/s, 25 DEG C of temperature；Modulus measurements parameter are as follows: strain 5%, 25 DEG C of temperature, frequency 0.1-100rad/s.Energy storage The gel strength of the general representative sample of modulus.Xanthan gum solution compound concentration is 0.1-2.0%, v/v；1.0% xanthan peptization The sodium chloride concentration contained in salting liquid is 1-5000mM.

Measurement result is shown in Table 3 and table 4.

The rheological properties of the different xanthan gum product of table 3 (0.3%)

The rheological properties of 4 engineering strain XC of table (△ FG::pBBRL) generation xanthan gum XC-L

Wherein a refers to that 1% xanthan gum is dissolved in the NaCl solution containing various concentration.Specific concentration is 1,10,100, 1000, the NaCl solution of 3000,5000mM.

The viscosity of different xanthan gum products is as shown in Figure 3 with shearing force change curve.

The viscosity (a) and modulus (b) change curve of the xanthan gum XC-L of various concentration is as shown in Figure 4；

Viscosity (a) of the xanthan gum XC-L under different NaCl concentrations and modulus (b) change curve are as shown in Figure 5.

The three-dimensional structure of xanthan gum is observed

Xanthan gum is dissolved in pure water or 5000mM sodium chloride solution, its concentration 1.0mg/mL is made, is dissolved overnight. It is diluted to 10 μ g/mL of final concentration with identical solvent, 2 μ L xanthan gum solutions is taken to be placed in fresh mica surface layer, drying at room temperature. It is observed with atomic force microscope (AFM, Burker ICON Dimension), the analysis of 1.8 software of Nanoscope Analysis The height of xanthan molecules, height represent the crosslinking degree of xanthan molecules, and the three-dimensional knot of xanthan molecules is judged with this Structure.Height is higher, and corsslinking molecular chain is more；Height is lower, and corsslinking molecular chain is fewer.The results are shown in Table 5.

5 xanthan gum product XC-L of table is compared with the strand height of XC-5

The gel state of xanthan gum XC-L is observed as shown in fig. 6, a refers to that the gel state of the XC-L of various concentration, b refer to 1.0% XC-L is in various concentration Na⁺In gel state；Photo is shot after being inverted 12h.

Xanthan gum XC-L (a) and microstructure and height line chart of the XC-5 (b) in NaCl solution are as shown in fig. 7, wherein 1 and 2 represent more coiled strands in a figure, and 1-3 represents double helix chain in b figure.

As shown in Table 2, have higher third by the xanthan gum XC-L that engineering strain XC (Δ FG::pBBRL) is generated Ketone acid content, close to saturation；And the acetone acid content for the xanthan gum product that wild type XC is generated under the conditions of different fermentations is not It is same and lower.Table 3 shows that xanthan gum XC-L has higher zero-shear viscosity and storage modulus, and storage modulus is greater than loss Modulus, it follows that XC-L is Weak Gels under low consistency conditions.The display of table 4 engineering strain XC (△ FG:: PBBRL after) generation xanthan gum XC-L is dissolved in water, with the increase of xanthan gum concentration, zero-shear viscosity and storage modulus gradually increase Add, after reaching 2% concentration, stronger gel state is presented in xanthan gum, has centainly self-maintaining.Similarly, in NaCl solution In, with Na⁺The increase of ion concentration, zero-shear viscosity and storage modulus gradually increase, and work as Na⁺When concentration reaches 5000mM, Zero-shear viscosity and storage modulus steeply rise, and illustrate that XC-L has very high gel strength in high level salt solution.In order to explain Two kinds of xanthan gum products have been carried out atomic force microscope by XC-L and the difference of other xanthan gum and the gel mechanism of XC-L Observation, as a result, it has been found that: in aqueous solution, there is single-stranded and double-stranded helical in XC-L and XC-5, but the height of XC-L is a little higher than XC-5 illustrates that XC-L may have weaker intramolecule interaction force and stronger intermolecular interaction；It is molten in salt In liquid, XC-L shows as multiply spiral, and XC-5 exists in the form of double helix chain, illustrates that XC-L is intermolecular in salting liquid Active force is further strengthened, and then gel state is presented.

In conclusion engineering strain XC (△ FG::pBBRL) generates xanthan gum XC-L in aqueous solution or salting liquid In gel state is presented, gel strength increases with the increase of xanthan gum concentration and ion concentration.Than spontaneous xanthan Glue has more superior thickening and gel characteristic, and is not limited by fermentation condition, can stability and high efficiency production.Therefore, the base Because engineered strain has extensive prospects for commercial application.

The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications It should be regarded as protection scope of the present invention.

Sequence table

<110>Nankai University

<120>one plants of engineering strains for producing gel state xanthan gum and its construction method and application

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 969

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 1

atggcgatct ggtcaacccg aagagcttct tcgtcaatgc gcgcggcatg agtgcagaag 60

ggttctggaa cgaggacagc aatctgttcg tcagtgccac acgacccggt gagcgcaact 120

tcctcccagg ctcgaacctg ccacgcgcct cttcctggtt catcgagccg gtgacgatgg 180

gcaattacat ctgcttcttc accgcgatcg tattgacgtt ctggcgctgg atgcggccgt 240

cgatgctgat tctgtctatt ggattgatcg gcttcatgat tgtggcatcc gacggccgac 300

tggctgccgg cacctgtgtg ctgatggtgc tgctgtcgcc gttattgaaa cggatggatc 360

agcggttggc gttcctgttg ttcctgtttg tgatcgcctc tgcctggctg ctggtgtgga 420

tgaccgggat tacggcctac caggacacca cgatggggcg catcttcttc actgtgaatt 480

cgatgaacaa tctatcgttc gagtcgtgga tgggcctgga ttttgcgcag gcctaccggt 540

atttcgacag cggtatttct tactttattg cttcgcagtc gattgtcggc gtgctggcgt 600

tcctgctgtc ttattcgttc ctgctgctga tgccgagcaa ggaagggcag ttgttcaaaa 660

accaggcgat gtttgccttt gcactgagcc tgttggtgtc taacggctat ttctcgatca 720

agacatcggc gctgtggtgg tttgtctgcg gctgcatgtg gcacctgatg ccagcagcgt 780

cagccgtgcc ggtgcgcgac gaaagcaagg aagatccaac ggacaacggc gtgcatgtgc 840

cgttgcccgc aggagcgccg cggtgaatac ggtgacaggg gcatcgggga cgtcggcgcc 900

tgtgcaggct gccggcgcgc gtgccttcgc gagcggccgt agccgcgatc cacgtatcga 960

tgcgaccaa 969

<210> 2

<211> 977

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 2

ggtgatcgat cgcaaactca tcggtacacc tgtctgggcg ctggctctct gcgcctttgc 60

catcgctgcc tgcattccca tgcgtgccgt gctggtgcgc cgcgccctgg atgttgggat 120

tgaaacgcaa gtgagacatt ttcagaatca tcagtcgatg tggcgtgttc gtgtgagtca 180

ccggcaaagg agatcggcgc aatgaaagtc gtgcatgtgg tccgccagtt ccatccgtcg 240

atcgggggga tggaggaagt cgtgctgaac gtggcacgtc agcatcaggc caacagtgcc 300

gacacggttg agatcgtgac gttggatcgt gtgttcaccg atccctctgc gcaactggcg 360

cagcacgagc tccatcaggg gttgtcgatc actcgcatcg gctatcgtgg ttcatcgcgg 420

tacccgatcg cgccgtcggt gctgggggcg atccgttcgg cggacgtggt gcatctgcat 480

ggcattgatt ttttctacga ctacctggcg ttgaccaagc cgctgcacgg caagccgatg 540

gtggtctcga cgcatggcgg gtttttccac actgcctatg cgtcgcgcat gaagcagatc 600

tggttccaga cgctgacgcg tacttctgcg ctggcctatg cgcgtgtgat cgccactagc 660

gagaatgacg gcgatctgtt cgccaaggtg gtcgcgccgt cgcgcttgcg ggtgatcgag 720

aacggtgtcg acgtggagaa gtatgcaggg cagggcgctc gagcgccggg acggaccatg 780

ctgtatttcg ggcgttggtc ggtcaacaag ggcctgatcg aaacgcttga attgctgcag 840

gctgcgctca cgcgtgatcc gcagtggcgg ttgatcatcg ccgggcgcga gtacgatttg 900

aatgaggcgg atctgcgcaa ggccatcgcc gaacgcggtt tgcaggacaa ggtgcagctg 960

agcatgtcgc catcgca 977

<210> 3

<211> 34

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 3

ggagttgtta attaaatggc gatctggtca accc 34

<210> 4

<211> 38

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 4

gagtttgcga tcgatcacct tggtcgcatc gatacgtg 38

<210> 5

<211> 38

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 5

cacgtatcga tgcgaccaag gtgatcgatc gcaaactc 38

<210> 6

<211> 31

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 6

gacatagtct agatgcgatg gcgacatgct c 31

<210> 7

<211> 21

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 7

cggtatttcg acagcggtat t 21

<210> 8

<211> 20

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 8

tctcctttgc cggtgactca 20

<210> 9

<211> 33

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 9

gaaatgaggt acccgtttga aggaggatcc ctg 33

<210> 10

<211> 32

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 10

gaacattaag cttgaaggct tcctgcgtgg tc 32

Claims

1. the genetic engineering bacterium of one plant of production gel state xanthan gum, which is characterized in that the genetic engineering bacterium is with Xanthomonas Campestris NK-01 is starting strain, and I gene gumF of acetyltransferase and acetyl group are knocked out in the starting strain II gene gumG of transferase, and it is overexpressed transketolases gene gumL；

2. genetic engineering bacterium according to claim 1, which is characterized in that the overexpression transketolases gene gumL is It is realized by being transferred to the multicopy expression plasmid pBBRL comprising transketolases gene gumL into the starting strain.

3. the preparation method of genetic engineering bacterium of any of claims 1 or 2, comprising the following steps:

1) the gumF gene and gumG gene in the starting strain are knocked out, the bacterial strain of gumF gene and gumG gene delection is obtained XC(△FG)；

2) transketolases is overexpressed in the gumF gene described in step 1) and the strain X C (△ FG) of gumG gene delection Gene gumL obtains genetic engineering bacterium.

4. preparation method according to claim 3, which is characterized in that knock out the gumF in the starting strain in step 1) Gene and gumG gene the following steps are included:

1.1) using the genome of the starting strain as template, the upstream homology arm segment that PCR amplification obtains gene gumF is carried out With the downstream homology arm segment of gene gumG；The nucleotide sequence such as SEQ ID of the upstream homology arm segment of the gene gumF Shown in No:1；The nucleotide sequence of the downstream homology arm segment of the gene gumG is as shown in SEQ ID No:2；

1.2) the downstream homology arm segment of the upstream homology arm segment for the gene gumF for obtaining step 1) and gene gumG It after being recombinated by overlap PCR, is connected in suicide plasmid pLO3, obtains seamless knockout plasmid plO3-FG；

1.3) plO3-FG of seamless knockout plasmid described in step 2) is transferred in starting strain and carries out striking for gumF and gumG gene Except the strain X C (△ FG) for obtaining gumF and gumG gene delection.

5. preparation method according to claim 3 or 4, which is characterized in that be overexpressed transketolases described in step 2) Gene gumL the following steps are included:

2.2) pBBRL of multicopy expression plasmid described in step 2.1) is transferred to the bacterial strain of the gumF and gumG gene delection Genetic engineering bacterium XC (△ FG::pBBRL) is obtained in XC (△ FG).

6. preparation method according to claim 5, which is characterized in that it is transferred to described in step 1.3) and is shifted for engagement, The quantity ratio of the starting strain and seamless knockout plasmid plO3-FG are 1:(1.5~2.5)；It is described engagement transfer temperature be 28~32 DEG C, the time of the engagement transfer is 8~12h.

7. a kind of method using the as claimed in claim 1 or 22 genetic engineering bacterium production xanthan gum, comprising the following steps:

S2) the activation bacterium solution is inoculated into fermentation medium and carries out fermented and cultured acquisition fermentation liquid；The fermented and cultured Temperature is 28~32 DEG C, and the time of the fermented and cultured is 60~84h；

8. the method according to the description of claim 7 is characterized in that step S1) described in activate the bacteria concentration OD of bacterium solution₆₀₀For 0.4~0.6.

9. according to the method described in claim 8, it is characterized in that, step S2) described in activation bacterium solution inoculum concentration be 8~ 12% (V/V).

10. the method according to the description of claim 7 is characterized in that the culture medium of the fermented and cultured is in terms of 1L, including following Component: cornstarch, 40~60g；Fish peptone, 3.5~4.5g；The water of calcium carbonate, 3.5~4.5g and surplus.