CN105002204B - One plant height produces the Gluconobater oxydans genetic engineering strain and its preparation method and application of 5-KGA - Google Patents

Abstract

The invention discloses a high-yielding 5-KGA gluconobacter oxidase genetically engineered bacterium and its preparation method and application. (1) The upstream and downstream fragments of the GA2DH gene and the PDC gene are respectively introduced into a recombination-integrated suicide plasmid to obtain a suicide plasmid respectively. Knock out the plasmids p△GOX1231 and p△GOX1081; (2) Transfer p△GOX1231 into the host bacteria, screen for antibiotic resistance, culture without resistance and sucrose in sequence, and then obtain the positive mutation of GA2DH gene knockout by colony PCR G.oxydans ZJU1; in the same way, transfer p△GOX1081 into G.oxydans ZJU1 to obtain the PDC gene knockout positive mutant G.oxydans ZJU2; (3) transfer sld AB, pqq ABCDE, tld D, P ₀₁₆₉ and cell The pigment bo ₃ oxidase cyo BACD gene is transferred into the Gluconobacter oxydans genetically engineered bacterium G.oxydans ZJU2 obtained in step (2). GA2DH is the main enzyme that oxidizes gluconic acid to form 2-KGA, while PDC is related to catalyzing the decarboxylation of pyruvate to form acetaldehyde. The knockout of GA2DH and PDC can solve the metabolic shunt during the formation of 5-KGA, reduce by-products, and realize High yield of 5‑KGA.

Description

A high-yield 5-KGA genetically engineered bacterium of Gluconobacter oxydans and its preparation method law and application

technical field

The present invention relates to a kind of Gluconobacter genetically engineered bacterium and application thereof, particularly overexpression of membrane-bound sorbitol dehydrogenase, coenzyme PQQ and respiratory chain terminal electron acceptor, increasing 5-keto-D-gluconic acid (5 -keto-D-gluconic acid, 5-KGA) engineering bacteria and its application, belonging to the technical field of genetic engineering and fermentation engineering.

Background technique

5-keto-D-gluconic acid (5-KGA) is an important intermediate compound. In the late 1940s, Gray synthesized Vc through 5-KGA and applied for patents US2421611 and US2421612. Later, there were patent reports on the synthesis of xylaric acid by 5-KGA, such as US5731467, and the synthesis of aromatic substances 4-hydroxy-5-methyl-2,3-dihydroxyfuranone, such as US4464409. More importantly, 5-KGA can prepare L-(+)-tartaric acid by chemical catalysis (Journal of American Chemical Society, 1933, 55, 3563, US2380196, WO9615095A1, GB2388368). Gluconobacter oxydans (Gluconobacter oxydans) 621H can tolerate high concentrations of glucose, and use glucose as a substrate to incompletely oxidize to gluconic acid (GA), and further oxidize to 2-keto-D-gluconic acid ( 2-keto-D-gluconic acid, 2-KGA) or 5-KGA.

The German Jülich Research Center carried out genetic modification on Gluconobacter oxydans DSM2343, such as the inactivation of the GA2DH gene (gluconate-2-dehydrogenase (gluconate-2-dehydrogenase) gene), the overexpression of the GA5DH gene, and the modification of the expression promoter, etc. The engineered strain G.oxydans MF1/pBBR1MCS5-P _tufB -ga5dh was constructed, and the final 5-KGA production reached 300mM, about 58.2g/L (Applied Microbiology and Biotechnology, 2006, 73(2), 443-451). Saichana of Yamaguchi University in Japan screened a heat-resistant Gluconobacter strain, and inserted a resistance gene to inactivate GA2DH. The final 5-KGA production was 190mM, about 36.86g/L (Appiled and Environmental Microbiology, 2009, 75(13), 4240- 4247). Li Boyi of Zhejiang University and others studied the fermentation process conditions of G.oxydans CGMCC 1.637. Under the conditions of constant pH 5.5 and dissolved oxygen control of 15%, glucose was added by flow, and the final 5-KGA output reached 75.5g/L, and the conversion rate exceeded 70%. (Acta Bioengineering Sinica, 2014, 30(9), 1486-1490); Tan Zhilei from Tianjin University of Science and Technology, etc. used 2-KGA-deficient Gluconobacter oxydans HGI-1 as the starting strain to study the effects of carbon and nitrogen sources on the synthesis of 5-KGA , in a 5L fermenter for batch fermentation scale-up test, the output of 5-KGA was 93.80g/L, and the average production rate was 1.56g/L·h.

The genome sequence of Gluconobacter oxydans 621H was published in 2005 (NC_006677), Nature Biotechnology, 23(2), 195-200. The two key enzymes involved in glucose metabolism to generate 5-KGA are membrane-bound glucose dehydrogenase (mGDH) and membrane-bound sorbitol dehydrogenase (SLDH). Using pyrroloquinoline quinone (PQQ) as a coenzyme, it transfers electrons to ubiquinone in the respiratory chain, and finally oxidizes through the terminal electron acceptor cytochrome bo ₃ oxidase (cytochrome bo ₃ oxidase) to transfer electrons from ubiquinone Transfer to molecular oxygen, and generate a proton gradient potential. In 2013, Meyer and Richhardt studied the role of coenzyme PQQ and cytochrome bo ₃ oxidase in the overexpression of membrane-bound proteins, and found that the amount of PQQ is an important factor affecting the oxidation of substrates by dehydrogenases, while cytochrome bo ₃ oxidizes Enzymes are rate-limiting factors for electron transport in the respiratory chain (Applied Microbiology and Biotechnology, 2013, 97, 3457-3466; Journal of Bacteriology, 2013, 195(18), 4210-4220). In 2013, Chen Jian and others applied for patent CN 103484417A, expressing SDH, SNDH and PQQ derived from K.vulgareWSH-001 in G.oxydans WSH-003, and producing 2-keto-L-gulonic acid (2-keto-L-gulonic acid (2 -keto-L-gulonicacid,2-KLG). In the same year, Tianjin Yuan Yingjin et al expressed sorbose dehydrogenase, sorbulose dehydrogenase and coenzyme PQQ in Ketogulonigenium vulgare at the same time, and produced 2-KLG from sorbose (Metabolic Engineering 2013, 19, 50-56).

Contents of the invention

Aiming at the problem of low yield of 5-KGA in the prior art, the present invention provides a high-yield 5-KGA-producing Gluconobacter oxydans genetically engineered bacterium and its preparation method and application,

A preparation method of a high-yield 5-KGA gluconobacter oxydans genetically engineered bacterium, comprising the steps of:

(1) Introduce the upstream and downstream fragments of the GA2DH gene and the PDC gene (pyruvate decarboxylase, gene) into the multiple cloning site of the recombination-integrated suicide plasmid pJKM, respectively, to obtain suicide knockout plasmids of the GA2DH gene The suicide knockout plasmid pΔGOX1081 of pΔGOX1231 and PDC gene; the recombinant integrated suicide plasmid has SacB gene, antibiotic marker and multiple cloning site;

(2) The obtained suicide knockout plasmid pΔGOX1231 was electrotransformed into the host bacteria, followed by antibiotic resistance screening, antibiotic-free culture and sucrose screening, and then the positive mutant of GA2DH gene knockout was obtained by colony PCR, named as G .oxydans ZJU1;

(3) The resulting suicide knockout plasmid pΔGOX1081 was electrotransformed into the passaged G.oxydans ZJU1, followed by antibiotic resistance selection, antibiotic-free culture and sucrose selection, and then the positive results of PDC gene knockout were obtained by colony PCR The mutant, that is, the genetically engineered bacterium of Gluconobacter oxydans, is named G.oxydans ZJU2;

(4) Transferring sldAB, pqqABCDE, tldD, P ₀₁₆₉ and cytochrome bo ₃ oxidase cyoBACD genes into the genetically engineered bacterium G. oxydans ZJU2 obtained in step (3).

In the present invention, the membrane-bound sorbitol dehydrogenase sldAB gene is overexpressed in the host, and coenzyme PQQ gene cluster and terminal electron acceptor cytochrome bo ₃ oxidase are fused and expressed. The genetically engineered bacteria G.oxydans ZJU2 was prepared based on the traceless gene system. By controlling pH and dissolved oxygen, the 5-KGA yield reached 102g/L and the average production rate was 1.7g/L·h under the condition of fed-batch fermentation. However, the yield of 5-KGA biosynthesis is relatively low and cannot meet the requirements of industrial applications, such as 5-KGA is used to catalyze the preparation of L-(+)-tartaric acid. In order to further increase the yield of 5-KGA, the present invention constructs a strain of overexpressing sldAB gene by means of genetic engineering on the basis of G.oxydans ZJU2, and at the same time fuses and expresses coenzyme PQQ and terminal electron acceptor cytochrome bo ₃ oxidase Bacteria, which can efficiently convert glucose into 5-KGA. The simultaneous transformation of coenzyme and respiratory chain metabolic engineering to produce 5-KGA in Gluconobacter oxydans has not been reported in China.

After G.oxydans ZJU1 is subcultured, the suicide plasmid will be automatically eliminated without affecting the next step of gene modification.

The base sequence of the SacB gene is shown in SEQ ID NO.1.

Preferably, the antibiotic marker is a karamycin resistance gene marker.

Preferably, the preparation method of the recombinant integrated suicide plasmid is as follows:

(1) Construction of the cloning plasmid pEASY-Blunt-SacB containing the SacB gene;

(2) Take the cloned plasmid pEASY-Blunt-SacB with the correct sequencing, clone the plasmid pEASY-Blunt-SacB and the suicide plasmid pK18mobGII by SspI single enzyme digestion, recover and connect, transform into E.coli DH5α, and screen to obtain the correct direction of SacB connection Positive clones were cultured to E. coli DH5α, and the integrated plasmid was extracted to obtain the recombinant integrated suicide plasmid pJKM.

The method for constructing the suicide plasmid pK18mobGII used in the present invention can be found in the literature New mobilizable vector suitable for gene replacement in Gram-negative bacteria and their use in mapping of the 3'end of the Xanthomonas campestris pv.Campestris gumoperon.Appl Environ Microbiol 1999,65:278 -282, by the constructor of the plasmid in the present invention (IELPI, L.Instituto de Investigaciones Bioquι′micas Fundacio′n Campomar, Facultad de Ciencias Exactas y Naturales, UBA, and CONICET, 1405 Buenos Aires, Argentina, Phone: 54 (1 ) 863-4011/19. Fax: 54(1) 865-2246. E-mail: LIELPI@iib.uba.ar.). Starting from the suicide plasmid pK18mobGII, a novel integrated suicide plasmid pJKM containing the SacB gene was constructed, which was applied to the gene traceless operation of Gluconobacter. The SacB gene encodes a fructan sucrase that can be secreted, which can hydrolyze sucrose to form high molecular weight fructan, so that Gram-negative bacteria appear lethal phenotypes on medium containing 5-10% sucrose, as Reverse screening marker, safe operation, non-toxic.

The method for constructing the recombinant plasmid pEASY-Blunt-SacB containing the SacB gene is as follows:

(1) using the base sequences shown in SEQ ID NO.2 and SEQ ID NO.3 as primers to amplify the SacB gene;

Upstream primer: 5'-AATATTcacatatacctgccgttcactat-3' (SEQ ID NO.2)

Downstream primer: 5'-AATATTccatcggcattttcttttgcg-3' (SEQ ID NO.3)

(2) Blunt-end seamless cloning was carried out with a blunt-end cloning kit (pEASY-Blunt Simple Cloning Kit, Quanshijin, Beijing) to obtain a recombinant plasmid pEASY-Blunt-SacB containing the SacB gene.

The host bacterium described in the step (2) of constructing the recombinant bacterium in the present invention is wild-type Gluconobacter oxydans G.oxydans DSM2343. The host bacterium does not need any operation, only the wild type can be used, the application range is wide, and the operation is safe.

Preferably, the sizes of the upstream and downstream fragments of the gluconate 2-dehydrogenase (gluconate 2-dehydrogenase, GA2DH) gene and the pyruvate dehydrogenase (pyruvate decarboxylase, PDC) gene are both 500-1200 bp. More preferably 800-1000bp. More preferably, the upper and lower gene fragments of the GA2DH gene are each 1000 bp; the upper and lower gene fragments of the PDC gene are each 800 bp.

According to the published genome sequence of Gluconobacter oxydans 621H (NC_006677), primers for the upstream and downstream homologous fragments of GA2DH and PDC genes were designed:

Primers for the upstream and downstream homologous fragments of the GA2DH gene:

1231_HindIII_F: 5'-ataAAGCTTagccaaaggcggaaagacggc-3' (SEQ ID NO.4)

1231_Fus_R: 5'-catttcaggggagaccgcttaaatgaagtggccgctggtcatc-3' (SEQ ID NO. 5);

1231_Fus_F: 5'-gatgaccagcggccacttcatttaagcggtctcccctgaaatg-3' (SEQ ID NO. 6)

1231_XbaI_R: 5'-ataTCTAGAcgccggcactttcttctacc-3' (SEQ ID NO.7);

The primer sequences for amplifying the upstream and downstream gene fragments of PDC are as follows:

1081_HindIII_F: 5'-cccAAGCTTctcgtctgggcgattcatg-3 (SEQ ID NO.8)

1081_Fus_R: 5'-Cctgaggtactgaaatcatgacaaagcgtctgatccttcc-3 (SEQ ID NO.9)

1081_Fus_F: 5'-Ggaaggatcagacgctttgtcatgatttcagtacctcagg-3 (SEQ ID NO.10)

1081_SalI_R: 5'-acgcGTCGACAggcatgagacctacctga-3 (SEQ ID NO. 11).

The target fragments were obtained by PCR, and then fused into the plasmid pJKM by fusion PCR to construct the corresponding suicide knockout plasmids pΔGOX1231 and pΔGOX1081.

Preferably, the antibiotic resistance screening is to spread the transformant on a seed medium plate of kanamycin 50 μg/m and cefoxitin 50 μg/m to obtain a genome-integrated transformant; the sucrose selection is antibiotic gene After the integrated transformant is cultured in a mannitol medium test tube, it is streaked on a seed medium plate containing 5-10% (preferably 10%) sucrose and cefoxitin to obtain a mutant strain freed from the suicide plasmid.

The anti-antibody culture is to culture the obtained recombinant at 30° C. overnight without anti-antibody.

In step (4), the method that sldAB, pqqABCDE, tldD, P ₀₁₆₉ and cytochrome bo ₃ oxidase cyoBACD gene is transferred to the gained Gluconobacter oxydans genetic engineering bacteria G.oxydans ZJU2 of step (3) preferably comprises the following steps:

(1) Obtain sldAB, pqqABCDE, tldD, P ₀₁₆₉ and cytochrome bo ₃ oxidase cyoBACD gene fragments by PCR;

(2) The sldAB gene and P ₀₁₆₉ were connected to the pBBR1MCS5 vector to construct the recombinant expression plasmid pBB5-P ₀₁₆₉ -sldAB; the pqqABCDE, tldD, cytochrome bo ₃ oxidase cyoBACD and the promoter P ₀₁₆₉ were connected to the vector pUCpr to construct the recombinant expression plasmid pUCpr -P ₀₁₆₉ -pqqABCDE-tldD -P ₀₁₆₉ -cyoBACD;

(3) Transfer the two recombinant expression plasmids into G.oxydans ZJU2 by electrotransformation.

Further preferably, according to the Gluconobacter oxydans 621H genome sequence (NC_006677) in GenBank, design primers:

SLDH_F: '-GC TCTAGA GGACTTTCAGTTCTGGAGGCTTTCACCA-3' (SEQ ID NO. 12)

SLDH_R:5'-CG GAATTC TCCCACCCGAAAAATGGAAAAAACG-3' (SEQ ID NO.13)

After the sldAB fragment of the SLDH gene was obtained by PCR, it was connected to the pEASY-Blunt cloning vector and sequenced. At the same time, the promoter P ₀₁₆₉ derived from the Gluconobacter oxydans GOX0169 gene was cloned into the pEASY-Blunt cloning vector and sequenced. After obtaining the correct transformant, double-enzyme Cut and connect to the broad host expression vector pBBR1MCS5, construct the expression plasmid pBB5-P ₀₁₆₉ -sldAB, transform the constructed recombinant expression plasmid into E.coli DH5α, and verify the positive transformants by colony PCR, that is, 103bp and 2771bp bands appear respectively;

Amplification primers for promoter P ₀₁₆₉ :

0169_SacI_F: 5'-ATA GAGCTC tgaaagcggctggcgcgt-3' (SEQ ID NO.14);

0169_XbaI_R: 5'-GC TCTAGA gcggaaggcgttataccctga-3' (SEQ ID NO. 15).

The PQQ gene clusters pqqABCDE, tldD, cytochrome bo ₃ oxidase cyoBACD and promoter P ₀₁₆₉ derived from the annotation of the G. oxydans DSM2343 genome were respectively designed to obtain the genes, and each fragment contained a correspondingly connected 15-20bp overlapping region, They were respectively connected to the pEASY-Blunt cloning vector and sequenced. After obtaining the correct fragments, they were seamlessly cloned using the full gold directional seamless cloning kit (Code: CU101) to construct the recombinant expression plasmid pUCpr-P ₀₁₆₉ -pqqABCDE-tldD- _P0169 -cyoBACD;

Amplification primers for fusion promoter P ₀₁₆₉ :

Add_0169_F: 5'-acactgtttaaacaccgtgaaagcggctggcgc-3' (SEQ ID NO.16)

pQQ_Fuse0169_R: 5'-acatccgcgcggaaggcgttatac-3' (SEQ ID NO.17);

Amplification primers fused to pqqABCDE:

pQQ_Fuse0169_F: 5'-ccttccgcgcggatgttcagg-3' (SEQ ID NO.18)

tldD_FusepQQ_R: 5'-ccggctagaagatggcctctc-3' (SEQ ID NO.19);

Amplification primers fused to tldD:

tldD_FusepQQ_F: 5'-gccatcttctagccggtctgttc-3' (SEQ ID NO. 20)

0169_FusetldD_R: 5'-ctttcaggatcttcttcatg-3' (SEQ ID NO.21);

Amplification primers fused to the second promoter P ₀₁₆₉ :

0169_FusetldD_F: 5'-tcgcgactgaaagcggctggc-3' (SEQ ID NO.22)

ADD_0169_R: 5'-cggtacccggggatcctgcggaaggcgttatac-3' (SEQ ID NO. 23);

Amplification primers for cyoBACD:

cyoBACD_XbaI_F: 5'-cgatTCTAGAactactgcaagccggaacgg-3' (SEQ ID NO. 24)

cyoBACD_SacI_R: 5'-actgGAGCTCaagggctggcaggatttctc-3' (SEQ ID NO. 25).

The two constructed recombinant expression plasmids pBB5-P ₀₁₆₉ -sldAB and pUCpr-P ₀₁₆₉ -pqqABCDE-tldD-P ₀₁₆₉ -cyoBACD were transformed into G.oxydans ZJU2 by two electrotransformation methods to construct recombinant engineering bacteria G.oxydansZJU2/ pBB5- _P0169 -sldAB/pUCpr- _P0169 -pqqABCDE-tldD- _P0169 -cyoBACD.

The present invention also provides a high-yielding 5-KGA-producing Gluconobacter oxydans genetically engineered bacterium prepared by the preparation method. The genetically engineered bacterium of the present invention can express sldAB, pqqABCDE-tldD and cyoBACD in cells, and be used for converting glucose to produce 5-KGA. The recombinant expression plasmids constructed in the present invention are all constitutive, and can be expressed synchronously during the growth of bacteria.

The present invention also provides an application of the above-mentioned high-yield 5-KGA-producing Gluconobacter oxydans genetically engineered bacterium in producing 5-KGA.

Preferably, the application includes the following steps:

The high-yield 5-KGA oxidative gluconobacteria genetically engineered bacteria were inoculated into a fermenter for fermentation and culture after being enlarged and cultivated, and the fermentation liquid was collected. Detect the production of 5-keto-D-gluconic acid in the fermentation broth.

The 5-keto-D-gluconic acid in the fermentation broth can be further separated and purified for the preparation of L-tartaric acid.

The composition of the fermentation medium is: corn steep liquor 7.5g/L; (NH ₄ ) ₂ SO ₄ 0.41g/L; (NH ₄ ) ₂ HPO ₄ 0.1g/L; MgSO ₄ ·7H ₂ O 0.07g/L ; CaCO ₃ 10-20g/L (add after separate sterilization); Glucose 100g/L, initial pH 5.0-5.5, 121°C, sterilization for 15min, final concentration of cefoxitin 50μg/mL.

The conditions of fermentation culture are: the conditions of fermentation culture are: 28～32°C, ventilation of 0.8～1.2vvm, tank pressure 0.02～0.04MPa, stirring to control dissolved oxygen concentration of 20%～40%, fermentation for 55～65h, fermentation Feed was controlled during the process according to the glucose concentration in the medium.

Still further preferably, the application includes the following steps:

Add 200 μL of the recombinant bacteria preserved in the glycerol tube to a 5 mL seed medium test tube, cultivate overnight at 30°C and 200 rpm, and transfer 50 mL of fresh seed medium to the logarithmic growth phase (about 10-14h) according to the inoculum size of 4% (V/V) , transfer 200mL fresh seed culture medium to the logarithmic growth phase according to 8% (V/V) inoculum amount; inoculate in 15L fermenter (working volume 9L) according to 8% (V/V) inoculum amount, 30 ℃, 1vvm Ventilation volume, tank pressure 0.03MPa, stirring speed to control dissolved oxygen, fermentation for 60h.

Seed medium (g/L): mannitol 25; Yeast extract 5; tryptone 3; agar 15 (solid medium); pH 5.5-6.0;

Glucose needs to be fed during the fermentation process, and the feeding conditions are: when the glucose concentration in the medium is lower than 10g/L, it is better when the glucose concentration is lower than 20g/L, and it is best to start when the glucose concentration is lower than 30g/L Supplement glucose, and control the concentration of residual sugar in the fermenter to not be lower than 30g/L, and the quantity of supplementation of glucose is not higher than 40% of the initial glucose amount.

Most preferably, the conditions of fermentation culture are: 30°C, 1vvm ventilation, tank pressure 0.03MPa, stirring to control the dissolved oxygen concentration to 20%-40%, according to the dissolved oxygen situation, the stirring speed is controlled at 200rpm-800rpm, and the fermentation is 60h During the fermentation process, the feeding is controlled according to the glucose concentration in the medium. When the glucose concentration is lower than 30g/L, glucose is added, and the residual sugar concentration in the fermenter is controlled to not be lower than 30g/L. The amount of glucose added is not 40% higher than the initial glucose amount.

Determination of the content of glucose, GA and 5-KGA in the present invention: after the fermentation broth sample is centrifuged at 12000 rpm, it is processed through a 0.22 μm filter membrane. GA and 5-KGA were determined by high performance liquid chromatography (HPLC). Agilent 1100 chromatograph, Showa Denko Shodex DE-613 chromatographic column, mobile phase: 2mM perchloric acid solution, flow rate: 0.5mL/min, injection volume: 10μL, 210nm ultraviolet detection. Glucose uses a biosensing analyzer SBA-40D.

The present invention knocks out gluconate 2-dehydrogenase (gluconate2-dehydrogenase, GA2DH) and pyruvate dehydrogenase (pyruvate decarboxylase, PDC) gene, a strain of recombinant genetically engineered bacteria G.oxydans ZJU2 is constructed, which solves the problem of GM microbial safety, and it can effectively convert glucose into 5-KGA; for further improving the output of 5-KGA, the present invention is based on G.oxydans ZJU2. On the basis of oxydans ZJU2, a strain of overexpressed sldAB gene was constructed by genetic engineering, and an engineering bacterium was fused to express coenzyme PQQ and terminal electron acceptor cytochrome bo ₃ oxidase at the same time, which can efficiently convert glucose into 5-KGA.

The beneficial effects of the present invention are as follows:

GA2DH is the main enzyme that oxidizes gluconic acid to form 2-keto-D-gluconic acid (2-KGA), while PDC is related to catalyzing the decarboxylation of pyruvate to form acetaldehyde. Therefore, GA2DH Knockout of PDC and PDC can solve the metabolic shunt during the formation of 5-KGA, reduce by-products, and achieve high yield of 5-KGA.

In the present invention, the GA2DH and PDC genes of G. oxydans DSM2343 are knocked out through gene traceless modification, and the recombinant bacterium G. oxydans ZJU2 is obtained. Further through the transformation of genetic engineering, the sldAB, pqqABCDE-tldD and cyoBACD genes were expressed in the prepared genetically engineered bacteria G.oxydans ZJU2, and the recombinant expression plasmids pBB5-P ₀₁₆₉ -sldAB and pUCpr-P ₀₁₆₉ -pqqABCDE-tldD-P ₀₁₆₉ were constructed - cyoBACD, whose main application is to increase the production of 5-KGA. In a 15L fermenter, fed-batch fermentation was used to control the dissolved oxygen process, and the 5-KGA output reached 162g/L, with a yield of 2.53g/L·h, which has the value of industrial application.

Description of drawings

Fig. 1 is a new integrated plasmid pJKM constructed.

Figure 2 is a schematic diagram of the principle and process of GA2DH gene knockout.

Fig. 3 is a graph showing the colony PCR screening results of the recombinant bacterium G.oxydans ZJU1.

Fig. 4 is a graph showing the colony PCR screening results of the recombinant strain G.oxydans ZJU2.

Fig. 5 is a curve diagram of the fermentation process of the genetically engineered bacteria G.oxydans ZJU2/pBB5-P ₀₁₆₉ -sldAB/pUCpr-P ₀₁₆₉ -pqqABCDE-tldD-P ₀₁₆₉ -cyoBACD.

Detailed ways

Seed medium (g/L): mannitol 25; Yeast extract 5; tryptone 3; agar 15 (solid medium); pH 5.5-6.0;

Fermentation medium (g/L): corn steep liquor 7.5; (NH ₄ ) ₂ SO ₄ 0.41; (NH ₄ ) ₂ HPO ₄ 0.1; MgSO ₄ ·7H ₂ O0.07; CaCO ₃ 10-20 (after separate sterilization Add); glucose 25-100 (according to need), initial pH 5.0-5.5, 121°C, sterilize for 15min, final concentration of cefoxitine 50μg/mL.

The molecular biology experiment methods in the following examples without specific conditions indicated were carried out according to conventional conditions, referring to the conditions described in the "Molecular Cloning Experiment Guide" (New York: Cold Spring Harbor Laboratory Press, 2001).

Example 1 Construction of novel integrative plasmid pJKM

Primers were designed according to the plasmid pSM20 (the base sequences are shown in SEQ ID NO.2 and SEQ ID NO.3), PCR amplification was used to obtain the SacB gene fragment, and the blunt-end cloning kit (pEASY-Blunt Simple Cloning Kit, full gold , Beijing) for blunt-end seamless cloning to obtain the recombinant plasmid pEASY-Blunt-SacB containing the SacB gene.

After the sequencing is correct, the SacB gene fragment is recovered by single-enzyme digestion with SspI, and connected with the pK18mobGII plasmid that has been digested with SspI, and transformed into E.coli DH5α. Colony PCR screening obtains positive clones with the correct connection direction of SacB, and the constructed integrated suicide Plasmid pJKM, the plasmid map is shown in Figure 1.

Example 2 Construction of Target Gene Knockout Suicide Plasmid

According to the genome sequence of Gluconobacter oxydans 621H in GenBank (NC_006677), primers were designed respectively, and the primer sequences are shown in SEQ ID NO.4～SEQ ID NO.7:

Get 1000 bp of the upper and lower gene fragments of GA2DH by PCR, and fuse them into the integrated plasmid by fusion PCR (use the upper and lower fragments obtained by PCR as templates, 1231_HindIII_F and 1231_XbaI_R as primers, and perform secondary PCR to obtain the fusion fragments connecting the upper and lower reaches) In pJKM (prepared in Example 1), a suicide knockout plasmid pΔGOX1231 was constructed and sequenced to verify correctness.

The primer sequences for amplifying the upstream and downstream gene fragments of PDC are shown in SEQ ID NO.8~SEQ ID NO.11. Using these primers, 800 bp of the upstream and downstream gene fragments of PDC were obtained by PCR, and the suicide knockout plasmid was constructed by the same method pΔGOX1081, and proceed to the next step after the sequence verification is correct.

The construction of embodiment 3 engineering bacteria G.oxydans ZJU2

The constructed knockout plasmid pΔGOX1231 was transformed into the recipient bacterium Gluconobacter oxydans DSM2343 (purchased from the German Biological Resource Center DSMZ) by electrotransformation, and coated with kanamycin 50 μg/mL and cefoxitin A 50 μg/mL seed medium plate was used for single-crossover screening to obtain transformants in which the suicide plasmid was integrated into the genome.

Then the obtained single exchange transformant was transferred to an anti-resistant seed medium test tube and cultured overnight, and streaked on a seed medium plate containing cefoxitin and 10% sucrose for the second round of screening to obtain a double exchange transformant. In the double-crossover transformants, the GA2DH gene-knockout recombinant strain G.oxydans ZJU1 was screened by colony PCR.

. The principle and process of GA2DH gene knockout are shown in Figure 2, and the colony PCR identification chart of the recombinant bacterium G.oxydans ZJU1 is shown in Figure 3. From the screening results in Figure 3, it can be seen that the recombinant bacterium G.oxydans ZJU1 has successfully GA2DH gene knockout in Gluconobacter oxydans DSM2343.

On this basis, the PDC gene was knocked out by the same method to obtain the engineering bacterium G.oxydans ZJU2, and the colony PCR identification diagram of the recombinant bacterium G.oxydans ZJU2 is shown in Figure 4. The screening results in Figure 4 show that the recombinant bacterium G. The PDC gene in Gluconobacter oxydans DSM2343 has been successfully knocked out in oxydans ZJU1.

The construction of embodiment 4 recombinant expression plasmids

(1) Construction of pBB5-P ₀₁₆₉ -sldAB plasmid

The sldAB gene primers were designed according to the genome sequence (NC_006677) of Gluconobacter oxydans 621H in GenBank, and its sequences are shown in SEQ ID NO.12 and SEQ ID NO.13:

The 2771bp fragment of sldAB of SLDH gene was obtained by PCR and cloned into pEASY-Blunt vector. After the sequencing was correct, the sldAB fragment was recovered by double digestion with EcoRI and XbaI and connected to the broad host expression vector pBBR1MCS5 to construct the plasmid pBB5-sldAB; design primers (the sequences of which are shown in SEQ ID NO.14 and SEQ ID NO.15) for PCR The promoter P ₀₁₆₉ fragment derived from the Gluconobacter oxydans GOX0169 gene was obtained and cloned into the pEASY-Blunt vector. After correct sequencing, the P ₀₁₆₉ fragment was recovered by double enzyme digestion and inserted into pBB5-sldAB to construct the expression plasmid pBB5-P ₀₁₆₉ -sldAB.

The constructed recombinant expression plasmid was transformed into E.coli DH5α, and positive transformants were verified by colony PCR, that is, 103bp and 2771bp bands appeared respectively.

(2) Construction of pUCpr-P ₀₁₆₉ -pqqABCDE-tldD-P ₀₁₆₉ -cyoBACD plasmid According to the genome sequence (NC_006677) of Gluconobacter oxydans 621H in GenBank, the primers for pqqABCDE, tldD, cyoBACD and promoter P ₀₁₆₉ genes were designed, in order to realize multigene Each gene fragment obtained by PCR contains an overlapping region of 15-20 bp respectively. After each fragment obtained by PCR was correctly sequenced, the whole gold directional seamless cloning kit was used ( Seamless Cloning and Assembly Kit, CU101) was connected to construct the recombinant expression plasmid pUCpr-P ₀₁₆₉ -pqqABCDE-tldD-P ₀₁₆₉ , and the cyoBACD gene was digested, recovered and connected to construct the expression plasmid pUCpr-P ₀₁₆₉ -pqqABCDE-tldD-P ₀₁₆₉ - cyoBACD (the primer sequences of P ₀₁₆₉ , pqqABCDE, tldD, P ₀₁₆₉ and cyoBACD are shown in SEQ ID NO.16-SEQ ID NO.25), wherein the operation is performed according to the kit instructions.

Example 5

Construction of G.oxydans ZJU2/pBB5-P ₀₁₆₉ -sldAB/pUCpr-P ₀₁₆₉ -pqqABCDE-tldD-P ₀₁₆₉ -cyoBACD Engineering Bacteria

The Gluconobacter oxydans ZJU2 constructed in Example 3 was in the electric transformation medium (mannitol 80g/L, yeast extract 15g/L, MgSO ₄ 7H ₂ O 2.5g/L, glycerol 0.5g/L, CaCl _1.5g /L) to OD ₆₀₀ of 0.4-0.6, centrifuged at 4°C, 6000×g for 4 minutes to collect the bacteria, resuspended with 30 mL ice-cold 10% (V/V) sterile glycerol solution, and repeated twice, the bacteria The body weight was suspended in 2 mL of ice-cold 10% (V/V) sterile glycerol solution, and aliquoted into 40 μL tubes as competent cells. The pBB5-P ₀₁₆₉ -sldAB plasmid was electrotransformed into G.oxydans ZJU2 competent cells, and the positive recombinant G.oxydans ZJU2/pBB5-P ₀₁₆₉ -sldAB was screened on a mannitol plate containing 50 μg/mL gentamicin.

The correct positive recombinant G.oxydans ZJU2/pBB5-P ₀₁₆₉ -sldAB will be obtained, and competent cells will be prepared according to the above method. By electroporation again, pUCpr-P ₀₁₆₉ -pqqABCDE-tldD-P ₀₁₆₉ -cyoBACD was transformed into G.oxydans ZJU2/pBB5-P ₀₁₆₉ -sldAB, and in the presence of 50 μg/mL gentamicin and 100 μg/mL ampicillin The positive recombinants were screened on the mannitol plate to obtain the recombinant engineering bacteria G.oxydans ZJU2/pBB5-P ₀₁₆₉ -sldAB/pUCpr-P ₀₁₆₉ -pqqABCDE-tldD-P ₀₁₆₉ -cyoBACD.

Example 6

G.oxydans ZJU2/pBB5-P ₀₁₆₉ -sldAB/pUCpr-P ₀₁₆₉ -pqqABCDE-tldD-P ₀₁₆₉ -cyoBACD to produce 5-KGA by fermentation

Seed medium (g/L): mannitol 25; Yeast extract 5; tryptone 3; pH 5.5-6.0; sterilized at 121°C for 20 minutes, final concentration of gentamicin 50 μg/mL and ampicillin 100 μg/mL.

Fermentation medium (g/L): corn steep liquor 7.5; (NH ₄ ) ₂ SO ₄ 0.41; (NH ₄ ) ₂ HPO ₄ 0.1; MgSO ₄ ·7H ₂ O0.07; CaCO ₃ 10-20 (after separate sterilization added); initial glucose 100, initial pH 5.0-5.5, 121°C, sterilized for 15 minutes, final concentration of gentamicin 50 μg/mL and final concentration of ampicillin 100 μg/mL.

Culture conditions: Add 200 μL of recombinant bacteria preserved in glycerol tubes into 5 mL seed medium test tubes, cultivate overnight at 30° C., 200 rpm, transfer 50 mL of fresh seed medium to the logarithmic growth phase (about 10 -14h), transfer 200mL fresh seed culture medium to the logarithmic growth phase according to 8% (V/V) inoculum amount; inoculate in 15L fermenter (working volume 9L) according to 8% (V/V) inoculum amount, 30 ℃ , 1vvm ventilation, tank pressure 0.03MPa, stirring speed control dissolved oxygen 20-60%. When the glucose concentration in the medium is lower than 30g/L, start to add glucose, and control the residual sugar concentration in the fermenter to not be lower than 30g/L. The supplementary amount of glucose is 700g. After 64 hours of fermentation, the yield of 5-KGA reached 162g/L, and the yield was 2.53g/L·h. The fermentation curve is shown in Figure 5.

Claims (8)

1. a plant height produces the preparation method of the Gluconobater oxydans genetic engineering strain of 5-KGA, which is characterized in that including as follows Step：

（1）The upstream and downstream segment of GA2DH genes and PDC genes is introduced more grams of recombination and integration type suicide plasmid pJKM respectively Grand site, the suicide type for respectively obtaining GA2DH genes knock out the suicide type knockout plasmid p of plasmid p △ GOX1231 and PDC genes △GOX1081；The recombination and integration type suicide plasmid carriesSacBGene, antibiotic marker and multiple cloning sites；The recombination The preparation method of integrated suicide plasmid pJKM is as follows：

（a）Structure containsSacBThe cloned plasmids p of geneEASY-Blunt-SacB；

（b）Take the correct cloned plasmids p of sequencingEASY-Blunt-SacB, pass throughSspISingle endonuclease digestion cloned plasmids pEASY- Blunt-SacBWith suicide plasmid pK18mobGII, recycles and connect, is transformed intoE. coliDH5 α, screening obtainSacBConnection Positive colony in the right direction, and cultivateE. coliDH5 α, extraction integrative plasmid is to get recombination and integration type suicide matter Grain pJKM；

（2）Gained suicide type knockout plasmid p △ GOX1231 are transferred in host strain, are trained successively through antibiotic resistance screening, nonreactive It supports and sucrose screening is named as then by Positive mutants of bacterium colony PCR acquisition GA2DH gene knockoutsG. oxydans ZJU1；Step（2）Described in host strain be wild type Gluconobacter oxvdansG. oxydans DSM2343；

（3）Gained suicide type knockout plasmid p △ GOX1081 are transferred to after passageG. oxydansIn ZJU1, successively through anti- Then raw element resistance screening, nonreactive culture and sucrose screening obtain Positive mutants of PDC gene knockouts, i.e., by bacterium colony PCR Gluconobater oxydans genetic engineering strain is obtained, is named asG. oxydansZJU2；

（4）It willsldAB、pqqABCDE、tldD、P₀₁₆₉And cytochromesbo ₃Oxidizing fermentcyoBACD genes are transferred to step（3）Gained Gluconobater oxydans genetic engineering strainG. oxydansIn ZJU2 to obtain the final product.

2. the preparation method of the Gluconobater oxydans genetic engineering strain of high yield 5-KGA according to claim 1, feature It is, the upstream and downstream clip size of GA2DH genes and PDC genes is 500-1200 bp.

3. the preparation method of the Gluconobater oxydans genetic engineering strain of high yield 5-KGA according to claim 1, feature It is, the antibiotic resistance screening is the mannitol culture medium flat plate that transformant is coated on to kanamycins and Cefoxitin, Obtain genome conformity type transformant；The sucrose screening is that antibiotic resistance gene integration transformation is trained through mannitol culture medium test tube After supporting, the mannitol culture medium flat plate containing 5 ~ 10% sucrose and Cefoxitin is lined, obtains the saltant type for being detached from suicide plasmid Bacterial strain.

4. the preparation method of the Gluconobater oxydans genetic engineering strain of high yield 5-KGA according to claim 1, feature It is, step（4）Include the following steps：

（1）PCR is obtainedsldAB、pqqABCDE、tldD、P₀₁₆₉And cytochromesbo ₃Oxidizing fermentcyoBACD genetic fragments；

（2）It willsldAB genes and P₀₁₆₉Structure recombinant expression plasmid pBB5-P is connect with pBBR1MCS5 carriers₀₁₆₉-sldAB；It willpqqABCDE、tldD, cytochromesbo ₃Oxidizing fermentcyoBACD and promoter P₀₁₆₉Structure recombinant expression is connect with carrier pUCpr Plasmid pUCpr-P₀₁₆₉-pqqABCDE-tldD-P₀₁₆₉-cyoBACD；

（3）Two kinds of recombinant expression plasmids are transferred to by electrotransformationG. oxydansIn ZJU2.

5. a kind of oxidation grape for the high yield 5-KGA that the preparation method described in claim 1 ~ 4 any claim is prepared Saccharic acid oxydans genetic engineering bacterium.

6. the Gluconobater oxydans genetic engineering strain of high yield 5-KGA as claimed in claim 5 is in production 5- ketone group-D- grapes Application in saccharic acid, which is characterized in that include the following steps：

The Gluconobater oxydans genetic engineering strain of the high yield 5-KGA is seeded to after expanding and cultivating in fermentation tank and is carried out Fermented and cultured collects zymotic fluid, the isolated 5- keto-D-gluconic acids from zymotic fluid.

7. applying according to claim 6, which is characterized in that the group of the fermentation medium of the fermented and cultured becomes：Corn Starch 7.5 g/L；(NH₄)₂SO₄0.41 g/L；(NH₄)₂HPO₄0.1 g/L；MgSO₄·7H₂O 0.07 g/L；CaCO₃ 10- 20 g/L；Glucose 100 g/L, initial pH 5.0-5.5,121 DEG C, sterilize 15 min, the western 50 μ g/ of spit of fland final concentration of cephalo mL。

8. applying according to claim 6, which is characterized in that the condition of the fermented and cultured is：28 ~ 32 DEG C, 0.8 ~ 1.2 The ventilatory capacity of vvm, it is 20% ~ 40% that tank, which presses 0.02 ~ 0.04 MPa, mixing control oxyty, and ferment 55 ~ 65h, in fermentation process Feed supplement is controlled according to the concentration of glucose in culture medium.