Gluconobacter oxydans for retarding metabolism of L-sorbose and application of gluconobacter oxydans

Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to a genetically engineered bacterium for retarding metabolism of L-sorbose. According to the invention, gluconobacter oxydans is used as a starting strain, and a synthetic gene GOX0814 of a PTS system transporter subunit IIA, a synthetic gene GOX0849 of NADPH dependent L-sorbose reductase and a synthetic gene GOX2071 of lactic dehydrogenase are knocked out on a genome. By knocking out the three genes, the consumption rate of the L-sorbose and the production of intermediate metabolites are reduced, and an L-sorbose production strain for retarding the metabolism of the L-sorbose is obtained. When the strain is fermented for 28 hours in a shake flask, the residual sugar concentration of L-sorbose in fermentation liquor is increased by 1.9 times or more than 1.9 times compared with the original strain, when the strain is fermented for 28 hours in a fermentation tank, the residual sugar concentration of L-sorbose is increased by 5.1 times or more than 5.1 times compared with the original strain, and the strain has important industrial application value for producing 2-KGA by applying a one-step fermentation method.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering, and in particular relates to a Gluconobacter oxidans strain capable of slowing down L-sorbose metabolism and an application thereof. Background Art

[0002] Vitamin C, also known as L-ascorbic acid, is an essential nutrient for humans and some animals. Vitamin C deficiency in humans can lead to scurvy. Vitamin C is also widely used in medicine, food, cosmetics and other industries due to its strong reducing properties. Currently, the industrial production of vitamin C usually adopts the "three-bacteria two-step" fermentation method, which uses D-sorbitol as a substrate and utilizes Gluconobacter oxidans ( Gluconobacter oxydans ) first converts D-sorbitol into L-sorbose, and then with the help of Ketogulobacterium ( Ketogulonicigenium vulgare ) and Bacillus megaterium ( Bacillus megaterium ) co-culture system, further converting L-sorbose into 2-keto-L-gulonic acid (2-KGA), and finally converting 2-KGA into vitamin C by chemical methods.

[0003] In view of the long fermentation cycle and high energy consumption of the traditional three-bacteria two-step fermentation method, researchers have developed a two-bacteria ( G. oxydans and K. vulgare ) One-step fermentation method. This process uses Gluconobacter oxydans to convert D-sorbitol into L-sorbose, and then L-sorbose is metabolized into 2-KGA by Bacillus ketogulonicus. However, in this process, there is a metabolic pathway for L-sorbose in Gluconobacter oxydans, which competes with the precursors for the synthesis of 2-KGA. Summary of the invention

[0004] In view of the above problems, in order to slow down the metabolism of sorbose by Gluconobacter oxydans, the present invention uses the Red homologous recombination method to seamlessly knock out three genes related to L-sorbose metabolism in Gluconobacter oxydans, constructs a strain of Gluconobacter oxydans that slows down the metabolism of L-sorbose, and achieves an increase in the amount of residual L-sorbose in the fermentation broth. The constructed strain has good industrial application prospects.

[0005] One of the technical solutions provided by the present invention is a method for slowing down the metabolism of L-sorbose, wherein the method is to knock out the synthetic gene of the PTS system transport protein subunit ⅡA in Gluconobacter oxydans GOX0814、 Synthetic gene of NADPH-dependent L-sorbose reductase GOX0849 , and lactate dehydrogenase synthetic genes GOX2071 achieved.

[0006] The second technical solution provided by the present invention is a genetically engineered bacterium of Gluconobacter oxydans that can slow down the metabolism of L-sorbose. The genetically engineered bacterium uses Gluconobacter oxydans as a host and knocks out the synthetic gene of the PTS system transport protein subunit ⅡA on the genome. GOX0814 , NADPH-dependent L-sorbose reductase synthesis gene GOX0849 , lactate dehydrogenase synthesis gene GOX2071 By knocking out these three genes, the consumption rate of L-sorbose was reduced, thereby increasing the production of L-sorbose.

[0007] Said GOX0814 Gene, nucleotide sequence is shown in the sequence listing SEQ ID NO.1; Said GOX0849 Gene, nucleotide sequence is shown in the sequence listing SEQ ID NO.2; Said GOX2071 Gene, nucleotide sequence is shown in the sequence listing SEQ ID NO.3; Preferably, the host bacteria of the genetically engineered bacteria is Gluconobacter oxydans G. oxydans 621H.

[0008] The third technical solution provided by the present invention is a method for constructing the above-mentioned genetically engineered bacteria. G. oxydans 621H carries out genome-level directional transformation, which specifically includes the following steps: (1) Knockout of the synthetic gene of PTS system transporter subunit ⅡA GOX0814 , slowing down the consumption of L-sorbose; (2) Knockout of the gene that synthesizes NADPH-dependent L-sorbose reductase GOX0849 , slowing down the consumption of L-sorbose; (3) Knockout of the synthetic gene of lactate dehydrogenase GOX2071 , slowing down the consumption of L-sorbose; The above construction order is not in particular order and can be adjusted according to needs.

[0009] The fourth technical solution provided by the present invention is the application of the genetically engineered bacteria described in the second technical solution, especially the application in the fermentation conversion of D-sorbitol into L-sorbose, and more particularly the application in slowing down the metabolism of L-sorbose and increasing the yield of 2-KGA; Furthermore, the method for fermenting and converting D-sorbitol using the above genetically engineered bacteria is as follows: 1. Shake flask fermentation (1) Primary seed culture: Take 300-400 µL of glycerol culture from a -80°C freezer tube and inoculate into 50 mL of fresh seed culture medium. Incubate at 220-250 rpm in a shaker at 28-30°C for 22-26 h. (2) Secondary seed culture: Transfer the primary seeds to a shake flask containing 50 mL of fresh seed culture medium at an inoculation ratio of 8-12%. Seal the flask with nine layers of gauze and culture at 220-250 rpm in a shaker at 28-30°C for 22-26 hours. (3) Shake flask fermentation: The secondary seeds were transferred to a shake flask containing fresh fermentation medium at an inoculation ratio of 8-12%, and cultured in a shaker at 220-250 rpm at 28-30°C. The fermentation cycle was 30-36 h. At 28 h of fermentation, the concentration of L-sorbose in the fermentation broth increased by about 1.9-2.2 times compared with the original strain.

[0010] 2. Fermentation in fermenter (1) Primary seed culture: Take 300-400 µL of glycerol culture from a tube stored at -80°C and inoculate it into 50 mL of fresh seed culture medium. Incubate the culture in a shaker at 220-250 rpm at 28-30°C for 22-26 h. (2) Secondary seed culture: Transfer the primary seeds to a shake flask containing 50 mL of fresh seed culture medium at an inoculation ratio of 8-12%. Seal the flask with nine layers of gauze and culture at 220-250 rpm in a shaker at 28-30°C for 22-26 hours. (3) Transfer the secondary seeds to the fermentation tank at an inoculation ratio of 8-12% (the fermentation medium liquid volume is 60%). During the fermentation process, the pH is maintained at around 6.8-7.0, the temperature is maintained at around 28-30°C, the rotation speed is controlled at around 800-900rpm, the ventilation is 2-3 L / min, the dissolved oxygen is maintained between 50-70%, and the fermentation cycle is about 35-40 hours; Among them, at 28 h of fermentation, the residual sugar concentration of L-sorbose in the fermentation broth increased by about 5.1-5.5 times compared with the original strain; Furthermore, the seed culture medium is composed of: D-sorbitol 15-25 g / L, corn steep liquor 2-4 g / L, beef extract 2-4 g / L, yeast powder 2-4 g / L, peptone 8-12 g / L, potassium dihydrogen phosphate 0.8-1.2 g / L, urea 0.8-1.2 g / L, anhydrous magnesium sulfate 0.1-0.3 g / L, calcium carbonate 0.8-1.2 g / L, defoamer 1-2 drops, the rest is water, pH 6.8-7.0.

[0011] Furthermore, the fermentation medium is composed of: 80-120 g / L of D-sorbitol, 8-12 g / L of corn steep liquor, 5-8 g / L of calcium carbonate, 1-2 drops of defoaming agent, and the rest is water, with a pH of 6.8-7.0.

[0012] Beneficial Effects The present invention G. oxydans 621H is the starting strain, and three genes related to sorbose bypass metabolism and transport were knocked out in the genome GOX0814 , GOX0849 , GOX2071 A strain that slows down the metabolism of L-sorbose is obtained. The residual sugar content of L-sorbose in the obtained strain is increased by about 1.9 to 2.2 times in shake flask fermentation and about 5.1 to 5.5 times in fermenter fermentation compared with the original strain. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 PCR validation of knockout GOX0814 Gene fragment Among them, M: Marker; 1: upstream homology arm; 2: promoter Ptuf; 3: tetracycline resistance gene; 4: terminator Ttuf; 5: downstream homology arm; 6: targeting fragment ΔGOX0814; 7: original length of GOX0814; 8: band length after knocking out GOX0814; 9: band length after removing the marker gene.

[0014] Figure 2 for G. oxydans 621H and G. oxydans Fermentation results in M1 5L fermenter.

[0015] Figure 3 for G. oxydans 621H and G. oxydans M1 shake flask fermentation results. DETAILED DESCRIPTION

[0016] The present invention is described below by specific embodiments. Unless otherwise specified, the test methods used in the present invention are methods known to those skilled in the art. In addition, the embodiments should be understood to be illustrative rather than limiting the scope of the present invention, and the essence and scope of the present invention are limited only by the claims. For those skilled in the art, various changes or modifications to the material composition and dosage in these embodiments, without departing from the essence and scope of the present invention, also belong to the protection scope of the present invention.

[0017] The host bacteria used in the present invention is Gluconobacter oxydans Gluconobacter oxydans621H, which is a prior art strain and has been published in many literatures, such as PRUST et al. in "Complete genome sequence of the acetic acid bacterium Gluconobacteroxydans "Reported in (PRUST C, HOFFMEISTERM, LIESEGANG H, et al. Complete genome sequence of the acetic acid bacteriumGluconobacter oxydans [J]. Nature biotechnology, 2005, 23(2): 195-200.).

[0018] The partial solution or culture medium involved in the present invention is as follows: 10% glycerol: Measure 20 mL of glycerol, dilute to 200 mL with ultrapure water, mix thoroughly, and sterilize.

[0019] Solid culture medium: D-sorbitol 15-25 g / L, corn steep liquor 2-4 g / L, beef extract 2-4 g / L, yeast powder 2-4 g / L, peptone 8-12 g / L, potassium dihydrogen phosphate 0.8-1.2 g / L, urea 0.8-1.2 g / L, anhydrous magnesium sulfate 0.1-0.3 g / L, calcium carbonate 0.8-1.2 g / L, agar powder 18-22 g / L.

[0020] The composition of the seed culture medium is: D-sorbitol 15-25 g / L, corn steep liquor 2-4 g / L, beef extract 2-4 g / L, yeast powder 2-4 g / L, peptone 8-12 g / L, potassium dihydrogen phosphate 0.8-1.2 g / L, urea 0.8-1.2 g / L, anhydrous magnesium sulfate 0.1-0.3 g / L, calcium carbonate 0.8-1.2 g / L, pH 6.8-7.0. Fermentation medium: D-sorbitol 80-120 g / L, corn steep liquor 8-12 g / L, calcium carbonate 5-8 g / L, defoamer 1-2 drops, pH 6.8-7.0.

[0021] The present invention will be further explained below through specific examples.

[0022] Example 1: Construction of genetically engineered bacteria of Gluconobacter oxydans for converting D-sorbitol into L-sorbose In this example, Gluconobacter oxydans Gluconobacter oxydans 621H was used as the starting strain, and the Red homologous recombination method was used to GOX0814 Gene, GOX0849 Gene, GOX2071 The gene is knocked out in a targeted manner to construct an engineered strain that converts D-sorbitol into L-sorbose.

[0023] by GOX0814 Taking gene knockout as an example, some of the experimental methods involved are as follows: 1. Preparation G. oxydans 621H strain competent 1) From G. oxydans The glycerol tube of strain 621H was streaked onto a solid culture medium plate and cultured at 30°C for 48 h until single colonies could be clearly identified; 2) Pick a single colony with good growth and inoculate it into the seed culture medium, and culture it at 30℃ and 220 rpm for 24 h; 3) Take the seed solution and transfer it to the seed culture medium at a 1% inoculum rate, and culture it at 30°C and 220 rpm for 8 h until the OD 600 to 0.6-1.0; 4) Centrifuge the centrifuge tube containing the bacterial solution at 4500 rpm for 10 min at 4°C and remove the supernatant; 5) Add 25 mL of 10% glycerol solution pre-cooled to 4°C to resuspend the cells so that the cells are fully dispersed.

[0024] 6) Centrifuge at 4500 rpm for 10 min at 4°C and discard the supernatant; 7) Repeat steps 5) and 6); 8) Add 1 mL of pre-cooled 10% glycerol solution, shake well, place in an ice bath, and dispense 100 μL per tube into 1.5 mL centrifuge tubes, keep in an ice bath, and store at -80℃ after aliquoting.

[0025] 2. pRedGx Plasmid Transformation (1) Construction of pRedGx plasmid Plasmid pKD46 contains the receptor P araBAD Promoter-regulated outside , page , grief The gene is induced by L-arabinose and carries an ampicillin resistance gene as a selection marker. Its replicon pSC101 is a low-copy temperature-sensitive replicon, which is convenient for plasmid elimination. However, the temperature-sensitive replicon is not suitable for Gluconobacter oxydans, and Gluconobacter oxydans will produce L-sorbose after using D-sorbitol, which may affect the induced expression of the arabinose operon. Therefore, using the pKD46 plasmid as the backbone, the pSC101 replicon was replaced with the pBBR1 Rep replicon, the P araBAD Promoter-regulated outside , page , grief Gene replacement lacI q -P trc Promoter-regulated outside , page , grief pRedGx was constructed by replacing the ampicillin resistance gene with the kanamycin resistance gene (for the specific construction method, refer to Liu LP, Yang X, Zhao XJ, Zhang KY, Li WC, Xie YY, JiaSR, Zhong C. A Lambda Red and FLP / FRT-Mediated Site-Specific Recombination System in Komagataeibacter xylinusand Its Application to Enhance the Productivity of Bacterial Cellulose. ACS Synth Biol. 2020, 9(11):3171-3180).

[0026] (2) Transformation of pRedGx plasmid Plasmid pRedGx can express Red recombinase in the strain through IPTG induction, thereby improving the efficiency of homologous recombination.

[0027] 1) Cool a 0.1 cm electroporation cuvette on ice, add 5-10 μL of pRedGx plasmid to 100 μL of competent cells, and incubate on ice for 5-10 min.

[0028] 2) Perform electroporation according to the instructions of the electroporator (Bio-Rad, USA). After the 1800 V electroporation, add 1 mL of pre-cooled seed culture medium immediately, culture at 30°C for 6 h, then centrifuge and spread onto a solid culture medium plate with the corresponding antibiotic (50 μg / mL kanamycin), culture at 30°C inverted, and transformants may appear in 48-72 h. The positive transformants screened by single colony PCR were named G. oxydans 621H / pRedGx.

[0029] 3. Construction of targeting fragments Build GOX0814 The upstream and downstream homology arms of Gluconobacter oxydans The 621H genome was used as a template and amplified by PCR using the primer pair GOX0814-F1 / GOX0814-R1. GOX0814 The upstream homology arm of the gene was obtained by PCR amplification using primer pair GOX0814-F2 / GOX0814-R2 GOX0814 The downstream homologous arm of the gene. G. oxydans H24 promoter P tuf (synthesized by GENEWISE, sequence as SEQ ID NO.4) was used as the promoter of tetracycline gene (marker gene, SEQ ID NO.5), and the primer pair GOX0814-FRT-tuf-F1 / GOX0814-FRT-tuf-R1 was used to obtain P with FRT site by PCR amplification. tuf Promoter fragment. Using pACYC184 plasmid as template, the tetracycline resistance gene fragment was obtained by PCR amplification using primer pair TCR-F / TCR-R. G. oxidant H24 terminator T tuf (synthesized by GENEWISE, sequence as SEQ ID NO.6) as the terminator of the tetracycline gene, and the T with FRT site was obtained by PCR amplification using the primer pair GOX0814-FRT-tuf-F2 / GOX0814-FRT-tuf-R2. tuf The upstream homology arm and promoter P obtained above were PCR-PCR-PCR-PCR-assisted with primer pair GOX0814-F1 / GOX0814-R2. tuf , tetracycline resistance gene, terminator T tuf , the downstream homology arm fragments were overlapped in the form of fusion PCR to obtain the target fragment Δ GOX0814 .

[0030] 4. Transformation of knockout fragments and verification of recombinant bacteria The obtained Δ GOX0814 Electroporation of fragments G. oxydans 621H / pRedGx competent cells (Note: When preparing competent cells containing pRedGx plasmids, the OD 600 When the OD value was 0.2-0.3, IPTG was added at a final concentration of 0.1 mM to induce the expression of the exogenous recombinase, and then the culture was continued until the OD value reached 0. 600 to 0.6-1.0), after recovery culture, it was spread on a solid culture medium plate containing tetracycline and inverted in a 30°C incubator. Transformants could be observed after 48-72 hours, and the primer pair GOX0814-F1 / GOX0814-R2 was used to perform single colony PCR to verify whether the target gene was knocked out.

[0031] The knockout results are as follows Figure 1 Knockout GOX0814 The theoretical length is as follows: upstream homology arm 553 bp, promoter P tuf 574 bp, tetracycline resistance gene 1125 bp, terminator T tuf400 bp, downstream homology arm 628 bp, targeting fragment Δ GOX0814 3075 bp, GOX0814 Original length 1252 bp, knockout GOX0814 The length of the latter band is 3075 bp, and the length of the band after removing the marker gene is 1108 bp.

[0032] 5. Loss of plasmid pRedGx Plasmid pCP20 contains the pSC101 temperature-sensitive replicon and the FLP recombinase encoding gene php Considering that the thermosensitive plasmid is not suitable for Gluconobacter oxydans, the pSC101 replicon was replaced with the pBBR1 Rep replicon with the same FRT site, based on the pCP20 backbone. lacI q -P trc Promoter-regulated php The pFLPGx plasmid was reconstructed by combining elements such as the gene (IPTG-induced expression) and the gentamicin resistance gene (for specific construction methods, refer to Liu LP, Yang X, Zhao XJ, Zhang KY, Li WC, Xie YY, Jia SR, Zhong C. A Lambda Red and FLP / FRT-Mediated Site-Specific Recombination System in Komagataeibacter xylinus and Its Application to Enhance the Productivity of Bacterial Cellulose. ACS Synth Biol. 2020, 9(11): 3171-3180). The pFLPGx plasmid can express FLP recombinase in the strain by IPTG induction, and can recognize the FRT site to achieve homologous recombination.

[0033] The pFLPGx plasmid was transformed into the successfully verified knockout bacteria, and the transformants were screened using solid culture medium plates containing gentamicin. Since the pFLPGx plasmid and the pRedGx plasmid contained the same replicon but carried different resistance genes, strains in which the pFLPGx plasmid was successfully transformed but the pRedGx plasmid was lost could be obtained based on the incompatibility of the plasmids.

[0034] 6. Loss of pFLPGx plasmid and removal of marker gene In the pFLPGx vector, both ends of the replicon carry FRT sites repeated in the same direction, which can be recognized by the FLP recombinase to excise the replicon. If the pFLPGx replicon is excised, the plasmid will not be able to survive in the host, thereby achieving the purpose of eliminating the pFLPGx plasmid. At the same time, since the resistance gene inserted in the genome also carries FRT sites on both sides, it can also be recognized by the FLP enzyme, thereby achieving the purpose of scarless knockout.

[0035] The strain obtained in step 5 was induced by IPTG. After 24 h of culture, it was diluted and spread on a solid culture medium plate without antibiotics. After a single colony grew, it was spotted on solid culture medium plates containing only gentamicin, only tetracycline, and no antibiotics. If the strain can only grow on the plate without antibiotics but cannot grow on the plate containing both gentamicin and tetracycline, it is the recombinant bacteria that has lost the pFLPGx plasmid and has eliminated tetracycline resistance.

[0036] GOX0849 , GOX2071 The knockout process of the two genes is the same as above (just replace the corresponding primers, see Table 1 for specific primers), thus constructing GOX0814 Gene, GOX0849 Gene 、 and GOX2071 Gene knockout engineered strains G. oxydans M1.

[0037] Table 1 Primers involved in strain construction

[0038] Example 2: Using the strain constructed in Example 1 above as a production strain to transform D-sorbitol Production strains: Original strains G. oxydans 621H and knockout constructs from Example 1 GOX0814 Gene, GOX0849 Gene, GOX2071 Genetically engineered bacteria are production strains G. oxydans M1 is the production strain; (1) Primary seed culture: Take 300 µL of glycerol from a tube stored at -80°C and inoculate it into 50 mL of fresh seed culture medium. Incubate the culture in a shaker at 30°C and 220 rpm for 24 h. (2) Secondary seed culture: Transfer the primary seeds to a shake flask containing 50 mL of fresh seed culture medium at a 10% inoculation ratio. Seal the flask with nine layers of gauze and culture at 220 rpm in a shaker at 30°C for 24 h. (3) The secondary seeds were transferred to a 5 L fermentation tank (liquid volume was 60%) at an inoculation ratio of 10% for cultivation. During the fermentation process, the pH was maintained at around 7.0, the temperature was maintained at around 30°C, the dissolved oxygen was maintained at around 55%, the rotation speed was controlled at around 850 rpm, the ventilation was controlled at around 2.4 L / min, and the fermentation cycle was about 36 h. The fermentation process curve is shown in Figure 2 shown.

[0039] Depend on Figure 2 It can be seen that both strains converted all D-sorbitol into L-sorbose at around 7 h, but since then G. oxydans 621H strain, G. oxydans The L-sorbose metabolism rate of the M1 strain was significantly slowed down; at 28 h, G. oxydans The residual L-sorbose concentration in the fermentation broth of M1 reached 32.25 g / L, which was higher than that of the original strain. G. oxydans 621H improves by 5.1 times. G. oxydans After M1 metabolizes L-sorbose, a higher concentration of L-sorbose can be accumulated in the fermentation broth, which is beneficial for the subsequent application in the two-bacteria one-step fermentation to produce 2-KGA.

[0040] The seed culture medium is composed of: 20 g / L D-sorbitol, 3 g / L corn steep liquor, 3 g / L beef extract, 3 g / L yeast powder, 10 g / L peptone, 1 g / L potassium dihydrogen phosphate, 1 g / L urea, 0.2 g / L anhydrous magnesium sulfate, 1 g / L calcium carbonate, 2 drops of defoaming agent, and the rest is water, with a pH of 7.0.

[0041] The fermentation medium comprises: 80 g / L D-sorbitol, 10 g / L corn steep liquor, 5 g / L calcium carbonate, 2 drops of defoaming agent, and the rest is water, with a pH of 7.0.

[0042] Example 3: Transformation of D-sorbitol using the strain constructed in Example 1 as a production strain Production strains: Original strains G. oxydans 621H and knockout constructs from Example 1 GOX0814 Gene, GOX0849 Gene, GOX2071 Genetically engineered bacteria G. oxydans M1 is the production strain; (1) Primary seed culture: Take 300 µL of glycerol from a tube stored at -80°C and inoculate into 50 mL of fresh seed culture medium. Incubate at 30°C in a shaker at 220 rpm for 24 h. (2) Secondary seed culture: Transfer the primary seeds to a shake flask containing 50 mL of fresh seed culture medium at a 10% inoculation ratio. Seal the flask with nine layers of gauze and culture at 220 rpm in a shaker at 30°C for 24 h. (3) Shake flask fermentation: Transfer the secondary seeds to a shake flask containing 50 mL of fresh fermentation medium at a 10% inoculation ratio and culture at 220 rpm in a shaker at 30°C. The fermentation cycle is 36 h. The fermentation process curve is as follows Figure 3 shown; Figure 2 Similarly, D-sorbitol was completely converted into L-sorbose at around 7 h, and then was consumed by the strain. G. oxydans 621H strain, G. oxydans The metabolic rate of L-sorbose in the M1 strain was significantly slowed down. At 28 h, the residual L-sorbose concentration in the fermentation broth was 38.2 g / L, which was about 1.9 times higher than that of the original strain. G. oxydans After M1 metabolizes L-sorbose, a higher concentration of L-sorbose can be accumulated in the fermentation broth, which is beneficial for the subsequent application in the two-bacteria one-step fermentation to produce 2-KGA.

[0043] The seed culture medium is composed of: 20 g / L D-sorbitol, 3 g / L corn steep liquor, 3 g / L beef extract, 3 g / L yeast powder, 10 g / L peptone, 1 g / L potassium dihydrogen phosphate, 1 g / L urea, 0.2 g / L anhydrous magnesium sulfate, 1 g / L calcium carbonate, 2 drops of defoaming agent, and the rest is water, with a pH of 7.0.

[0044] The fermentation medium comprises: 80 g / L D-sorbitol, 10 g / L corn steep liquor, 5 g / L calcium carbonate, 2 drops of defoaming agent, and the rest is water, with a pH of 7.0.

[0045] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the patent. It should be pointed out that, for ordinary technicians in this field, the above-mentioned implementation methods can also be modified, combined and improved without departing from the concept of this patent, which all belong to the protection scope of this patent. Therefore, the protection scope of this patent shall be based on the claims.

Claims

1. A method for slowing down the metabolism of L-sorbose, characterized in that: The method is to knock out the synthetic gene of the PTS system transport protein subunit ⅡA in Gluconobacter oxydans GOX0814, Synthetic gene of NADPH-dependent L-sorbose reductase GOX0849 , and lactate dehydrogenase synthetic genes GOX2071 achieved.

2. A genetically engineered strain of Gluconobacter oxydans that slows down the metabolism of L-sorbose, characterized in that: The genetically engineered bacteria uses Gluconobacter oxydans as a host and knocks out the synthetic gene of the PTS system transport protein subunit ⅡA on the genome. GOX0814 , NADPH-dependent L-sorbose reductase synthesis gene GOX0849 , and lactate dehydrogenase synthetic genes GOX2071 Obtained.

3. The genetically engineered bacterium according to claim 2, characterized in that: Said GOX0814 Gene, the nucleotide sequence of which is shown in the sequence table SEQ ID NO.1; GOX0849 Gene, the nucleotide sequence is shown in the sequence table SEQ ID NO.2; GOX2071 The nucleotide sequence of the gene is shown in the sequence listing SEQ ID NO.

3.

4. The genetically engineered bacterium according to claim 2, characterized in that: The host bacteria of the genetically engineered bacteria is Gluconobacter oxydans ( G. oxydans )621H.

5. The use of the genetically engineered bacteria according to any one of claims 2 to 4, characterized in that: It is used to slow down the metabolism of L-sorbose.

6. The use of the genetically engineered bacteria according to any one of claims 2 to 4, characterized in that: It is used to slow down the metabolism of L-sorbose and increase the yield of 2-KGA.

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