CN112680484B - A method for producing 3,4-dihydroxybutyric acid using a double bacteria co-cultivation system - Google Patents

Abstract

The invention discloses a method for producing 3,4-dihydroxybutyric acid by utilizing a double-bacteria co-cultivation system. 3,4-dihydroxybutyric acid was produced by fermentation in a two-bacteria co-cultivation system. Experiments confirm that the method provided by the invention can make the 3,4-dihydroxybutyric acid production amount reach 3.26g/L, and the yield is 0.47g 3,4-dihydroxybutyric acid/1g xylose, effectively solving the currently reported problem. The production of 3,4-dihydroxybutyric acid by a single genetically engineered bacterium has the problem of low yield and low yield, and provides a new idea for the application of Gluconobacter oxydans in the biological production of chemicals that require complex synthetic pathways, new method.

Description

A method for producing 3,4-dihydroxybutyric acid using a double bacteria co-cultivation system

technical field

The invention relates to a method for producing 3,4-dihydroxybutyric acid by fermentation, in particular to a co-cultivation system composed of Gluconobacter oxydans and engineering Escherichia coli, and using xylose as a substrate to ferment and produce 3,4-dihydroxybutyric acid The butyric acid method belongs to the field of biotechnology.

Background technique

3,4-Dihydroxybutyric acid and its dehydration product 3-hydroxybutyrolactone are important platform compounds (Gao H et al., 2017, Bioresour. Technol., 245:794-800), which are widely used in polymers, Production of Solvents and Nutrient Additives (Wang J et al., 2017, Metab. Eng., 41:39-45). The chemical production of 3,4-dihydroxybutyric acid and 3-hydroxybutyrolactone has the disadvantages of harsh and violent reaction conditions, expensive substrates, and the need for heavy metals or rare metals to participate in the reaction (KumarP et al., 2005, Tetrahedron: Asymmetry, 16:2717-2721). Because the production of chemicals by microbial fermentation has the advantages of mild reaction conditions, less pollution, and cheap substrates, the production of 3,4-dihydroxybutyric acid by microbial fermentation has important research value and broad market prospects.

Escherichia coli has the characteristics of simple culture requirements, rapid growth, clear genetic background, and easy genetic engineering operation. It has been successfully used in metabolic engineering to produce 3,4-dihydroxybutyric acid. Due to the toxic effects of intermediate products, high redox pressure, and reversibility of the reaction process, the production of 3,4-dihydroxybutyric acid by recombinant Escherichia coli has low yield and low yield. According to reports, the domestic research group has achieved the production of 3,4-dihydroxybutyric acid by expressing exogenous xylose dehydrogenase, aldehyde dehydrogenase and other metabolic engineering transformations in Escherichia coli, and using xylose as a substrate. 0.38g/L (Gao H et al., 2017, Bioresour. Technol., 245: 794-800); there are foreign reports on the production of 3,4-dihydroxybutyric acid using glucose and glycolic acid as substrates. Metabolic engineering of bacillus glyoxylate cycle, 3,4-dihydroxybutyric acid production reached 0.7g/L (Dhamankar H et al., 2014, Metab.Eng., 25:72-81).

T et al.,2006,J.Bacteriol.,188:7668-7676)，限制了氧化葡萄糖酸杆菌在需要复杂代谢途径的化学品合成方面的应用。Gluconobacter oxydans is a strictly aerobic bacteria (Prust C et al., 2005, Nat. Biotechnol., 23: 195-200), its cell membrane has a variety of membrane-bound dehydrogenases, which can participate in a variety of oxidation reactions, This makes it an important strain for industrial applications (Kiefler I et al., 2017, Appl. Microbiol. Biotechnol., 101:5453-5467). For example, G. oxydans is widely used in industry for the production of gluconic acid, xylonic acid, sorbose and dihydroxyacetone from glucose, xylose, sorbitol and glycerol, respectively. However, Gluconobacter oxydans has defects such as direct secretion of oxidation products into the extracellular space, incomplete TCA cycle in vivo, and difficulty in metabolic engineering (Prust C et al., 2005, Nat. Biotechnol., 23: 195-200;

T et al., 2006, J. Bacteriol., 188:7668-7676), limiting the use of G. oxydans in the synthesis of chemicals that require complex metabolic pathways.

After searching, it was found that a co-cultivation system of Gluconobacter oxydans and engineered Escherichia coli was established by utilizing the characteristics of E. The method of producing 3,4-dihydroxybutyric acid by fermentation of sugar as substrate has not been reported yet.

SUMMARY OF THE INVENTION

Aiming at the problems of the existing method for producing 3,4-dihydroxybutyric acid using recombinant Escherichia coli, the product yield is low, the yield is low, the intermediate product has a toxic effect on the strain, and the reaction process is reversible. The invention provides a method for high-yielding 3,4-dihydroxybutyric acid using xylose as a substrate by using a double bacteria co-cultivation system composed of Gluconobacter oxydans and Escherichia coli.

The method for producing 3,4-dihydroxybutyric acid using a double bacteria co-cultivation system according to the present invention, the steps are:

(1) Construction of a double-bacteria co-cultivation system of Gluconobacter oxydans and engineered Escherichia coli

Take the activated Gluconobacter oxydans, centrifuge at 6000 rpm and wash 1-2 times with physiological saline, then resuspend to OD=3 with physiological saline, for subsequent use; take the activated engineering Escherichia coli, centrifuge at 6000 rpm/separate and use Wash 1-2 times with normal saline, then resuspend to OD=2 with normal saline, for later use; mix the above-mentioned two resuspended bacterial solutions according to the volume ratio of 1～3:1～2, inoculate in the co-cultivation fermentation medium and then inoculate it in the co-cultivation fermentation medium. Make the final OD of the bacterial liquid to be 0.2±0.1, and construct a double-bacteria co-cultivation system of Gluconobacter oxydans and engineered Escherichia coli;

in:

The wild-type Gluconobacter oxydans 621H is selected from the above-mentioned Gluconobacter oxydans; the bacterium is a Gram-negative bacterium and grows strictly aerobic. The activation medium of Gluconobacter oxydans 621H is sorbitol complex medium, and its formula is: sorbitol 73g/L, yeast powder 18.4g/L, (NH ₄ ) ₂ SO ₄ 1.5g/L, KH ₂ PO ₄ 1.5g/L, MgSO ₄ ·7H ₂ O 0.47g/L; autoclave at 121°C for 20 minutes.

The above-mentioned engineering Escherichia coli was selected as Escherichia coli 6KI, and its genotype is Escherichia coli W3110(DE3)ΔxylAΔyjhHΔyagEΔyiaEΔyqdDΔxynR::xylD&kdcA; the bacteria are Gram-negative bacteria, which grow aerobic or facultative anaerobic, and the bacteria are better The culture temperature is 37±1°C and can be grown in LB medium. The activation medium of the engineered Escherichia coli 6KI is LB medium, and its formula is: peptone 10 g/L; yeast powder 5 g/L; NaCl 10 g/L; and autoclaving at 121° C. for 20 minutes.

The above-mentioned co-cultivation fermentation medium formula is: xylose 7±2g/L, glucose 5±1g/L, yeast powder 5±1g/L, peptone 10±2g/L, NaCl 10±2g/L; under the condition of 121°C Autoclave for 20 minutes.

(2) Fermentation production of 3,4-dihydroxybutyric acid by double bacteria co-culture system

The obtained double bacteria co-cultivation system was fermented and cultured at 28±2°C and the shaking speed was 200±20rpm. When the total OD of the system was 0.7 to 0.8, the final concentration of 1mM IPTG was added for induction, and every 3 hours. Manually add 10M NaOH to adjust the pH of the medium to 6.8-7.0, and ferment and culture for 54-65 hours until the sugar consumption is completely terminated, that is, a fermentation broth containing 3,4-dihydroxybutyric acid is obtained.

In the above-mentioned method for producing 3,4-dihydroxybutyric acid using a dual bacteria co-cultivation system: the two resuspended bacterial solutions of the Gluconobacteroxydans 621H and the engineering Escherichia coli 6KI are preferably mixed in a volume ratio of 1:1 and then inoculated into the co-cultivation In the fermentation medium, the final OD of the bacterial liquid was 0.2, and a double-bacteria co-culture system of Gluconobacter oxydans and engineered Escherichia coli was constructed.

In the above-mentioned method for producing 3,4-dihydroxybutyric acid using a dual bacteria co-cultivation system: the co-cultivation fermentation medium formula is preferably: xylose 7g/L, glucose 5g/L, yeast powder 5g/L, peptone 10g /L, NaCl 10g/L.

In the above-mentioned method for producing 3,4-dihydroxybutyric acid using a double bacteria co-cultivation system: the OD ratio of two bacterial liquids of Gluconobacter oxydans 621H and engineering Escherichia coli 6KI in the double bacteria co-cultivation system is preferably equal to 3:2 , and preferably fermented and cultured at 28° C. and under the conditions of a shaker rotation speed of 200 rpm.

The construction method of above-mentioned engineering Escherichia coli 6KI is:

Taking Escherichia coli W3110 (DE3) as the starting strain, the strain was continuously genetically modified by using Red recombination technology (Datsenko KA et al., 2000, Proc.Natl.Acad.Sci.US A., 97:6640-6645), Proceed as follows:

(1) The xylose isomerase gene xylA and the 2-keto-3-deoxyxylonate aldolase genes yjhH and yagE were knocked out in Escherichia coli W3110(DE3) by genetic engineering, blocking the strain from using xylose and the endogenous pathway of xylonic acid to construct strain Escherichia coli 3K.

(2) Knock out the xylonic acid operon transcriptional repressor gene xynR in Escherichia coli 3K by genetic engineering, block the regulation of the transcriptional repressor XynR on the xylonic acid operon, and enhance the xylonic acid dehydratase and xylonic acid The expression of transporter protein enhances the synthesis and metabolism of the target product, and the strain Escherichia coli 4K is constructed.

(3) Knock out the NADPH-dependent aldehyde reductase gene yqhD in Escherichia coli 4K by genetic engineering, block the reduction of 3,4-dihydroxybutyraldehyde to form 1,2,4-butanetriol, and construct strain Escherichia coli 5K .

(4) The glyoxylate reductase gene yiaE was knocked out in Escherichia coli 5K by genetic engineering, weakening the reduction reaction of the intermediate product 2-keto-3-deoxyxylonic acid, enhancing the synthesis and metabolism of the target product, and constructing the strain Escherichia coli 6K .

(5) Commercially insert the xylonic acid dehydratase gene xylD from Caulobacter crescentus and the branched-2-keto acid decarboxylase gene kdcA from Lactococcus lactis by genetic engineering The plasmid pACYCDuet-1 (Fig. 1) was used to amplify the gene expression cassette by PCR, construct an operon and knock it into the original xynR site of the Escherichia coli 6K genome to construct an engineered Escherichia coli 6KI.

The detection methods for the above-mentioned 3,4-dihydroxybutyric acid and other substances are: using a biosensor analyzer (SBA-40D) to detect glucose; using a high performance liquid chromatograph (HPLC) Shimadzu 20-AT to detect xylose, the analysis conditions are: Differential refractive index detector; Bio-Rad Aminex HPX-87P analytical column (300×7.8mm), column temperature 75℃, mobile phase is pure water, flow rate 0.7mL/min, injection volume 5μL; HPLC is used to detect xylose acid, 2-keto-3-deoxyxylonic acid, 3,4-dihydroxybutyraldehyde, 1,2,4-butanetriol and 3,4-dihydroxybutyric acid under the following conditions: UV detector, detection The wavelength is 210 nm; Bio-RadAminex HPX-87H analytical column (300×7.8 mm), the column temperature is 30° C., the mobile phase is 0.1% formic acid, the flow rate is 0.4 mL/min, and the injection volume is 5 μL.

The present invention develops a co-cultivation of Gluconobacter oxydans and Escherichia coli to produce 3,4-dihydroxyl by taking advantage of the easy engineering of Escherichia coli combined with the incomplete oxidizing ability of G. oxydans and the direct secretion of oxidation products into the extracellular space. The new method of butyric acid production provides a new idea for the biological production of 3,4-dihydroxybutyric acid, and provides new guidance for the industrial application of Gluconobacter oxydans. In the above-mentioned two-bacteria co-cultivation system, G. oxidans first oxidizes xylose to xylonic acid, then Escherichia coli converts xylic acid to 3,4-dihydroxybutyraldehyde, and finally, G. oxidans converts 3,4-dihydroxybutyraldehyde. Dihydroxybutyraldehyde is oxidized to 3,4-dihydroxybutyric acid. The method solves the current problem of producing 3,4-dihydroxybutyric acid by a single strain, and realizes high-efficiency and high-yield production of 3,4-dihydroxybutyric acid. The method provided by the invention can make the production amount of 3,4-dihydroxybutyric acid reach 3.26g/L, and the yield is 0.47g 3,4-dihydroxybutyric acid/1g xylose, and the result is shown in FIG. 2 .

The outstanding features and beneficial effects that the present invention has are:

(1) The present invention provides a new idea for the production of 3,4-dihydroxybutyric acid, which overcomes the toxic effect, high redox pressure, and reversibility of the reaction process of the intermediate product in the process of producing 3,4-dihydroxybutyric acid by a single strain And other issues.

(2) The present invention can make the production amount of 3,4-dihydroxybutyric acid reach 3.26g/L, and has the excellent characteristics of high yield and high production efficiency, and its output is the current biological method to produce 3,4-dihydroxybutyrate. The highest yield of acid.

(3) The present invention provides new guidance for the application of Gluconobacter oxydans in the field of biosynthesis requiring complex pathways.

Description of drawings

Figure 1: pACYCDuet-xylD-kdcA plasmid map.

Figure 2: The process curve of fed-batch fermentation in the co-culture system with glucose and xylose as substrates.

Detailed ways

The content of the present invention will be described in detail below with reference to the specific drawings and embodiments. The following examples are only preferred embodiments of the present invention. It should be noted that the following descriptions are only for explaining the present invention, and do not limit the present invention in any form. Any simple modifications, equivalent changes and modifications made all fall within the scope of the technical solutions of the present invention.

In the following examples, the used Gluconobacter oxydans 621H was purchased from the Global Biological Resource Center (ATCC), and the strain number is: ATCC 621H. The reagents used in the method of the present invention were purchased from Sinopharm Chemical Reagent Co., Ltd. and Sigma-Aldrich (Shanghai) Trading Co., Ltd.; the plasmids pKD4 (CGSC7632), pKD46 (CGSC7669) and pCP20 (CGSC14177) used for knockout were purchased from Yale University Plasmid Gene Collection. Other materials, reagents, etc. used, unless otherwise specified, are obtained from commercial sources.

Example 1: Production of 3,4-dihydroxybutyric acid by fermenting xylose as a substrate using a double-bacteria co-cultivation system composed of Gluconobacter oxydans and Escherichia coli

(1) Construction of a double-bacteria co-cultivation system of Gluconobacter oxydans and engineered Escherichia coli

Take the activated wild-type Gluconobacter oxydans 621H, centrifuge at 6000 rpm for 10 minutes, wash the precipitate with normal saline 1-2 times, and then resuspend it to OD=3 with normal saline for later use; take the activated engineered Escherichia coli Escherichia coli 6KI, centrifuged at 6000 rpm for 10 minutes, washed the precipitate 1-2 times with normal saline, and then resuspended with normal saline to OD=2, for later use; the above two resuspended bacteria were mixed according to the volume ratio of 1:1 After inoculation in the co-cultivation fermentation medium, the final OD of the bacterial liquid is 0.2, and a double-bacteria co-cultivation system of Gluconobacter oxydans and engineered Escherichia coli is constructed;

in:

The above-mentioned wild-type Gluconobacter oxydans 621H is a Gram-negative bacterium and grows strictly aerobic. The optimal culture temperature of the bacterium is 26°C, and it can grow in a sorbitol complex medium. The activation medium of Gluconobacter oxydans 621H is sorbitol complex medium, and its formula is: sorbitol 73g/L, yeast powder 18.4g/L, (NH ₄ ) ₂ SO ₄ 1.5g/L, KH ₂ PO ₄ 1.5g/L, MgSO ₄ ·7H ₂ O 0.47g/L; autoclave at 121°C for 20 minutes.

The genotype of the above engineered Escherichia coli 6KI is Escherichia coli W3110(DE3)ΔxylAΔyjhHΔyagEΔyiaEΔyqdDΔxynR::xylD&kdcA; the bacterium is a gram-negative bacterium that grows aerobic or facultatively anaerobic, and the optimal culture temperature of the bacterium is 37± 1°C, can be grown in LB medium. The activation medium of the engineered Escherichia coli 6KI is LB medium, and its formula is: peptone 10 g/L; yeast powder 5 g/L; NaCl 10 g/L; and autoclaving at 121° C. for 20 minutes.

The above co-cultivation fermentation medium formula is: xylose 7g/L, glucose 5g/L, yeast powder 5g/L, peptone 10g/L, NaCl 10g/L; autoclave at 121°C for 20 minutes. The double bacteria co-culture fermentation system is 50 mL.

The above-mentioned activation modes of Gluconobacter oxydans 621H and Escherichia coli 6KI are:

(1) Plate culture: The strain Escherichia coli 6KI was streaked onto an LB plate containing agar with a mass-to-volume ratio of 1.5-1.8%, and cultured at 37±1°C for 12±1 hours; the strain G.oxydans621H was streaked Lined onto a sorbitol complex medium plate containing agar with a mass-to-volume ratio of 1.5-1.8%, and cultured at 30±1°C for 36±1 hours.

(2) First-class seeds: Under aseptic conditions, pick a single colony on the LB plate in step (1) with a sterile toothpick, then inoculate it into 5 mL of LB liquid medium, shake at 37±1°C with a shaker Cultivate for 12±1 hours; under sterile conditions, pick a single colony on the plate of step (1) sorbitol complex medium with a sterile toothpick, then inoculate it into 5mL sorbitol complex liquid medium, 30± Incubate at 1°C for 24±1 hours.

(3) Secondary seeds: under aseptic conditions, take the Escherichia coli 6KI bacterial liquid cultured in step (2) with an inoculum volume of 1 to 2%, inoculate into 50 mL of LB liquid medium, 37 ± 1 ℃ of shaking and culturing in a shaker for 12±1 hours to obtain the activated Escherichia coli 6KI bacterial liquid; under aseptic conditions, take the G.oxydnas621H bacterial liquid cultivated in step (2) and inoculate it with a volume ratio of 1.5 to 2% amount, inoculated into 50 mL of sorbitol complex liquid medium, and cultured at 30±1°C for 24±1 hours to obtain the activated wild-type Gluconobacter oxydans 621H bacterial liquid.

(2) Fermentation production of 3,4-dihydroxybutyric acid by double bacteria co-culture system

The obtained double-bacteria co-cultivation system was fermented and cultured at 28°C and the shaking speed was 200rpm. When the total OD of the system was 0.8, the final concentration of 1mM IPTG was added for induction, and 10M NaOH was manually added every 3 hours to adjust the culture. The pH of the base is adjusted to 6.8, and the fermentation is carried out for 54 to 60 hours until the sugar consumption is completely terminated, that is, a fermentation broth containing 3,4-dihydroxybutyric acid is obtained.

Example 2: Production of 3,4-dihydroxybutyric acid by fermenting xylose as a substrate using a double-bacteria co-cultivation system composed of Gluconobacter oxydans and Escherichia coli

(1) Construction of a double-bacteria co-cultivation system of Gluconobacter oxydans and engineered Escherichia coli

Take the activated wild-type Gluconobacter oxydans 621H, centrifuge at 6000 rpm for 10 minutes, wash the precipitate with normal saline 1-2 times, and then resuspend it to OD=3 with normal saline for later use; take the activated engineered Escherichia coli Escherichia coli 6KI, centrifuged at 6000 rpm/centrifugation for 10 minutes, washed the precipitate 1-2 times with normal saline, and then resuspended with normal saline to OD=2 for later use; the above two resuspended bacterial solutions were mixed according to the volume ratio of 3:2 After inoculation in the co-cultivation fermentation medium, the final OD of the bacterial liquid is 0.3, and a double-bacteria co-cultivation system of Gluconobacter oxydans and engineered Escherichia coli is constructed;

in:

The above-mentioned wild-type Gluconobacter oxydans 621H is a Gram-negative bacterium and grows strictly aerobic. The optimal culture temperature of the bacterium is 26°C, and it can grow in a sorbitol complex medium. The activation medium of Gluconobacter oxydans 621H is sorbitol complex medium, and its formula is: sorbitol 73g/L, yeast powder 18.4g/L, (NH ₄ ) ₂ SO ₄ 1.5g/L, KH ₂ PO ₄ 1.5g/L, MgSO ₄ ·7H ₂ O 0.47g/L; autoclave at 121°C for 20 minutes.

The genotype of the above engineered Escherichia coli 6KI is Escherichia coli W3110(DE3)ΔxylAΔyjhHΔyagEΔyiaEΔyqdDΔxynR::xylD&kdcA; the bacterium is a gram-negative bacterium that grows aerobic or facultatively anaerobic, and the optimal culture temperature of the bacterium is 37± 1°C, can be grown in LB medium. The activation medium of the engineered Escherichia coli 6KI is LB medium, and its formula is: peptone 10 g/L; yeast powder 5 g/L; NaCl 10 g/L; and autoclaving at 121° C. for 20 minutes.

The above co-cultivation fermentation medium formula is: xylose 9g/L, glucose 6g/L, yeast powder 6g/L, peptone 12g/L, NaCl 12g/L; autoclave sterilization at 121°C for 20 minutes. The double bacteria co-culture fermentation system is 50 mL.

(2) Fermentation production of 3,4-dihydroxybutyric acid by double bacteria co-culture system

The obtained double-bacteria co-cultivation system was fermented and cultured at 30 °C and a shaking table rotation speed of 220 rpm. When the total OD of the system was 0.8, the final concentration of 1 mM IPTG was added for induction, and 10 M NaOH was manually added every 3 hours to adjust the culture. The pH of the base is adjusted to 6.9, and the fermentation is carried out for 54 to 65 hours until the sugar consumption is completely terminated, that is, a fermentation broth containing 3,4-dihydroxybutyric acid is obtained.

The above-mentioned activation methods of Gluconobacter oxydans and Escherichia coli 6KI are the same as those described in Example 1.

Example 3: Production of 3,4-dihydroxybutyric acid by fermenting xylose as a substrate using a double-bacteria co-cultivation system composed of Gluconobacter oxydans and Escherichia coli

(1) Construction of a double-bacteria co-cultivation system of Gluconobacter oxydans and engineered Escherichia coli

Take the activated wild-type Gluconobacter oxydans 621H, centrifuge at 6000 rpm for 10 minutes, wash the precipitate with normal saline 1-2 times, and then resuspend it to OD=3 with normal saline for later use; take the activated engineered Escherichia coli Escherichia coli 6KI, centrifuged at 6000 rpm/centrifugation for 10 minutes, washed the precipitate 1-2 times with normal saline, and then resuspended with normal saline to OD=2, for later use; the above two resuspended bacterial solutions were mixed according to the volume ratio of 1:2 After inoculation in the co-cultivation fermentation medium, the final OD of the bacterial liquid is 0.2, and a double-bacteria co-cultivation system of Gluconobacter oxydans and engineered Escherichia coli is constructed;

in:

The above-mentioned wild-type Gluconobacter oxydans 621H is a Gram-negative bacterium and grows strictly aerobic. The optimal culture temperature of the bacterium is 26°C, and it can grow in a sorbitol complex medium. The activation medium of Gluconobacter oxydans 621H is sorbitol complex medium, and its formula is: sorbitol 73g/L, yeast powder 18.4g/L, (NH ₄ ) ₂ SO ₄ 1.5g/L, KH ₂ PO ₄ 1.5g/L, MgSO ₄ ·7H ₂ O 0.47g/L; autoclave at 121°C for 20 minutes.

The genotype of the above engineered Escherichia coli 6KI is Escherichia coli W3110(DE3)ΔxylAΔyjhHΔyagEΔyiaEΔyqdDΔxynR::xylD&kdcA; the bacterium is a gram-negative bacterium that grows aerobic or facultatively anaerobic, and the optimal culture temperature of the bacterium is 37± 1°C, can be grown in LB medium. The activation medium of the engineered Escherichia coli 6KI is LB medium, and its formula is: peptone 10 g/L; yeast powder 5 g/L; NaCl 10 g/L; and autoclaving at 121° C. for 20 minutes.

The above co-cultivation fermentation medium formula is: xylose 5g/L, glucose 4g/L, yeast powder 4g/L, peptone 8g/L, NaCl 8g/L; autoclave sterilization at 121°C for 20 minutes. The double bacteria co-culture fermentation system is 50 mL.

(2) Fermentation production of 3,4-dihydroxybutyric acid by double bacteria co-culture system

The obtained double bacteria co-cultivation system was fermented and cultured at 26 ° C and the shaking speed was 180 rpm. When the total OD of the system was 0.7, the final concentration of 1 mM IPTG was added for induction, and 10 M NaOH was manually added every 3 hours to adjust the culture. The pH of the base is adjusted to 7.0, and the fermentation is carried out for 57 to 62 hours until the sugar consumption is completely terminated, that is, a fermentation broth containing 3,4-dihydroxybutyric acid is obtained.

The above-mentioned activation methods of Gluconobacter oxydans and Escherichia coli 6KI are the same as those described in Example 1.

Example 4: Construction of genetically engineered strain Escherichia coli 6KI

Taking Escherichia coli W3110 (DE3) as the starting strain, the strain was continuously genetically modified by using Red recombination technology (Datsenko KA et al., 2000, Proc.Natl.Acad.Sci.US A., 97:6640-6645), The general steps are as follows:

(1) The xylose isomerase gene xylA and the 2-keto-3-deoxyxylonate aldolase genes yjhH and yagE were knocked out in Escherichia coli W3110(DE3) by genetic engineering, blocking the strain from using xylose and the endogenous pathway of xylonic acid to construct strain Escherichia coli 3K.

(2) Knock out the xylonic acid operon transcriptional repressor gene xynR in Escherichia coli 3K by genetic engineering, block the regulation of the transcriptional repressor XynR on the xylonic acid operon, and enhance the xylonic acid dehydratase and xylonic acid The expression of transporter protein enhances the synthesis and metabolism of the target product, and the strain Escherichia coli 4K is constructed.

(3) Knock out the NADPH-dependent aldehyde reductase gene yqhD in Escherichia coli 4K by genetic engineering, block the reduction of 3,4-dihydroxybutyraldehyde to form 1,2,4-butanetriol, and construct strain Escherichia coli 5K .

(4) The glyoxylate reductase gene yiaE was knocked out in Escherichia coli 5K by genetic engineering, weakening the reduction reaction of the intermediate product 2-keto-3-deoxyxylonic acid, enhancing the synthesis and metabolism of the target product, and constructing the strain Escherichia coli 6K .

(5) Commercially insert the xylonic acid dehydratase gene xylD from Caulobacter crescentus and the branched-2-keto acid decarboxylase gene kdcA from Lactococcus lactis by genetic engineering The plasmid pACYCDuet-1 (Fig. 1) was used to amplify the gene expression cassette by PCR, construct an operon and knock it into the original xynR site of the Escherichia coli 6K genome to construct an engineered Escherichia coli 6KI.

The genes involved in the above-mentioned Escherichia coli 6KI genetically engineered strains and related transformation methods have been partially disclosed in patent CN201710611460.1, and the transformation methods are described in patent CN201710611460.1.

The specific operation method is:

(1) Construction of gene knockout engineering bacteria

(1) Knockout method: The gene knockout method used in the present invention is the Red recombination knockout technology (Datsenko KA etal., Proc.Natl.Acad.Sci.US A., 2000, 97:6640-6645). In this method, the plasmids pKD4 (CGSC7632), pKD46 (CGSC7669) and pCP20 (CGSC14177) used for the knockout were purchased from the Yale University Plasmid Gene Collection.

(2) Obtainment of mutant fragments: The nucleotide mutant fragments used in the above method have two homology arms upstream and downstream of the target knockout gene and a kanamycin resistance gene expression cassette. The nucleotide mutation fragment of the target gene is obtained by recombinant PCR.

Taking xylA knockout as an example, use xylA upstream primer 1/xylA downstream primer 1 to amplify the upstream homology arm of the target gene with Escherichia coliK12 genome as template; use xylA upstream primer 2/xylA downstream primer 2 to amplify the card with plasmid pKD4 as template Kanamycin resistance gene expression cassette; use xylA upstream primer 3/xylA downstream primer 3 to amplify the downstream homology arm of the target gene with Escherichia coli K12 genome as a template; two homology arms and kanamycin resistance gene expression cassette Recombinant PCR was performed to obtain the xylA mutant fragment. The mutated fragments of the remaining genes yjhH, yagE, xynR, yqhD and yiaE were obtained in the same manner as the above method, and the primer sequences of the nucleotide mutant fragments used are as follows:

xylA upstream primer-5'-GTAGTTAGAGGACAGTTTTAATAAG-3'

xylA downstream primer-5'-AGCTCCAGCCTACACATTGAACTCCATAA-3'

xylA upstream primer two 5'-ATTATGGAGTTCAATGTGTAGGCTGGAGCT-3'

xylA downstream primer two 5'-CTGCACAGTTAGCCGATGGGAATTAGCCATG-3'

xylA upstream primer three 5'-ATGGCTAATTCCCATCGGCTAACTGTGCAG-3'

xylA downstream primer three 5'-TCTGGCCGGCAATACCCAATGCTTT-3'

yjhH upstream primer-5'-ATACGCGCAATACATTTACCGATAAAA-3'

yjhH downstream primer-5'-CTCCAGCCTACACTACCTCAGTTTC-3'

yjhH upstream primer two 5'-GGAAACTGAGGTAGTGTAGGCTGGA-3'

yjhH downstream primer two 5'-ATGAGTTTCTCCATGGGAATTAGCC-3'

yjhH upstream primer three 5'-GGCTAATTCCCATGGAGAAACTCATGT-3'

yjhH downstream primer three 5'-TTCATCTGGATGTCCAGTTCGTAAT-3'

yagE upstream primer-5'-CTCCATAAACGGGTTCTTATGCCTT-3'

yagE downstream primer-5'-CTCCAGCCTACACGAGATCTCCTTG-3'

yagE upstream primer two 5'-GCAAGGAGATCTCGTGTAGGCTGGA-3'

yagE downstream primer two 5'-GTTATCGTCCGGCATGGGAATTAGC-3'

yagE upstream primer three 5'-GGCTAATTCCCATGCCGGACGATAA-3'

yagE downstream primer three 5'-TCTGCATGCCGATCTCCCAATGCCC-3'

xynR upstream primer-5'-AACGTGAAGTTCCTGCACTGTCT-3'

xynR downstream primer-5'-GAAGCAGCTCCAGCCTACACAATGCTGGCATGTCCACGC-3'

xynR upstream primer two 5'-AGCGTGGACATGCCAGCATTGTGTAGGCTGGAGCTGCTTC-3'

xynR downstream primer two 5'-CTACGAGCCGGTCTAACGGCATGGGAATTAGCCATGGTCCA-3'

xynR upstream primer three 5'-GGACCATGGCTAATTCCCATGCCGTTAGACCGGCTCGT-3'

xynR downstream primer three 5'-CTTTGTGGACTACGAGGAGGGA-3'

yqhD upstream primer-5'-CCATACAACAAACGCACATCGGGCA-3'

yqhD downstream primer-5'-AGCTCCAGCCTACACTACTTGCTCCCTTTG-3'

yqhD upstream primer two 5'-CAAAGGGAGCAAGTAGTGTAGGCTGGAGC-3'

yqhD downstream primer two 5'-TGAGGCGTAAAAAGCATGGGAATTAGCCATGG-3'

yqhD upstream primer three 5'-ATGGCTAATTCCCATGCTTTTTACGCCCTCA-3'

yqhD downstream primer three 5'-CTGAGGCATTTTCAGGGCTTTGCCG-3'

yiaE upstream primer-5'-CGGGTGGTCACGACCTGAACATGC-3'

yiaE downstream primer-5'-TCCAGCCTACACGCTTCTCTCCATT-3'

yiaE upstream primer two 5'-ATGGAGAGAAGCGTGTAGGCTGGAG-3'

yiaE downstream primer two 5'-CGCAGTCGCGGCATGGGAATTAGC-3'

yiaE upstream primer three 5'-GCTAATTCCCATGCCGCGACTGCGT-3'

yiaE downstream primer three 5'-CCAAAGTGGTAACGACGCCAGCTGA-3'

(3) Competent preparation and electrotransformation: After the pKD46 plasmid was chemically introduced into the target strain, the recombinant strain was screened on a LB solid plate carrying 50 μg/mL spectinomycin resistance, and cultured and competently prepared after verification. The recombinant strains were cultured in 50 mL LB liquid medium with 50 μg/mL spectinomycin resistance, incubated at 30°C for 30 minutes, induced by adding IPTG with a final concentration of 0.5 mM, and centrifuged at 6000 rpm for 10 minutes when the OD reached 0.5-0.6. After removing the supernatant, the cells were washed twice with 40 mL of 4°C pre-cooled pure water, and then washed once with 40 mL of 40 mL of 4°C pre-cooled 10% glycerol. The cells collected by centrifugation were resuspended in 150 μL of 10% glycerol, mixed with 100 ng/μL of nucleotide mutant fragments, and distributed into two pre-cooled 2 mm electroporation cups. Use an electroporator under the conditions of a voltage of 2200V, a capacitance of 25μF, and a resistance of 200Ω. Use 500 μL of LB liquid to resuspend the bacterial solution, incubate at 37°C for 40 minutes in a shaker at 180 rpm, and coat a plate containing 50 μg/mL kanamycin resistance. After culturing in a 37°C incubator for 12-13 hours, pick a single colony. PCR verification.

(4) Elimination of kanamycin resistance gene: The strain verified by PCR was chemically introduced into pCP20 plasmid, incubated at 30°C, 180 rpm shaker for 40 minutes, and then coated with LB containing 40 μg/mL chloramphenicol resistance After culturing for 16-17 hours in a 30°C incubator, a single colony was picked for verification. The verified strains were inoculated into anti-anti-LB liquid medium, placed at 42°C, cultured on a shaker at 180 rpm for 2 generations, and then streaked at 37°C. Pick the same colony and spot it on LB non-antibody plate, 40 μg/mL chloramphenicol-resistant LB plate and 50 μg/mL kanamycin-resistant plate respectively, and culture for 12-13 hours. Select colonies that grow on non-resistant plates but do not grow on resistant plates for strain PCR verification. The correct strain will be verified and kept ready for use.

The knocked-out xylose isomerase xylA gene sequence is 1323 bases in length, and its nucleotide sequence is shown in SEQ ID NO. The length of the gene sequence is 906 bases, and its nucleotide sequence is shown in SEQ ID NO.2; the length of the knockout 2-keto-3-deoxyxylonate aldolase yagE gene sequence is 909 bases, Its nucleotide sequence is shown in SEQ ID NO.3; the knockout xylon acid operon transcription inhibitor xynR gene sequence is 759 bases in length, and its nucleotide sequence is shown in SEQ ID NO.4; knockout The removed NADPH-dependent aldehyde reductase yqhD gene sequence is 1164 bases in length, and its nucleotide sequence is shown in SEQ ID NO. 5; the knocked out glyoxylate reductase gene yiaE gene sequence is 975 bases in length , and its nucleotide sequence is shown in SEQ ID NO.6.

(2) Construction of gene knock-in engineered bacteria

(1) Gene cloning: The original pET28a-xylD and pET28a-kdcA vectors carrying the xylonic acid dehydratase gene xylD and the branched-2-keto acid decarboxylase gene kdcA respectively were provided by Universal Biosystems (Anhui) Co., Ltd. (Chuzhou, China) China, Anhui) synthesis. The nucleotide sequence of xylD dehydratase gene xylD is shown in SEQ ID NO.7; the nucleotide sequence of branched-chain-2-keto acid decarboxylase gene kdcA is shown in SEQ ID NO.8. Primers were designed based on the nucleotide sequence of xylD, and a synthetic primer was used to amplify the xylD fragment with pET28a-xylD as a template, and the fragment contained restriction sites BamHI and SacI. The amplification primer sequences are as follows:

Upstream primer 5'-ATATGGATCCGATGCGTAGTGCCCT-3', carrying a BamHI site;

The downstream primer, 5'-ATGCGAGCTCTTAATGATTATGGCG-3', carries a SacI site.

(2) The fragment amplified by PCR in step (1) is recovered and subjected to restriction enzyme digestion reaction and nucleotide electrophoresis with plasmid pACYCDuet-1 under the restriction enzymes BamHI and SacI. The recovered fragment xylD and pACYCDuet-1 were ligated to obtain a recombinant plasmid pACYCDuet-xylD.

(3) Design primers based on the kdcA nucleotide sequence, and use synthetic primers to amplify with pET28a-kdcA as a template to obtain a kdcA fragment containing restriction sites BglII and XhoI. The amplification primer sequences are as follows:

Upstream primer 5'-ATATAGATCTCATGTACACCGTTGGCG-3', carrying a BglII site;

The downstream primer, 5'-ATATCTCGAGTTACTTATTCTGTTC-3', carries an XhoI site.

(4) The fragment amplified by PCR in step (3) is recovered and subjected to restriction enzyme digestion reaction and nucleotide electrophoresis with plasmid pACYCDuet-xylD under the restriction enzymes BglII and XhoI. The recovered fragment kdcA and pACYCDuet-xylD were ligated to obtain the recombinant plasmid pACYCDuet-xylD-kdcA.

(5) Design primers with the sequence of recombinant plasmid pACYCDuet-xylD-kdcA, and use synthetic primers to amplify the gene knock-in expression cassette with plasmid pACYCDuet-xylD-kdcA as a template. All gene knock-in amplification primer sequences are as follows:

Upstream primer-5'-CTGGATCTGCGCCTGTTGCCCCGA-3', xynR upstream homology arm amplification primer;

Downstream primer - 5'-CCTAATGCAGGAGTCGCATAAATGCTGGCATGTCCACGCT-3', xynR upstream homology arm amplification primer;

Upstream primer two 5'-AGCGTGGACATGCCAGCATTTATGCGACTCCTGCATTAGG-3', gene knock-in expression cassette amplification primer;

Downstream primer two 5'-GAAGCAGCTCCAGCCTACACCAAAAAACCCCTCAAGACCC-3', gene knock-in expression cassette amplification primer;

Upstream primer three 5'-GGGTCTTGAGGGGTTTTTTGGTGTAGGCTGGAGCTGCTTC-3', kanamycin resistance gene expression cassette amplification primer;

Downstream primer three 5'-CTACGAGCCGGTCTAACGGCATGGGAATTAGCCATGGTCC-3', kanamycin resistance gene expression cassette amplification primer;

Upstream primer four 5'-GGACCATGGCTAATTCCCATGCCGTTAGACCGGCTCGTAG-3', xynR downstream homology arm amplification primer;

Downstream primer four 5'-CGCTTGACCCGGAGCTGCAGACCCT-3', xynR downstream homology arm amplification primer;

(6) Perform four-segment recombination PCR on the xynR upstream and downstream homology arms, gene knock-in expression cassette, and kanamycin resistance gene expression cassette nucleotides amplified by PCR in step (5) to obtain gene knock-in mutant fragments .

(7) The gene knock-in method is consistent with the gene knock-out method, and the mutant fragments are obtained in the steps (1) to (6), and the engineered strain Escherichia coli 6KI is obtained after the gene knock-in of Escherichia coli 6K.

Wherein, the length of the expressed xylonate dehydratase xylD gene sequence is 1788 bases, and its nucleotide sequence is as shown in SEQ ID NO.7; the length of the expressed branched-chain-2-keto acid decarboxylase kdcA gene sequence is 1644 bases, and its nucleotide sequence is shown in SEQ ID NO.8. The map of the expression vector pACYCDuet-xylD-kdcA is shown in Figure 1.

The above-mentioned recombinant Escherichia coli 6KI is a gram-negative bacterium that grows aerobic or facultatively anaerobic, and the optimal culture temperature is 37±1°C. It can grow on LB medium and can be used with Wild-type G. oxydans combined to construct a co-culture system to produce 3,4-dihydroxybutyric acid from xylose substrate.

sequence listing

<110> Shandong University

<120> A method for producing 3,4-dihydroxybutyric acid using a double bacteria co-cultivation system

<141>2021-01-13

<160>8

<210> 1

<211> 1323

<212> DNA

<213> Escherichia coli

<221> Xylose isomerase xylA gene

<222>(1)…(1323)

<400> 1

atgcaagcct attttgacca gctcgatcgc gttcgttatg aaggctcaaa atcctcaaac 60

ccgttagcat tccgtcacta caatcccgac gaactggtgt tgggtaagcg tatggaagag 120

cacttgcgtt ttgccgcctg ctactggcac accttctgct ggaacggggc ggatatgttt 180

ggtgtggggg cgtttaatcg tccgtggcag cagcctggtg aggcactggc gttggcgaag 240

cgtaaagcag atgtcgcatt tgagtttttc cacaagttac atgtgccatt ttattgcttc 300

cacgatgtgg atgtttcccc tgagggcgcg tcgttaaaag agtacatcaa taattttgcg 360

caaatggttg atgtcctggc aggcaagcaa gaagagagcg gcgtgaagct gctgtgggga 420

acggccaact gctttacaaa ccctcgctac ggcgcgggtg cggcgacgaa cccagatcct 480

gaagtcttca gctgggcggc aacgcaagtt gttacagcga tggaagcaac ccataaattg 540

ggcggtgaaa actatgtcct gtggggcggt cgtgaaggtt acgaaacgct gttaaatacc 600

gacttgcgtc aggagcgtga acaactgggc cgctttatgc agatggtggt tgagcataaa 660

cataaaatcg gtttccaggg cacgttgctt atcgaaccga aaccgcaaga accgaccaaa 720

catcaatatg attacgatgc cgcgacggtc tatggcttcc tgaaacagtt tggtctggaa 780

aaagagatta aactgaacat tgaagctaac cacgcgacgc tggcaggtca ctctttccat 840

catgaaatag ccaccgccat tgcgcttggc ctgttcggtt ctgtcgacgc caaccgtggc 900

gatgcgcaac tgggctggga caccgaccag ttcccgaaca gtgtggaaga gaatgcgctg 960

gtgatgtatg aaattctcaa agcaggcggt ttcaccaccg gtggtctgaa cttcgatgcc 1020

aaagtacgtc gtcaaagtac tgataaatat gatctgtttt acggtcatat cggcgcgatg 1080

gatacgatgg cactggcgct gaaaattgca gcgcgcatga ttgaagatgg cgagctggat 1140

aaacgcatcg cgcagcgtta ttccggctgg aatagcgaat tgggccagca aatcctgaaa 1200

ggccaaatgt cactggcaga tttagccaaa tatgctcagg aacatcattt gtctccggtg 1260

catcagagtg gtcgccagga acaactggaa aatctggtaa accattatct gttcgacaaa 1320

taa 1323

<210> 2

<211> 906

<212> DNA

<213> Escherichia coli

<221> 2-keto-3-deoxyxylonate aldolase yjhH gene

<222>(1)…(906)

<400> 2

atgaaaaaat tcagcggcat tattccaccg gtatccagca cgtttcatcg tgacggaacc 60

cttgataaaa aggcaatgcg cgaagttgcc gacttcctga ttaataaagg ggtcgacggg 120

ctgttttatc tgggtaccgg tggtgaattt agccaaatga atacagccca gcgcatggca 180

ctcgccgaag aagctgtaac cattgtcgac gggcgagtgc cggtattgat tggcgtcggt 240

tccccttcca ctgacgaagc ggtcaaactg gcgcagcatg cgcaagccta cggcgctgat 300

ggtatcgtcg ccatcaaccc ctactactgg aaagtcgcac cacgaaatct tgacgactat 360

taccagcaga tcgcccgtag cgtcacccta ccggtgatcc tgtacaactt tccggatctg 420

acgggtcagg acttaacccc ggaaaccgtg acgcgtctgg ctctgcaaaa cgagaatatc 480

gttggcatca aagacaccat cgacagcgtt ggtcacttgc gtacgatgat caacacagtt 540

aagtcggtac gcccgtcgtt ttcggtattc tgcggttacg atgatcattt gctgaatacg 600

atgctgctgg gcggcgacgg tgcgataacc gccagcgcta actttgctcc ggaactctcc 660

gtcggcatct accgcgcctg gcgtgaaggc gatctggcga ccgctgcgac gctgaataaa 720

aaactactac aactgcccgc tatttacgcc ctcgaaacac cgtttgtctc actgatcaaa 780

tacagcatgc agtgtgtcgg gctgcctgta gagacatatt gcttaccacc gattcttgaa 840

gcatctgaag aagcaaaaga taaagtccac gtgctgctta ccgcgcaggg cattttacca 900

gtctga 906

<210>3

<211> 909

<212> DNA

<213> Escherichia coli

<221> 2-keto-3-deoxyxylonate aldolase yagE gene

<222> (1)…(909)

<400> 3

atgccgcagt ccgcgttgtt cacgggaatc attccccctg tctccaccat ttttaccgcc 60

gacggccagc tcgataagcc gggcaccgcc gcgctgatcg acgatctgat caaagcaggc 120

gttgacggcc tgttcttcct gggcagcggt ggcgagttct cccagctcgg cgccgaagag 180

cgtaaagcca ttgcccgctt tgctatcgat catgtcgatc gtcgcgtgcc ggtgctgatc 240

ggcaccggcg gcaccaacgc ccgggaaacc atcgaactca gccagcacgc gcagcaggcg 300

ggcgcggacg gcatcgtggt gatcaacccc tactactgga aagtgtcgga agcgaacctg 360

atccgctatt tcgagcaggt ggccgacagc gtcacgctgc cggtgatgct ctataacttc 420

ccggcgctga ccgggcagga tctgactccg gcgctggtga aaaccctcgc cgactcgcgc 480

agcaatatta tcggcatcaa agacaccatc gactccgtcg cccacctgcg cagcatgatc 540

cataccgtca aaggtgccca tccgcacttc accgtgctct gcggctacga cgatcatctg 600

ttcaataccc tgctgctcgg cggcgacggg gcgatatcgg cgagcggcaa ctttgccccg 660

caggtgtcgg tgaatcttct gaaagcctgg cgcgacgggg acgtggcgaa agcggccggg 720

tatcatcaga ccttgctgca aattccgcag atgtatcagc tggatacgcc gtttgtgaac 780

gtgattaaag aggcgatcgt gctctgcggt cgtcctgtct ccacgcacgt gctgccgccc 840

gcctcgccgc tggacgagcc gcgcaaggcg cagctgaaaa ccctgctgca acagctcaag 900

ctttgctga 909

<210> 4

<211> 759

<212> DNA

<213> Escherichia coli

<221> xylonic acid operon transcriptional repressor xynR gene

<222> (1)…(759)

<400> 4

atgccgatta ttcagtctgt tgaacgtgcg ttgcagatcc tcgacctgtt caacgagcag 60

gccaccgagc ttaagatcac cgacatcagc aaactgatgg ggctgagcaa gagtaccctc 120

cactcgctgc taaaaaccct gcagcttcac ggctatatcg atcagaaccc ggagaacggc 180

aagtatcgcc tcggcatgaa gctggtcgag cgcggccatt ttgtcgtggg ctccatcgat 240

attcggcaga aggcaaaagg ctggctgacg gagctgtccc ggcggaccgg gcagaccacc 300

catctgggga tcctggacgg gcgtgaaggg gtctatatcg agaagattga aggcaagctg 360

gccgccatcg cctattcacg catcggccgc cgcctgccgg tgcacgccac cgccatcggc 420

aaggtgttga ttgcctggct gggcgaggcc gagctgaacg ccctgctgga gggctatcag 480

tacactacct ttacgcccgc caccctcgcg tctcgcgaag ccttaatgag cgccctggcg 540

cagacccgcg agcaaggcta cgccctggac agcgaagaga acgagcaggg cgtgcgctgc 600

gtggcggtgc cggtgtggaa ccacgagtcc cgcgtcatcg ccgccctgag cctgtcgacg 660

ctgacctccc gcgtggacga cgcggagctg gctaatttcc gcgagcagct tcagcaggcc 720

gggctcgcgc tctcgcgcgc gctgggctac ccggcctga 759

<210> 5

<211> 1164

<212> DNA

<213> Escherichia coli

<221> NADPH-dependent aldehyde reductase yqhD gene

<222>(1)…(1164)

<400> 5

atgaacaact ttaatctgca caccccaacc cgcattctgt ttggtaaagg cgcaatcgct 60

ggtttacgcg aacaaattcc tcacgatgct cgcgtattga ttacctacgg cggcggcagc 120

gtgaaaaaaa ccggcgttct cgatcaagtt ctggatgccc tgaaaggcat ggacgtgctg 180

gaatttggcg gtattgagcc aaacccggct tatgaaacgc tgatgaacgc cgtgaaactg 240

gttcgcgaac agaaagtgac tttcctgctg gcggttggcg gcggttctgt actggacggc 300

accaaattta tcgccgcagc ggctaactat ccggaaaata tcgatccgtg gcacattctg 360

caaacgggcg gtaaagagat taaaagcgcc atcccgatgg gctgtgtgct gacgctgcca 420

gcaaccggtt cagaatccaa cgcaggcgcg gtgatctccc gtaaaaccac aggcgacaag 480

caggcgttcc attctgccca tgttcagccg gtatttgccg tgctcgatcc ggtttatacc 540

tacaccctgc cgccgcgtca ggtggctaac ggcgtagtgg acgcctttgt acacaccgtg 600

gaacagtatg ttaccaaacc ggttgatgcc aaaattcagg accgtttcgc agaaggcatt 660

ttgctgacgc taatcgaaga tggtccgaaa gccctgaaag agccagaaaa ctacgatgtg 720

cgcgccaacg tcatgtgggc ggcgactcag gcgctgaacg gtttgattgg cgctggcgta 780

ccgcaggact gggcaacgca tatgctgggc cacgaactga ctgcgatgca cggtctggat 840

cacgcgcaaa cactggctat cgtcctgcct gcactgtgga atgaaaaacg cgataccaag 900

cgcgctaagc tgctgcaata tgctgaacgc gtctggaaca tcactgaagg ttccgatgat 960

gagcgtattg acgccgcgat tgccgcaacc cgcaatttct ttgagcaatt aggcgtgccg 1020

acccacctct ccgactacgg tctggacggc agctccatcc cggctttgct gaaaaaactg 1080

gaagagcacg gcatgaccca actgggcgaa aatcatgaca ttacgttgga tgtcagccgc 1140

cgtatatacg aagccgcccg ctaa 1164

<210> 6

<211> 975

<212> DNA

<213> Escherichia coli

<221> Glyoxylate reductase gene yiaE gene

<222> (1)…(975)

<400>6

atgaagccgt ccgttatcct ctacaaagcc ttacctgatg atttactgca acgcctgcaa 60

gagcatttca ccgttcacca ggtggcaaac ctcagcccac aaaccgtcga acaaaatgca 120

gcaatttttg ccgaagctga aggtttactg ggttcaaacg agaatgtaaa tgccgcattg 180

ctggaaaaaa tgccgaaact gcgtgccaca tcaacgatct ccgtcggcta tgacaatttt 240

gatgtcgatg cgcttaccgc ccgaaaaatt ctgctgatgc acacgccaac cgtattaaca 300

gaaaccgtcg ccgatacgct gatggcgctg gtgttgtcta ccgctcgtcg ggttgtggag 360

gtagcagaac gggtaaaagc aggcgaatgg accgcgagca taggcccgga ctggtacggc 420

actgacgttc accataaaac actgggcatt gtcgggatgg gacggatcgg catggcgctg 480

gcacaacgtg cgcactttgg cttcaacatg cccatcctct ataacgcgcg ccgccaccat 540

aaagaagcag aagaacgctt caacgcccgc tactgcgatt tggatactct gttacaagag 600

tcagatttcg tttgcctgat cctgccgtta actgatgaga cgcatcatct gtttggcgca 660

gaacaattcg ccaaaatgaa atcctccgcc attttcatta atgccggacg tggcccggtg 720

gttgacgaaa atgcactgat cgcagcattg cagaaaggcg aaattcacgc tgccgggctg 780

gatgtcttcg aacaagagcc actgtccgta gattcgccgt tgctctcaat ggccaacgtc 840

gtcgcagtac cgcatattgg atctgccacc catgagacgc gttatggcat ggccgcctgt 900

gccgtggata atttgattga tgcgttacaa ggaaaggttg agaagaactg tgtgaatccg 960

cacgtcgcgg actaa 975

<210> 7

<211> 1788

<212> DNA

<213> Caulobacter crescentus

<221> Nucleotide sequence of xylonate dehydratase gene xylD

<222> (1)…(1788)

<400> 7

atgcgtagtg ccctgagtaa tcgtaccccg cgccgttttc gtagccgcga ttggtttgat 60

aatccggatc atattgatat gaccgcactg tatctggaac gctttatgaa ttatggcatt 120

accccggaag aactgcgtag tggtaaaccg attattggca ttgcccagac cggtagtgat 180

attagtccgt gtaatcgcat tcatctggat ctggtgcagc gtgttcgcga tggcattcgc 240

gatgccggtg gcattccgat ggaatttccg gttcatccga tttttgaaaa ttgccgtcgt 300

ccgaccgccg cactggatcg caatctgagc tatctgggcc tggttgaaac cctgcatggt 360

tatccgattg atgcagttgt tctgaccacc ggctgcgata aaaccacccc ggccggtatt 420

atggcagcaa ccaccgtgaa tattccggcc attgttctga gcggcggtcc gatgctggat 480

ggttggcatg aaaatgaact ggtgggcagc ggcaccgtta tttggcgcag tcgtcgcaaa 540

ctggccgcag gcgaaattac cgaagaagag tttattgatc gtgcagcaag tagtgcaccg 600

agcgccggcc attgtaatac catgggtaca gcaagcacca tgaatgcagt ggccgaagca 660

ctgggcctga gtctgaccgg ctgcgccgct attccggccc cttatcgtga acgtggccag 720

atggcatata aaaccggcca gcgcattgtt gatctggcat atgatgatgt gaaaccgctg 780

gatattctga ccaaacaggc atttgaaaat gccattgcac tggttgcagc cgccggtggc 840

agcaccaatg cacagccgca tattgttgcc atggcccgtc atgccggcgt ggaaattacc 900

gcagatgatt ggcgcgccgc atatgatatt ccgctgattg tgaatatgca gccggcaggc 960

aaatatctgg gtgaacgttt tcatcgcgca ggtggtgccc cggcagtgct gtgggaactg 1020

ctgcagcagg gtcgcctgca tggcgatgtt ctgaccgtga ccggcaaaac catgagtgaa 1080

aatctgcagg gccgcgaaac cagcgatcgc gaagttattt ttccgtatca tgaaccgctg 1140

gccgaaaaag ccggttttct ggttctgaaa ggcaatctgt ttgattttgc aattatgaaa 1200

agcagtgtga ttggtgaaga atttcgtaaa cgctatctga gtcagccggg tcaggaaggt 1260

gtgtttgaag cccgtgccat tgtttttgat ggcagcgatg attatcataa acgtattaat 1320

gacccggccc tggaaattga tgaacgttgc attctggtta ttcgcggtgc cggcccgatt 1380

ggctggccgg gtagtgcaga agtggtgaat atgcaaccgc cggatcatct gctgaaaaaa 1440

ggcattatga gcctgccgac cctgggtgac ggtcgccaga gcggtacagc agatagtccg 1500

agcattctga atgccagccc ggaaagcgcc attggtggcg gcctgagttg gctgcgcacc 1560

ggtgacacca ttcgcattga tctgaatacc ggccgctgcg atgccctggt tgatgaagca 1620

accattgcag cccgtaaaca ggatggtatt ccggcagttc cggccaccat gaccccgtgg 1680

caggaaatct atcgtgcaca tgccagccag ctggataccg gtggtgttct ggaatttgca 1740

gtgaaatatc aggatctggc cgcaaaactg ccgcgccata atcattaa 1788

<210> 8

<211> 1644

<212> DNA

<213> Lactococcus lactis

<221> Branched-chain-2-ketoacid decarboxylase gene kdcA nucleotide sequence

<222>(1)…(1644)

<400> 8

atgtacaccg ttggcgatta tctgctggat cgtctgcatg aactgggtat tgaagaaatt 60

tttggtgttc cgggtgacta taatctgcag tttctggatc agattattag tcgcgaagat 120

atgaaatgga ttggtaatgc caatgaactg aatgcaagtt atatggccga tggttatgcc 180

cgcaccaaaa aagcagcagc ctttctgacc acctttggcg tgggtgaact gagtgcaatt 240

aatggtctgg ccggtagtta tgccgaaaat ctgccggtgg ttgaaattgt tggcagtccg 300

accagcaaag ttcagaatga tggtaaattt gtgcatcata ccctggcaga tggcgatttt 360

aaacatttta tgaaaatgca cgagccggtg accgcagccc gtaccctgct gaccgcagaa 420

aatgcaacct atgaaattga tcgtgtgctg agtcagctgc tgaaagaacg caaaccggtt 480

tatattaatc tgccggttga tgttgccgcc gccaaagcag aaaaaccggc cctgagtctg 540

gaaaaagaaa gcagcaccac caataccacc gaacaggtta ttctgagcaa aattgaagaa 600

agcctgaaaa atgcacagaa accggttgtt attgcaggcc atgaagtgat tagctttggt 660

ctggaaaaaa ccgtgaccca gtttgttagc gaaaccaaac tgccgattac caccctgaat 720

tttggtaaaa gtgcagtgga tgaaagcctg ccgagttttc tgggcatcta taatggcaaa 780

ctgagtgaaa ttagtctgaa aaatttcgtg gaaagcgcag attttattct gatgctgggt 840

gttaaactga ccgatagcag caccggcgcc tttacccatc atctggatga aaataagatg 900

attagcctga atatcgatga aggtattatt tttaacaagg tggttgaaga tttcgatttt 960

cgtgcagtgg tgagtagtct gagtgaactg aaaggcattg aatatgaagg tcagtatatt 1020

gataagcagt atgaagagtt tattccgagt agcgccccgc tgagtcagga tcgcctgtgg 1080

caggccgttg aaagtctgac ccagagtaat gaaaccattg ttgccgaaca gggtaccagc 1140

tttttcggcg caagtaccat ttttctgaaa agtaatagcc gctttatcgg ccagccgctg 1200

tggggtagta ttggttatac ctttccggca gccctgggca gccagattgc agataaagaa 1260

agccgtcatc tgctgtttat tggcgatggt agtctgcagc tgaccgttca ggaactgggt 1320

ctgagcattc gtgaaaaact gaatccgatt tgttttatta tcaacaacga cggctatacc 1380

gtggaacgtg aaattcatgg tccgacccag agttataatg atattccgat gtggaattac 1440

agcaaactgc cggaaacctt tggcgcaacc gaagatcgtg ttgttagtaa aattgtgcgt 1500

accgaaaatg aatttgttag cgtgatgaaa gaagcacagg ccgatgttaa tcgtatgtat 1560

tggattgaac tggtgctgga aaaagaggat gcaccgaaac tgctgaaaaa gatgggcaaa 1620

ctgtttgccg aacagaataa gtaa 1644

Claims (4)

1. A method for producing 3, 4-dihydroxybutyric acid by using a double-bacterium co-culture system comprises the following steps:

(1) double-bacterium co-culture system for constructing gluconobacter oxydans and engineering escherichia coli

Taking activated gluconobacter oxydans, centrifuging at 6000 rpm, washing for 1-2 times by using normal saline, and then re-suspending by using normal saline until OD is 3 for later use; taking activated engineering escherichia coli, centrifuging at 6000 rpm, washing for 1-2 times by using normal saline, and then re-suspending by using normal saline until OD is 2 for later use; mixing the two kinds of heavy suspension bacteria liquid according to a volume ratio of 1-3: 1-2, inoculating the mixture into a co-culture fermentation medium, and enabling the final OD of the bacteria liquid to be 0.2 +/-0.1, so as to construct a double-bacteria co-culture system of the gluconobacter oxydans and the engineering escherichia coli; wherein,

the Gluconobacter oxydans is selected from wild type Gluconobacter oxydans 621H;

the engineering Escherichia coli is engineering Escherichia coli6KI, the genotype of which is Escherichia coli W3110(DE3) delta xylA delta yjhH delta yagE delta yaiaE delta yqdD delta xynR, xylD & kdcA; the construction method of the engineering Escherichia coli6KI comprises the following steps: escherichia coli W3110(DE3) is used as an original strain, and the strain is subjected to the following continuous gene modification by adopting Red recombination technology: 1) knocking out xylose isomerase genes xylA and 2-keto-3-deoxyxylonic acid aldolase genes yjhH and yagE in Escherichia coli W3110(DE3) by adopting a genetic engineering means, blocking endogenous ways of strains for utilizing xylose and xylonic acid, and constructing a strain Escherichia coli 3K; 2) knocking out xylonic acid operon transcription inhibiting factor gene xynR in Escherichia coli 3K by adopting a gene engineering means, blocking the regulation of the xylonic acid operon by the transcription inhibiting factor xynR, enhancing the expression of xylonic acid dehydratase and xylonic acid transport protein, enhancing the anabolism of a target product, and constructing a strain Escherichia coli 4K; 3) knocking out an NADPH dependent aldehyde reductase gene yqhD in Escherichia coli 4K by adopting a genetic engineering means, blocking 3, 4-dihydroxy butyraldehyde reduction to form 1,2, 4-butanetriol, and constructing a strain Escherichia coli 5K; 4) knocking out glyoxylate reductase gene yiAE in Escherichia coli 5K by adopting a gene engineering means, weakening the reduction reaction of an intermediate product 2-keto-3-deoxyxylonic acid, enhancing the anabolism of a target product, and constructing a strain Escherichia coli 6K; 5) inserting xylonic acid dehydratase gene xylD from corynebacterium parvum (Caulobacter creescens) and branched-2-keto acid decarboxylase gene kdcA from Lactococcus lactis into a commercialized plasmid pACYCDuet-1 by adopting a genetic engineering means, amplifying a gene expression frame by PCR, constructing an operon, and knocking the operon into an Escherichia coli6K genome original xynR site to construct an engineering Escherichia coli strain Escherichia coli6 KI;

the formula of the co-culture fermentation medium is as follows: 7 +/-2 g/L of xylose, 5 +/-1 g/L of glucose, 5 +/-1 g/L of yeast powder, 10 +/-2 g/L of peptone and 10 +/-2 g/L of NaCl;

(2) fermentation production of 3, 4-dihydroxybutyric acid by utilizing double-bacterium co-culture system

And (3) carrying out fermentation culture on the obtained double-bacterium co-culture system at the temperature of 28 +/-2 ℃ and the rotation speed of a shaking table of 200 +/-20 rpm, adding 1mM IPTG (isopropyl thiogalactoside) at the final concentration for induction when the total OD of the system is 0.7-0.8, manually adding 10M NaOH every 3 hours to adjust the pH of a culture medium to 6.8-7.0, and carrying out fermentation culture for 54-65 hours until the sugar consumption is completely stopped, thus obtaining the fermentation liquor containing 3, 4-dihydroxybutyric acid.

2. The method for producing 3, 4-dihydroxybutyric acid using a two-strain co-culture system according to claim 1, characterized in that: the two heavy suspension bacterial liquids of Gluconobacter oxydans621H and engineering Escherichia coli6KI are mixed according to the volume ratio of 1:1 and then inoculated into a co-culture fermentation medium, and the final OD of the bacterial liquid is 0.2, so that the double-bacterium co-culture system of the Gluconobacter oxydans and the engineering Escherichia coli is constructed.

3. The method for producing 3, 4-dihydroxybutyric acid using a dual-bacterial co-culture system according to claim 1, wherein: the formula of the co-culture fermentation medium is as follows: 7g/L of xylose, 5g/L of glucose, 5g/L of yeast powder, 10g/L of peptone and 10g/L of NaCl.

4. The method for producing 3, 4-dihydroxybutyric acid using a dual-bacterial co-culture system according to claim 1, wherein: the OD ratio of two bacterial liquids of Gluconobacter oxydans621H and engineering Escherichia coli6KI in the double-bacterial co-culture system is equal to 3:2, and fermentation culture is carried out at 28 ℃ and the rotation speed of a shaking table of 200 rpm.

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