CN112680484A

CN112680484A - Method for producing 3, 4-dihydroxybutyric acid by using double-bacterium co-culture system

Info

Publication number: CN112680484A
Application number: CN202110057551.1A
Authority: CN
Inventors: 高超; 张一鹏; 马翠卿; 许平
Original assignee: Shandong University
Current assignee: Shandong University
Priority date: 2021-01-15
Filing date: 2021-01-15
Publication date: 2021-04-20
Anticipated expiration: 2041-01-15
Also published as: CN112680484B

Abstract

The invention discloses a method for producing 3, 4-dihydroxybutyric acid by using a double-bacterium co-culture system, which comprises the steps of (1) constructing a double-bacterium co-culture system of gluconobacter oxydans and engineering Escherichia coli 6KI, and (2) fermenting and producing the 3, 4-dihydroxybutyric acid by using the double-bacterium co-culture system. Experiments prove that the method provided by the invention can enable the production amount of 3, 4-dihydroxybutyric acid to reach 3.26g/L and the yield to be 0.47g of 3, 4-dihydroxybutyric acid/1 g of xylose, effectively solves the problems of low yield and low yield of 3, 4-dihydroxybutyric acid produced by single genetic engineering bacteria reported at present, and provides a new thought and a new method for the application of gluconobacter oxydans in the biological production of chemicals requiring complex synthetic routes.

Description

Method for producing 3, 4-dihydroxybutyric acid by using double-bacterium co-culture system

Technical Field

The invention relates to a method for producing 3, 4-dihydroxybutyric acid by fermentation, in particular to a method for producing 3, 4-dihydroxybutyric acid by fermentation by taking xylose as a substrate through a co-culture system consisting of gluconobacter oxydans and engineering escherichia coli, belonging to the technical field of biology.

Background

3, 4-dihydroxybutyric acid and its dehydration product, 3-hydroxybutyrolactone, are important platform compounds (Gao H et al, 2017, BioResourcer. Technol.,245: 794-one 800) and are widely used in the production of polymers, solvents and nutritional additives (Wang J et al, 2017, Metab. Eng.,41: 39-45). The chemical method for producing 3, 4-dihydroxybutyric acid and 3-hydroxybutyrolactone has the defects of harsh and severe reaction conditions, expensive substrate and the need of heavy metal or rare metal to participate in the reaction (Kumar P et al, 2005, Tetrahedron: Asymmetry,16: 2717-2721). Because the microbial fermentation method for producing chemicals has the advantages of mild reaction conditions, less pollution, cheap substrate and the like, the microbial fermentation method for producing 3, 4-dihydroxybutyric acid has important research value and wide market prospect.

Escherichia coli has the characteristics of simple culture requirement, rapid growth, clear genetic background, easy genetic engineering operation and the like, and has been successfully used for producing 3, 4-dihydroxybutyric acid by metabolic engineering modification. Because the intermediate product has the problems of toxic action, high oxidation-reduction pressure, reversible reaction process and the like, the yield of the 3, 4-dihydroxybutyric acid produced by the recombinant escherichia coli is low, and the yield is low. It is reported that the domestic subject group realizes the production of 3, 4-dihydroxybutyric acid by expressing exogenous xylose dehydrogenase, aldehyde dehydrogenase and other metabolic engineering transformation in Escherichia coli and taking xylose as a substrate, and the yield is 0.38g/L (Gao H et al, 2017, Bioresource, technol.,245: 794-; the production of 3, 4-dihydroxybutyric acid by using glucose and glycolic acid as substrates is reported abroad, and the yield of 3, 4-dihydroxybutyric acid reaches 0.7g/L by metabolic engineering modification of the glyoxylic acid cycle of escherichia coli (Dhamankar H et al, 2014, Metab. Eng.,25: 72-81).

Gluconobacter oxydans is a strict aerobic bacterium (Prust C et al, 2005, nat. Biotechnol.,23:195- & 200), the cell membrane of which has various membrane-bound dehydrogenases and can participate in various oxidation reactions, making it an important industrial application strain (Kiefler I et al, 2017, appl. Microbiol. Biotechnol.,101:5453- & 5467). For example, gluconobacter oxydans is widely used industrially for the production of glucose from glucose, xylose, sorbitol and glycerol, respectivelyAcids, xylonic acids, sorbose and dihydroxyacetone. However, gluconobacter oxydans has the defects that the oxidation product is directly secreted to the extracellular space, the TCA cycle in vivo is incomplete, and the metabolic engineering of the gluconobacter oxydans is difficult to modify (Prust C et al, 2005, nat. Biotechnol.,23: 195-200;

t et al, 2006, j. bacteriol, 188: 7668-.

Through retrieval, a co-culture system of gluconobacter oxydans and engineering escherichia coli is established by utilizing the characteristics that escherichia coli is easy to engineer and combines the incomplete oxidation capability of gluconobacter oxydans and oxidized products are directly secreted to the outside, and a method for producing 3, 4-dihydroxybutyric acid by fermenting with xylose as a substrate is not reported yet.

Disclosure of Invention

Aiming at the problems that the existing method for producing 3, 4-dihydroxybutyric acid by using recombinant escherichia coli has low product yield, low yield, toxic action of intermediate products on bacterial strains, reversible reaction process and the like. The invention provides a method for high-yield production of 3, 4-dihydroxybutyric acid by using a dual-bacterium co-culture system consisting of gluconobacter oxydans and escherichia coli and taking xylose as a substrate.

The 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 bacterium is a gram-negative bacterium and grows aerobically strictly, the preferable culture temperature of the bacterium is 28 +/-1 ℃, and the bacterium can grow in a sorbitol complex culture medium. The activating culture medium of the gluconobacter oxydans621H is a sorbitol complex culture medium, and the formula of the sorbitol complex culture medium is as follows: sorbitol 73g/L, yeast powder 18.4g/L, (NH)₄)₂SO₄ 1.5g/L，KH₂PO₄ 1.5g/L，MgSO₄·7H₂O is 0.47 g/L; autoclaved at 121 ℃ for 20 minutes.

The engineering Escherichia coli is engineering Escherichia coli 6KI, the genotype of which is Escherichia coli W3110(DE3) delta xylA delta yjhH delta yagE delta yaiaE delta yqdD delta xynR, xylD & kdcA; the bacteria are gram-negative bacteria, aerobically or facultatively grow, the culture temperature of the bacteria is 37 +/-1 ℃, and the bacteria can grow in LB culture medium. The activating culture medium of the engineering Escherichia coli Escherichia coli 6KI is an LB culture medium, and the formula is as follows: peptone 10 g/L; 5g/L of yeast powder; NaCl 10 g/L; autoclaving at 121 ℃ for 20 min.

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; autoclaving at 121 ℃ for 20 min.

(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.

In the method for producing 3, 4-dihydroxybutyric acid by using the double-bacterium co-culture system, the following steps are carried out: the two kinds of heavy suspension bacterial liquids of Gluconobacter oxydans621H and engineering Escherichia coli 6KI are preferably mixed according to the volume ratio of 1:1, then inoculated into a co-culture fermentation medium, and the final OD of the bacterial liquid is 0.2, so that the dual-bacteria co-culture system of the Gluconobacter oxydans and the engineering Escherichia coli is constructed.

In the method for producing 3, 4-dihydroxybutyric acid by using the double-bacterium co-culture system, the following steps are carried out: the formula of the co-culture fermentation medium is preferably as follows: 7g/L of xylose, 5g/L of glucose, 5g/L of yeast powder, 10g/L of peptone and 10g/L of NaCl.

In the method for producing 3, 4-dihydroxybutyric acid by using the double-bacterium co-culture system, the following steps are carried out: the OD ratio of two bacterial liquids of Gluconobacter oxydans621H and engineering Escherichia coli 6KI in the double-bacterial co-culture system is preferably equal to 3:2, and fermentation culture is preferably carried out at 28 ℃ and the rotation speed of a shaking table of 200 rpm.

The construction method of the engineering Escherichia coli 6KI comprises the following steps:

escherichia coli W3110(DE3) was used as the starting strain, and the strain was subjected to continuous genetic engineering by the Red recombination technique (Datsenko KA et al, 2000, Proc. Natl. Acad. Sci. U S A.,97: 6640-:

(1) a genetic engineering means is adopted to knock out xylose isomerase genes xylA and 2-keto-3-deoxyxylonic acid aldolase genes yjhH and yagE in Escherichia coli W3110(DE3), so that endogenous pathways of strains for utilizing xylose and xylonic acid are blocked, and a strain Escherichia coli 3K is constructed.

(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 (figure 1) by adopting a genetic engineering means, amplifying a gene expression frame by PCR, constructing an operon, and knocking the operon into an original xynR site of an Escherichia coli 6K genome to construct an engineering Escherichia coli strain Escherichia coli 6 KI.

The detection method of the 3, 4-dihydroxybutyric acid and other substances comprises the following steps: detecting glucose by adopting a biosensor analyzer (SBA-40D); detecting xylose by using a High Performance Liquid Chromatograph (HPLC) Shimadzu 20-AT under the analysis conditions that: a differential refractive detector; a Bio-Rad Aminex HPX-87P analytical column (300X 7.8mm), the column temperature is 75 ℃, the mobile phase is pure water, the flow rate is 0.7mL/min, and the injection volume is 5 mu L; detecting xylonic acid, 2-keto-3-deoxyxylonic acid, 3, 4-dihydroxy butyraldehyde, 1,2, 4-butanetriol and 3, 4-dihydroxy butyric acid by adopting HPLC, wherein the analysis conditions are as follows: an ultraviolet detector with the detection wavelength of 210 nm; Bio-Rad Aminex HPX-87H analytical column (300X 7.8mm), column temperature 30 ℃, mobile phase 0.1% formic acid, flow rate 0.4mL/min, injection volume 5 u L.

The invention develops a new method for producing 3, 4-dihydroxybutyric acid by co-culturing the gluconobacter oxydans and the escherichia coli by utilizing the characteristics that the escherichia coli is easy to engineer and combines the incomplete oxidation capability of the gluconobacter oxydans and the oxidized product is directly secreted to the outside of cells, provides a new idea for producing the 3, 4-dihydroxybutyric acid by a biological method and provides new guidance for the industrial application of the gluconobacter oxydans. The gluconobacter oxydans in the dual-bacterium co-culture system firstly oxidizes the xylose into the xylonic acid, then the escherichia coli converts the xylonic acid into the 3, 4-dihydroxy butyraldehyde, and finally the gluconobacter oxydans oxidizes the 3, 4-dihydroxy butyraldehyde into the 3, 4-dihydroxy butyric acid. The method solves the problem of producing 3, 4-dihydroxybutyric acid by using a single strain at present, and realizes the high-efficiency and high-yield production of 3, 4-dihydroxybutyric acid. The method of the present invention enables the production of 3, 4-dihydroxybutyric acid to be 3.26g/L, and the yield to be 0.47g of 3, 4-dihydroxybutyric acid/1 g of xylose, as shown in FIG. 2.

The invention has the outstanding characteristics and beneficial effects that:

(1) the invention provides a new idea for producing 3, 4-dihydroxybutyric acid, and solves the problems that an intermediate product has toxic action, high oxidation-reduction pressure, reversible reaction process and the like in the process of producing 3, 4-dihydroxybutyric acid by using a single strain.

(2) The invention can lead the production of the 3, 4-dihydroxybutyric acid to reach 3.26g/L, and has the excellent characteristics of high yield and high production efficiency, and the yield is the highest yield of the 3, 4-dihydroxybutyric acid produced by the current biological method.

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

Drawings

FIG. 1: plasmid map of pACYCDuet-xylD-kdcA.

FIG. 2: the co-culture system takes glucose and xylose as substrates and adopts a fed-batch fermentation process curve.

Detailed Description

The present invention will be described in detail with reference to the following detailed drawings and examples. The following examples are only preferred embodiments of the present invention, and it should be noted that the following descriptions are only for explaining the present invention and not for limiting the present invention in any form, and any simple modifications, equivalent changes and modifications made to the embodiments according to the technical spirit of the present invention are within the scope of the technical solution of the present invention.

In the following examples, gluconobacter oxydans621H was purchased from the global biological resource center (ATCC) under the strain numbers: ATCC 621H. Reagents used in the method of the present invention were purchased from national pharmaceutical group chemical reagents ltd and sigma aldrich trade ltd; plasmids pKD4(CGSC7632), pKD46(CGSC7669) and pCP20(CGSC14177) were purchased from the university of Yale plasmid Gene Collection. Other materials, reagents and the like used, unless otherwise specified, are commercially available.

Example 1: fermentation production of 3, 4-dihydroxybutyric acid by using xylose as substrate in dual-bacterium co-culture system consisting of gluconobacter oxydans and escherichia coli

Centrifuging activated wild Gluconobacter oxydans621H at 6000 rpm for 10 min, washing the precipitate with physiological saline for 1-2 times, and then resuspending the precipitate with physiological saline until OD is 3 for later use; taking activated engineering Escherichia coli 6KI, centrifuging at 6000 rpm for 10 minutes, washing the precipitate for 1-2 times by using normal saline, and then re-suspending the precipitate until OD is 2 by using the normal saline for later use; mixing the two heavy suspension bacterial liquids according to the volume ratio of 1:1, inoculating the mixture into a co-culture fermentation medium, and enabling the final OD of the bacterial liquid to be 0.2, so as to construct a double-bacterium co-culture system of the gluconobacter oxydans and the engineering escherichia coli;

wherein:

the wild Gluconobacter oxydans621H is gram-negative bacteria and strictly grows aerobically, the preferred culture temperature of the bacteria is 26 ℃, and the bacteria can grow in a sorbitol complex culture medium. The activating culture medium of the gluconobacter oxydans621H is a sorbitol complex culture medium, and the formula of the sorbitol complex culture medium is as follows: sorbitol 73g/L, yeast powder 18.4g/L, (NH)₄)₂SO₄1.5g/L，KH₂PO₄ 1.5g/L，MgSO₄·7H₂O is 0.47 g/L; autoclaved at 121 ℃ for 20 minutes.

The genotype of the engineering Escherichia coli 6KI is Escherichia coli W3110(DE3) delta xylA delta yjhH delta yagE delta yaedelta yqdD delta xynR, xylD & kdcA; the bacteria are gram-negative bacteria, aerobically or facultatively grow, the culture temperature of the bacteria is 37 +/-1 ℃, and the bacteria can grow in LB culture medium. The activating culture medium of the engineering Escherichia coli Escherichia coli 6KI is an LB culture medium, and the formula is as follows: peptone 10 g/L; 5g/L of yeast powder; NaCl 10 g/L; autoclaving at 121 ℃ for 20 min.

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; autoclaving at 121 ℃ for 20 min. The double-bacterium co-culture fermentation system is 50 mL.

The activating modes of the gluconobacter oxydans621H and the Escherichia coli 6KI are as follows:

(1) plate culture: marking Escherichia coli 6KI strain on an LB plate containing agar with the mass-volume ratio of 1.5-1.8%, and culturing at 37 +/-1 ℃ for 12 +/-1 hours; streaking the strain G.oxydans621H on a sorbitol complex culture medium plate containing agar with the mass volume ratio of 1.5-1.8%, and culturing at 30 +/-1 ℃ for 36 +/-1 hours.

(2) First-stage seed: under the aseptic condition, picking a single colony on the LB plate in the step (1) by using an aseptic toothpick, then inoculating the single colony into 5mL of LB liquid culture medium, and carrying out shaking culture on a shaker at the temperature of 37 +/-1 ℃ for 12 +/-1 hours; picking a single colony on the sorbitol complex medium plate in the step (1) by using a sterile toothpick under the sterile condition, then inoculating the single colony into 5mL of sorbitol complex liquid medium, and culturing for 24 +/-1 hours at the temperature of 30 +/-1 ℃.

(3) Secondary seeds: under the aseptic condition, inoculating the Escherichia coli 6KI bacterial liquid cultured in the step (2) into 50mL of LB liquid culture medium in an inoculation amount of 1-2% by volume ratio, and carrying out shake culture on a shaker at the temperature of 37 +/-1 ℃ for 12 +/-1 hours to obtain activated Escherichia coli 6KI bacterial liquid; under the aseptic condition, inoculating the G.oxydnas621H bacterial liquid cultured in the step (2) into 50mL of sorbitol complex liquid culture medium by the inoculation amount of 1.5-2% in volume ratio, and culturing at 30 +/-1 ℃ for 24 +/-1 hours to obtain the activated wild type Gluconobacter oxydans621H bacterial liquid.

And (3) carrying out fermentation culture on the obtained double-bacterium co-culture system at the temperature of 28 ℃ and the rotation speed of a shaking table of 200rpm, adding 1mM IPTG (isopropyl-beta-thiogalactoside) at the final concentration for induction when the total OD of the system is 0.8, manually adding 10M NaOH every 3 hours to adjust the pH of a culture medium to 6.8, and carrying out fermentation culture for 54-60 hours until the sugar consumption is completely stopped, thus obtaining the fermentation liquor containing 3, 4-dihydroxybutyric acid.

Example 2: fermentation production of 3, 4-dihydroxybutyric acid by using xylose as substrate in dual-bacterium co-culture system consisting of gluconobacter oxydans and escherichia coli

Centrifuging activated wild Gluconobacter oxydans621H at 6000 rpm for 10 min, washing the precipitate with physiological saline for 1-2 times, and then resuspending the precipitate with physiological saline until OD is 3 for later use; taking activated engineering Escherichia coli 6KI, centrifuging at 6000 rpm for 10 minutes, washing the precipitate for 1-2 times by using normal saline, and then re-suspending the precipitate until OD is 2 by using the normal saline for later use; mixing the two heavy suspension bacterial liquids according to the volume ratio of 3:2, inoculating the mixture into a co-culture fermentation medium, and enabling the final OD of the bacterial liquid to be 0.3, so as to construct a double-bacterium co-culture system of the gluconobacter oxydans and the engineering escherichia coli;

wherein:

The formula of the co-culture fermentation medium is as follows: 9g/L of xylose, 6g/L of glucose, 6g/L of yeast powder, 12g/L of peptone and 12g/L of NaCl; autoclaving at 121 ℃ for 20 min. The double-bacterium co-culture fermentation system is 50 mL.

And (3) carrying out fermentation culture on the obtained double-bacterium co-culture system at the temperature of 30 ℃ and the rotation speed of a shaking table of 220rpm, adding 1mM IPTG (isopropyl-beta-thiogalactoside) at the final concentration for induction when the total OD of the system is 0.8, manually adding 10M NaOH every 3 hours to adjust the pH of a culture medium to 6.9, 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.

The above-mentioned Gluconobacter oxydans and Escherichia coli 6KI activation method was the same as that described in example 1.

Example 3: fermentation production of 3, 4-dihydroxybutyric acid by using xylose as substrate in dual-bacterium co-culture system consisting of gluconobacter oxydans and escherichia coli

Centrifuging activated wild Gluconobacter oxydans621H at 6000 rpm for 10 min, washing the precipitate with physiological saline for 1-2 times, and then resuspending the precipitate with physiological saline until OD is 3 for later use; taking activated engineering Escherichia coli 6KI, centrifuging at 6000 rpm for 10 minutes, washing the precipitate for 1-2 times by using normal saline, and then re-suspending the precipitate until OD is 2 by using the normal saline for later use; mixing the two heavy suspension bacterial liquids according to the volume ratio of 1:2, inoculating the mixture into a co-culture fermentation medium, and enabling the final OD of the bacterial liquid to be 0.2, so as to construct a double-bacterium co-culture system of the gluconobacter oxydans and the engineering escherichia coli;

wherein:

The formula of the co-culture fermentation medium is as follows: 5g/L of xylose, 4g/L of glucose, 4g/L of yeast powder, 8g/L of peptone and 8g/L of NaCl; autoclaving at 121 ℃ for 20 min. The double-bacterium co-culture fermentation system is 50 mL.

And (3) carrying out fermentation culture on the obtained double-bacterium co-culture system at 26 ℃ under the condition of the rotation speed of a shaking table of 180rpm, adding 1mM IPTG (isopropyl-beta-thiogalactoside) at the final concentration for induction when the total OD of the system is 0.7, manually adding 10M NaOH every 3 hours to adjust the pH of a culture medium to 7.0, and carrying out fermentation culture for 57-62 hours until the sugar consumption is completely stopped, thus obtaining the fermentation liquor containing 3, 4-dihydroxybutyric acid.

Example 4: construction of gene engineering strain Escherichia coli 6KI

The gene related to the Escherichia coli 6KI gene engineering strain and a related transformation method are disclosed in part by a patent CN201710611460.1, and the transformation method is disclosed in a patent CN 201710611460.1.

The specific operation method comprises the following steps:

construction of Gene knockout engineering bacteria

(1) The knockout method comprises the following steps: the gene knockout method used in the present invention is the Red recombination knockout technology (Datsenko KA et al, Proc. Natl. Acad. Sci. U.S. A.,2000,97: 6640-6645). In this method, plasmids pKD4(CGSC7632), pKD46(CGSC7669) and pCP20(CGSC14177) used for knockout were purchased from the plasmid gene collection center of Yale university.

(2) Obtaining of mutant fragments: the nucleotide mutant fragment used in the method has two homologous arms at the upstream and the downstream of the target knockout gene and a kanamycin resistance gene expression cassette. The nucleotide mutant fragment of the target gene was obtained by recombinant PCR.

Taking xylA knockout as an example, an upstream primer I of xylA/a downstream primer I of xylA is adopted, and an Escherichia coli K12 genome is taken as a template to amplify an upstream homology arm of a target gene; amplifying a kanamycin resistance gene expression cassette by adopting an xylA upstream primer II/a xylA downstream primer II and taking a plasmid pKD4 as a template; amplifying a downstream homologous arm of a target gene by adopting an xylA upstream primer three/a xylA downstream primer three and taking an Escherichia coli K12 genome as a template; and carrying out recombinant PCR on the two homologous arms and a kanamycin resistance gene expression cassette to obtain the xylA mutant fragment. The mutant fragments of the remaining genes yjhH, yagE, xynR, yqhD and yiaE were obtained in a manner consistent with the above method, wherein the primer sequences of the nucleotide mutant fragments used were 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 I5'-CTCCATAAACGGGTTCTTATGCCTT-3'

yagE downstream primer one 5'-CTCCAGCCTACACGAGATCTCCTTG-3'

yagE upstream primer two 5'-GCAAGGAGATCTCGTGTAGGCTGGA-3'

yagE downstream primer two 5'-GTTATCGTCCGGCATGGGAATTAGC-3'

yagE upstream primer III 5'-GGCTAATTCCCATGCCGGACGATAA-3'

yagE downstream primer III 5'-TCTGCATGCCGATCTCCCAATGCCC-3'

xynR upstream primer I5'-AACGTGAAGTTCCTGCACTGTCT-3'

xynR downstream primer I5'-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 one 5'-CCATACAACAAACGCACATCGGGCA-3'

yqhD downstream primer one 5'-AGCTCCAGCCTACACTACTTGCTCCCTTTG-3'

yqhD upstream primer two 5'-CAAAGGGAGCAAGTAGTGTAGGCTGGAGC-3'

yqhD downstream primer two 5'-TGAGGCGTAAAAAGCATGGGAATTAGCCATGG-3'

yqhD upstream primer three 5'-ATGGCTAATTCCCATGCTTTTTACGCCTCA-3'

yqhD downstream primer three 5'-CTGAGGCATTTTTCAGGGCTTTGCCG-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) Competence preparation and electrotransformation: after the pKD46 plasmid is chemically introduced into a target strain, a solid LB plate carrying 50 mug/mL spectinomycin resistance is adopted to screen a recombinant strain, and after verification, the recombinant strain is cultured and is subjected to competence preparation. Culturing the recombinant strain by adopting an LB liquid culture medium carrying 50 mu g/mL spectinomycin resistance 50mL, culturing at 30 ℃ for 30 minutes, adding IPTG (isopropyl-beta-thiogalactoside) with the final concentration of 0.5mM for induction, and carrying out 6000-10-minute centrifugal strain collection when OD is 0.5-0.6. After removing the supernatant, the cells were washed with 40mL of 4 ℃ precooled pure water for 2 times, and then with 40mL of 4 ℃ precooled 10% glycerol for 1 time. The thalli after centrifugal collection is re-suspended by 150 mu L10% glycerol, and 100 ng/mu L nucleotide mutation fragment is added to be mixed evenly, and then the mixture is subpackaged into 2 precooled electric rotary cups with 2 mm. The voltage is 2200V, the capacitance is 25 muF, and the resistance is 200 omega by using an electrotransformation machine. And (3) adopting 500 mu L of LB liquid to resuspend the bacterial liquid, incubating the bacterial liquid in a shaker at 37 ℃ for 40 minutes, coating a plate containing 50 mu g/mL kanamycin resistance, culturing the plate in an incubator at 37 ℃ for 12-13 hours, and selecting a single colony for PCR verification.

(4) Kanamycin resistance gene elimination: introducing the bacterial strain with correct PCR verification into pCP20 plasmid by a chemical method, incubating the bacterial strain in a shaker at 30 ℃ and 180 ℃ for 40 minutes, coating an LB plate containing 40 mu g/mL chloramphenicol resistance, culturing the bacterial strain in an incubator at 30 ℃ for 16-17 hours, and picking out a single bacterial colony for verification. Inoculating the strains which are verified to be correct to an anti-LB liquid culture medium, placing the strains at 42 ℃, carrying out shake culture for 2 generations at 180 ℃, and carrying out streak culture at 37 ℃. The same colony is picked and spotted on an LB non-resistant plate, a 40 mu g/mL chloramphenicol resistant LB plate and a 50 mu g/mL kanamycin resistant plate respectively, and cultured for 12-13 hours. Colonies that grew on non-resistant plates, but not on resistant plates, were selected for strain PCR validation. And (5) preserving the strains which are verified to be correct for later use.

The length of the knocked-out xylose isomerase xylA gene sequence is 1323 basic groups, and the nucleotide sequence is shown as SEQ ID NO. 1; the length of the knocked-out 2-keto-3-deoxyxylonic acid aldolase yjhH gene sequence is 906 bases, and the nucleotide sequence is shown as SEQ ID NO. 2; the length of the knocked-out 2-keto-3-deoxyxylonic acid aldolase yagE gene sequence is 909 bases, and the nucleotide sequence is shown as SEQ ID NO. 3; the length of the knocked-out xylonic acid operon transcription inhibiting factor xynR gene sequence is 759 basic groups, and the nucleotide sequence is shown as SEQ ID NO. 4; the length of the knocked-out NADPH dependent aldehyde reductase yqhD gene sequence is 1164 bases, and the nucleotide sequence is shown as SEQ ID NO. 5; the length of the knocked-out glyoxylate reductase gene yiaE gene sequence is 975 basic groups, and the nucleotide sequence is shown as SEQ ID NO. 6.

(II) construction of Gene knock-in engineering bacteria

(1) Cloning genes: the original pET28a-xylD and pET28a-kdcA vectors, which carry xylonate dehydratase gene xylD and branched-2-keto acid decarboxylase gene kdcA, respectively, were synthesized by Universal biosystems (Anhui) Inc. (Chuzhou, China, Anhui). The nucleotide sequence of the xylonic acid dehydratase gene xylD is shown as SEQ ID NO. 7; the nucleotide sequence of the branched-2-keto acid decarboxylase gene kdcA is shown as SEQ ID NO. 8. Designing a primer by using the xylD nucleotide sequence, and amplifying by using a synthesized primer and using pET28a-xylD as a template to obtain a xylD fragment, wherein the fragment contains restriction sites BamHI and SacI. The amplification primer sequences are as follows:

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

the downstream primer 5'-ATGCGAGCTCTTAATGATTATGGCG-3', carrying a SacI site.

(2) The fragment obtained by PCR amplification in step (1) is recovered and subjected to enzyme digestion reaction and nucleotide electrophoresis with plasmid pACYCDuet-1 under restriction enzymes BamHI and SacI. And carrying out a connection reaction on the recovered fragment xylD and pACYCDuet-1 to obtain a recombinant plasmid pACYCDuet-xylD.

(3) Primers were designed based on the nucleotide sequence of kdcA, and a kdcA fragment containing restriction sites BglII and XhoI was amplified using pET28a-kdcA as a template using synthetic primers. The amplification primer sequences are as follows:

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

the downstream primer 5'-ATATCTCGAGTTACTTATTCTGTTC-3', carrying an XhoI site.

(4) Recovering the fragment obtained by PCR amplification in the step (3), and carrying out enzyme digestion reaction and nucleotide electrophoresis on the fragment and the plasmid pACYCDuet-xylD under restriction enzymes BglII and XhoI. And carrying out ligation reaction on the recovered fragment kdcA and pACYCDuet-xylD to obtain a recombinant plasmid pACYCDuet-xylD-kdcA.

(5) Designing a primer by using a recombinant plasmid pACYCDuet-xylD-kdcA sequence, and amplifying a gene knock-in expression cassette by using a synthetic primer by using the plasmid pACYCDuet-xylD-kdcA as a template. All knock-in amplification primer sequences were as follows:

an upstream primer I5'-CTGGATCTGCGCCTGTTGGCCCCGA-3', an xynR upstream homology arm amplification primer;

a downstream primer I5'-CCTAATGCAGGAGTCGCATAAATGCTGGCATGTCCACGCT-3', an XynR upstream homology arm amplification primer;

a second upstream primer 5'-AGCGTGGACATGCCAGCATTTATGCGACTCCTGCATTAGG-3', a gene knock-in expression cassette amplification primer;

a second downstream primer 5'-GAAGCAGCTCCAGCCTACACCAAAAAACCCCTCAAGACCC-3', a gene knock-in expression cassette amplification primer;

an upstream primer of three 5'-GGGTCTTGAGGGGTTTTTTGGTGTAGGCTGGAGCTGCTTC-3', a kanamycin resistance gene expression cassette amplification primer;

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

an upstream primer of four 5'-GGACCATGGCTAATTCCCATGCCGTTAGACCGGCTCGTAG-3', an XynR downstream homology arm amplification primer;

a downstream primer of four 5'-CGCTTGACCCGGAGCTGCAGACCCT-3', an xynR downstream homology arm amplification primer;

(6) and (3) carrying out four-segment recombinant PCR on the xynR upstream and downstream homologous arms, the gene knock-in expression cassette and the kanamycin resistance gene expression cassette nucleotide obtained by PCR amplification in the step (5) to obtain a gene knock-in mutant segment.

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

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

The recombinant Escherichia coli 6KI is a gram-negative bacterium, aerobically or facultatively grows at a preferable culture temperature of 37 +/-1 ℃, can grow on an LB culture medium, and can be used for constructing a co-culture system by combining with wild type G.oxydans to produce 3, 4-dihydroxybutyric acid by using a xylose substrate.

Sequence listing

<110> Shandong university

<120> method for producing 3, 4-dihydroxybutyric acid by using double-bacterium co-culture 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-deoxyxylonic acid 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-deoxyxylonic acid 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 transcription repression factor 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> nucleotide sequence of branched-2-keto acid decarboxylase gene kdcA

<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

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

wherein the Gluconobacter oxydans is selected from wild type Gluconobacter oxydans 621H; the engineering Escherichia coli is engineering Escherichia coli 6KI, the genotype of which is Escherichia coli W3110(DE3) delta xylA delta yjhH delta yagE delta yaiaE delta yqdD delta xynR, xylD & kdcA; 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. The method for producing 3, 4-dihydroxybutyric acid using a dual-bacterial co-culture system according to claim 1, wherein: the two heavy suspension bacterial liquids of Gluconobacter oxydans621H and engineering Escherichia coli 6KI 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 coli 6KI in the double-bacterial co-culture system is equal to 3:2, and fermentation culture is carried out at the temperature of 28 ℃ and the rotating speed of a shaking table of 200 rpm.