CN114806998A - Ralstonia engineering bacterium for producing glucose and fermentation production method - Google Patents

Abstract

The application relates to the field of genetic engineering, in particular to a ralstonia bacterium engineering bacterium for producing glucose and a fermentation production method. The invention knocks out glucokinase gene (in) in Ralstonia H16glk) Realizes the use of fructose, glycerol and carbon dioxide (CO) ₂ ) Accumulation of product glucose as sole carbon source. Then by blocking the Entner-Doudoroff (ED) pathway and pentose phosphate poly ([ R)]Carbon flux splitting by-3-Hydroxybutanoic acid) (PHB) Synthesis pathwayFlow, increasing the supply of the product precursors glucose-6-phosphate and glucose-1-phosphate in cells of mutant Ralstonia species. By using fructose, glycerol and CO ₂ The highest yield of glucose reached 140.8, 73.9 and 253.3 mg/L respectively when the fermentation was carried out with the sole carbon source.

Description

Ralstonia engineering bacterium for producing glucose and fermentation production method

Technical Field

The invention relates to the field of genetic engineering, in particular to a ralstonia engineering bacterium for producing glucose and a fermentation production method.

Background

Climate change is causing a new global food crisis, and the heavy use of fossil fuels releases greenhouse gases (such as CO) ₂ ) Is a major factor contributing to the appearance of extreme weather. Therefore, the CO in the atmosphere is eliminated by technical means ₂ Meanwhile, the grain crisis is relieved. Glucose, an energy substance, is a monosaccharide synthesized by green plants through photosynthesis, and is also a basic constituent unit of substances in food and feed materials. Meanwhile, the glucose can be widely added into medicines and seasonings. With the development of synthetic biological technology, glucose production by metabolic engineering means has become possible.

At present, reports of glucose production by means of metabolic engineering are found in Escherichia coli and Saccharomyces cerevisiae as host strains. Carbon sources include xylose, arabinose, and arabinose which can be directly utilizedCO converted into acetic acid by electrochemical method and then utilized by bacterial strain ₂ . By knocking out a glucose metabolism pathway of escherichia coli, the synthesis of glucose can be realized by taking xylose and arabinose as carbon sources. In the metabolic engineering research of producing glucose by using saccharomyces cerevisiae as a host strain, as saccharomyces cerevisiae cannot directly utilize CO ₂ First, CO is electrochemically introduced ₂ Converting into acetic acid which can be used by the saccharomyces cerevisiae, and then synthesizing glucose by the saccharomyces cerevisiae by taking the acetic acid as a direct carbon source, wherein CO is indirectly utilized ₂ The process increases the complexity of the production process and increases the application cost. At present, no report is available for producing glucose by using fructose and a cheap renewable carbon source of glycerol as a carbon source.

Ralstonia H16 (Cupriavidus necatorH16) Is a facultative autotrophic gram-negative bacterial strain. It lacks the complete glycolytic pathway and pentose phosphate pathway due to its deletion of 6-phosphofructokinase and 6-phosphogluconate dehydrogenase. Metabolism of glucose-6-phosphate via the Entner-Doudoroff (ED) pathway pyruvate then enters the Krebs cycle or supplies poly ([ R ] s)]-3-hydroxybutyric acid). As a candidate strain with potential 'cell agriculture', the Ralstonia H16 can utilize a wide range of organic substances as carbon sources (such as fructose, glycerol and the like), and more importantly, the Ralstonia H16 can directly use CO through Calvin-Benson-Bassham (CBB) circulation ₂ As a carbon source. The ralstonia H16 has a natural PHB synthetic pathway, and the partial carbon flux can be diverted to synthesize other high value-added compounds through the reconstruction of a metabolic pathway. At present, the metabolic engineering of Ralstonia H16 has been successfully realized by glycerol or CO ₂ Synthesis of isobutanol, methyl ketone, trehalose and mannose as carbon sources.

Disclosure of Invention

To realize the utilization of renewable materials and greenhouse gas CO ₂ The invention provides a method for producing glucose from fructose, glycerol and CO ₂ The ralstonia engineering bacteria are used for producing glucose as carbon sources.

It is still another object of the present invention to provide a method for producing glucose by high-efficiency fermentation.

The engineered bacterium of the Ralstonia for producing glucose is a mutant Ralstonia which knocks out glucose kinase gene of the Ralstonia and key genes in an Entner-Doudoroff (ED) path and a poly ([ R ] -3 hydroxybutyric acid) (PHB) synthesis path,

wherein, the glucose kinase gene to be knocked out is a glucokinase coding gene glk (H16 _ B2564), and the nucleotide sequence is shown as SEQ ID NO: 1 is shown in the specification;

the key gene of the ED route is an encoding gene of glucose-6-phosphate dehydrogenasezwf1（H16_A0316）、zwf2(H16 _ B1501) andzwf3(H16 _ B2566) having the nucleotide sequence set forth in SEQ ID NO: 2. SEQ ID NO: 3 and SEQ ID NO: 4 is shown in the specification;

the key gene of the PHB synthetic pathway is poly ([ R)]-3 hydroxybutyrate) polymerase encoding genephaC1(H16 _ A1437), acetyl-CoA acetyltransferase encoding GenephaA(H16 _ A1438) and acetoacetyl-CoA reductase-encoding genesphaB1(H16 _ a 1439), the nucleotide sequence of which is set forth in SEQ ID NO: 5. SEQ ID NO: 6 and SEQ ID NO: shown at 7.

The method for constructing the glucose-producing ralstonia engineering bacteria comprises the following steps:

the knocked-out glucokinase gene is a glucokinase coding geneglk(H16 _ B2564) constructing mutant ralstonia underpan cells;

key genes in ED pathway and PHB synthetic pathway are knocked out,

wherein the glucose kinase gene to be knocked out is a glucose kinase coding geneglk(H16 _ B2564) having the nucleotide sequence set forth in SEQ ID NO: 1 is shown in the specification;

the key gene of the ED route is an encoding gene of glucose-6-phosphate dehydrogenasezwf1（H16_A0316）、zwf2(H16 _ B1501) andzwf3(H16 _ B2566) having the nucleotide sequence set forth in SEQ ID NO: 2. SEQ ID NO: 3 and SEQ ID NO: 4 is shown in the specification;

the key gene of the PHB synthetic pathway is poly ([ R)]-3 hydroxybutyric acid) polymerizationEnzyme-encoding genephaC1(H16 _ A1437), acetyl-CoA acetyltransferase encoding GenephaA(H16 _ A1438) and acetoacetyl-CoA reductase-encoding genesphaB1(H16 _ a 1439), the nucleotide sequence of which is set forth in SEQ ID NO: 5. SEQ ID NO: 6 and SEQ ID NO: shown at 7.

Another object of the present invention is to provide a method for producing glucose by fermentation, comprising the steps of: and (3) fermenting the glucose-producing ralstonia engineering strain in a shake flask to obtain glucose.

The method for producing glucose by fermentation according to the present invention, wherein fructose, glycerol or CO is used ₂ The shake flask fermentation is carried out on the culture medium which is the only carbon source.

The method for producing glucose by fermentation comprises the following steps of: 3.5 g/L Na ₂ HPO ₄ ，1.5 g/L KH ₂ PO ₄ ，1.0 g/L (NH ₄ ) ₂ SO ₄ ，80 mg/L MgSO ₄ ⋅7H ₂ O，1 mg/L CaSO ₄ ⋅2H ₂ O，0.56 mg/L NiSO ₄ ⋅7H ₂ O, 0.4 mg/L ferric citrate, 200 mg/L NaHCO ₃ Adding fructose and glycerol at a ratio of 4 g/L or introducing H ₂ :O ₂ :CO ₂ Mixed gas of =8:1:1 as a carbon source.

The method for producing glucose by fermentation according to the present invention, wherein the final concentration of kanamycin antibiotic added to the fermentation medium is 200 mg/L.

The method for producing glucose by fermentation is characterized in that the shake flask fermentation condition is 30 ℃ and 200 rpm.

The technical scheme of the application has the advantages that:

1. the advantage of using the engineered strain of ralstonia H16 for glucose synthesis is that it cannot use glucose and does not require modification of the strain to achieve product accumulation. Meanwhile, Ralstonia H16 has a broad substrate spectrum. Can utilize organic substances such as fructose, glycerol and the like as carbon sources to synthesize products, and has high-efficiency CO ₂ Immobilization capacity, i.e. the ability to directly utilize CO ₂ To carry outThe synthesis of glucose without the assistance of electrochemical means. The application constructs a strain by using renewable substrates (fructose and glycerol) and directly using CO through a metabolic engineering means ₂ Ralstonia H16 engineered strain for the synthesis of glucose as carbon source. Based on the technology, the method not only can solve the problem of increasingly severe global food shortage, but also is a method for efficiently eliminating greenhouse gases in the atmosphere.

2. To construct an engineered strain for glucose accumulation, first, the glucokinase involved in the first step of glucose metabolism is inactivated. Whereas the wild type strain of Ralstonia H16 was unable to utilize glucose as a carbon source. Therefore, the invention firstly identifies the activity of glucokinase in the strain. Expression of Zymomonas mobilis in wild-type strains using free plasmids (Zymomonas mobilis) The GLF, the source of the glucose transporter independent of energy, realizes that the Ralstonia H16 can proliferate by using glucose as a unique carbon source. This indicates that glucokinase gene is present in the genome of the strain and that the expressed protein has catalytic activity. A glucokinase gene (of Ralstonia H16) was foundglk) And knocked out to realize the accumulation of the catalytic reaction substrate (glucose). The results show that the knockoutglkThe mutant strain realizes the effects of using fructose, glycerol and CO ₂ Directly synthesizes glucose as a unique carbon source.

3. By single-knocking out or combined knocking out key genes in ED path and PHB synthetic path to cut off the shunting of glucose synthetic precursor glucose 6-phosphate in each path, the product precursor supply in chassis cells of mutant Ralstonia is improved, and fructose, glycerol and CO are utilized ₂ The glucose yield of the optimal strain reaches 140.8, 73.9 and 253.3 mg/L respectively when the culture medium serving as the sole carbon source ferments to produce the glucose.

Drawings

FIG. 1 shows glucose utilization and growth of cells by wild strains and engineered bacteria of Ralstonia;

FIG. 2 shows the accumulation of glucose when grown in different carbon sources after glk knock-out by Ralstonia;

FIG. 3 shows the results of glucose synthesis by wild strains of Ralstonia and the engineered strains of the present application using fructose as carbon source;

FIG. 4 shows fructose consumption results of wild strains of Ralstonia and engineered strains of the present application;

FIG. 5 shows the results of measurement of cell growth when glucose was synthesized using fructose as a carbon source by a wild strain of Ralstonia and an engineered strain of the present application;

FIG. 6 shows the results of glucose synthesis by wild strains of Ralstonia and the engineered strains of the present application using glycerol as a carbon source;

FIG. 7 shows glycerol sugar consumption results of wild strains of Ralstonia and engineered strains of the present application;

FIG. 8 shows the results of measurement of cell growth when glucose was synthesized using glycerol as a carbon source by a wild strain of Ralstonia and an engineered strain of the present application;

FIG. 9 shows the utilization of CO by wild strains of Ralstonia and engineered strains of the present application ₂ Results of glucose synthesis for the carbon source;

FIG. 10 shows the utilization of CO by wild strains of Ralstonia and engineered strains of the present application ₂ The result of the measurement of cell growth when glucose was synthesized as a carbon source.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. The materials, reagents and the like used are commercially available unless otherwise specified.

Example 1 Glucokinase in Ralstonia H16glkFunctional identification

Ralstonia H16 can utilize fructose, glycerol and CO ₂ Growth was performed as a carbon source. However, this bacterium cannot utilize glucose. Previous studies have demonstrated that the genome of this bacterium contains all the enzyme genes for glucose metabolism.

Based on the expression, the free plasmid pBBR1-MCS2 which can be autonomously replicated in the ralstonia H16 is used as a vector to heterologously express the zymomonas mobilis (Zymomonas mobilis) Energy-independent glucose transporter GLF. To contain noglfA fermentation experiment with 10 g/L glucose as the sole carbon source was carried out using pBBR1-MCS2 empty vector as a control for the gene expression cassette. The results are shown in FIG. 1. Ralstonia expressing the glucose transporter has a glucose utilization ability not seen in the control strain. After a lag period of about 2 days, the dry cell weight reached 2.9 g/L after 7 days of fermentation. This result not only indicates that glucose could not be utilized by Ralstonia H16 due to the lack of glucose transporter, but also indicates that Ralstonia H16 contains a catalytically active glucokinase to catalyze the first step of glucose utilization by the bacterial cells, i.e., glucose to glucose-6-phosphate. Based on genome data of Ralstonia H16, we found a glucokinase geneglkAnd knock out it. The results show that the knockoutglkThe accumulation of glucose in the latter period is shown in FIG. 2. 4 g/L of fructose is used as a carbon source, and the yield of glucose of the engineering strain is 24.7 mg/L; the yield of engineering strain glucose is 47.5 mg/L by taking 4 g/L of glycerol as a carbon source; with CO ₂ As a carbon source, the yield of the glucose of the engineering strain is 180.1 mg/L.

EXAMPLE 2 construction of Ralstonia H16∆glk∆zwf∆PHB engineering strain

The mutant ralstonia chassis cell is a mutant strain for knocking out glucokinase genes, ED pathway key genes and PHB synthesis pathway key genes. The glucose kinase gene to be knocked out isglkThe key gene of the ED pathway is a coding gene of glucose-6-phosphate dehydrogenasezwf1（H16_A0316）、zwf2(H16 _ B1501) andzwf3(H16 _ B2566) wherein the key gene for PHB synthetic pathway is poly ([ R)]-3 hydroxybutyrate) polymerase encoding genephaC1(H16 _ A1437), acetyl-CoA acetyltransferase encoding GenephaA(H16 _ A1438) and acetoacetyl-CoA reductase-encoding genesphaB1（H16_A1439）。

First, construction of Ralstonia H16∆glkBacterial strains

Will be provided withglkThe gene target knock-out vector pK18-glk was introduced into the host Ralstonia H16 by a conjugation transfer method. First round of screening is carried out by using kanamycin to obtain a knockout vector which is successfully integrated into a strain baseA host strain on the genome. On the basis, sucrose is used for the second round of screening to obtain successful knockoutglkGene Ralstonia H16 ΔglkAnd (3) strain. The specific method comprises the following steps:

1. build beltglkKnock-out plasmid pK18-glk of upstream and downstream homology arms of gene sequence

According toglkThe gene sequence (H16 _ B2564) was used to design synthetic primers. The primers designed above were used to amplify separately the genome of Ralstonia H16 using the templateglkThe homologous arms of the upstream and downstream genes have the fragment size of about 500 bp. The obtained fragment was ligated to pK18mobSacB plasmid using Gibson Assembly ligationEcoRI/SmaAnd (c) a site I. The plasmid is transformed into an Escherichia coli S17-1 host by electric shock, an LB solid plate (kanamycin 50 mug/mL) is coated, and positive clones are screened after overnight culture at 37 ℃, wherein the target fragment is 1000 bp. Designated as pK 18-glk.

2. Construction of Ralstonia H16 Strain with integration of pK18-glk plasmid into the genome

Escherichia coli S17-1 strain carrying pK18-glk plasmid and Ralstonia H16 strain were inoculated to LB liquid medium for overnight culture, and corresponding antibiotics (kanamycin 50 μ g/mL or gentamicin 10 μ g/mL) were added, respectively. The cells were centrifuged at 4600 rpm for 8 min to collect the cells, and the cells were washed three times with LB medium. Coli S17-1 strain harboring pK18-glk plasmid and Ralstonia H16 strain were mixed at a ratio of 3:1, spotted on LB plates, and incubated overnight at 30 ℃. The overnight incubated mixed bacteria washed with LB were spread on LB solid plates (kanamycin 200. mu.g/mL and gentamicin 10. mu.g/mL). After the plate was cultured at 30 ℃ for 48 hours, positive clones were selected, and the target fragment was a kanamycin-resistant gene fragment of 500 bp in length. The positive clones were cultured overnight at 30 ℃ in 1.5 mL of EP tubes supplemented with LB liquid medium.

3. Construction ofglkRalstonia H16 strain with successfully knocked-out gene

Diluting the bacterial liquid to 10 ^-2 Spread on LB solid plates supplemented with 100 g/L sucrose, and induced cultured at 30 ℃ for 48 hours. Performing colony PCR detection, screening the target gene and successfully knockingThe size of the target fragment was 1000 bp, except for the clones. The successfully constructed strain was inoculated in LB liquid medium, named Ralstonia H16glk。

Secondly, construction of Ralstonia H16∆glk∆zwfBacterial strains

In turn willzwfGene target knockout vectors pK18-zwf1, pK18-zwf2 and pK18-zwf3 were introduced into Ralstonia H16 by the method of conjugation and transfer∆glkIn a host. And (3) carrying out first round screening by using kanamycin to obtain a host strain with a knockout vector successfully integrated into a strain genome. On the basis, sucrose is used for the second round of screening to obtain successful knockoutglkGene Ralstonia H16 Δglk∆zwfAnd (3) strain. The specific method comprises the following steps:

1. build beltzwf1、zwf2Andzwf3knock-out plasmids pK18-zwf1, pK18-zwf2 and pK18-zwf3 of upstream and downstream homology arms of gene sequence

According tozwf1（H16_A0316）、zwf2(H16 _ B1501) andzwf3(H16 _ B2566) Gene sequence synthetic primers were designed. The primers designed above were used to amplify separately the genome of Ralstonia H16 using the templateglkThe homologous arms of the upstream and downstream genes have the fragment size of about 500 bp. The obtained fragment was ligated to pK18mobSacB plasmid using Gibson Assembly ligationEcoRI/SmaAnd (c) a site I. The plasmid is transformed into an Escherichia coli S17-1 host by electric shock, an LB solid plate (kanamycin 50 mug/mL) is coated, and positive clones are screened after overnight culture at 37 ℃, wherein the target fragment is 1000 bp. Named pK18-zwf1, pK18-zwf2 and pK18-zwf 3.

2. Construction of Ralstonia H16 Strain with integration of pK18-zwf1 plasmid into the genome

Escherichia coli S17-1 strain harboring pK18-zwf1 plasmid and Ralstonia sp H16∆glkThe strain is inoculated with LB liquid culture medium for overnight culture, and corresponding antibiotics (kanamycin 50 mug/mL or gentamicin 10 mug/mL) are respectively added. The cells were centrifuged at 4600 rpm for 8 min to collect the cells, and the cells were washed three times with LB medium. Escherichia coli S17-1 strain harboring pK18-zwf1 plasmid and Ralstonia sp H16∆glkThe strains are mixed according to the proportion of 3:1After the incubation, the cells were spotted on LB plates and incubated overnight at 30 ℃. The overnight incubated mixed bacteria washed with LB were spread on LB solid plates (kanamycin 200. mu.g/mL and gentamicin 10. mu.g/mL). After the plate was cultured at 30 ℃ for 48 hours, positive clones were selected, and the target fragment was a kanamycin-resistant gene fragment of 500 bp in length. The positive clones were cultured overnight at 30 ℃ in 1.5 mL of EP tubes supplemented with LB liquid medium.

3. Construction ofzwf1Ralstonia H16 strain with successfully knocked-out gene

Diluting the bacterial liquid to 10 ^-2 Spread on LB solid plates supplemented with 100 g/L sucrose, and induced cultured at 30 ℃ for 48 hours. And carrying out colony PCR detection, and screening clone with the target gene successfully knocked out, wherein the size of the target fragment is 1000 bp. The successfully constructed strain was inoculated in LB liquid medium, named Ralstonia H16glk∆zwf1。

4. Construction ofzwf2Andzwf3ralstonia H16 strain with successfully knocked-out gene

According to the above steps, respectively using Ralstonia H16∆glk∆zwf1And Ralstonia H16∆glk∆ zwf1∆zwf2As a host, carrying outzwf2Andzwf3the knock-out of (3). Finally obtaining the Ralstonia H16∆glk∆zwfAnd (3) strain.

Thirdly, constructing the Ralstonia H16∆glk∆zwf∆PHB strains

The phaC1AB1 operon (containingphaC1、phaAAndphaB1gene) target knockout vector pK18-PHB is introduced into Ralstonia H16 by using combined transfer method∆glk∆zwfIn a host. And (3) carrying out first round screening by using kanamycin to obtain a host strain with a knockout vector successfully integrated into a strain genome. On the basis, sucrose is used for the second round of screening to obtain successful knockoutglkGenetic Ralstonia H16∆glk∆zwf∆PHB strain. The specific method comprises the following steps:

1. construction of a knock-out plasmid pK18-phaC1AB1 with upstream and downstream homology arms to the phaC1AB1 operon sequence

According to the phaC1AB1 operonphaC1（H16_A1437）、phaA(H16 _ A1438) andphaB1(H16 _ A1439)) sequence design of synthetic primers. The upstream and downstream homology arms of the operon gene were amplified using the above designed primers with the genome of Ralstonia H16 as a template, and the fragment size was about 500 bp. The obtained fragment was ligated to pK18mobSacB plasmid using Gibson Assembly ligationEcoRI/SmaAnd (c) a site I. The plasmid is transformed into an Escherichia coli S17-1 host by electric shock, an LB solid plate (kanamycin 50 mug/mL) is coated, and positive clones are screened after overnight culture at 37 ℃, wherein the target fragment is 1000 bp. Designated pK18-phaC1AB 1.

2. Construction of Ralstonia H16 with pK18-phaC1AB1 plasmid integrated into the genome∆glk∆zwfBacterial strains

Escherichia coli S17-1 strain harboring pK18-phaC1AB1 plasmid and Ralstonia H16∆glk∆zwfThe strain is inoculated with LB liquid culture medium for overnight culture, and corresponding antibiotics (kanamycin 50 mug/mL or gentamicin 10 mug/mL) are respectively added. The cells were centrifuged at 4600 rpm for 8 min to collect the cells, and the cells were washed three times with LB medium. Escherichia coli S17-1 strain harboring pK18-phaC1AB1 plasmid and Ralstonia H16∆glk∆zwfThe strains were mixed at a ratio of 3:1, spotted on LB plates and incubated overnight at 30 ℃. The overnight incubated mixed bacteria washed with LB were spread on LB solid plates (kanamycin 200. mu.g/mL and gentamicin 10. mu.g/mL). After the plate was cultured at 30 ℃ for 48 hours, positive clones were selected, and the target fragment was a kanamycin-resistant gene fragment of 500 bp in length. The positive clones were cultured overnight at 30 ℃ in 1.5 mL of EP tubes supplemented with LB liquid medium.

3. Construction ofphaC1、phaAAndphaB1ralstonia H16 strain with successfully knocked-out gene

Diluting the bacterial liquid to 10 ^-2 Spread on LB solid plates supplemented with 100 g/L sucrose, and induced cultured at 30 ℃ for 48 hours. And carrying out colony PCR detection, and screening clone with the target gene successfully knocked out, wherein the size of the target fragment is 1000 bp. The successfully constructed strain was inoculated in LB liquid medium, named Ralstonia H16glk∆zwf∆PHB. The aforementioned Ralstonia spThe mutants are shown in Table 1.

TABLE 1

Bacterial strains	Traits
		Ralstonia H16	Wild strain
Alternaria rosenbergii H16glk	glkGene knockout strain
		Alternaria rosenbergii H16glk∆zwf	glk、zwf1、zwf2 and zwf3Gene knockout strain
Alternaria rosenbergii H16glk∆PHB	glk、phaC1、phaAAndphaB1gene knockout strain
		Alternaria rosenbergii H16glk∆zwf∆PHB	glk、zwf1、zwf2、zwf3、phaC1、phaAAndphaB1gene knockout strain

Example 3 production of glucose by fermentation Using fructose as carbon Source Using an engineered Strain of Ralstonia for glucose production

Will Luo Er Si TongBacteria H16 and Ralstonia rosenbergii H16 Δ producing glucoseglkAlstonia bacterium H16glk∆zwfAlternaria rolfsii H16glk∆zwf∆The PHB engineering strain is inoculated in 50 mL LB liquid culture medium and rejuvenated at 30 ℃ and 200 rpm for 16 hours.

Preparing a fermentation medium with fructose as a unique carbon source: 3.5 g Na was accurately weighed ₂ HPO ₄ ，1.5 g KH ₂ PO ₄ ，1.0 g (NH ₄ ) ₂ SO ₄ ，80 mg MgSO ₄ ⋅7H ₂ O，1 mg CaSO ₄ ⋅2H ₂ O，0.56 mg NiSO ₄ ⋅7H ₂ O, 0.4 mg ferric citrate and 200 mg NaHCO ₃ And 4 g of fructose, using water as a solvent to fix the volume to 1L, and sterilizing for 30 minutes by high-pressure moist heat sterilization at 115 ℃ to obtain the fermentation culture medium using the fructose as the only carbon source.

The bacteria Ralstonia H16 and the glucose-producing Ralstonia H16glkAlstonia bacterium H16glk∆zwfAlternaria rolfsii H16glk∆zwf∆The PHB engineering strain is transferred into 200 mL fermentation medium according to the inoculation amount of 1 percent and is subjected to shake flask culture at 30 ℃ and 200 rpm overnight. To be OD ₆₀₀ When the concentration reaches more than 1.0, the mixture is centrifuged at 4600 rpm for 8 min to collect thalli. After three times of PBS washing, the cells were suspended by fermentation medium and OD was adjusted ₆₀₀ Approximatively 1.0, shake flask fermentations were carried out at 30 ℃ at 200 rpm. The glucose concentration and fructose concentration in the medium were measured by sampling at regular intervals.

Bacterial liquid OD ₆₀₀ And (3) determination: and measuring the absorption value of the bacterial liquid under the wavelength of 600 nm by using a visible spectrophotometer. The dry cell weight was calculated according to the formula reported in the literature: cell Dry weight (g/L)/OD ₆₀₀ And = 0.363 g/L.

Glucose concentration and fructose concentration measurement: and detecting glucose and fructose in the sample by using a high performance liquid chromatograph. The detector is Shimadzu parallax refraction detector, and the chromatographic column is Waters solar-Pak ^TM (300 mm. times.6.5 mm), column temperature 70 ℃. The mobile phase is ultrapure water, the flow rate is 0.5 mL/min, and the sample volume is 10 muL.

The results of the glucose concentration measurements are shown in figure 3,single knockout when fructose is used as the sole carbon sourceglkThe gene can synthesize glucose, and the yield is 24.7 mg/L after fermentation for 7 days. Ralstonia H16 Δ in mutant strainsglk∆zwf∆The PHB engineering strain is an optimal glucose production strain. After 4 days of fermentation, the maximum yield is 140.8 mg/L. The fructose concentration detection results are shown in fig. 4, and fructose can be completely consumed 1 day after the wild strain of ralstonia H16. Alternaria rosenbergii H16glk∆zwf∆The PHB engineered strain had the slowest fructose consumption and was completely depleted on day 5.

When fructose is used as the sole carbon source, fructose consumption occurs mainly via the ED pathway. Thus, in knockoutglkGene-based knockout of key genes in ED pathwayzwf1、zwf2、zwf3Then the glucose-1-phosphate and the glucose-3-phosphate are accumulated, thereby increasing the supply of the precursor substance for synthesizing the product glucose and further improving the yield of the glucose. But when in knockoutglkGene-based related gene for knocking out PHB synthetic pathwayphaC1、phaAAndphaB1thereafter, the glucose yield decreased by 42.9%. This is probably due to inhibition of the growth of the bacterial cells after knocking off the synthetic pathway (as shown in FIG. 5). Meanwhile, the literature reports that the removal of PHB synthesis pathway causes the loss of carbon flux in the form of pyruvic acid, which may also be one of the reasons for the decrease of glucose yield. In knock-outglkAfter double knockout of key genes of ED pathway and PHB synthesis pathway on the basis of genes, although the growth of thalli is seriously influenced to cause slow consumption of carbon source (as shown in figure 5), the loss of carbon flow can be blocked, thereby obtaining the best yield of glucose.

Example 4 production of glucose by fermentation Using an engineered Strain of Ralstonia for glucose production with Glycerol as carbon Source

The bacteria Ralstonia H16 and the glucose-producing Ralstonia H16glkAlstonia bacterium H16glk∆zwfAlternaria rolfsii H16glk∆zwfThe Δ PHB engineered strain was inoculated in 50 mL LB liquid medium and rejuvenated at 30 ℃ with 200 rpm for 16 hours.

Preparing a fermentation medium with glycerol as a sole carbon source: 3.5 g Na was accurately weighed ₂ HPO ₄ ，1.5 g KH ₂ PO ₄ ，1.0 g (NH ₄ ) ₂ SO ₄ ，80 mg MgSO ₄ ⋅7H ₂ O，1 mg CaSO ₄ ⋅2H ₂ O，0.56 mg NiSO ₄ ⋅7H ₂ O, 0.4 mg ferric citrate and 200 mg NaHCO ₃ And 4 g of glycerol, using water as a solvent to fix the volume to 1L, and sterilizing by high-pressure moist heat sterilization at 115 ℃ for 30 minutes to obtain the fermentation culture medium using the glycerol as the only carbon source.

The bacteria Ralstonia H16 and the glucose-producing Ralstonia H16glkAlstonia bacterium H16glk∆zwfAlternaria rolfsii H16glk∆zwfThe strain of PHB engineering strain is inoculated to 200 mL of fermentation medium according to the inoculum size of 1%, and shake-flask culture is carried out at 30 ℃ and 200 rpm overnight. To be OD ₆₀₀ When the concentration reaches more than 1.0, the mixture is centrifuged at 4600 rpm for 8 min to collect thalli. After three times of PBS washing, the cells were suspended by fermentation medium and OD was adjusted ₆₀₀ Approximatively 1.0, shake flask fermentations were carried out at 30 ℃ at 200 rpm. The glucose concentration and the glycerol concentration in the culture medium were measured by sampling at regular intervals.

Bacterial liquid OD ₆₀₀ And (3) determination: and measuring the absorption value of the bacterial liquid under the wavelength of 600 nm by using a visible spectrophotometer. The dry cell weight was calculated according to the formula reported in the literature: cell Dry weight (g/L)/OD ₆₀₀ And = 0.363 g/L.

Glucose concentration and glycerol concentration measurement: and detecting glucose and glycerol in the sample by using a high performance liquid chromatograph. The detector is Shimadzu parallax refraction detector, and the chromatographic column is Waters solar-Pak ^TM (300 mm. times.6.5 mm), column temperature 70 ℃. The mobile phase is ultrapure water, the flow rate is 0.5 mL/min, and the sample volume is 10 muL.

The results of glucose concentration measurements are shown in FIG. 6, when glycerol is used as the sole carbon source, glucose can be synthesized by single-knock-out of the glk gene, and the yield is 47.5 mg/L after 6 days of fermentation. Ralstonia H16 Δ in mutant strainsglk∆zwfThe engineering strain is the optimal glucose producing strain. After 6 days of fermentation, the maximum yield was 73.9 mg/L. Sweet tasteThe results of oil concentration measurements are shown in FIG. 7, where glycerol consumption rates of the wild strain and the mutant strain of Ralstonia H16 were similar, and glycerol was completely consumed after 3 days.

When glycerol is used as a carbon source, ralstonia bacteria convert glycerol into glycolytic intermediate dihydroxyacetone phosphate, which is then converted into phosphoenolpyruvate, which is then converted into pyruvate, which is then used for the synthesis of PHB or organic acids. Meanwhile, dihydroxyacetone phosphate is metabolized via gluconeogenesis pathway to synthesize glucose precursors of glucose, glucose-1-phosphate and glucose-6-phosphate. Because the ED pathway of the ralstonia sp is weaker when glycerol is used as a carbon source, the yield of glucose is only improved by 0.5 times after the zwf gene is knocked out, fructose is mainly metabolized through the ED pathway, and the lifting effect of knocking out the zwf gene on a product is more obvious when the fructose is used as the carbon source. Similar to fructose as carbon source, the decrease in glucose production following the knock-out PHB synthesis pathway can be attributed to lower bacterial mass (as shown in figure 8) and loss of carbon flux. Meanwhile, when glycerol is used as a carbon source, the carbon flux is mainly directed to the synthesis pathway of PHB by glycolysis, and therefore, the cut of the ED pathway cannot completely avoid the loss of the carbon flux. As shown in FIG. 7, it has been reported that the low glycerol utilization rate of the Ralstonia strain is low, and the wild type strain can consume 4 g/L of glycerol within 3 days, while the wild strain can consume the same amount of fructose within 1 day. Therefore, the utilization capacity of the strain to fructose and glycerol is greatly different. As shown in FIG. 7, there was no significant difference in the glycerol metabolism rates of the wild-type and mutant strains of Ralstonia, which may be related to the natural glycerol utilization characteristics thereof, so that no significant difference in carbon source utilization was exhibited between the respective mutant strains.

Example 5 engineering of Ralstonia with glucose production with CO ₂ Production of glucose by fermentation of carbon source

The bacteria Ralstonia H16 and the glucose-producing Ralstonia H16glkAlstonia bacterium H16glk∆zwfAlternaria rolfsii H16glk∆zwfInoculating the Tab PHB engineering strain in 50 mL LB liquid mediumRejuvenation was carried out at 30 ℃ for 16 hours at 200 rpm.

Preparing a fermentation medium with glycerol as a sole carbon source: 3.5 g Na was accurately weighed ₂ HPO ₄ ，1.5 g KH ₂ PO ₄ ，1.0 g (NH ₄ ) ₂ SO ₄ ，80 mg MgSO ₄ ⋅7H ₂ O，1 mg CaSO ₄ ⋅2H ₂ O，0.56 mg NiSO ₄ ⋅7H ₂ O, 0.4 mg ferric citrate and 200 mg NaHCO ₃ The volume is fixed to 1L by using water as a solvent, and the mixture is sterilized by moist heat under high pressure at 115 ℃ for 30 minutes.

The Alstonia sp H16 and the Alstonia sp H16 producing glucoseglkAlstonia bacterium H16glk∆zwfAlternaria rolfsii H16glk∆zwfThe strain of PHB engineering strain is inoculated to 200 mL of fermentation medium according to the inoculum size of 1%, and shake-flask culture is carried out at 30 ℃ and 200 rpm overnight. To be OD ₆₀₀ When the concentration reaches more than 1.0, the mixture is centrifuged at 4600 rpm for 8 min to collect thalli. After three times of PBS washing, the cells were suspended by fermentation medium and OD was adjusted ₆₀₀ Approximatively 1.0, anaerobic flask shake flask fermentations were carried out at 30 ℃ at 200 rpm. The mixed gas (H) is filled every day ₂ :O ₂ :CO ₂ =8:1: 1) and the glucose concentration in the medium was measured by sampling at regular intervals.

Bacterial liquid OD ₆₀₀ And (3) determination: and measuring the absorption value of the bacterial liquid under the wavelength of 600 nm by using a visible spectrophotometer. The dry cell weight was calculated according to the formula reported in the literature: cell Dry weight (g/L)/OD ₆₀₀ And = 0.363 g/L.

And (3) measuring the glucose concentration: and detecting the glucose in the sample by using a high performance liquid chromatograph. The detector is Shimadzu parallax refraction detector, and the chromatographic column is Waters solar-Pak ^TM (300 mm. times.6.5 mm), column temperature 70 ℃. The mobile phase is ultrapure water, the flow rate is 0.5 mL/min, and the sample volume is 10 muL.

The results of glucose concentration measurements are shown in FIG. 9, in which CO is added ₂ When the glk gene is knocked out as the only carbon source, glucose can be synthesized, and the yield is 180.1 mg/L after fermentation for 64 days. Ralstonia H16 Δ in mutant strainsglk∆zwfThe PHB engineering strain is most preferredA glucose producing strain. After 64 days of fermentation, the maximum yield was 73.9 mg/L.

Use of CO by Ralstonia ₂ In the case of carbon sources, truncation of the ED pathway can increase the yield of glucose. This indicates that CO is being utilized ₂ In the case of a carbon source, glucose-6-phosphate can be metabolized via the ED pathway. After knocking out the PHB synthetic pathway, the growth of the cells was affected (as shown in FIG. 10), but the glucose yield was improved. This indicates the presence of CO ₂ As a sole carbon source, the carbon flow in PHB-deficient strains may be lost in the form of pyruvate, but the amounts of glucose-1-phosphate and glucose-6-phosphate, precursors for glucose synthesis, are still greatly increased, thereby promoting the synthesis of products. However, from the aspect of product yield, single knockoutglkKnock-out ofglkAndzwfknock-out ofglkAnd PHB synthetic pathway, combinatorial knock-outsglk、zwfAnd PHB were 80.0, 91.8, 231.0 and 252.2 mg/(g of dry cell weight), respectively. This suggests that knock-out of the PHB synthesis pathway has a greater effect on glucose production than the ED pathway. Reported in the literature as CO ₂ As a carbon source, glucose-6-phosphate dehydrogenase activity in Ralstonia is inhibited, resulting in a poor carbon metabolism of the ED pathway. While the increased yield of the strain that truncates the ED pathway may be due more to its higher bacterial mass (as shown in figure 10).

The above examples are only for explaining the technical solutions of the present application, and do not limit the scope of protection of the present application.

Sequence listing

<110> Beijing animal husbandry and veterinary institute of Chinese academy of agricultural sciences

<120> glucose-producing ralstonia engineered bacterium and fermentation production method

<160> 7

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1014

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

atggccacca ctgcatcctc ctgcgctgac ttcccccgcc tgctcggcga tgtgggcggc 60

accaacgtgc gctttgcact ggaaaccgca ccaatgcgga tcggcccggt gaccgcgctg 120

aaggtggccg atttcccgtc gctcgaagca gccctgcgcc aatacctcga cggactttcc 180

gcgtcgggca agccggtgcc gcgccacgcg gcgatcggcc tggccaaccc ggtgaccggc 240

gaccaggtcc ggctcaccaa ccacaactgg tcgttctcga tcgacggcat gcggcgcgcg 300

ctcgggctgc agacgctggt ggccatcaac gacttcaccg cgctggcgct ggccctgccg 360

tacctgcctg ccgacgggct ggtgcccgtg cgtgccggca ccgcggtgcg caccgcgccg 420

ctcgcgctgg tcgggccggg caccgggctg ggcgtttccg gcctggtgcc ggcgccgggc 480

ggagcagccg tggcgctggc cggcgaaggc ggccatatcg agctgatgcc cgacactgac 540

gacgaatgga tcgcctggcg cgccgcccat cgcaacgttg gccgggtgtc ggcggagagg 600

ctcctgtgcg gcagcggcct gtcgcatatc catgccgcgc tggccgcaga aacgggtacg 660

cttttgctcg cgccgctgtt gccggagcag gtcaccaccg gcgccttcga gcgccacgac 720

ccgctgtgcc agcgcgccat ggcggtgttc ttcggcctgc tcggctcggt tgcggccgac 780

atcgccctgg tgctgggcgc acgtggcggg gtctacctgg gcggcggcat cctgccacgc 840

ttcgtgcctg cgctgcaggc ctcggccttc gccgagcgct ttgtcgccaa gggccgcatg 900

cgcggctggc tggaggccgt gccggtccac gtcatcaccg ccagccaccc cgcgctgccg 960

ggactggcgc gcgcactggc cgagcagctc gacggccgcg gccggaacgc atag 1014

<210> 2

<211> 1488

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atgcaaacca cccgaaccgc ctcttcttcc ggcactgccg acgcgcggcc actggacctc 60

gtcatcttcg gcggcgccgg cgacctgtcg gcacgcaagc tgctgccatc gctctacatg 120

tgccaccgcg acggcaacct gcccgacggc acccgcatca tcggcgtggg ccggcaccgc 180

tgggaccgcg aggcctttgt caattttgcc gatgaaagcg cacaaccctt tgtcgaagcg 240

cgctatctcg agccggccaa atggcagacc ttcctgcagc ggctggattt cgtgcacgtt 300

gacgcgtcgc agtccgggga ctatccggcg ctggccgcgc aactgcgcca ggaggcgctg 360

cgcatctatt acatggcgat gccgccgggc ttgttcgcgg ccacctgcga caaccttgcc 420

agccatggcc tgatcgccaa cgacacccgg ctggtgctgg aaaaaccgct gggcgtggac 480

ctggcctcgg ccatcggcat cggcgaggtg gtcagccgct acttcagcga ggaccgcacc 540

taccggatcg accactacct gggcaaggaa acggtgcaga acctgatggc gctgcgcttc 600

ggcaattcga tcttcgagcc gctgtggcgc acgccgttcg tgcgcagcgt gcagatcacc 660

gtggccgaga ccgtgggcgt gggcacgcgc ggcggcttct acgacgaggc cggcgccatg 720

cgcgacatgg tgcagaacca cctgctgcag ctggtcagca tcctggcgat ggagccgccg 780

gcagcgctca cctccgatgc ggtgcgcgat gagaagctca aggtgctgcg ctcgctgcgg 840

ccgatgtcgc ccgaggacgt acgccgcaat accgtgcgcg gccagtacac cgcgggcgcc 900

atcgccggcg agctggtgcg cggctacctg caggaagaag gcattccgcc agacagccgc 960

accgagacct tcgtcgcgat gcgcgccgag ctcggcacgt ggcgctggaa caaggtgccg 1020

ttctacctgc gcactggcaa gcgcatgcag gagcgcgtga ccgaggtggt gatcaacttc 1080

gccgaggtgc cgcattcgat cttcgatgcc ggcagcacgc tgcagcccaa ccgcatggtg 1140

atccggctgc agccggaaga gtcggtgcgg ctgacgctga tggtcaagca gccaggcgag 1200

ggcatgaagc tgaagccgct gagcctggcg ctgaacctgg actcggcctt caccacccgg 1260

cgcgccgaag cctacgagcg cctgctgctc gacgtgatac gcggacggct ggcgctgttc 1320

gtgcggcgcg acgaactgca ggccgcctgg acctgggtcg acccgatcct ggaagcgtgg 1380

cgccagcagg acgaaggccc gcgcccctat accgccggca cctggggccc ggcggcttcg 1440

tcggcgttca tggcgcgcga aggcgtgcag tggtctgagg aggcgtga 1488

<210> 3

<211> 1497

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atgatggcta gcccccctgc cggcgctccg gcacctgaac ctgctccaga gttcgatttc 60

gtactgttcg gcgccaccgg cgacctggcg cggcgcaagc tgctgccggc actgttcgat 120

gcccacgccg ccggttcgct gcatccgcgc gggcgcatcc tggcactggg cagccagccg 180

ctgtcgcatg acgcctacct ggcgatgcta aacgacgaag tgctgccagc gctggccggc 240

aacgccgcgg ccgcagcgtg gcagggcttt ctcgaccgca tcgtgtacct gcaggtcgat 300

gccaacgccg atgccggctt cggcgcgctg gccgagctgg tcaatgcgcg cgcggcgcca 360

gtggtggtgt tctacctggc cacggccccg cacctgttcg tcaccatctg cgagcaactg 420

gcgcgcgtgg gcctgaccgg cccgcgcagc cgcatcgtgc tggaaaaacc gctcgggcac 480

gacctggcct cgtccgaggc catcaacgcg gaagtggcgc ggcactttgc cgaggaccag 540

atctaccgga tcgaccacta cctgggcaag gagtcggtgc agaacctgat ggcggtgcgc 600

tttggcaacg tgctgttcga gccgctgtgg cggcgcgaat gggtgcgcca ggtgcagatc 660

accatcgccg aagagctggg cgtggagcgc cgcggcaact tctatgacgg catcggcgcg 720

ctgcgcgaca tggtgcagaa ccacctgctg cagctgctgt gcatggtggc gatggagccg 780

cccacgagcc tgtcggaaga cgccatccgc gatgagaagc tcaagatcct gaaggcgctg 840

cggccgatcc ggccggaaga cgtggccgaa aaggtggtgc gtggccagta ctcccgtggc 900

gccgcgggcg gcgacccggt gcccggctat gccagtgaac ccggcatcgc gcccgacagc 960

cgcaccgaga ccttcgtcgc tatccgcgcc gagatcgcca actggcgctg ggccggggtg 1020

ccgttctacc tgcgtaccgg caagcgcatg cagtcgcgcg tggcggagat cgtaatccat 1080

ttccatgacg tgccctaccc gctgttcccg cacccgctgg gaatcatgtc cggcaaccgg 1140

ctggtgatca cgctgcagcc cgaggagagc atccggctgt atttcctgac caagcagccg 1200

ggcgacacgc aggcgctgat tcccgcgtcc ctcgacctgc agctgaacac tgccgcccca 1260

cgtgtacggc gcgtgggcgc gtacgagcgc ctgctgctcg acgtgatccg cggccggctg 1320

ggcctgttcg tgcggcgcga tgagcaggta caggcgtggc gctgggtcgc gcccatcctg 1380

aagacgtggg agaactcgcc cacgccgccc aagccttaca ccgccggcac ctggggcccg 1440

gcagcctcca gtgccctgct gtcgcgagac ggcatggcct ggcatgaaga gatgtag 1497

<210> 4

<211> 1458

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

atgcctgatt tcgacatggt gctgttcggc ggcaccggcg acctggcgcg gcgcaagctg 60

ctgccgtccc tgttcgatgc ccaccacgcc ggcctgctgc atccggccgc acgcatcctc 120

gccaccggca gccagccgct ctcgaccgcg gactacctcg cctcgctgga gcagaccgtg 180

cgccccgggc tagcccatgc gccgcccgag tcatgggaca agttctgcgc gcgcattgtc 240

tacgtgcagg ccgatgcacg cgtgcccgcg catttcgatg cgctcgcgga gcaggtgctg 300

gcacgcaagc ctgaagtggt ggtgtgctat ctcgccaccg cgccgcacct gttcgtgtcg 360

atctgcgagc agctggcacg caccggcctg aacacgcgcc aggcgcccaa cgtgcgcatc 420

gtgctggaaa aaccgctggg gcatgacctc gaatccaacg aggcaatcaa ctcggcagtg 480

gcgaagttct tcgccgaaga gcagatctac cgcatcgatc attacctcgg caaggagtcg 540

gtgcagaacc tgatggcgat ccgcttcggc aacgcgctgt tcgagccgct gtggcggcgc 600

gagtgggtgc aggacgtgca gatcaccatc gccgaagaac tcggcgtgga aacgcgcggc 660

gacttctacg accgcaccgg cgcgctgcgc gacatggtgc agaaccactt gctgcagctc 720

ttgtgcatgg tggcgatgga gccgccggcc agcctcagcg aagatgccat ccgcgacgag 780

aagatcaaga tcctgaaggc gctcaagccg atcacgccgc aggacgtggc cgagaagacc 840

gtgcgcggcc agtaccgctc cggtgccatc ggcggcaagc cggtgccggg ctacctggaa 900

gaagcgggca tcgcgcccga cagccgcacc gagacctttg tcgcgatcaa ggccgagatc 960

gccaactggc gctgggaagg cgtgccgttc tacctgcgca ccggcaagcg tatgcagtcg 1020

cgcgtggccg agatcgtgat ccatttccgc gacgtgccgc acgcgatctt cccgcggccg 1080

ctgacgctgt cgccgcagaa ccggctggtg atccagctgc agccggaaga aagcatccgc 1140

ctgtactgcc tggtcaagca gcctggcgac acgctggagc tgacgccgac ctcgctcgac 1200

ctggactttg ccaactcgtt caaggtgcgc cgcgccggcg cctacgaacg cttgctgctc 1260

gacgtgatcc gcggccggct gggcctgttc gtgcgccgcg atgaacaagt gcaggcctgg 1320

cgctgggtcg aacccatcat cgacacctgg gatgccagca ccgtgccgcc caagccctat 1380

accgccggca cctggggacc ggccgcgtcg tcggcgctga tgtcgcgcga cggcgggctg 1440

tggcacgagg aagcctga 1458

<210> 5

<211> 1770

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

atggcgaccg gcaaaggcgc ggcagcttcc acgcaggaag gcaagtccca accattcaag 60

gtcacgccgg ggccattcga tccagccaca tggctggaat ggtcccgcca gtggcagggc 120

actgaaggca acggccacgc ggccgcgtcc ggcattccgg gcctggatgc gctggcaggc 180

gtcaagatcg cgccggcgca gctgggtgat atccagcagc gctacatgaa ggacttctca 240

gcgctgtggc aggccatggc cgagggcaag gccgaggcca ccggtccgct gcacgaccgg 300

cgcttcgccg gcgacgcatg gcgcaccaac ctcccatatc gcttcgctgc cgcgttctac 360

ctgctcaatg cgcgcgcctt gaccgagctg gccgatgccg tcgaggccga tgccaagacc 420

cgccagcgca tccgcttcgc gatctcgcaa tgggtcgatg cgatgtcgcc cgccaacttc 480

cttgccacca atcccgaggc gcagcgcctg ctgatcgagt cgggcggcga atcgctgcgt 540

gccggcgtgc gcaacatgat ggaagacctg acacgcggca agatctcgca gaccgacgag 600

agcgcgtttg aggtcggccg caatgtcgcg gtgaccgaag gcgccgtggt cttcgagaac 660

gagtacttcc agctgttgca gtacaagccg ctgaccgaca aggtgcacgc gcgcccgctg 720

ctgatggtgc cgccgtgcat caacaagtac tacatcctgg acctgcagcc ggagagctcg 780

ctggtgcgcc atgtggtgga gcagggacat acggtgtttc tggtgtcgtg gcgcaatccg 840

gacgccagca tggccggcag cacctgggac gactacatcg agcacgcggc catccgcgcc 900

atcgaagtcg cgcgcgacat cagcggccag gacaagatca acgtgctcgg cttctgcgtg 960

ggcggcacca ttgtctcgac cgcgctggcg gtgctggccg cgcgcggcga gcacccggcc 1020

gccagcgtca cgctgctgac cacgctgctg gactttgccg acacgggcat cctcgacgtc 1080

tttgtcgacg agggccatgt gcagttgcgc gaggccacgc tgggcggcgg cgccggcgcg 1140

ccgtgcgcgc tgctgcgcgg ccttgagctg gccaatacct tctcgttctt gcgcccgaac 1200

gacctggtgt ggaactacgt ggtcgacaac tacctgaagg gcaacacgcc ggtgccgttc 1260

gacctgctgt tctggaacgg cgacgccacc aacctgccgg ggccgtggta ctgctggtac 1320

ctgcgccaca cctacctgca gaacgagctc aaggtaccgg gcaagctgac cgtgtgcggc 1380

gtgccggtgg acctggccag catcgacgtg ccgacctata tctacggctc gcgcgaagac 1440

catatcgtgc cgtggaccgc ggcctatgcc tcgaccgcgc tgctggcgaa caagctgcgc 1500

ttcgtgctgg gtgcgtcggg ccatatcgcc ggtgtgatca acccgccggc caagaacaag 1560

cgcagccact ggactaacga tgcgctgccg gagtcgccgc agcaatggct ggccggcgcc 1620

atcgagcatc acggcagctg gtggccggac tggaccgcat ggctggccgg gcaggccggc 1680

gcgaaacgcg ccgcgcccgc caactatggc aatgcgcgct atcgcgcaat cgaacccgcg 1740

cctgggcgat acgtcaaagc caaggcatga 1770

<210> 6

<211> 1182

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

atgactgacg ttgtcatcgt atccgccgcc cgcaccgcgg tcggcaagtt tggcggctcg 60

ctggccaaga tcccggcacc ggaactgggt gccgtggtca tcaaggccgc gctggagcgc 120

gccggcgtca agccggagca ggtgagcgaa gtcatcatgg gccaggtgct gaccgccggt 180

tcgggccaga accccgcacg ccaggccgcg atcaaggccg gcctgccggc gatggtgccg 240

gccatgacca tcaacaaggt gtgcggctcg ggcctgaagg ccgtgatgct ggccgccaac 300

gcgatcatgg cgggcgacgc cgagatcgtg gtggccggcg gccaggaaaa catgagcgcc 360

gccccgcacg tgctgccggg ctcgcgcgat ggtttccgca tgggcgatgc caagctggtc 420

gacaccatga tcgtcgacgg cctgtgggac gtgtacaacc agtaccacat gggcatcacc 480

gccgagaacg tggccaagga atacggcatc acacgcgagg cgcaggatga gttcgccgtc 540

ggctcgcaga acaaggccga agccgcgcag aaggccggca agtttgacga agagatcgtc 600

ccggtgctga tcccgcagcg caagggcgac ccggtggcct tcaagaccga cgagttcgtg 660

cgccagggcg ccacgctgga cagcatgtcc ggcctcaagc ccgccttcga caaggccggc 720

acggtgaccg cggccaacgc ctcgggcctg aacgacggcg ccgccgcggt ggtggtgatg 780

tcggcggcca aggccaagga actgggcctg accccgctgg ccacgatcaa gagctatgcc 840

aacgccggtg tcgatcccaa ggtgatgggc atgggcccgg tgccggcctc caagcgcgcc 900

ctgtcgcgcg ccgagtggac cccgcaagac ctggacctga tggagatcaa cgaggccttt 960

gccgcgcagg cgctggcggt gcaccagcag atgggctggg acacctccaa ggtcaatgtg 1020

aacggcggcg ccatcgccat cggccacccg atcggcgcgt cgggctgccg tatcctggtg 1080

acgctgctgc acgagatgaa gcgccgtgac gcgaagaagg gcctggcctc gctgtgcatc 1140

ggcggcggca tgggcgtggc gctggcagtc gagcgcaaat aa 1182

<210> 7

<211> 741

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

atgactcagc gcattgcgta tgtgaccggc ggcatgggtg gtatcggaac cgccatttgc 60

cagcggctgg ccaaggatgg ctttcgtgtg gtggccggtt gcggccccaa ctcgccgcgc 120

cgcgaaaagt ggctggagca gcagaaggcc ctgggcttcg atttcattgc ctcggaaggc 180

aatgtggctg actgggactc gaccaagacc gcattcgaca aggtcaagtc cgaggtcggc 240

gaggttgatg tgctgatcaa caacgccggt atcacccgcg acgtggtgtt ccgcaagatg 300

acccgcgccg actgggatgc ggtgatcgac accaacctga cctcgctgtt caacgtcacc 360

aagcaggtga tcgacggcat ggccgaccgt ggctggggcc gcatcgtcaa catctcgtcg 420

gtgaacgggc agaagggcca gttcggccag accaactact ccaccgccaa ggccggcctg 480

catggcttca ccatggcact ggcgcaggaa gtggcgacca agggcgtgac cgtcaacacg 540

gtctctccgg gctatatcgc caccgacatg gtcaaggcga tccgccagga cgtgctcgac 600

aagatcgtcg cgacgatccc ggtcaagcgc ctgggcctgc cggaagagat cgcctcgatc 660

tgcgcctggt tgtcgtcgga ggagtccggt ttctcgaccg gcgccgactt ctcgctcaac 720

ggcggcctgc atatgggctg a 741

Claims (6)

1. A ralstonia engineering bacterium for producing glucose is characterized in that the engineering bacterium is ralstonia which knocks out key genes in glucokinase gene, ED path and poly ([ R ] -3 hydroxybutyric acid) synthetic path,

wherein the glucokinase gene is a glucokinase coding geneglkThe nucleotide sequence is shown as SEQ ID NO: 1 is shown in the specification;

is knockedThe key gene of the ED pathway is the coding gene of glucose-6-phosphate dehydrogenasezwf1、zwf2Andzwf3the genezwf1The nucleotide sequence of (a) is shown as SEQ ID NO: 2, the genezwf2The nucleotide sequence of (a) is shown as SEQ ID NO: 3, the genezwf3The nucleotide sequence of (a) is shown as SEQ ID NO: 4 is shown in the specification;

knocked-out poly ([ R)]-3-Hydroxybutanoic acid) synthetic pathway is poly ([ R)]-3 hydroxybutyrate) polymerase encoding genephaC1Acetyl coenzyme A acetyltransferase coding genephaAAnd acetoacetyl-CoA reductase-encoding genephaB1The poly ([ R)]-3 hydroxybutyrate) polymerase encoding genephaC1The nucleotide sequence of (a) is shown as SEQ ID NO: 5, the acetyl coenzyme A acetyl transferase coding genephaAThe nucleotide sequence of (a) is as shown in SEQ ID NO: 6, the acetoacetyl-CoA reductase encoding genephaB1The nucleotide sequence of (a) is shown as SEQ ID NO: shown at 7.

2. A method of increasing glucose yield in ralstonia, said method comprising the steps of:

knocking out glucose kinase gene of Ralstonia and constructing mutant Ralstonia chassis cells;

key genes in an ED pathway and a poly ([ R ] -3-hydroxybutyric acid) synthesis pathway are knocked out,

wherein the glucokinase gene is a glucokinase coding geneglkThe nucleotide sequence is shown as SEQ ID NO: 1 is shown in the specification;

the key gene of the knocked-out ED pathway is a coding gene of glucose-6-phosphate dehydrogenasezwf1、zwf2Andzwf3the genezwf1The nucleotide sequence of (a) is as shown in SEQ ID NO: 2, the genezwf2The nucleotide sequence of (a) is shown as SEQ ID NO: 3, the genezwf3The nucleotide sequence of (a) is shown as SEQ ID NO: 4 is shown in the specification;

knocked-out poly ([ R)]-3-Hydroxybutanoic acid) synthetic pathway is poly ([ R)]-3 hydroxybutyrate) polymerase encoding genephaC1Acetyl coenzyme A acetyltransferase coding genephaAAnd acetoacetyl-CoA reductionEnzyme-encoding genephaB1The poly ([ R)]-3 hydroxybutyrate) polymerase encoding genephaC1The nucleotide sequence of (a) is shown as SEQ ID NO: 5, the acetyl coenzyme A acetyl transferase coding genephaAThe nucleotide sequence of (a) is shown as SEQ ID NO: 6, the acetoacetyl-CoA reductase encoding genephaB1The nucleotide sequence of (a) is shown as SEQ ID NO: shown at 7.

3. A method for producing glucose by fermentation, comprising the step of shake flask fermentation of the engineered Ralstonia bacterium for producing glucose of claim 1.

4. The method of claim 3, wherein the fermentation is performed using a medium with different carbon sources, wherein the medium with different carbon sources has the following formula: 3.5 g/L Na ₂ HPO ₄ ，1.5 g/L KH ₂ PO ₄ ，1.0 g/L (NH ₄ ) ₂ SO ₄ ，80 mg/L MgSO ₄ ⋅7H ₂ O，1 mg/L CaSO ₄ ⋅2H ₂ O，0.56 mg/L NiSO ₄ ⋅7H ₂ O, 0.4 mg/L ferric citrate, 200 mg/L NaHCO ₃ Adding fructose and glycerol at a ratio of 4 g/L or introducing H ₂ :O ₂ :CO ₂ Mixed gas of =8:1:1 as a carbon source.

5. The method of claim 4, wherein the medium containing the different carbon source contains a final concentration of 200 mg/L kanamycin antibiotic.

6. The method for the fermentative production of glucose according to claim 3, wherein the fermentation conditions are a temperature of 30 ℃ and a rotation speed of 200 rpm.

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