KR101483012B1 - Method for production of 3-hydroxypropionic acid using recombinant E. coli with high yield - Google Patents

Abstract

The present invention relates to a method for producing 3-hydroxypropionic acid in high yield by using recombinant E. coli inactivated or overexpressed with a related enzyme involved in 3-HP biosynthesis as a host and using glycerol, xylose or glucose as a substrate will be.

Description

Method for production of 3-hydroxypropionic acid using recombinant Escherichia coli at a high yield (Method for production of 3-hydroxypropionic acid using recombinant E. coli with high yield)

The present invention relates to a method for producing 3-hydroxypropionic acid with high yield using recombinant E. coli, and more particularly, to a method for producing 3-hydroxypropionic acid by using recombinant E. coli inactivated or overexpressed with a related enzyme involved in 3-HP biosynthesis, Glycerol, xylose or glucose as a substrate to produce 3-hydroxypropionic acid in high yield.

3-hydroxypropionic acid (3-HP) or a salt thereof is mainly used as an antistatic agent for a crosslinking agent, a metal lubricant and a fabric of a polymer coating agent, and is an important building block in organic synthesis Can be used. The key compounds synthesized from 3-HP as a precursor include 1,3-propanediol, acrlici acid, methyl acrylate, acrylamide, ethyl 3-HP, malonic acid malonic acid, propiolactone, and acrylonitrile.

3-HP is used as a precursor for the synthesis of various materials as described above, and has various application fields. The global market size is about 3.63 million tons per year. 3-HP is important enough to occupy the third position among the 12 platform compounds for regenerative biomass products currently on the US DOE list.

However, there is not much research on the biosynthetic pathway of 3-HP which plays an important and useful role as described above, and most of the researches that have been conducted are syntheses of 3-HP using glycerol as a substrate. However, little research has been conducted on the technology for biosynthesis of 3-HP from glucose or xylose in addition to glycerol.

Korean Patent No. 10-1048151 (registered on July 04, 2011), which is registered by the present inventors, discloses that "glycerol is replaced with 3-hydroxypropionic aldehyde (3-hydroxypropionic acid) which is a precursor of 3-hydroxypropionic acid DHAB2 and DHAB3 among the subunits DHAB1, DHAB2 and DHAB3 constituting the glycerol dehydratase derived from Lactobacillus brevis, respectively, in order to convert the starting codon TTG (3-HPA) And glycerol dehydratase (DHAB) derived from Lactobacillus brevis, in which GTG is mutated to ATG, respectively, and glycerol dehydratase reactivase derived from Lactobacillus brevis are expressed; - transformed to express an enzyme that converts hydroxypropionaldehyde to 3-hydroxypropionic acid; A method of adding the glycerol and the bacteria, glucose is produced a 3-hydroxy-propionic acid from glycerol, characterized in that culturing in a medium the presence enough not to deplete the culture is "the substrate.

In the present invention, a method of producing 3-HP at a high yield by using glycerol, xylose or glucose as a substrate is developed and provided.

The first aspect of the present invention is a glycerol dehydratase which converts glycerol to 3-hydroxypropionaldehyde (3-HPA) which is a precursor of 3-hydroxypropionic acid. ; DHAB) is expressed; Is transformed to express an aldehyde dehydrogenase which converts 3-hydroxypropionaldehyde to 3-hydroxypropionic acid; A gene encoding a glycerol kinase and / or a glycerol dehydrogenase, or a gene encoding the glycerol kinase and / or glycerol dehydrogenase, respectively, may not be activated, so that part of the gene is disrupted so that its function is lost, Transformed; And culturing the recombinant E. coli in a medium supplemented with glycerol. The present invention also provides a method for producing 3-hydroxypropionic acid.

The second aspect of the present invention is a glycerol dehydratase which converts glycerol to 3-hydroxypropionaldehyde (3-HPA) which is a precursor of 3-hydroxypropionic acid. ; DHAB) is expressed; Is transformed to express an aldehyde dehydrogenase which converts 3-hydroxypropionaldehyde to 3-hydroxypropionic acid; A gene encoding a glycerol kinase and / or a glycerol dehydrogenase, or a gene encoding the glycerol kinase and / or glycerol dehydrogenase, respectively, may not be activated, so that part of the gene is disrupted so that its function is lost, Transformed; The glycerol-3-phosphatase and glycerol-3-phosphate dehydrogenase, which convert dihydroxyacetone phosphate into glycerol, are designed to express glycerol-3-phosphate dehydrogenase Converted; And culturing the recombinant E. coli in a medium supplemented with xylose. The present invention also provides a method for producing 3-hydroxypropionic acid.

The third aspect of the present invention is a glycerol dehydratase which converts glycerol to 3-hydroxypropionaldehyde (3-HPA) which is a precursor of 3-hydroxypropionic acid. ; DHAB) is expressed; Is transformed to express an aldehyde dehydrogenase which converts 3-hydroxypropionaldehyde to 3-hydroxypropionic acid; A gene encoding a glycerol kinase and / or a glycerol dehydrogenase, or a gene encoding the glycerol kinase and / or glycerol dehydrogenase, respectively, may not be activated, so that part of the gene is disrupted so that its function is lost, Transformed; The glycerol-3-phosphatase and glycerol-3-phosphate dehydrogenase, which convert dihydroxyacetone phosphate into glycerol, are designed to express glycerol-3-phosphate dehydrogenase Converted; And culturing the recombinant E. coli in a medium supplemented with glucose. The present invention also provides a method for producing 3-hydroxypropionic acid.

On the other hand, in the first, second, or third aspect of the present invention, the Escherichia coli preferably is selected from the group consisting of 1,3-propanediol dehydrogenase ( yqhD ) It is preferable that the gene encoding the gene is further transformed so that a part of the gene is disrupted so that the function thereof is lost or the whole gene is removed.

On the other hand, in the first, second or third aspect of the present invention, the recombinant E. coli preferably has a self-contained aldehyde dehydrogenase (hereinafter referred to as " aldehyde dehydrogenase " ) encoding gene is removed, Pseudomonas her labor Rouge (Pseudomonas aeruginosa- derived aldehyde dehydrogenase gene is preferably introduced.

On the other hand, in the first, second or third aspect of the present invention, it is preferable that the medium is added with coenzyme B ₁₂ . Glycerol having hydratase is an enzyme that converted glycerol to 3-hydroxy propionaldehyde, in the present invention, Lactobacillus brevis (Lactobacillus brevis . Glycerol dehydratase is B ₁₂ -dependent because B ₁₂ is required for the reaction.

On the other hand, in the first, second or third aspect of the present invention, the Escherichia coli is further transformed so that glycerol dehydratase reactivase is expressed, wherein the expressed glycerol dehydra Because it can maintain the activity of glycerol dehydratase (DHAB) constantly.

In the present invention, glycerol kinase ( glpK ), glycerol dehydrogenase ( gldA ) genes involved in glycerol metabolism are treated with lambda red recombinase to obtain high concentration of 3-HP from glycerol. Method. In order to inhibit the production of 1,3-PDO (propanediol), another metabolite that can be produced from 3-hydroxypropionaldehyde (3-HPA), a precursor of 3-HP, The 1,3-propanediol dehydrogenase ( yqhD ) gene was disrupted by the same method. This multiple strains of BL21 star (DE3) established by Δ glpK Δ yqhD / pELDRR / pCEa strain and BL21 star (DE3) Δ glpK Δ gldA / pELDRR / pCEa strain of 3-HP yield 3.6 times respectively as compared to existing strains , And 5.2 times, respectively.

On the other hand, in the case of the existing strains, the activity of the aldehyde dehydrogenase gene, which functions to convert 3-HPA to 3-HP, is significantly lower than that of glycerol dehydratase, 3-HPA, which is a metabolic pathway, is accumulated in the strain. In the present invention, Escherichia coli (E. coli) Pseudomonas lover than the high activity Kinase (aldehyde dehydrogenase) through to the hydro-derived aldehyde was used in the existing industrial Rouge (Pseudomonas The yield was increased 1.27 times by introducing the aldehyde dehydrogenase gene derived from the aeruginosa .

Thereafter, glycerol-3-phosphatase and glycerol-3-phosphatase, which further convert dihydroxyacetone phosphate to glycerol to produce 3-HP using xylose as a substrate, The glycerol-3-phosphate dehydrogenase gene was cloned into pCDFduet-1 vector and transformed. This, the BL21 star (DE3) of the strains built with Δ glp K Δyqh D / pELDRR / pCaGPDGPP strain and BL21 star (DE3) Δ glp K Δ gld A / pELDRR / pCaGPDGPP strain is 13.2 to 3-HP from xylose each g / L and 22.2 g / L, respectively.

Figure 1, on the other hand, shows the metabolic pathway in the recombinant E. coli of the present invention for 3-HP production. In Fig. 1, the gene indicated by "X" means that the gene is completely or partially disrupted and its activity is not exerted, and the gene indicated by the blue ellipse is a newly expressed or overexpressed gene.

In the present invention, in order to convert glycerol to 3-hydroxypropionaldehyde (3-HPA) which is a precursor of 3-hydroxypropionic acid, glycerol dehydratase ; DHAB). Since Escherichia coli does not have this enzyme, clones derived from other microorganisms should be cloned. Cloning and expression in E. coli of a variety of microbial glycerol dehydratases can be performed from common sense in the art with reference to U.S. Pat. No. 7,067,300 (Suryang Kwak, Yong-Cheol Park, Jin-Ho Seo: Biosynthesis of 3-hydroxypropionic acid from glycerol in recombinant Escherichia coli expressing Lactobacillus brevis dhaB and dhaR gene clusters and Escherichia coli K-12 aldH, 2012 online (Subramanian Mohan Raj, Sunghoon Park: Production of 3-hydroxypropionic acid from glycerol by recombinant Escherichia coli BL21 strain, Process Biochemistry, 2008, 1440-1446). As an example, as used in Korean Registered Patent No. 10-1048151 (registered on July 04, 2011), which the present inventors have registered, Lactobacillus brevis brevis can be used. However, when an enzyme derived from another microorganism is expressed in Escherichia coli, it is necessary to convert the initiation codon of the enzyme into an initiation codon (ATG) that can be initiated in Escherichia coli. And thus the detailed description thereof will be omitted.

In the present invention, glycerol-3-phosphatase and glycerol-3-phosphate dehydrogenase are used to produce glycerol from dihydroxyacetone phosphate (DHAP) -phosphate dehydrogenase. Since Escherichia coli does not have this enzyme, it must be cloned and expressed from other microorganisms (M. Hartlep, A.-P. Zeng : Study of two-stage processes for the microbial production of 1,3-propanediol from glucose, Appl Microbiol Biotechnol, 2002, 60: 60-66). As an example, Saccharomyces cerevisiae can be used. The cloning and expression of the gene in E. coli can be easily carried out from the common knowledge and skill in the art as described above, so that description thereof will be omitted.

In the meantime, the method of transformation used in the present invention can be carried out by a known method known in the genetic engineering system, so that detailed description thereof will be omitted.

According to the first, second and third embodiments of the present invention, industrially useful 3-hydroxypropionic acid (3-HP) can be produced from glycerol, xylose and glucose in high yields, respectively.

Figure 1 shows the metabolic pathway in the present recombinant E. coli for 3-HP production.
2 is a schematic diagram of gene disruption using the lambda red recombinase method.
3 is a vector map of the vector pCPa72 constructed in the present invention.
4 is a vector map of the vector pCaGPDGPP constructed in the present invention.
FIG. 5 shows the results of the fed-batch culture of the four strains constructed in the present invention (FIGS. 5A, 5A, 5C and 5D). 5a: BL21 star (DE3) / pELDRR / pCEa, 5b: BL21 star (DE3) ΔglpK / pELDRR / pCEa, 5c: BL21 star (DE3) Δ glpK Δ gldA / pELDRR / pCEa, 5d: BL21 star (DE3) Δ glpK DELTA yqhD / pELDRR / pCEa.
Fig. 6 is a result of comparing the non-enzyme activity of an aldehyde dehydrogenase derived from E. coli with an aldehyde dehydrogenase derived from Pseudomonas aeruginosa.
Figure 7 is introduced through the hydro-azepin to the Pseudomonas Ke Rouge labor-derived aldehyde BL21 star (DE3) Δ glpK Δ is a fed-batch culture the results of the yqhD / pELDRR / pCPa72 strain.
Figure 8 is a batch culture results in the BL21 star (DE3) Δ glpK / pCaGPDGPP strain.
9 is BL21 star (DE3) Δ glp KΔ is a fed-batch culture results of gldA / pELDRR / pCaGPDGPP strain.
10 is BL21 star (DE3) Δ glp K Δ is a fed-batch culture the results of the yqhD / pELDRR / pCaGPDGPP strain.
11 is BL21 star (DE3) in the glucose medium Δ glpK Δ yqhD / pELDRR / pCa72 strain (Fig. 11a), BL21 star (DE3) Δ glpK Δ gldA / pELDRR / pCa72 strain (Fig. 11b), BL21 star (DE3) Δ glpK Δ yqhD / pELDRR / pCaGPDGPP strain (Fig. 11c), a batch culture results in the BL21 star (DE3) Δ glpK ΔgldA / pELDRR / pCaGPDGPP strain (Fig. 11d).

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the scope of the present invention is not limited to the following embodiments, and includes modifications of equivalent technical ideas.

[ Example  1: Production of recombinant Escherichia coli]

1) Glycerol Kinase ( glycerol kinase ), Glycerol dehydrogenase ( glycerol  dehydrogenase), 1,3- Propanediol  Dehydrogenase (1,3- propanediol  dehydrogenase) gene

Glycerol kinase of the parent strain BL21 star (DE3) (glycerol kinase , glpK), through dihydro having glycerol kinase (glycerol dehydrogenase, gldA), having come through dihydro-1,3-dimethyl propane dehydratase (1,3-propanediol dehydrogenase, yqhD ). For the disruption of the gene, the lambda red recombinase method (Kirill A. Datsenko, Barry L. Wanner and Wanner: One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products, PNAS, 2000, 12: 6640-6645). 2 is a schematic diagram of gene disruption using the lambda red recombinase method.

First, kanamycin-resistance cassette pKD13 (using the primers listed in Table 1 to obtain (kanamycin resistance cassettes) Kirill A. Datsenko , Barry L. Wanner and Wanner: One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products, PNAS, 2000, 12: 6640-6645) was used as a template for polymerase chain reaction (PCR). The thus-obtained kanamycin-resistant cassette pKD46 (Kirill A. Datsenko, Barry L. Wanner and Wanner: One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products, PNAS, 2000, 12: 6640-6645) Genes ( glpK , gldA , yqhD ) were disrupted by transformation into E. coli BL21 star (DE3). In order to confirm that the target gene was disrupted by colony PCR using the primers listed in Table 1, pCP20 (Kirill A. Datsenko, Barry L. Wanner and Wanner: One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products, PNAS, 2000, 12: 6640-6645) was introduced to remove the kanamycin resistance cassette (Fig. 2).

[Table 1]

Glycerol kinase, glycerol dehydrogenase, a strain used for the disruption of the 1,3-propanediol dehydrogenase gene, a primer, a vector

Experimental results, BL21 star (DE3) Δ glpK, BL21 star (DE3) Δ Δ gldA glpK, BL21 star (DE3) Δ Δ glpK yqhD The strain was constructed.

 2) Pseudomonas Aruzinosa ( Pseudomonas aeruginosa Originating Aldehyde  Dehydrogenase ( aldehyde dehydrogenase ) gene Cloning

Pseudomonas Alejino (Pseudomonas aeruginosa) Encoding an aldehyde dehydrogenase derived from an aldehyde dehydrogenasePSPA _3072The gene was amplified by PCR and then introduced into the pCDFDuet-1 vector. As a result, pCDFDuet-1-PSPA _3072 (pCPa72) vector was constructed (Table 2) (Figure 3). 3 is a vector map of the constructed vector pCPa72.

[Table 2]

The strains, primers, vectors used for gene introduction of aldehyde dehydrogenase

* The underlined sequence is the restriction enzyme site.

 3) Glycerol-3-phosphatase ( glycerol -3- 포스화제 ) And glycerol-3- Phosphate  Dehydrogenase ( glycerol -3- phosphate dehydrogenase ) Of the gene Cloning

Saccharomyces cerevisiae) D-452-2-derived glycerol-3-phosphatase (glycerol-3-phosphatase) and glycerol-3-phosphate dehydro through dehydratase (glycerol-3-phosphate dehydrogenase) and GPP2 the GPD1 encoding each The gene was amplified by PCR and cloned into MCS2 (Multi Cloning Site 2) of pCPa72 vector constructed in 2) of Example 1 above. As a result, the pCPa72-GPD1-GPP2 (pCaGPDGPP) vector was constructed (Table 3, Fig. 4). Figure 4 is a vector map of the constructed vector pCaGPDGPP.

[Table 3]

A strain, a primer, and a vector used for introducing glycerol-3-phosphatase and glycerol-3-phosphate dehydrogenase gene

* The underlined sequence is the restriction enzyme site.

 4) Construction of recombinant E. coli through transformation

The vector constructed in 2) and 3) of Example 1 above was mixed with the vector of pET29-dhaB-dhaR (pELDRR) (refer to Reference 7 in Korean Patent No. 10-1048151) 1) to construct recombinant E. coli. The strains and plasmids used were as follows (Table 4, Table 5).

[Table 4]

Strain used for transformation

[Table 5]

The plasmid used for transformation

* Kan ^r : Kanamycin resistance gene, Sm ^r : Streptomycin / Spectinomycin resistance gene

[ Example  2: Recombinant Escherichia coli to confirm the gene disruption effect Oil price formula  Culture - using glycerol as substrate -]

(DE3) / pELDRR / pCEa, b: BL21 star (DE3) ? GlpK (DE3) in order to examine the gene disruption effect in 1) / pELDRR / pCEa, c: BL21 star (DE3) ? glpK ? gldA / pELDRR / pCEa, d: BL21 star (DE3) ? glpK ? yqhD / pELDRR / pCEa were carried out by pH-stat fed-batch culture. The pCEa vector introduced into the four strains was the vector described in Reference 7 in Korean Patent No. 10-1048151, which was registered by the inventors of the present invention. The pCEa vector was obtained by using the E. coli K12-derived aldehyde dehydrogenase gene Cloned and inserted.

Fermentation was performed in a 1 L volume in a 2.5 L jar fermenter. The culture medium was a Riesenberg medium supplemented with 20 g / L glucose. Kanamycin 50 mg / L and streptomycin 50 mg / L were used as antibiotics. During the culture, air was supplied at a rate of 1 vvm, stirring was performed at 12,000 ~ 13,000 rpm, and incubation temperature was 37 ° C before induction of expression and 25 ° C after induction of expression. The pH was adjusted to 6.8 by feeding 28% ammonia water.

From the point when the sugar was depleted in the medium, a feeding solution consisting of 800 g / L glucose and 14 g / L MgSO ₄ · 7H ₂ O was supplied. When the OD ₆₀₀ value reached 100-120, expression was induced by adding 0.2 μM IPTG (isopropyl-β-D-thio-galactoside) and 20 μM coenzyme B ₁₂ (glycerol dehydratase cofactor). After induction of expression, coenzyme B ₁₂ I cut off the light to prevent the decomposition of. In addition, 60 g / L of glycerol, a substrate of 3-HP, was dumped upon induction of expression.

The result of culturing was as shown in Fig. BL21 star (DE3) Δ glpK / pELDRR / pCEa strain is suppressed from being glycerol is used in the cell growth by being glpK is crushed, the old BL21 star (DE3) 3-HP production was increased approximately 2-fold compared to pELDRR / pCEa strain, the BL21 star (DE3) Δ glpK / pELDRR / Δ glpK Δ yqhD / pELDRR / pCEa strain pCEa strains was crushed for further yqhD gene is to reduce the production of 1,3-propanediol (propanediol) 3-HP production from about Respectively. 5 (a) to 5 (d) show the results (a, b, c, d) of the fermentation of the four strains.

[room Sime  3: Pseudomonas Aruzinosa  Derived Aldehyde Dehydrogenase  Checking the effect of gene transfer - using glycerol as substrate -]

Pseudomonas aeruginosa administered in Example 1, 2) In order to confirm the genealogical effect of aldehyde dehydrogenase derived from E. coli, the fermentation was carried out and the activity of aldehyde dehydrogenase was measured.

Titration was done in a slightly modified version of Bergmeyer and Gawehn (1974). A 200 μL sample consisting of 50 mM potassium phosphate buffer (pH 7.0), 0.4 mM NAD ⁺ , 5 mM MgCl _2, 0.4 mM DTT and 50 mM 3-HPA was transferred to a microplate reader (Molecular Devices Co., Sunnyvale , CA, USA) and the amount of NAD ⁺ reduced at 30 ° C and 340 nm was measured. One unit of ALDH was defined as the amount of enzyme required to reduce 1 micromolar of NAD ⁺ to NADH per minute. Specific enzyme activity (U / mg protein) values were calculated by dividing the ALDH titer by the cell protein concentration Respectively.

The culture conditions of the fed-batch culture were the same as the fed-batch culture conditions described in Example 2 above.

The results of the titration and fed-batch culture were as shown in Figs. 6 and 7. FIG. 6 is a graphE. coli) Derived from aldehyde dehydrogenase and aldehyde dehydrogenase derived from Pseudomonas aeruginosa. FIG. 7 is a graph showing the effect of BL21 star (DE3) -adrenoceptor-derived aldehyde dehydrogenase derived from Pseudomonas aeruginosaΔ glpK Δ yqhD/ pELDRR / pCPa72 strain.

Pseudomonas Alejino The resulting aldehyde dehydrogenase is referred to as < RTI ID = 0.0 > Showed about 1.42-fold higher potency as compared to the aldehyde dehydrogenase derived from E. coli . Pseudomonas Ke Rouge through dihydro having aldehyde of industrial origin result of the introduction of the strain azepin fed-batch culture, the E. coli (E. coli) The 3-HP production compared to the aldehyde dehydro strain introduced through the dehydratase derived from 57.38 g / L, and the yield (M 3 -HP / M glycerol) was also increased by about 27% to 0.93.

[ Example  4: glycerol-3-phosphatase ( glycerol -3- 포스화제 ) And glycerol-3- Force Pate Dehydrogenase ( glycerol -3- phosphate dehydrogenase ) Confirmation of gene transfer effect - Xylose  use-]

To confirm the effect of introducing glycerol-3-phosphatase and glycerol-3-phosphate dehydrogenase genes GPD1 and GPP2 in 3) of Example 1, batch culture and fed-batch culture were carried out.

The batch culture was carried out in a volume of 100 mL. The medium used was Riesenberg medium supplemented with 10 g / L of xylose and 5 g / L of yeast extract. Streptomycin 50 mg / L was used as an antibiotic. Induction of the introduced enzyme was performed by adding 0.2 Mm of IPTG when the OD ₆₀₀ value became 0.8 ~ 1. The cells were cultured at 37 ° C until expression induction and at 25 ° C after expression, and cultured at 250 rpm for oxygen agitation.

On the other hand, fed-batch culture was performed in a 1 L volume in a 2.5 L jar fermenter. The medium used was Regenberg medium supplemented with 20 g / L of xylose and 5 g / L of yeast extract, and 50 mg / L of kanamycin and 50 mg / L of streptomycin were used as antibiotics. The preculture was the same as the batch culture condition except that 50 mg / L of kanamycin and 50 mg / L of streptomycin were added and cultured for 12 hours without expression induction. During the culture, air was supplied at a rate of 1 vvm, stirring was performed at 12,000 ~ 13,000 rpm, and the incubation temperature was 37 ° C before expression induction and 25 ° C after expression induction. The pH was adjusted to 6.74 to 6.82 by feeding 28% ammonia water.

From the point where the sugar is depleted in the medium, a feeding solution composed of 600 g / L xylose and 14 g / L MgSO ₄ · 7H ₂ O is constantly fed at a rate of 0.75 g / L · h . When the OD ₆₀₀ value was 3.0 ~ 8.0, 20 μM of IPTG and coenzyme B ₁₂ (cofactor of glycerol dehydratase) were added to induce expression, and after induction of expression, light was blocked to prevent degradation of coenzyme B ₁₂ .

Xylene were BL21 star (DE3) Δ glpK / batch culture results in pCaGPDGPP strain pattern to accumulate glycerol is produced unlike before 8 and seemed, GPD1, GPP2 introduced conducted by Los medium. Figure 8 is a batch culture results in the BL21 star (DE3) Δ glpK / pCaGPDGPP strain.

Thus by introducing a plasmid in pCaGPDGPP star BL21 (DE3) Δ Δ gldA glpK / pELDRR and BL21 star (DE3) Δ glp K Δ yqh D / pELD RR was subjected to fed-batch culture according. The result is 9, seemed to Figure 10 glp K, yqh D-deficient mutant recombinant E. coli was producing 3-HP of 13.2 g / L for 120 hours, glpK, gldA deficient mutant recombinant E. coli was 22.2 g / L for 223 hours Of 3-HP. 9 is BL21 star (DE3) is a fed-batch culture results in Δ glp KΔ g l dA / pELDRR / pCaGPDGPP strain. 10 is BL21 star (DE3) Δ glp K Δ is a fed-batch culture the results of the yqhD / pELDRR / pCaGPDGPP strain.

[ Example  5: Using glucose as a substrate, 3- HP  production]

Batch cultures were performed to produce 3-HP from glucose. The batch culture was carried out in a volume of 100 mL. The medium used was Riesenberg medium supplemented with 10 g / L of glucose and 5 g / L of yeast extract. Streptomycin 50 mg / L was used as an antibiotic. Induction of the introduced enzyme was performed by adding 0.2 Mm of IPTG when the OD ₆₀₀ value became 0.8 ~ 1. The cells were cultured at 37 ° C until expression induction and at 25 ° C after expression, and cultured at 250 rpm for oxygen agitation.

Conducted by the glucose medium BL21 star (DE3) Δ glpK Δ yqh pELDRR / pCa72, BL21 star (DE3) Δ glpK Δ gldA / pELDRR / pCa72, BL21 star (DE3) Δ glpK Δ yqhD / pELDRR / pCaGPDGPP, BL21 star (DE3) The results of the batch culture of Δ glpK Δ gldA / pELDRR / pCaGPDGPP strain were as shown in FIGS. Figure 11a is from the glucose medium BL21 star (DE3) is a batch culture results in Δ glpK ΔyqhD / pELDRR / pCa72 strain. Figure 11b is in the glucose medium BL21 star (DE3) Δ glpK Δ is a batch culture results in the gldA / pELDRR / pCa72 strain. Figure 11c is in the glucose medium BL21 star (DE3) Δ glpK Δ is a batch culture results in yqhD / pELDRR / pCaGPDGPP strain. Figure 11d is in the glucose medium BL21 star (DE3) is a batch culture results in Δ glpK ΔgldA / pELDRR / pCaGPDGPP strain.

The experimental results, GPD and the GPP introduction BL21 star (DE3) Δ glpK ΔyqhD / pELDRR / pCaGPDGPP and BL21 star (DE3) Δ glpK Δ gldA / pELDRR / pCaGPDGPP only 3-HP has been producing strain, BL21 star (DE3) Δ Δ yqhD glpK / pELDRR / pCa72, BL21 star (DE3) Δ Δ gldA glpK / pELDRR / pCa72 strain did not produce 3-HP, because the path to create a glycerol.

On the other hand, BL21 star (DE3) Δ glpK Δ yqhD / pELDRR / pCaGPDGPP and BL21 star (DE3) Δg l pKΔ gldA / pELDRR / pCaGPDGPP strain is 0.7 g / L, 0.6 g of 3-HP from glucose of 10 g / L, respectively / L.

Claims (7)

delete Glycerol dehydratase (DHAB), which converts glycerol to 3-hydroxypropionaldehyde (3-HPA), which is a precursor of 3-hydroxypropionic acid, Being;
Is transformed to express an aldehyde dehydrogenase which converts 3-hydroxypropionaldehyde to 3-hydroxypropionic acid;
In order that the glycerol kinase does not exhibit activity or that both the glycerol kinase and the glycerol dehydrogenase do not exhibit activity, the gene encoding each of them is disrupted so that the function thereof is lost. Or all of the gene is removed;
The glycerol-3-phosphatase and glycerol-3-phosphate dehydrogenase, which convert dihydroxyacetone phosphate into glycerol, are designed to express glycerol-3-phosphate dehydrogenase Converted;
Wherein the recombinant Escherichia coli is cultured in a medium supplemented with xylose.
delete 3. The method of claim 2,
The recombinant Escherichia coli,
The aldehyde dehydrogenase coding gene, which is responsible for converting 3-HPA to 3-HP, is removed, and an aldehyde dehydrogenase gene derived from Pseudomonas aeruginosa is introduced ≪ RTI ID = 0.0 > 3-hydroxypropionic < / RTI > acid.
3. The method of claim 2,
The above-
In order to prevent 1,3-propanediol dehydrogenase ( yqhD ) from exhibiting activity, a gene coding for the gene may be partially disrupted such that its function is lost, ≪ RTI ID = 0.0 > 3-hydroxypropionic < / RTI > acid.
3. The method of claim 2,
The above-
Wherein the coenzyme B ₁₂ is added.
3. The method of claim 2,
The above-
Glycerol dehydratase < / RTI > reactivase is further transformed to express the glycerol dehydratase reactivase.

Images