Method for synthesizing D-psicose from glucose by modifying escherichia coli

Abstract

The invention provides a method for synthesizing D-psicose from glucose by modifying escherichia coli. The modification comprises the following steps: (1) introducing an exogenous D-psicose-6-phosphate epimerase gene alsE with an SUMO tag and an exogenous D-psicose-6-phosphate phosphatase gene a6PP with an SUMO tag, and constructing a metabolic pathway for synthesizing D-psicose from glucose; (2) a phosphofructokinase A gene pfkA, a phosphofructokinase B gene pfkB and a UDP-galactose-4-epimerase gene galE are knocked out, so that side metabolic shunt is reduced, accumulation of a D-psicose synthesis precursor is increased, and the synthesis efficiency of D-psicose is improved; according to the present invention, a 6-glucose phosphate isomerase gene pgi and a phosphogluconate dehydratase gene edd are knocked out, and exogenous NADP + dependent glutamate dehydrogenase gene gdh1 and NAD + dependent glutamate dehydrogenase gene gdh2 are introduced so as to regulate intracellular cofactor balance and restore cell growth, such that the bacterial strain can effectively synthesize the target product while the bacterial strain has good growth performance;

Description

Technical Field

[0001] This invention belongs to the field of Escherichia coli metabolic processes, specifically relating to a method for synthesizing D-allulose from glucose by modifying Escherichia coli. Background Technology

[0002] In recent years, with the increasing prevalence of metabolic diseases such as obesity, diabetes, and heart complications, the World Health Organization has begun to call for a reduction in the intake of high-calorie foods. Replacing sugar with low-calorie sweeteners can slow down metabolic imbalances at their source, thereby reducing the occurrence of these diseases. Sweeteners can be divided into artificial sweeteners and natural sweeteners. Artificial sweeteners have been found to pose health risks, such as excessive appetite regulation and inducing neuritis. Natural sweeteners, on the other hand, are favored by consumers and researchers due to their green, environmentally friendly, low-calorie, and palatable characteristics. Natural sweeteners mainly refer to sweet organic compounds that can be directly extracted from nature and are primarily found in the secondary metabolic network of plants. Typical natural sweeteners include fructose, xylitol, and steviol glycosides. These natural sweeteners not only satisfy people's pursuit of deliciousness but also have physiological functions such as regulating calorie intake, alleviating inflammation, and regulating lipid metabolism, showing broad market prospects in functional foods and healthcare.

[0003] However, the process of preparing large-scale natural sweeteners is cumbersome and costly. With the development of synthetic biology, researchers have begun to explore the use of microorganisms to synthesize natural sweeteners. In recent years, this method has been widely applied to the synthesis of natural sweeteners due to its green nature, high selectivity, and simple reaction conditions. This technology mainly utilizes common strains such as *E. coli* and *Saccharomyces cerevisiae* for gene editing and other biotechnological methods to efficiently convert substrates such as glucose and fructose into target products. The biosynthesis of D-allulose, which has become increasingly popular in recent years, has also benefited from this technological trend. However, current biosynthetic systems still face several bottlenecks: insufficient catalytic activity, thermal stability, and operational stability of key enzymes; uneven distribution of metabolic flux along pathways, resulting in low conversion rates; and poor reusability of whole-cell catalysts or immobilized enzymes. Summary of the Invention

[0004] To address the aforementioned problems, this invention aims to provide a method for synthesizing D-allulose from glucose by modifying Escherichia coli.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: A method for synthesizing D-allulose from glucose by modifying Escherichia coli, which uses Escherichia coli as a chassis host bacterium and achieves the synthesis of D-allulose from glucose through genetic modification and fermentation culture; the genetic modification includes: (1) introducing an exogenous D-allulose-6-phosphate epimerase gene. alsEand exogenous D-allulose-6-phosphate phosphatase gene a6PP (1) Construct a metabolic pathway for the synthesis of D-allulose from glucose; (2) Knock out the phosphofructokinase A gene. pfkA Phosphofructokinase B gene pfkB and UDP-galactose-4-episomerase gene galE This reduces the diversion of byproducts, increases the accumulation of D-allulose precursors, and improves the synthesis efficiency of D-allulose. Furthermore, the method includes the following steps: 1) Genes simultaneously carrying D-allulose-6-phosphate epimerase alsE and D-allulose-6-phosphate phosphatase gene a6PP The recombinant plasmid was transformed into the chassis host bacterium Escherichia coli E.coli JM109 (DE3) was used to construct and obtain a recombinant strain. E.coli ( sumo-alsE, a6PP ); 2) In recombinant strains E.coli ( sumo-alsE, a6PP Based on this, the phosphofructokinase A gene was knocked out sequentially. pfkA Phosphofructokinase B gene pfkB UDP-galactose-4-episomerase gene galE Recombinant strains were obtained by constructing E.coli ( sumo-alsE , a6PP , Δ pfkA , Δ pfkB , Δ galE ); 3) Recombinant strains E.coli ( sumo-alsE , a6PP , Δ pfkA , Δ pfkB , Δ galE Fermentation culture in a glucose-containing medium enables the synthesis of D-allulose from glucose; Furthermore, the D-allulose-6-phosphate epimerase gene alsE The NCBI gene number is 948595; the D-allulose-6-phosphate epimerase gene alsE A SUMO tag is added to the 5' end of the gene, and the UniProtKB number of the SUMO tag is P55853; the D-allulose-6-phosphate phosphatase gene a6PP The NCBI sequence number is BF9343_0892; the phosphofructokinase A gene pfkA The NCBI gene number is 948412; the phosphofructokinase B gene pfkBThe NCBI gene number is 946230; the UDP-galactose-4-epimerase gene galE The NCBI gene number is 945354; Furthermore, the fermentation culture method is as follows: selecting recombinant strains E.coli ( sumo-alsE , a6PP ,Δ pfkA , Δ pfkB , Δ galE Single colonies of the bacteria were inoculated into 4 mL of LB broth containing 50 μg / mL kanamycin and incubated overnight at 37°C and 220 rpm. Subsequently, a 2 vol% inoculum was transferred to 50 mL of LB broth containing 50 μg / mL kanamycin and 10 g / L glucose and incubated at 37°C and 220 rpm until the bacterial OD reached the target value. 600 When the concentration reaches 0.7, add IPTG to a final concentration of 0.2 mM, and then transfer to a shaker at 30°C and 220 rpm for fermentation.

[0006] A method for synthesizing D-allulose from glucose by modifying Escherichia coli, which uses Escherichia coli as a chassis host bacterium and achieves the synthesis of D-allulose from glucose through genetic modification and fermentation culture; the genetic modification includes: (1) introducing an exogenous D-allulose-6-phosphate epimerase gene. alsE and exogenous D-allulose-6-phosphate phosphatase gene a6PP (1) Construct a metabolic pathway for the synthesis of D-allulose from glucose; (2) Knock out the phosphofructokinase A gene. pfkA Phosphofructokinase B gene pfkB and UDP-galactose-4-episomerase gene galE To reduce the diversion of byproducts, increase the accumulation of D-allulose synthesis precursors, and improve the synthesis efficiency of D-allulose; (3) Knock out glucose-6-phosphate isomerase gene pgi and phosphoglucose dehydratase gene edd Then introduce exogenous NADP + Glutamate-dependent dehydrogenase gene gdh1 and NAD + Glutamate-dependent dehydrogenase gene gdh2 In order to regulate the balance of intracellular cofactors and restore cell growth, the strain can be ensured to have good growth performance while effectively synthesizing the target product. Furthermore, the method includes the following steps: 1) Genes simultaneously carrying D-allulose-6-phosphate epimerase alsE and D-allulose-6-phosphate phosphatase gene a6PP The recombinant plasmid was transformed into the chassis host bacterium Escherichia coliE.coli JM109 (DE3) was used to construct and obtain a recombinant strain. E.coli ( sumo-alsE, a6PP ); 2) In recombinant strains E.coli ( sumo-alsE, a6PP Based on this, the phosphofructokinase A gene was knocked out sequentially. pfkA Phosphofructokinase B gene pfkB UDP-galactose-4-episomerase gene galE Recombinant strains were obtained by constructing E.coli ( sumo-alsE , a6PP , Δ pfkA , Δ pfkB , Δ galE ); 3) In recombinant strains E.coli ( sumo-alsE , a6PP , Δ pfkA , Δ pfkB , Δ galE Based on this, the glucose-6-phosphate isomerase gene was knocked out. pgi Then knock out the phosphoglucose dehydratase gene edd Recombinant strains were obtained by constructing E.coli ( sumo-alsE , a6PP , Δ pfkA , Δ pfkB , Δ galE , Δ pgi , Δ edd ); will simultaneously carry NADP + Glutamate-dependent dehydrogenase gene gdh1 and NAD + Glutamate-dependent dehydrogenase gene gdh2 Transformation of recombinant plasmids into recombinant strains E.coli ( sumo-alsE , a6PP , Δ pfkA , Δ pfkB , Δ galE , Δ pgi , Δ edd Recombinant strains were obtained by constructing [the appropriate method / system]. E.coli ( sumo-alsE , a6PP , gdh1 , gdh2, Δ pfkA , Δ pfkB , Δ galE , Δ pgi ,Δ edd); 4) Recombinant strains E.coli ( sumo-alsE , a6PP , gdh1 , gdh2, Δ pfkA , Δ pfkB , Δ galE , Δ pgi , Δ edd Fermentation culture in a glucose-containing medium enables the synthesis of D-allulose from glucose; Furthermore, the D-allulose-6-phosphate epimerase gene alsE The NCBI gene number is 948595; the D-allulose-6-phosphate epimerase gene alsE A SUMO tag is added to the 5' end of the gene, and the UniProtKB number of the SUMO tag is P55853; the D-allulose-6-phosphate phosphatase gene a6PP The NCBI sequence number is BF9343_0892; the phosphofructokinase A gene pfkA The NCBI gene number is 948412; the phosphofructokinase B gene pfkB The NCBI gene number is 946230; the UDP-galactose-4-epimerase gene galE The NCBI gene number is 945354; the glucose-6-phosphate isomerase gene pgi The NCBI gene number is 948535; the phosphoglucose dehydrase gene edd The NCBI gene number is 946362; the NADP... + Glutamate-dependent dehydrogenase gene gdh1 The NCBI gene number is 854557; the NAD... + Glutamate-dependent dehydrogenase gene gdh2 The NCBI gene number is 851311; Furthermore, the fermentation culture method is as follows: selecting recombinant strains E.coli ( sumo-alsE , a6PP , gdh1 , gdh2, Δ pfkA , Δ pfkB , Δ galE , Δ pgi , Δ eddSingle colonies of the bacteria were inoculated into 4 mL of LB broth containing 50 μg / mL kanamycin and incubated overnight at 37°C and 220 rpm. Subsequently, a 2 vol% inoculum was transferred to 50 mL of LB broth containing 50 μg / mL kanamycin and 10 g / L glucose and incubated at 37°C and 220 rpm until the bacterial OD reached the target value. 600 When the concentration reaches 0.7, add IPTG to a final concentration of 0.2 mM, and then transfer to a shaker at 30°C and 220 rpm for fermentation.

[0007] In step (2) above, the phosphofructokinase A gene pfkA Phosphofructokinase B gene pfkB UDP-galactose-4-episomerase gene galE 6-phosphate glucose isomerase gene pgi The knockout was performed using the λ-Red homologous recombination method on the phosphoglucose dehydratase gene. edd The knockout was performed using CRISPR-Cas9 technology.

[0008] The λ-Red homologous recombination method is performed as follows: The host strain carrying the pKD46 plasmid is cultured at 30°C to the logarithmic growth phase, followed by the preparation of electrotransformation competent cells; using the pKD13 plasmid as a template, specific primers containing homologous arms on both sides are used to amplify and obtain FRT-Kan plasmid. R -FRT linear targeting fragments were amplified and then purified for later use. The purified linear DNA fragments were electroporated into the prepared competent cells, and homologous recombination was mediated by λ-Red recombinase induced by arabinose expression via pKD46 plasmid. The revived bacterial culture was then plated onto cells containing Kansas. R On the screening plates, incubate at 30°C to screen for Kan R And Amp R Candidate clones were obtained; the resulting candidate clones were cultured at 42°C to eliminate the pKD46 plasmid. Subsequently, the pCP20 plasmid was introduced into the candidate clones with eliminated pKD46 plasmid, and under culture conditions at 30°C, the Kan between the FRT sites was cleaved using the Flp recombinase expressed by the pCP20 plasmid. R Resistance box; the colonies were incubated again at 42°C to eliminate the pCP20 plasmid; the colonies were then inoculated onto antibiotic-free plates and Kansas plates using the streak plating method. R Flat plate and Cm R Incubate on plates. A validated knockout strain should be able to grow on antibiotic-free plates, while in Kansas... R With Cm R They cannot grow on the plate.

[0009] The specific primers used in gene knockout using the λ-Red homologous recombination method are shown below:

[0010] The operation of CRISPR-Cas9 technology is as follows: First, a guide RNA targeting site (N20) is designed based on the target gene sequence, and a gRNA expression plasmid pEcgRNA-N20 is constructed based on this targeting site. Simultaneously, using 500bp sequences upstream and downstream of the target gene as homologous arms, a linear donor DNA of 1000bp in total length is constructed. To establish the CRISPR-Cas gene editing system, the pEcCas plasmid expressing the Cas protein is first introduced into the target host strain via electroporation. Subsequently, strains that have successfully carried the pEcCas plasmid and have been induced with arabinose are prepared as electroporation competent cells. The pEcgRNA-N20 plasmid and the donor DNA are co-electroporated into these competent cells at a 1:4 molar ratio to initiate the CRISPR-mediated gene transfer process. Finally, rhamnose, glucose, and sucrose are used sequentially for induction to induce plasmid loss. After induction, colonies are streaked and inoculated onto cells containing Spc. R and Kan R On selective culture plates, the successful loss of plasmids is verified by the colony growth phenotype. Colonies that can only grow on non-resistance plates and cannot grow on either type of resistance plate confirm that both plasmids have been successfully eliminated.

[0011] In CRISPR-Cas9 technology, it is used to construct targeted edd The primer sequences for the gene guide RNA (gRNA) are: edd-N20-F (5'-TAGTGATGATCGATGAGAAAGTGG-3'); edd-N20-R (5'-AAACCCACTTTCTCATCGATCATC-3').

[0012] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention achieves the biosynthesis of D-allulose using inexpensive glucose as a substrate, with a green and environmentally friendly process. By knocking out key competing metabolic genes, the generation of byproducts is inhibited, and the carbon metabolic flux is directed towards the synthesis of the target product, thereby improving the efficiency of D-allulose synthesis. Furthermore, by knocking out regulatory genes and introducing glutamate dehydrogenase genes, cell growth defects caused by multiple gene knockouts are salvaged, enabling the strain to restore good growth performance while effectively synthesizing D-allulose, thus enriching the biosynthetic metabolic regulation strategy of D-allulose. Attached Figure Description

[0013] Figure 1 Diagram of a cell factory in recombinant Escherichia coli synthesizing D-allulose.

[0014] Figure 2 : A map of the recombinant plasmid. A represents the recombinant plasmid pRSFDuet- sumo - alsE - a6PP B is the recombinant plasmid pACYCDuet- gdh1 - gdh2 .

[0015] Figure 3 A is a recombinant strain E.coli Fermentation product diagram of -pRSFDute-1; B represents recombinant strain. E.coli ( sumo- alsE , a6PP) A diagram of the fermentation products.

[0016] Figure 4 A is a recombinant strain E.coli ( sumo-alsE , a6PP , Δ pfkA , Δ pfkB The fermentation product diagram is shown in Figure 1; B represents the recombinant strain. E.coli ( sumo-alsE , a6PP , Δ pfkA , Δ pfkB , Δ galE A diagram of the fermentation products.

[0017] Figure 5 A is a recombinant strain E.coli ( sumo-alsE , a6PP , Δ pfkA , Δ pfkB , Δ galE , Δ pgi The fermentation product diagram is shown in Figure 1; B represents the recombinant strain. E.coli ( sumo-alsE , a6PP , Δ pfkA , Δ pfkB , Δ galE ,Δ pgi , Δ edd The fermentation product diagram is shown; C represents the recombinant strain. E.coli ( sumo-alsE , a6PP , gdh1 , gdh2 , Δ pfkA , Δ pfkB , Δ galE , Δ pgi , Δ edd A diagram of the fermentation products. Detailed Implementation

[0018] The technical solution of the present invention will be further described in the following sections with reference to specific embodiments, but the scope of protection of the present invention is not limited to the listed embodiments.

[0019] The sumo- involved in the embodiments alsE Genes alsE The gene is obtained by adding a SUMO tag to the 5' end; where, alsE The gene was derived from Escherichia coli K-12 strain MG1655 substrain, with NCBI gene number 948595; the SUMO tag was derived from Caenorhabditis elegans, with UniProtKB number P55853.

[0020] The embodiments involve a6PP The gene is derived from Bacteroides fragilis, and its NCBI sequence number is BF9343_0892.

[0021] The embodiments involve pfkA The gene is derived from the substrain MG1655 of Escherichia coli K-12, with an NCBI gene number of 948412.

[0022] The embodiments involve pfkB The gene is derived from the Escherichia coli K-12 strain MG1655 substrain, with its NCBI gene number 946230.

[0023] The embodiments involve galE The gene is derived from the Escherichia coli K-12 strain MG1655 substrain, with its NCBI gene number 945354.

[0024] The embodiments involve pgi The gene is derived from the Escherichia coli K-12 strain MG1655 substrain, with its NCBI gene number 948535.

[0025] The embodiments involve edd The gene is derived from the Escherichia coli K-12 strain MG1655 substrain, with its NCBI gene number 946362.

[0026] The embodiments involve gdh1 The gene is derived from Saccharomyces cerevisiae, and its NCBI gene number is 854557.

[0027] The embodiments involve gdh2 The gene is derived from Saccharomyces cerevisiae, and its NCBI gene number is 851311.

[0028] The LB liquid culture medium formulation involved in the examples is as follows: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, and the remainder is water; pH 7.0. Example 1

[0029] Will sumo-alsE The gene is inserted between the NcoⅠ and HindⅢ restriction sites in the pRSFDuet-1 plasmid, and simultaneously... a6PP The gene was inserted between the NdeⅠ and KpnⅠ restriction sites of the plasmid to construct the recombinant plasmid pRSFDuet-1-sumo- alsE - a6PP ( Figure 2 A) The recombinant plasmid pRSFDuet-1-sumo- alsE - a6PP Transformed into E. coli E.coli JM109(DE3) was constructed to obtain a recombinant strain. E.coli (sumo-alsE, a6PP). The pRSFDuet-1 plasmid was transformed into E. coli. E.coli JM109 (DE3) was used to construct and obtain a recombinant strain. E.coli -pRSFDute-1.

[0030] Fermentation verification: Selecting recombinant strains E.coli (sumo-alsE, a6PP) and recombinant strains E.coli Single colonies of -pRSFDute-1 were inoculated into 4 mL of LB broth containing 50 μg / mL kanamycin and incubated overnight at 37°C and 220 rpm. Subsequently, 2 vol% inoculum was transferred to 50 mL of LB broth containing 50 μg / mL kanamycin and 10 g / L glucose, respectively, and incubated at 37°C and 220 rpm until the bacterial OD reached the target cell count. 600 When the concentration reached 0.7, IPTG was added to a final concentration of 0.2 mM, and the mixture was then transferred to a shaker at 30°C and 220 rpm for fermentation. The total fermentation time was 60 hours, with samples taken every 12 hours to determine the cell OD. 600 Simultaneously, high-performance liquid chromatography (HPLC) was used to analyze the fermentation products, employing Waters Sugar-Pak... TM The chromatographic column and refractive index detector (RID) were used with water as the mobile phase, a flow rate of 0.4 mL / min, a column temperature of 85 ℃, and a run time of 20 min to quantitatively detect the contents of glucose and D-alokulose, respectively, under the same detection conditions.

[0031] The results are as follows Figure 3 As shown. Recombinant strain E.coli ( sumo-alsE , a6PPThe fermentation products of this compound include D-allulose, with the yield reaching a maximum of approximately 0.27 g / L after 48 hours of fermentation. At this point, the space-time yield is approximately 0.006 g / (L·h), the yield is approximately 0.098 g / g, and the cell density (OD) is [not specified]. 600nm The value is approximately 1.3. Meanwhile, the recombinant strain... E.coli D-allulose was not detected in the fermentation products of -pRSFDute-1. These results demonstrate the feasibility of the D-allulose synthesis pathway constructed in this invention. Example 2

[0032] This embodiment describes the recombinant strain. E.coli ( sumo-alsE , a6PP The endogenous metabolic network of carbon source metabolism is rationally designed and reconstructed. By knocking out key genes in competing metabolic pathways, the accumulation of intracellular fructose-6-phosphate (F-6-P) is increased, and the metabolic flux of downstream F-6-P pathways is weakened, thereby reducing ineffective carbon diversion and achieving efficient and targeted transport and utilization of carbon source metabolism to target product synthesis pathways.

[0033] In recombinant strains E.coli ( sumo-alsE , a6PP Based on this, the λ-red homologous recombination method was used to sequentially knock out pfkA Genes and pfkB Genes were used to construct recombinant strains. E.coli ( sumo-alsE , a6PP , Δ pfkA , Δ pfkB Further research on recombinant strains... E.coli ( sumo-alsE , a6PP , Δ pfkA , Δ pfkB Based on this, the λ-red homologous recombination method was used to knock out... galE Genes were used to construct recombinant strains. E.coli ( sumo-alsE , a6PP , Δ pfkA , Δ pfkB , Δ galE ).

[0034] Select recombinant strains E.coli ( sumo-alsE , a6PP , Δ pfkA , Δ pfkB ) and recombinant strains E.coli ( sumo-alsE , a6PP , Δ pfkA , Δ pfkB, Δ galE Single colonies of the bacteria were fermented for verification, and the fermentation culture conditions, cell growth index determination methods and fermentation product detection conditions were completely consistent with those in Example 1.

[0035] The results are as follows Figure 4 As shown. Recombinant strain E.coli ( sumo-alsE , a6PP , Δ pfkA , Δ pfkB The D-allulose yield reached its maximum of approximately 0.5 g / L after 36 hours of fermentation, with a space-time yield of approximately 0.014 g / (L·h), a yield of approximately 0.40 g / g, and a cell density (OD) of approximately [missing value]. 600nm The value is approximately 0.8. This is compared to the value of the unknocked variant. pfkA and pfkB strains of genes E.coli ( sumo- alsE , a6PP ), E.coli ( sumo-alsE , a6PP , Δ pfkA , Δ pfkB Cell density (OD) 600nm While the yield of D-allulose decreased significantly, the space-time yield, production rate, and overall yield all increased significantly. This indicates that knockout... pfkA and pfkB Following gene modification, bacterial growth was weakened, and carbon flux shifted significantly from "cell growth" to "glycan production," resulting in a substantial increase in D-allulose yield, efficiency, and spacetime productivity. (Recombinant strain) E.coli ( sumo-alsE , a6PP , Δ pfkA , Δ pfkB , Δ galE The D-allulose yield reached its maximum of approximately 0.39 g / L after 24 hours of fermentation, with a space-time yield of approximately 0.016 g / (L·h), a yield of approximately 0.54 g / g, and a cell density (OD) of approximately [missing value]. 600nm The value is approximately 1.0. This indicates that further knocking is needed. galE After gene therapy, the side metabolite diversion is further reduced, while partially restoring bacterial growth, achieving a better overall balance in terms of D-allulose spacetime yield, productivity, and cell density. Example 3

[0036] In the recombinant strains that have been constructed E.coli ( sumo-alsE , a6PP , Δ pfkA , Δ pfkB , Δ galEBased on this, further genetic engineering was performed on the recombinant strain to compensate for the adverse effects of gene knockout on cell growth, restore normal cell growth and vitality, and ensure that the recombinant strain can effectively synthesize the target product while having good growth performance.

[0037] First, in the recombinant strain E.coli ( sumo-alsE , a6PP , Δ pfkA , Δ pfkB , Δ galE Based on this, the λ-red homologous recombination method was used to knock out... pgi Genes were used to construct recombinant strains. E.coli ( sumo-alsE , a6PP , Δ pfkA , Δ pfkB , Δ galE , Δ pgi ).

[0038] Then, in the recombinant strain E.coli ( sumo-alsE , a6PP , Δ pfkA , Δ pfkB , Δ galE , Δ pgi Based on this, CRISPR-Cas9 technology is used to knock out edd Genes were used to construct recombinant strains. E.coli ( sumo-alsE , a6PP , Δ pfkA , Δ pfkB , Δ galE , Δ pgi , Δ edd ).

[0039] Finally, gdh1 The gene is inserted between the BamHI and EcoRI restriction sites in the pACYCDuet-1 plasmid, while simultaneously... gdh2 The gene was inserted between the NdeⅠ and KpnⅠ restriction sites of the plasmid to construct the recombinant plasmid pACYCDuet-1- gdh1 - gdh2 ( Figure 2 B); the recombinant plasmid pACYCDuet-1- gdh1 - gdh2 Transformation into recombinant strains E.coli ( sumo-alsE , a6PP , Δ pfkA , Δ pfkB , Δ galE , Δ pgi, Δ edd Recombinant strains were obtained by constructing [the appropriate method / system]. E.coli ( sumo- alsE , a6PP , gdh1 , gdh2 , Δ pfkA , Δ pfkB , Δ galE , Δ pgi , Δ edd ).

[0040] Select recombinant strains E.coli ( sumo-alsE , a6PP , Δ pfkA , Δ pfkB , Δ galE , Δ pgi ), recombinant strains E.coli ( sumo-alsE , a6PP , Δ pfkA , Δ pfkB , Δ galE , Δ pgi , Δ edd ) and recombinant strains E.coli ( sumo-alsE , a6PP , gdh1 , gdh2 , Δ pfkA , Δ pfkB , Δ galE , Δ pgi , Δ edd Single colonies of the bacteria were fermented for verification, and the fermentation culture conditions, cell growth index determination methods and fermentation product detection conditions were completely consistent with those in Example 1.

[0041] The results are as follows Figure 5 As shown. Recombinant strain E.coli ( sumo-alsE , a6PP , Δ pfkA , Δ pfkB , Δ galE ,Δ pgi Cell density (OD) after 60 hours of fermentation 600nm The value reached a maximum of approximately 2.4, but D-allulose was not detected in the fermentation products throughout the entire fermentation process. This indicates that knockout... pgi Following gene sequencing, the conversion of G-6-P to F-6-P was blocked, and the supply of D-allulose precursors was interrupted. A large amount of carbon source flowed to the ED / PP pathway, with the ED pathway consuming most of the carbon source and providing energy for the cell, supporting highly efficient bacterial proliferation, characterized by "bacterial growth without sugar production." Recombinant strainE.coli ( sumo-alsE , a6PP , Δ pfkA ,Δ pfkB , Δ galE , Δ pgi , Δ edd The D-allulose yield reached its maximum of approximately 0.07 g / L after 48 hours of fermentation, with a space-time yield of approximately 0.001 g / (L·h), a yield of approximately 0.18 g / g, and a cell density (OD) of approximately [missing value]. 600nm The value is approximately 0.8. This indicates that during the knockout process... pgi Knockout on the basis of genes edd The gene blocks the flow of carbon flux to the ED pathway, forcing more carbon sources into the PP pathway to increase NADPH synthesis; however, the energy supply function of the ED pathway is completely deprived, leading to severe inhibition of cell growth, and only a trace amount of D-allulose can be synthesized through a bypass conversion of intermediate products from the PP pathway. Recombinant strain E.coli ( sumo-alsE , a6PP , gdh1 , gdh2 ,Δ pfkA , Δ pfkB , Δ galE , Δ pgi , Δ edd The D-allulose yield reached its maximum of approximately 0.18 g / L after 24 hours of fermentation, with a space-time yield of approximately 0.008 g / (L·h), a yield of approximately 0.2 g / g, and a cell density (OD) of approximately [missing value]. 600nm The value is approximately 1.8. This indicates overexpression. gdh1 and gdh2 Following gene knockout, by regulating the intracellular NADPH / NADH balance, excess NADPH produced by the PP pathway is converted into NADH and enters the respiratory chain for energy, effectively alleviating the metabolic disorders caused by multiple gene knockout; while restoring some cell growth, the efficient synthesis of D-allulose is also restored.

[0042] The above descriptions are all preferred embodiments of the present invention. All equivalent changes and modifications made in accordance with the claims of the present invention should be included within the scope of the present invention.

Claims

1. A method for synthesizing D-allulose from glucose by modifying Escherichia coli, characterized in that: Using Escherichia coli as the chassis host bacterium, the synthesis of glucose into D-allulose was achieved through genetic modification and fermentation culture; the genetic modification included: (1) introducing an exogenous D-allulose-6-phosphate epimerase gene. asE and exogenous D-allulose-6-phosphate phosphatase gene a6PP (1) Construct a metabolic pathway for the synthesis of D-allulose from glucose; (2) Knock out the phosphofructokinase A gene. pfkA Phosphofructokinase B gene pfkB and UDP-galactose-4-episomerase gene Gale This reduces the diversion of byproducts, increases the accumulation of D-allulose precursors, and improves the synthesis efficiency of D-allulose.

2. The method for synthesizing D-allulose from glucose by modifying Escherichia coli according to claim 1, characterized in that: Read more: 1) Genes simultaneously carrying D-allulose-6-phosphate epimerase asE and D-allulose-6-phosphate phosphatase gene a6PP The recombinant plasmid was transformed into the chassis host bacterium Escherichia coli E.coli JM109 (DE3) was used to construct and obtain a recombinant strain. E.coli ( sumo-alse, a6PP ); 2) In recombinant strains E.coli ( sumo-alse, a6PP Based on this, the phosphofructokinase A gene was knocked out sequentially. pfkA Phosphofructokinase B gene pfkB UDP-galactose-4-episomerase gene Gale Recombinant strains were constructed and obtained. E.coli ( sumo-alsE , a6PP , Δ pfkA , Δ pfkB , Δ Gale ); 3) Recombinant strains E.coli ( sumo-alsE , a6PP , Δ pfkA , Δ pfkB , Δ Gale Fermentation culture in a glucose-containing medium enables the synthesis of D-allulose from glucose.

3. The method for synthesizing D-allulose from glucose by modifying Escherichia coli according to claim 2, characterized in that: The D-allulose-6-phosphate epimerase gene asE The NCBI gene number is 948595; the D-allulose-6-phosphate epimerase gene asE A SUMO tag is added to the 5' end of the gene, and the UniProtKB number of the SUMO tag is P55853; the D-allulose-6-phosphate phosphatase gene a6PP The NCBI sequence number is BF9343_0892; the phosphofructokinase A gene pfkA The NCBI gene number is 948412; the phosphofructokinase B gene pfkB The NCBI gene number is 946230; the UDP-galactose-4-epimerase gene Gale The NCBI gene number is 945354.

4. A method for synthesizing D-allulose from glucose by modifying Escherichia coli according to claim 2, characterized in that: The fermentation culture method is as follows: selecting recombinant strains E.coli ( sumo-alsE , a6PP , Δ pfkA , Δ pfkB , Δ Gale Single colonies of the bacteria were inoculated into 4 mL of LB broth containing 50 μg / mL kanamycin and incubated overnight at 37°C and 220 rpm. Subsequently, a 2 vol% inoculum was transferred to 50 mL of LB broth containing 50 μg / mL kanamycin and 10 g / L glucose and incubated at 37°C and 220 rpm until the bacterial OD reached the target cell count. 600 When the concentration reaches 0.7, add IPTG to a final concentration of 0.2 mM, and then transfer to a shaker at 30°C and 220 rpm for fermentation.

5. The method for synthesizing D-allulose from glucose by modifying Escherichia coli according to claim 1, characterized in that: The genetic modification also includes: knocking out the glucose-6-phosphate isomerase gene. pgi and phosphoglucose dehydratase gene edd Then introduce exogenous NADP + Glutamate-dependent dehydrogenase gene gdh1 and NAD + Glutamate-dependent dehydrogenase gene gdh2 This is to regulate the balance of intracellular cofactors, restore cell growth, and ensure that the strain can effectively synthesize the target product while maintaining good growth performance.

6. The method for synthesizing D-allulose from glucose by modifying Escherichia coli according to claim 5, characterized in that: Read more: 1) Genes simultaneously carrying D-allulose-6-phosphate epimerase asE and D-allulose-6-phosphate phosphatase gene a6PP The recombinant plasmid was transformed into the chassis host bacterium Escherichia coli E.coli JM109 (DE3) was used to construct and obtain a recombinant strain. E.coli ( sumo-alse, a6PP ); 2) In recombinant strains E.coli ( sumo-alse, a6PP Based on this, the phosphofructokinase A gene was knocked out sequentially. pfkA Phosphofructokinase B gene pfkB UDP-galactose-4-episomerase gene Gale Recombinant strains were constructed and obtained. E.coli ( sumo-alsE , a6PP , Δ pfkA , Δ pfkB , Δ Gale ); 3) In recombinant strains E.coli ( sumo-alsE , a6PP , Δ pfkA , Δ pfkB , Δ Gale Based on this, the glucose-6-phosphate isomerase gene was knocked out. pgi Then knock out the phosphoglucose dehydratase gene edd Recombinant strains were constructed and obtained. E.coli ( sumo-alsE , a6PP , Δ pfkA , Δ pfkB ,Δ Gale , Δ pgi , Δ edd ); will simultaneously carry NADP + Glutamate-dependent dehydrogenase gene gdh1 and NAD + Glutamate-dependent dehydrogenase gene gdh2 Transformation of recombinant plasmids into recombinant strains E.coli ( sumo-alsE , a6PP , Δ pfkA , Δ pfkB ,Δ Gale , Δ pgi , Δ edd Recombinant strains were obtained by constructing [the appropriate method / system]. E.coli ( sumo-alsE , a6PP , gdh1 , gdh2, Δ pfkA , Δ pfkB , Δ Gale , Δ pgi ,Δ edd ); 4) Recombinant strains E.coli ( sumo-alsE , a6PP , gdh1 , gdh2, Δ pfkA , Δ pfkB , Δ Gale ,Δ pgi , Δ edd Fermentation culture in a glucose-containing medium enables the synthesis of D-allulose from glucose.

7. The method for synthesizing D-allulose from glucose by modifying Escherichia coli according to claim 6, characterized in that: The D-allulose-6-phosphate epimerase gene asE The NCBI gene number is 948595; the D-allulose-6-phosphate epimerase gene asE A SUMO tag is added to the 5' end of the gene, and the UniProtKB number of the SUMO tag is P55853; the D-allulose-6-phosphate phosphatase gene a6PP The NCBI sequence number is BF9343_0892; the phosphofructokinase A gene pfkA The NCBI gene number is 948412; the phosphofructokinase B gene pfkB The NCBI gene number is 946230; the UDP-galactose-4-epimerase gene Gale The NCBI gene number is 945354; the glucose-6-phosphate isomerase gene pgi The NCBI gene number is 948535; the phosphoglucose dehydrase gene edd The NCBI gene number is 946362; the NADP... + Glutamate-dependent dehydrogenase gene gdh1 The NCBI gene number is 854557; the NAD... + Glutamate-dependent dehydrogenase gene gdh2 The NCBI gene number is 851311.

8. A method for synthesizing D-allulose from glucose by modifying Escherichia coli according to claim 6, characterized in that: The fermentation culture method is as follows: selecting recombinant strains E.coli ( sumo-alsE , a6PP , gdh1 , gdh2, Δ pfkA , Δ pfkB , Δ Gale , Δ pgi , Δ edd Single colonies of the bacteria were inoculated into 4 mL of LB broth containing 50 μg / mL kanamycin and incubated overnight at 37°C and 220 rpm. Subsequently, a 2 vol% inoculum was transferred to 50 mL of LB broth containing 50 μg / mL kanamycin and 10 g / L glucose and incubated at 37°C and 220 rpm until the bacterial OD reached the target cell count. 600 When the concentration reaches 0.7, add IPTG to a final concentration of 0.2 mM, and then transfer to a shaker at 30°C and 220 rpm for fermentation.

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