Method for efficiently synthesizing D-psicose from cane sugar by escherichia coli through double PTS transport

Abstract

The invention discloses a method for efficiently synthesizing D-psicose from cane sugar by escherichia coli through double PTS transfer. E.coliJM109 (DE3) is used as a chassis host bacterium, and a D-psicose synthesis path based on a sucrose PTS transport system is established through co-expression of alsE, a6PP, scrA and scrB genes; by introducing the glpX [K29A] gene, a carbon source is driven to flow from a D-fructose PTS transport system to a D-psicose synthesis path; through knockout of pfkA and rpiA genes and inhibition of expression of fbaA genes, an endogenous glycolysis pathway and a phosphopentose pathway are adjusted, and the carbon flow entering tricarboxylic acid circulation is adjusted, and finally, the purpose that the D-psicose is efficiently synthesized from sucrose by escherichia coli through double PTS transport is finally achieved. Genetic engineering modification is carried out on wild escherichia coli, D-psicose is efficiently synthesized from sucrose by using a sucrose PTS and fructose PTS system, and a new scheme is provided for synthesis of D-psicose.

Description

Technical Field

[0001] The present invention belongs to the field of metabolic engineering, and specifically relates to transforming Escherichia coli by utilizing genetic engineering technology to obtain a recombinant Escherichia coli strain, which can efficiently synthesize D-psicose from sucrose by utilizing a double PTS transport pathway. Background Art

[0002] The rare monosaccharide D-psicose is a low-calorie sweetener with approximately 70% of the sweetness of sucrose and only 0.3% of its energy. Therefore, it is considered an ideal replacement for high-fructose corn syrup (HFCS) and sucrose in the food and beverage industry. Furthermore, D-psicose exhibits strong anti-hyperglycemic, anti-hyperlipidemic, and antioxidant properties, thus holding great potential for pharmaceutical applications. Traditional chemical production methods are increasingly restricted due to environmental concerns and demanding production conditions. In contrast, biological methods are considered a more promising alternative for the synthesis of D-psicose.

[0003] Recently, commercial production of D-allose has typically employed an enzymatic approach based on the Izumoring strategy, in which D-allose-3-epimerase (DAEase) is the key biocatalyst for the reversible isomerization of D-fructose to D-psicose, achieving a conversion rate of approximately 30%. To improve catalytic efficiency, numerous studies have focused on discovering new epimerases from diverse species, implementing genetic mutations at the active site, and developing efficient immobilization techniques. It has also been reported that the addition of borate can shift the isomerization equilibrium in a positive direction, as borate has a greater binding affinity for D-psicose than for D-fructose. In contrast to the Izumoring strategy, an alternative approach, known as phosphorylation-dephosphorylation, was first proposed for the in vitro synthesis of D-psicose from starch. In this context, two key enzymes were discovered and selected: psicose-6-phosphate 3-epimerase (AlsE) and psicose-6-phosphate phosphatase (A6PP), which convert fructose-6-phosphate (F-6-P) to psicose-6-phosphate (A-6-P) and dephosphorylate A-6-P to D-psicose. Because the dephosphorylation process is irreversible, this method could theoretically achieve higher substrate conversion rates and product yields. However, whether synthesizing D-psicose based on the Izumoring method or the phosphorylation-dephosphorylation method, enzyme production and immobilization are challenges.

[0004] The technology for synthesizing D-allose via microbial fermentation is making significant progress, attributed to its simpler operational procedures and milder conditions compared to enzymatic methods. Sucrose, a widely available and economical disaccharide, is gaining increasing prominence as a raw material for chemical production. Given that excessive consumption of nutritive sweeteners increases the risk of obesity and hyperglycemia, the biosynthesis of non-nutritive D-psicose using sucrose as a substrate is becoming increasingly important. In the CscABK system, the hydrolysis rate exceeds the phosphorylation rate of sugars, leading to the accumulation of D-glucose and D-fructose in the culture medium. Consequently, the carbon source consumption rate of the engineered cells is reduced due to carbon metabolism repression (CCR). In contrast, in the PTS system, a sucrose-specific permease (ScrA) facilitates the transport and phosphorylation of sucrose to form sucrose-6-phosphate (S-6-P), which is then hydrolyzed by sucrose-6-phosphate hydrolase (ScrB) to glucose-6-phosphate (G-6-P) and D-fructose. Compared with the CscABK pathway, the sucrose PTS can circumvent the effects of the D-glucose CCR and simplify genetic manipulation, providing an opportunity to improve the efficiency of D-allose production in cell factories. Summary of the Invention

[0005] The present invention aims to address the above problems and provide a method for Escherichia coli to efficiently synthesize D-psicose from sucrose using double PTS transport.

[0006] To achieve the above object, the present invention adopts the following technical solutions: A method for efficiently synthesizing D-psicose from sucrose by Escherichia coli using double PTS transport is disclosed. The method uses E. coli JM109 (DE3) as a chassis host bacterium, co-expresses a D-psicose-6-phosphate epimerase gene, a D-psicose-6-phosphatase gene, a sucrose-specific permease gene, and a sucrose-6-phosphate hydrolase gene, thereby establishing a D-psicose synthesis pathway based on the sucrose PTS transport system; introduces a fructose-1,6-bisphosphatase gene to drive the carbon source from the D-fructose PTS transport system to the D-psicose synthesis pathway; and knocks out the 6-phosphofructokinase gene and the ribose-5-phosphate isomerase gene and inhibits the expression of the fructose-bisphosphate aldolase gene to regulate the endogenous glycolysis pathway and the pentose phosphate pathway and adjust the carbon flow entering the tricarboxylic acid cycle, ultimately achieving efficient synthesis of D-psicose from sucrose by E. coli using double PTS transport.

[0007] Furthermore, the D-psicose-6-phosphate epimerase gene is alsE, and the UniProtKB number of alsE is P32719; the D-psicose-6-phosphatase gene is a6PP, and the NCBI number of a6PP is BF9343_0892; the sucrose-specific permease gene is scrA, and the UniProtKB number of scrA is A0A0H3FUP0; the sucrose-6-phosphate hydrolase gene is scrB, and the UniProtKB number of scrB is A0A0H3FTU2; the fructose-1,6-bisphosphatase gene is selected from glpX[K29A], yggF, and fbp, and the nucleotide sequence of glpX[K29A] is SEQ ID NO.1, the UniProtKB number of yggF is P21437, and the UniProtKB number of fbp is P0A993; the above-mentioned 6-phosphofructokinase gene is selected from pfkA and pfkB, the UniProtKB number of pfkA is P0A796, and the UniProtKB number of pfkB is P06999; the above-mentioned ribose-5-phosphate isomerase gene is rpiA, and the UniProtKB number of rpiA is P0A7Z0; the above-mentioned fructose-bisphosphate aldolase gene is fbaA, and the UniProtKB number of fbaA is P0AB71.

[0008] Furthermore, the above steps include: 1) Using E. coli JM109 (DE3) as the host strain, the pfkA and rpiA genes were knocked out by homologous recombination technology to obtain a pfkA / rpiA double gene knockout recombinant strain; 2) Construction of recombinant plasmids carrying the alsE, a6PP, scrA, and scrB genes, as well as a recombinant plasmid carrying the fbaA gene antisense RNA and the glpX[K29A] gene; 3) Recombinant plasmids carrying the alsE, a6PP, scrA, and scrB genes, as well as a recombinant plasmid carrying the fbaA antisense RNA and the glpX[K29A] gene, were simultaneously transformed into the pfkA / rpiA double-knockout recombinant strain LJARGa, which can efficiently synthesize D-psicose from sucrose using a dual PTS transporter.

[0009] Furthermore, the order of the above knockout is: first knock out the pfkA gene, then knock out the rpiA gene.

[0010] Furthermore, the recombinant plasmid carrying the alsE, a6PP, scrA and scrB genes simultaneously uses the pACYCDuet-1 plasmid as a backbone plasmid.

[0011] Furthermore, the recombinant plasmid carrying both the fbaA gene antisense RNA and the GlpX[K29A] gene uses the pETDuet-1 plasmid as a backbone plasmid.

[0012] Furthermore, the nucleotide sequence of the fbaA gene antisense RNA is selected from SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4.

[0013] Furthermore, the above method also includes inoculating the recombinant strain LJARGade into a fermentation medium with sucrose as a substrate for fermentation culture.

[0014] Furthermore, the formula of the fermentation medium is: sucrose 7-9 g / L, yeast extract 5 g / L, tryptone 10 g / L, sodium chloride 5 g / L, and the balance is water; the fermentation culture is carried out in a shake flask, and the shake flask fermentation method is: a single colony of LJARGa is inoculated into 4 mL of LB liquid medium, cultured overnight at 37°C and 220 rpm, and then transferred to a 250 mL shake flask containing 50 mL of fermentation medium, and cultured at 37°C and 220 rpm until the OD 600 When the value reached 0.6-0.8, IPTG was added to a final concentration of 0.21 mM and fermented at 30°C and 220 rpm.

[0015] Furthermore, the formula of the fermentation medium is as follows: sucrose 28.9 g / L, sodium chloride 5 g / L, yeast extract 24 g / L, tryptone 21.7 g / L, manganese chloride 3 mM, and the balance is water; the fermentation culture is carried out in a fermenter, and the fermentation method is as follows: a single colony of LJARGa is inoculated into 4 mL of LB liquid medium, cultured overnight at 37°C and 220 rpm, and then transferred to a 5 L fermenter containing 3 L of fermentation medium, and cultured at 37°C and 220 rpm until the OD 600 When the value reaches 0.6-0.8, IPTG is added at a final concentration of 0.21 mM and fermented at 30°C. During the entire fermentation process, the temperature is maintained at 30°C, the pH value of the fermentation medium is stabilized at 7, and the dissolved oxygen level is maintained at 25%-35%. Sucrose is added at the 20th hour of fermentation, and the total amount of sucrose fed is 55.2 g / L.

[0016] Compared with the prior art, the present invention has the following significant effects: The present invention proposes a new method for efficiently converting sucrose into D-psicose through fermentation in Escherichia coli. Using E. coli JM109 (DE3) as the host strain, a D-psicose-6-phosphate epimerase gene, a D-psicose-6-phosphatase gene, a sucrose-specific permease gene, and a sucrose-6-phosphate hydrolase gene are co-expressed to establish a D-psicose synthesis pathway based on the sucrose PTS transport system. The fructose-1,6-bisphosphatase gene is introduced to drive the flow of carbon sources from the D-fructose PTS transport system to the D-psicose synthesis pathway. Furthermore, the 6-phosphofructokinase and ribose-5-phosphate isomerase genes are knocked out through homologous recombination technology, and the expression of the fructose-bisphosphate aldolase gene is inhibited using antisense RNA technology. This modulates the endogenous glycolysis and pentose phosphate pathways, as well as the carbon flux into the tricarboxylic acid cycle. Ultimately, recombinant E. coli is obtained that can efficiently synthesize D-psicose from sucrose using dual PTS transport. The present invention transforms wild Escherichia coli JM109 (DE3) by genetic engineering, and efficiently synthesizes D-psicose from sucrose through the double PTS system-dependent transport of sucrose and D-fructose, providing a new solution for the synthesis of D-psicose. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 : Schematic diagram of the cell factory that synthesizes D-psicose from sucrose.

[0018] Figure 2 Advantages of the PTS sucrose transport system in cell factories. A, Schematic diagram of sucrose transport in E. coli (non-PTS) (CscABK system) and E. coli (PTS) (ScrAB system). B, Analysis of sucrose assimilation capacity in E. coli (non-PTS). C, Analysis of sucrose assimilation capacity in E. coli (PTS). D, D-psicose synthesis by shake flask fermentation in E. coli (PTS-SAP).

[0019] Figure 3 Functional characterization of FBPase. A, SDS-PAGE analysis; Lane 1: Standard protein marker; Lane 2: Soluble protein from E. coli (Control); Lane 3: Soluble protein from E. coli (YggF); Lane 4: Soluble protein from E. coli (Fbp); Lane 5: Soluble protein from E. coli (GlpX). B, The ability of crude enzyme solutions from E. coli (YggF), E. coli (Fbp), and E. coli (GlpX) to convert F-1,6-BP to F-6-P. C, D-psicose synthesis by shake flask fermentation of E. coli (PTS-SAPY), E. coli (PTS-SAPF), and E. coli (PTS-SAPG).

[0020] Figure 4 : Regulation of the EMP pathway, PP pathway, and carbon flux. A, D-psicose synthesis by shake flask fermentation of LJGA. B, D-psicose synthesis by shake flask fermentation of LJGAB. C, mRNA levels of the zwf gene in E. coli (PTS-SAPG) and LJGA. D, D-psicose synthesis by shake flask fermentation of LJGAR. E, Schematic diagram of antisense RNA (asRNA) inhibition of fbaA expression. F, D-psicose synthesis by shake flask fermentation of LJARGa100, LJARGa150, and LJARGa300.

[0021] Figure 5 : Synthesis of D-psicose by fed-batch fermentation in LJARGa300 fermenter. DETAILED DESCRIPTION

[0022] In order to make the contents of the present invention easier to understand, the technical solutions of the present invention are further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.

[0023] In the present invention, the UniProtKB number of sucrose-6-phosphate hydrolase cscA is P40714; The UniProtKB number of the sucrose permease gene cscB is P30000; The UniProtKB number of the fructokinase gene cscK is P40713; The UniProtKB number of the sucrose-specific permease gene scrA is A0A0H3FUP0; The UniProtKB number of the sucrose-6-phosphate hydrolase gene scrB is A0A0H3FTU2; The UniProtKB number of the D-psicose-6-phosphate epimerase gene alsE is P32719; The NCBI accession number of the D-psicose-6-phosphatase gene a6PP is BF9343_0892; The nucleotide sequence of the fructose-1,6-bisphosphatase gene mutant glpX[K29A] is shown in SEQ ID NO. 1; The UniProtKB number of the fructose-1,6-bisphosphatase gene yggF is P21437; The UniProtKB number of the fructose-1,6-bisphosphatase gene fbp is P0A993; The UniProtKB number of the 6-phosphofructokinase gene PfkA is P0A796; The UniProtKB number of the 6-phosphofructokinase gene PfkB is P06999; The UniProtKB number of the ribose-5-phosphate isomerase gene RpiA is P0A7Z0; The UniProtKB number of the fructose-bisphosphate aldolase gene fbaA is P0AB71.

[0024] In the present invention, the fbaA gene antisense RNA is regulated by the Pdc promoter, and the nucleotide sequence is selected from SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4.

[0025] In the present invention, the titer of D-psicose in the fermentation broth refers to the content of D-psicose in a unit volume of the fermentation broth.

[0026] In the present invention, the yield of D-psicose in the fermentation broth refers to the mass ratio of unit mass of substrate converted into D-psicose in the fermentation broth.

[0027] In the present invention, the cell density (OD 600 ) was monitored by ultraviolet spectrophotometer; the titer concentration of sugar substances was quantitatively analyzed by high performance liquid chromatography (HPLC) system; the qualitative verification of sugar substances was carried out by liquid chromatography-mass spectrometry system equipped with a dual electrospray ionization source.

[0028] In the present invention, the enzymatic activity of fructose-1,6-bisphosphatases (FBPases), including GlpX, YggF, and Fbp, was determined by high-sensitivity fluorescence assay using an F-6-P assay kit. The enzyme activity unit (U) was defined as the amount of enzyme that catalyzes the conversion of 1 μmol F-1,6-BP to F-6-P per minute.

[0029] In the present invention, pRSFDuet-1 plasmid, pACYCDuet-1 plasmid, and pETDuet-1 plasmid were all purchased from Novagen.

[0030] In the present invention, the formula of the LB liquid culture medium is: 5 g / L yeast extract, 10 g / L tryptone, 5 g / L sodium chloride, and the balance is water; pH = 7.4-7.6.

[0031] Example 1: ① Using the pRSFDuet-1 plasmid as the backbone plasmid, clone the cscA gene between the Nde I and Xho I sites, the cscB gene between the Nco I and Bam HI sites, and the cscK gene between the Sac I and Hind III sites to obtain the pRSFDuet-cscA-cscB-cscK plasmid. Transform the pRSFDuet-cscA-cscB-cscK plasmid into E. coli JM109(DE3) to obtain the recombinant E. coli (non-PTS) strain.

[0032] ② Using the pRSFDuet-1 plasmid as the backbone plasmid, clone the scrA gene between the Nco I and BamH I sites, and clone the scrB gene between the Nde I and Kpn I sites, generating the pRSFDuet-scrA-scrB plasmid. Transform pRSFDuet-scrA-scrB into E. coli JM109(DE3) to generate the recombinant E. coli strain (PTS).

[0033] A single colony of the recombinant strain E. coli (non-PTS) or E. coli (PTS) was inoculated into 4 mL of LB liquid medium and cultured overnight at 37°C and 220 rpm. The inoculum was then transferred to a 250 mL shake flask containing 50 mL of LB liquid medium containing 8 g / L sucrose at a 2% (v / v) inoculum and cultured at 37°C and 220 rpm until the OD 600 When the value reached 0.6-0.8, IPTG was added to a final concentration of 0.21 mM, and the culture was incubated at 30°C and 220 rpm for 80 h. The fermentation broth was analyzed by HPLC. For E. coli (non-PTS), sucrose was rapidly hydrolyzed within 36 h of fermentation, resulting in cumulative amounts of D-glucose and D-fructose reaching 2.5 g / L and 4 g / L in the fermentation broth, respectively. Due to the co-inhibitory effect of D-glucose, the consumption of D-fructose was delayed, and complete consumption of total sugars by E. coli (non-PTS) was observed after 72 h of fermentation. For E. coli (PTS), sucrose was rapidly hydrolyzed within 36 h of fermentation, resulting in cumulative amounts of D-fructose reaching 2.1 g / L in the fermentation broth. Complete consumption of total sugars by E. coli (non-PTS) was observed after 72 h of fermentation.

[0034] ③ Using the pACYCDuet-1 plasmid as the backbone plasmid, the alsE gene was cloned between the Nco I and Hind III sites, the a6PP gene was cloned between the Nde I and Kpn I sites, the scrA gene was cloned between the Hind III and Afl II sites, and the scrB gene was cloned between the Kpn I and Avr II sites to obtain the pACYCDuet-alsE-a6PP-scrA-scrB plasmid. The pETDuet-1 plasmid and the pACYCDuet-alsE-a6PP-scrA-scrB plasmid were simultaneously transformed into E. coli JM109(DE3) to generate the recombinant strain E. coli (PTS-SAP), thereby constructing a novel synthetic pathway from sucrose to D-allose: the sucrose hydrolysis products glucose-6-phosphate (G-6-P) and D-fructose in the cytoplasm are converted into fructose-6-phosphate (F-6-P) by glucose-6-phosphate isomerase (Pgi) and fructose kinase (Mak), respectively, thereby irreversibly synthesizing D-psicose.

[0035] A single colony of the recombinant E. coli (PTS-SAP) strain was inoculated into 4 mL of LB liquid medium and cultured overnight at 37°C and 220 rpm. The inoculum was then transferred to a 250 mL shake flask containing 50 mL of LB liquid medium containing 7.4 g / L sucrose at a concentration of 2% (v / v) and cultured at 37°C and 220 rpm until the OD 600When the value reached 0.6-0.8, IPTG was added to a final concentration of 0.21 mM. The culture was incubated at 30°C and 220 rpm for 80 hours. HPLC analysis of the fermentation broth showed that the titer of D-psicose in the E. coli (PTS-SAP) fermentation broth was 0.3 g / L, and the yield of D-psicose was 0.041 g / g. Compared to the E. coli cell factory (CscA, CscB, CscK, AlsE, A6PP) constructed by the inventors in [Zheng, LJ; Chen, WX; Zheng, SH; Ullah, I.; Zheng, HD; Fan, LH; Guo, Q. Biosynthesis of nonnutritive monosaccharide D-allulose by metabolically engineered Escherichia coli from nutritive disaccharidesucrose. Biotechnol. Bioeng. 2024, 121, 3684-3693.], the titer and yield of D-psicose increased by 43% and 64%, respectively. This highlights the beneficial effect of the sucrose PTS system on D-psicose production.

[0036] Example 2: ①Transform the pETDuet-1 plasmid into E. coli JM109 (DE3) to obtain the recombinant strain E. coli (Control).

[0037] ② Using the pETDuet-1 plasmid as a backbone plasmid, clone the yggF gene between the Nde I and Kpn I sites of the backbone plasmid to generate the pETDuet-yggF plasmid. Transform the pETDuet-yggF plasmid into E. coli JM109(DE3) to obtain the recombinant E. coli (YggF) strain. Transform the pACYCDuet-alsE-a6PP-scrA-scrB plasmid into E. coli (YggF) to obtain the recombinant E. coli (PTS-SAPY) strain.

[0038] ③ Using the pETDuet-1 plasmid as a backbone plasmid, clone the fbp gene between the Nco I and BamH I sites to generate the pETDuet-fbp plasmid. Transform the pETDuet-fbp plasmid into E. coli JM109(DE3) to obtain the recombinant E. coli (Fbp) strain. Transform the pACYCDuet-alsE-a6PP-scrA-scrB plasmid into E. coli (Fbp) to obtain the recombinant E. coli (PTS-SAPF) strain.

[0039] ④ Using the pETDuet-1 plasmid as the backbone plasmid, clone the glpX[K29A] gene between the Nde I and Kpn I sites of the backbone plasmid to generate the pETDuet-glpX[K29A] plasmid. Transform pETDuet-glpX[K29A] into E. coli JM109(DE3) to obtain the recombinant strain E. coli(GlpX[K29A]). Transform the pACYCDuet-alsE-a6PP-scrA-scrB plasmid into E. coli(GlpX[K29A]) to obtain the recombinant strain E. coli(PTS-SAPG).

[0040] A single colony of the recombinant strain E. coli (Control), E. coli (YggF), E. coli (Fbp), or E. coli (GlpX[K29A]) was inoculated into 4 mL of LB liquid medium and cultured overnight at 37°C and 220 rpm. The inoculum was then transferred to a 250 mL shake flask containing 50 mL of LB liquid medium at a 2% (v / v) inoculum. IPTG was added to a final concentration of 0.21 mM, and the cells were induced at 30°C and 220 rpm for 24 h. The cells were then pulverized using a cell ultrasonic disruptor, and the resulting suspension was centrifuged at 8000 rpm and 4°C for 10 min. The supernatant was collected to obtain a soluble protein solution (i.e., crude enzyme solution) for crude enzyme activity analysis and SDS-PAGE analysis. Soluble expression of fructose-1,6-bisphosphatase YggF, fructose-1,6-bisphosphatase Fbp, and fructose-1,6-bisphosphatase gene mutant GlpX[K29A] in Figure 3 A was verified; the in vitro enzyme catalysis reaction was carried out using F-1,6-BP as a substrate, and the resulting F-6-P concentration was as follows Figure 3 As shown in B, the catalytic activities of fructose-1,6-bisphosphatase YggF, fructose-1,6-bisphosphatase Fbp, and fructose-1,6-bisphosphatase gene mutant GlpX[K29A] were verified.

[0041] A single colony of the recombinant strain E. coli (PTS-SAPY) or E. coli (PTS-SAPF) or E. coli (PTS-SAPG) was inoculated into 4 mL of LB liquid medium and cultured overnight at 37°C and 220 rpm. The inoculum was then transferred to a 250 mL shake flask containing 50 mL of LB liquid medium containing 8 g / L sucrose at a 2% (v / v) inoculum and cultured at 37°C and 220 rpm until the OD 600When the value reached 0.6-0.8, IPTG was added to a final concentration of 0.21 mM and fermented at 30°C and 220 rpm for 80 hours. The fermentation broth was then analyzed by HPLC. The results showed that the D-psicose titer in the E. coli (PTS-SAPG) fermentation broth was the highest, at 0.5 g / L, representing increases of 106% and 79% compared to those in E. coli (PTS-SAPY) and E. coli (PTS-SAPF), respectively.

[0042] Example 3: Given that F-6-P plays a key role in the EMP and PP pathways, which are crucial for cell growth, it is necessary to strategically regulate endogenous metabolic pathways to achieve a balance between product yield and biomass yield. In the EMP pathway, the ATP-dependent phosphofructokinases PfkA and PfkB catalyze the phosphorylation of F-6-P to F-1,6-BP, which is then cleaved by FbaA into dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G-3-P). Here, this example modulates the endogenous glycolysis pathway and the pentose phosphate pathway and adjusts the carbon flux entering the tricarboxylic acid cycle by knocking out the 6-phosphofructokinase gene, the ribose-5-phosphate isomerase gene, and inhibiting the expression of the fructose-bisphosphate aldolase gene.

[0043] ① Based on E. coli (PTS-SAPG), the pfkA gene was knocked out using λ-red homologous recombination technology to obtain the recombinant strain LJGA.

[0044] ② Based on LJGA, the pfkB gene was knocked out using λ-red homologous recombination technology to obtain the recombinant strain LJGAB.

[0045] A single colony of the recombinant strain LJGA or LJGAB was inoculated into 4 mL of LB liquid medium and cultured overnight at 37°C and 220 rpm. The inoculum was then transferred to a 250 mL shake flask containing 50 mL of LB liquid medium containing 8 g / L sucrose at a 2% (v / v) inoculum and cultured at 37°C and 220 rpm until the OD 600 The value reached 0.6-0.8, IPTG was added at a final concentration of 0.21 mM, and the culture was carried out at 30°C and 220 rpm for 80 h. The fermentation broth was analyzed by HPLC. The results showed that the titer of D-psicose in the LJGA fermentation broth was 1.1 g / L, and the yield of D-psicose was 0.125 g / g. Compared with LJGA, the titer of D-psicose in the LJGAB fermentation broth was reduced by 41.8%. Within 36 h of fermentation, the sucrose consumption rate decreased from 0.36 g / L / h to 0.16 g / L / h, and the specific cell growth rate decreased from 0.12 h -1 Down to 0.09h -1This indicates that the limited growth capacity of LJGAB may lead to a decrease in the expression of enzymes involved in the synthesis pathway, thereby affecting the production of D-psicose.

[0046] The mRNA levels of the zwf gene in the recombinant strains E. coli (PTS-SAPG) and LJGA were measured. The results showed that knocking out the pfkA gene led to a 12.4-fold increase in the mRNA level of zwf, a key gene in the PP pathway.

[0047] ③ Based on LJGA, the rpiA gene was knocked out using λ-red homologous recombination technology to obtain the recombinant strain LJGAR.

[0048] A single colony of the recombinant strain LJGAR was inoculated into 4 mL of LB liquid medium and cultured overnight at 37°C and 220 rpm. The inoculum was then transferred to a 250 mL shake flask containing 50 mL of LB liquid medium containing 8 g / L sucrose at a 2% (v / v) inoculum and cultured at 37°C and 220 rpm until the OD 600 When the value reached 0.6-0.8, IPTG was added to a final concentration of 0.21 mM and fermented at 30°C and 220 rpm for 80 hours. The fermentation broth was then analyzed by HPLC. The results showed that compared with LJGA, the cell density of LJGAR was reduced by 30.0%, but the titer of D-psicone in the fermentation broth increased to 1.9 g / L, and the yield of D-psicose was 0.210 g / g.

[0049] ④ Based on E. coli JM109 (DE3), the pfkA and rpiA genes were knocked out in sequence using λ-red homologous recombination technology to obtain the pfkA / rpiA double gene knockout recombinant strain.

[0050] ⑤ Using the pETDuet-1 plasmid as the backbone plasmid, the glpX[K29A] gene was cloned between the Nde I and Kpn I sites, and the fbaA gene antisense RNA (SEQ ID NO. 2) was cloned between the Nco I and Sph I sites to generate the pETDuet-G-a100 plasmid. The pACYCDuet-alsE-a6PP-scrA-scrB plasmid and the pETDuet-G-a100 plasmid were simultaneously transformed into the pfkA / rpiA double knockout recombinant strain to generate the recombinant strain LJARGa100.

[0051] ⑥ Using the pETDuet-1 plasmid (Novagen) as the backbone plasmid, the glpX[K29A] gene was cloned between the Nde I and Kpn I sites, and the fbaA gene antisense RNA (SEQ ID NO. 3) was cloned between the Nco I and Sph I sites to generate the pETDuet-G-a150 plasmid. The pACYCDuet-alsE-a6PP-scrA-scrB plasmid and the pETDuet-G-a150 plasmid were simultaneously transformed into the pfkA / rpiA double knockout recombinant strain to generate the recombinant strain LJARGa150.

[0052] ⑦ Using the pETDuet-1 plasmid (Novagen) as the backbone plasmid, the glpX[K29A] gene was cloned between the Nde I and Kpn I sites, and the fbaA gene antisense RNA (SEQ ID NO. 4) was cloned between the Nco I and Sph I sites to generate the pETDuet-G-a300 plasmid. The pACYCDuet-alsE-a6PP-scrA-scrB plasmid and the pETDuet-G-a300 plasmid were simultaneously transformed into the pfkA / rpiA double knockout recombinant strain to generate the recombinant strain LJARGa300.

[0053] A single colony of the recombinant strain LJARGa100, LJARGa150, or LJARGa300 was inoculated into 4 mL of LB liquid medium and cultured overnight at 37°C and 220 rpm. The inoculum was then transferred to a 250 mL shake flask containing 50 mL of LB liquid medium containing 8 g / L sucrose at a 2% (v / v) inoculum and cultured at 37°C and 220 rpm until the OD 600 When the value reached 0.6-0.8, IPTG was added to a final concentration of 0.21 mM and fermented at 30°C and 220 rpm for 80 hours. The fermentation broth was then analyzed by HPLC. The results showed that the LJARGa300 fermentation broth had the highest D-allose titer, reaching 2.3 g / L, a 351.0% increase compared to E. coli (PTS-SAPG), and a D-psicose yield of 0.26 g / g.

[0054] Example 4: A single colony of the recombinant strain LJARGa300 was inoculated into 4 mL of LB liquid medium and cultured overnight at 37°C and 220 rpm. The inoculum was then transferred to a 5 L fermentor containing 3 L of fermentation medium (28.9 g / L sucrose, 5 g / L sodium chloride, 24 g / L yeast extract, 21.7 g / L tryptone, 3 mM manganese chloride, and the balance was water; initial pH = 7) at a 5% (v / v) inoculum volume and cultured at 37°C and 220 rpm until the OD600 When the value reached 0.6-0.8, IPTG was added to a final concentration of 0.21 mM. Fermentation was carried out at 30°C for 50 hours, and the fermentation broth was analyzed by HPLC. Throughout the fermentation process, the dissolved oxygen concentration was automatically controlled within the range of 25%-35% by the stirring rate and aeration supply cascade. The pH of the culture medium was stably maintained at 7 by 50% (v / v) orthophosphoric acid and 30% (v / v) ammonia. Sucrose was added at the 20th hour of fermentation, with a total sucrose feed of 55.2 g / L. The results showed that the D-allose titer in the LJARGa300 fermentation broth was 12.81 g / L, and the D-psicose yield was 0.23 g / g.

[0055] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A method for efficiently synthesizing D-psicose from sucrose in Escherichia coli using dual PTS transport, characterized by: by E. coli Using JM109 (DE3) as the chassis host, a D-psicose synthesis pathway based on the sucrose PTS transport system was established by co-expressing the D-psicose-6-phosphate epimerase gene, the D-psicose-6-phosphatase gene, the sucrose-specific permease gene, and the sucrose-6-phosphate hydrolase gene. The fructose-1,6-bisphosphatase gene was introduced to drive the carbon source flow from the D-fructose PTS transport system to the D-psicose synthesis pathway. By knocking out the 6-phosphofructokinase gene and the ribose-5-phosphate isomerase gene and inhibiting the expression of the fructose-bisphosphate aldolase gene, the endogenous glycolysis pathway and the pentose phosphate pathway were regulated, and the carbon flow entering the tricarboxylic acid cycle was adjusted, ultimately enabling Escherichia coli to efficiently synthesize D-psicose from sucrose using dual PTS transport.

2. The method according to claim 1, wherein: The D-psicose-6-phosphate epimerase gene is alsE , alsE The UniProtKB number is P32719; the D-psicose-6-phosphatase gene is 6PP , 6PP The NCBI number is BF9343_0892; the sucrose-specific permease gene is scA , scA The UniProtKB number is A0A0H3FUP0; the sucrose-6-phosphate hydrolase gene is scrB , scrB The UniProtKB number is A0A0H3FTU2; the fructose-1,6-bisphosphatase gene is selected from glpX[K29A] 、 yggF 、 fbp , glpX[K29A] The nucleotide sequence is SEQ ID NO.1, yggF The UniProtKB number is P21437, fbp The UniProtKB number is P0A993; the 6-phosphofructokinase gene is selected from pfq 、 pfB , pfq The UniProtKB number is P0A796, pfB The UniProtKB number is P06999; the ribose-5-phosphate isomerase gene is rpiA , rpiA The UniProtKB number is P0A7Z0; the fructose-bisphosphate aldolase gene is fbx , fbx The UniProtKB number is P0AB71.

3. The method according to claim 2, wherein: The following steps are involved: 1) E. coli JM109 (DE3) was used as the chassis host strain, and homologous recombination technology was used to knock out pfq and rpiA Genes, pfkA / rpiA Double gene knockout recombinant strain; 2) Build and carry alsE 、 6PP 、 scA and scrB Recombinant plasmids carrying genes and fbx Gene antisense RNA and glpX[K29A] Recombinant plasmid of gene; 3) will carry alsE 、 6PP 、 scA and scrB Recombinant plasmids carrying genes and fbx Gene antisense RNA and glpX[K29A] The recombinant plasmid of the gene was simultaneously transformed into pfkA / rpiA The double gene knockout recombinant strain obtained the recombinant strain LJARGa, which can efficiently synthesize D-psicose from sucrose using double PTS transport.

4. The method according to claim 3, wherein: The order of knockout is: first knock out pfq Gene, then knock out rpiA Gene.

5. The method according to claim 3, wherein: Said carrying alsE 、 6PP 、 scA and scrB The recombinant plasmid of the gene uses pACYCDuet-1 plasmid as the backbone plasmid.

6. The method according to claim 3, wherein: Said carrying fbx Gene antisense RNA and GlpX [K29A] The recombinant plasmid of the gene uses pETDuet-1 plasmid as the backbone plasmid.

7. The method according to claim 3, wherein: described fbx The nucleotide sequence of the gene antisense RNA is selected from SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.

4.

8. The method according to claim 3, wherein: The method further comprises inoculating the recombinant strain LJARGade into a fermentation medium with sucrose as a substrate for fermentation culture.

9. The method according to claim 8, characterized in that: The fermentation medium is formulated as follows: 7-9 g / L sucrose, 5 g / L yeast extract, 10 g / L tryptone, 5 g / L sodium chloride, and the balance is water. The fermentation is carried out in a shake flask, and the shake flask fermentation method is as follows: a single colony of LJARGa is inoculated into 4 mL LB liquid medium, cultured overnight at 37°C and 220 rpm, and then transferred to a 250 mL shake flask containing 50 mL fermentation medium and cultured at 37°C and 220 rpm until the OD reaches 0. 600 When the value reaches 0.6-0.8, IPTG is added to a final concentration of 0.21 mM and fermented at 30°C and 220 rpm.

10. The method according to claim 8, characterized in that: The fermentation medium is formulated as follows: 28.9 g / L sucrose, 5 g / L sodium chloride, 24 g / L yeast extract, 21.7 g / L tryptone, 3 mM manganese chloride, and the balance is water. The fermentation is carried out in a fermentor. The fermentation method is as follows: a single colony of LJARGa is inoculated into 4 mL LB liquid medium, cultured overnight at 37°C and 220 rpm, and then transferred to a 5 L fermentor containing 3 L fermentation medium and cultured at 37°C and 220 rpm until the OD reaches 0. 600 When the value reached 0.6~0.8, IPTG was added at a final concentration of 0.21mM and fermented at 30℃. During the entire fermentation process, the temperature was maintained at 30℃, the pH value of the fermentation medium was stabilized at 7, and the dissolved oxygen level was maintained at 25%~35%. Sucrose was added at the 20th hour of fermentation, and the total amount of sucrose fed was 55.2g / L.

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