A method for escherichia coli to efficiently synthesize d-allulose by using a nutritive sweetener-sucrose

Abstract

The present application relates to the field of metabolic engineering, and provide a method for high-efficiency synthesis of D-allulose by Escherichia coli using nutritional sweetener-sucrose, wherein D-allulose is produced by recombinant Escherichia coli using cheap and traditional nutritional sweetener, and wherein Escherichia coli JM109(DE3) is used as a chassis host, wherein a path for producing D-allulose from sucrose is constructed by introducing exogenous proteins sucrose-6-phosphate hydrolase CscA, sucrose permease CscB, fructokinase CscK, D-allulose-6-phosphate epimerase AlsE and D-allulose-6-phosphate phosphatase A6PP into wild-type Escherichia coli to realize co-expression, and wherein the substrate utilization rate is improved by introducing ptsG-F and knocking out ptsG, fruA, ptsI and ptsH to reprogram the sugar transmembrane transport pathway.

Description

Technical Field

[0001] This invention relates to the field of metabolic engineering, specifically to a method for Escherichia coli to efficiently synthesize D-allulose using the nutritional sweetener sucrose. By using genetic engineering technology to modify Escherichia coli, a recombinant strain of Escherichia coli is obtained, which can effectively assimilate the nutritional sweetener sucrose to efficiently synthesize D-allulose. Background Technology

[0002] Sweeteners can be divided into two main categories based on their nutritional composition: non-nutritive sweeteners and nutritious sweeteners. Non-nutritive sweeteners, also known as non-caloric sweeteners, provide no energy and are considered an excellent alternative to sucrose or other traditional nutritious sweeteners. Studies have confirmed that the use of non-nutritive sweeteners in food and beverages is beneficial for controlling obesity and regulating blood sugar. D-allulose is one such non-nutritive sweetener, a rare six-carbon monosaccharide with extremely low calories. It also possesses unique physiological properties, such as anti-inflammatory, antioxidant, and therapeutic effects against atherosclerosis.

[0003] The scarcity of D-allulose in nature has fueled considerable interest in its commercial production. Microbial fermentation is a low-cost and environmentally friendly method for synthesizing high-value chemicals. Currently, the mainstream methods for fermenting D-allulose production are based on the Izumoring reversible isomerization principle, the aldol condensation principle, and the phosphoric acid dephosphorylation principle. Due to the reversibility of the Izumoring principle, the conversion rate of D-fructose to D-allulose is approximately 30%, while the maximum theoretical conversion rate of D-glucose to D-allulose via a cascade of reversible isomerization is only about 20%. In the aldol condensation principle, the 1-phosphate ketose formed by the condensation of dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde (D-GA) generates a certain proportion of the byproduct D-sorbose mixed in with D-allulose after dephosphorylation, increasing separation costs. In comparison, the phosphorylation-dephosphorylation principle is superior for the synthesis of D-allulose. D-allulose-6-phosphate epimerases AlsE and A6PP can irreversibly convert fructose-6-phosphate (F-6-P) to D-allulose. This strategy combines reversible isomerization with irreversible dephosphorylation, thereby improving substrate utilization and avoiding the generation of byproducts.

[0004] Nutritional sweeteners include monosaccharides (such as D-fructose, D-glucose, and D-galactose), disaccharides (such as sucrose and maltose), and polyols (such as sugar alcohols). Sucrose is widely used in the food and medical fields, serving as an additive to enrich food flavor and as a drug carrier to promote absorption. However, excessive sucrose intake poses significant risks of tooth decay and obesity, and is unfriendly to diabetic patients. Although the use of sucrose as a sweetener is controversial, it is abundant in renewable resources such as sugarcane and sugar beets, and its purification process is mature and inexpensive, making it an ideal carbon source for microbial fermentation. Furthermore, many reported D-allulose production strategies utilize D-fructose or D-glucose as substrates, both of which can be obtained from the hydrolysis of sucrose. Escherichia coli is a highly advantageous chassis host with a clear research background, strong metabolic plasticity, rapid growth, and simple culture conditions. However, more than half of E. coli strains cannot utilize sucrose as a carbon source. Therefore, theoretically, establishing an E. coli cell factory that utilizes sucrose to produce D-allulose has enormous potential for industrial application. Summary of the Invention

[0005] Therefore, to address the aforementioned problems, this invention provides a method for the efficient synthesis of D-allulose by *Escherichia coli* using the nutritional sweetener sucrose, through the introduction of exogenous genes. cscA , cscB , cscK , AlsE and A6PP The preliminary synthetic pathway of D-allulose was constructed by introducing p tsG-F and knockout ptsG , fruA , ptsI and ptsH To recode the transport pathways of D-glucose and D-fructose produced by sucrose hydrolysis, and to achieve the co-utilization of sucrose hydrolysis products, through... pfkA and zwf By inactivating the D-allulose synthesis pathway, scaling up the culture and optimizing cell growth, a cell factory for the efficient synthesis of D-allulose using sucrose as a substrate was successfully constructed.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: E. coli JM109 (DE3) can synthesize D-allulose from sucrose, which first requires overexpression of exogenous proteins sucrose-6-phosphate hydrolase CscA, sucrose permease CscB, fructose kinase CscK, D-allulose-6-phosphate epimerase AlsE, and D-allulose-6-phosphate phosphatase A6PP. This is achieved through the introduction of... ptsG-F and knockout ptsG , fruA , ptsI and ptsHThis reprogrammed the transmembrane sugar transport pathway, thereby improving substrate utilization. To further modulate the carbon flux of the Embden-Meyerhof-Parnas (EMP) and pentose phosphate (PP) pathways, knockout was performed. pfkA and zwf This allows for the redistribution of carbon flux. Scale-up fermentation was conducted in a 5 L microbioreactor, enabling E. coli to efficiently synthesize D-allulose from sucrose.

[0007] Furthermore, the above-mentioned method for the efficient synthesis of D-allulose by Escherichia coli using the nutritional sweetener sucrose specifically includes the following steps:

[0008] (1) In E.coli Overexpression of the exogenous proteins sucrose-6-phosphate hydrolase CscA, sucrose permease CscB, fructose kinase CscK, D-allulose-6-phosphate epimerase AlsE, and D-allulose-6-phosphate phosphatase A6PP in JM109 (DE3) disrupted the pathway for E. coli to synthesize D-allulose using sucrose as a substrate. Recombinant strains were obtained. E.coli (CscA,CscB, CscK, AlsE, A6PP);

[0009] (2) Based on step (1), by knocking out ptsG , fruA , ptsI and ptsH At the same time, introduce ptsG-F and cscK , obtained strain E.coli (ΔPtsG, ΔFruA, ΔPtsI, ΔPtsH, PtsG-F, CscK) We recoded the intracellular transport pathways for D-glucose and D-fructose, producing F-6-P for D-allulose synthesis, while eliminating the CCR effect, thus achieving the co-utilization of D-glucose and D-fructose. By introducing the sucrose hydrolysis pathway and the D-allulose synthesis pathway, we obtained strains... E. coli (ΔPtsG, ΔFruA, ΔPtsI, ΔPtsH, CscA, CscB, CscK, AlsE, A6PP, PtsG-F) improved the substrate utilization rate for the synthesis of D-allulose from sucrose;

[0010] (3) Based on step (2), the direction of intracellular carbon flux is repositioned by knocking out key genes in the EMP pathway. pfkA It inhibits the massive conversion of F-6-P to F-1,6-BP for metabolism in the cell center; and retains... pfkB It is beneficial to cell growth; knockout zwfThis blocked the carbon flux in the PP pathway, further enhancing the D-allulose synthesis pathway;

[0011] (4) Based on step (3), the recombinant strain E. coli (ΔPtsG, ΔFruA, ΔPtsI, ΔPtsH, ΔPfkA, ΔZwf, CscA, CscB, CscK, AlsE, A6PP, PtsG-F) were used to scale up the fermentation in a 5 L microbioreactor, select a more nutrient-rich culture medium for fermentation, optimize cell growth, and further improve the fermentation efficiency of the cell factory for D-allulose synthesis.

[0012] Furthermore, the gene overexpressed by E. coli JM109 (DE3) in step (1) above... cscA , cscB , cscK , alsE and a6PP ,in cscA , cscB and cscK All of them were derived from Escherichia coli strain W, with UniProt sequence numbers P40714, P30000 and P40713, respectively; AlsE Derived from Escherichia coli K12 strain, UniProt sequence number P32719. a6PP It comes from Bacteroides fragilis NCTC 9343, NCBI sequence number BF9343_0892.

[0013] Furthermore, the gene knocked out in E. coli JM109 (DE3) in step (2) above... ptsG , fruA, ptsI and ptsH The UniProt sequence numbers are P69786, P20966, P08839, and P0AA04, respectively. The exogenous protein PtsG-F overexpressed in E. coli is derived from a mutant of PtsG in E. coli strain K12, in which the 12th amino acid valine (V) is replaced by phenylalanine (F). The UniProt sequence is referenced at P69786.

[0014] Furthermore, the gene knocked out in E. coli JM109 (DE3) in step (3) above... pfkA and zwf The UniProt sequence numbers were P0A796 and P0AC53, respectively. The knockout principle was based on λ-red homologous recombination. Recombinant strains were obtained. E. coli(ΔPtsG, ΔFruA, ΔPtsI, ΔPtsH, ΔPfkA, ΔZwf, CscA, CscB, CscK, AlsE, A6PP,PtsG-F).

[0015] Furthermore, the recombinant strain in step (4) above E. coli (ΔPtsG, ΔFruA, ΔPtsI, ΔPtsH, ΔPfkA, ΔZwf, CscA, CscB, CscK, AlsE, A6PP, PtsG-F) were cultured in a 5 L microbioreactor containing 5-6 g / L glycerol, 24-25 g / L yeast extract, 12-14 g / L peptone, 2.31-2.35 g / L KH2PO4, 12.54-12.6 g / L K2HPO4, and sucrose.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: In the present invention, by overexpressing the exogenous gene in wild-type JM109 (DE3), a pathway for the cellular assimilation of sucrose to synthesize D-allulose was successfully constructed, and then by introducing... ptsG-F and knockout ptsG , fruA , ptsI and ptsH This reprogrammed the transmembrane sugar transport pathway, thereby improving substrate utilization. To further modulate the carbon flux of the Embden-Meyerhof-Parnas (EMP) and pentose phosphate (PP) pathways, knockout was performed. pfkA and zwf This achieves a redistribution of carbon flux. The resulting strain... E. coli The enzymes (ΔPtsG, ΔFruA, ΔPtsI, ΔPtsH, ΔPfkA, ΔZwf, CscA, CscB, CscK, AlsE, A6PP, PtsG-F) were scaled up and fermented in a 5 L microbioreactor, thereby achieving efficient synthesis of D-allulose from sucrose by Escherichia coli, with a yield of 11.15 g / L. Attached Figure Description

[0017] Figure 1 Diagram of a cell factory;

[0018] Figure 2 Functional analysis diagram of the D-allose synthesis pathway;

[0019] (a) E. coli Fermentation diagram of (CscA, CscB, CscK, AlsE, A6PP) products;

[0020] (b) LC-MS analysis of the fermentation broth after 84 h;

[0021] Figure 3 A schematic diagram illustrating the effect of the non-phosphate D-fructose transporter (PtsG-F) on glucose PTS;

[0022] (a) E. coli (ΔPtsG) Fermentation diagram in LB medium containing D-glucose and D-fructose;

[0023] (b) E. coli (ΔPtsG, ΔFruA, PtsG-F, CscK) Fermentation diagram in LB medium containing D-glucose and D-fructose;

[0024] (c) E. coli (ΔPtsG) Fermentation diagram in LB medium containing D-glucose and D-xylose;

[0025] (d) E. coli (ΔPtsG, PtsG-F) Fermentation diagram in LB medium containing D-glucose and D-xylose;

[0026] Figure 4 A schematic diagram illustrating the co-utilization of sucrose hydrolysis products in order to eliminate the CCR effect;

[0027] (a) Schematic diagram of the CCR effect triggered by D-glucose PTS;

[0028] (b) E. coli Fermentation diagram of (ΔPtsG, ΔFruA, ΔPtsI, ΔPtsH, PtsG-F, CscK) in LB medium containing D-glucose and D-fructose;

[0029] (c) E. coli (ΔPtsG, ΔFruA, ΔPtsI, ΔPtsH, CscA, CscB, CscK, AlsE, A6PP, PtsG-F) Product fermentation diagram;

[0030] Figure 5 A schematic diagram illustrating the reorientation of carbon flux in the EMP and PP pathways to enhance the synthesis of D-allulose.

[0031] (a) E. coli (ΔPtsG, ΔFruA, ΔPtsI, ΔPtsH, ΔPfkA, CscA, CscB, CscK,AlsE, A6PP, PtsG-F) Product fermentation diagram;

[0032] (b) E. coli(ΔPtsG, ΔFruA, ΔPtsI, ΔPtsH, ΔPfkA, ΔZwf, CscA, CscB, CscK, AlsE, A6PP, PtsG-F) Product fermentation diagram;

[0033] Figure 6 Schematic diagram for optimizing D-allulose production through scale-up fermentation;

[0034] (a) Fermentation parameters;

[0035] (b) E. coli (ΔPtsG, ΔFruA, ΔPtsI, ΔPtsH, ΔPfkA, ΔZwf, CscA,CscB, CscK, AlsE, A6PP, PtsG-F) Fermentation diagram of products in sucrose-containing TB medium. Detailed Implementation

[0036] To make the content of this invention easier to understand, the following is combined with... Figures 1 to 6 The following detailed descriptions of specific embodiments further illustrate the technical solutions described in this invention, but this invention is not limited thereto. Example 1

[0037] Overexpression of exogenous genes in wild-type Escherichia coli JM109 (DE3) (purchased from: BeNa Culture Collection) cscA , cscB , cscK , AlsE and A6PP Recombinant Escherichia coli was obtained. E. coli ( CscA, CscB, CscK, AlsE, A6PP) The cells were pre-cultured overnight in 4 mL LB tubes containing 100 μg / mL ampicillin, 50 μg / mL kanamycin, and 30 μg / mL chloramphenicol at 37°C and 220 rpm. Then, the cells were seeded into 250 mL shake flasks containing 50 mL LB medium and 8.26 g / L sucrose for fermentation. When the cell density reached OD200... 600 When the protein content reached approximately 0.6, 0.2 mM IPTG was added to induce protein expression, and then the fermentation temperature was adjusted to 30°C. Samples were taken every 12 h, and the samples were analyzed using high-performance liquid chromatography (HPLC) equipped with a refractive index detector (RID) and a Sugar-Pak I column (6.5 × 300 mm). 2High-performance liquid chromatography (HPLC) analysis was performed using a Chromaster system (Hitachi, Japan) from Waters Corporation (USA). Ultrapure water was used as the mobile phase at a flow rate of 0.5 mL / min. At 84 h of fermentation, samples were validated for D-allulose using LC-MS on an Agilent 1260 and 6520 system equipped with dual electrospray ionization sources. Specific parameters were as follows: nebulizer pressure, 40 psi; capillary voltage, +3.5 kV; nitrogen drying gas, 10 L / min; drying gas temperature, 350 °C; acquisition frequency, 1 m / s; acquisition mode, 2 GHz; dynamic spread mode, 100-350 m / z.

[0038] Sucrose was completely hydrolyzed to D-glucose and D-fructose within 36 h of fermentation. The yield of D-allulose was 0.21 g / L, and the yield of sucrose was approximately 0.025 g / g, indicating that the synthetic pathway was successfully constructed. The product was also identified by LC-MS analysis. Example 2

[0039] Reprogramming sugar transport pathways to achieve co-utilization of sucrose hydrolysis products

[0040] Substrate utilization was improved by reprogramming the transmembrane sugar transport pathway through the introduction of ptsG-F and the knockout of ptsG, fruA, ptsI, and ptsH. A DNA fragment carrying two flipper enzyme recognition sites (FRTs) and a kanamycin resistance gene was electroporated into JM109 (DE3)-pKD46 cells. Cells were cultured at 30 °C and induced with 5 g / L L-arabinose. As a result, the kanamycin resistance fragment was homologously integrated into the *E. coli* genome, replacing the target gene, and pKD46 was subsequently removed. FLP recombinase recognizing the FRT site was expressed using pCP20 to remove the kanamycin resistance gene from the genome. Finally, pCP20 was heated and discarded to obtain an antibiotic-free strain.

[0041] After introducing the sucrose assimilation pathway and the D-allulose synthesis pathway, recombinant strains of *E. coli* (ΔPtsG,ΔFruA, ΔPtsI, ΔPtsH, CscA, CscB, CscK, AlsE, A6PP, PtsG-F) were obtained. The yield of D-allulose reached 0.59 g / L, with a yield of 0.072 g / g. Figure 2 Compared to the previous method, the titer increased by approximately 1.9 times, indicating that reprogramming the sugar transport pathway to improve substrate utilization is effective for the production of D-allulose. Example 3

[0042] E. coli(ΔPtsG, ΔFruA, ΔPtsI, ΔPtsH, ΔPfkA, ΔZwf, CscA, CscB, CscK, AlsE, A6PP, PtsG-F) were incubated in 3L TB medium containing 100 μg / mL ampicillin, 50 μg / mL kanamycin, and 30 μg / mL chloramphenicol sucrose. TB medium consisted of 5 g / L glycerol, 24 g / L yeast extract, 12 g / L peptone, 2.31 g / L KH₂PO₄, and 12.54 g / L K₂HPO₄. Fermentation data are as follows: Figure 6 As shown in Figure a, dissolved oxygen was maintained at 20-40% by adjusting the stirring speed and gas flow rate. The pH was controlled at 7.0 using phosphate (50%, v / v) and ammonia (50%, v / v). The cell incubation temperature was set at 37 °C, but was changed to 30 °C after the addition of IPTG to optimize protein expression. Figure 6 The data in b indicate that cell growth is significantly enhanced, and the final density OD 600 It is 5.64, compared to Figure 5 The density in cell b was approximately 3.5 times higher. The titer of D-allulose reached 11.15 g / L, while the yield on sucrose was 0.208 g / g. The changes in D-allulose yield and cell density during the construction process are shown in the table below:

[0043]

[0044] Although the invention has been specifically shown and described in conjunction with preferred embodiments, those skilled in the art will understand that various changes in form and detail may be made to the invention without departing from the spirit and scope of the invention as defined in the appended claims, all of which shall be within the scope of protection of the invention.

[0045] CscA

[0046] MTQSRLHAAQNALAKLHERRGNTFYPHFHLAPPAGWMNDPNGLIWFNDRYHAFYQHHPMSEHWGPMHWGHATSDDMIHWQHEPIALAPGDENDKDGCFSGSAVDDNGVLSLIYTGHVWLDGAGNDDAIREVQCLATSRDGIHFEKQGVILTPPEGIMHFRDPKVWREADTWWMVVGAKDPGNTGQILLYRGSSLREWTFDRVLAHADAGESYMWECPDFFSLGDQHYLMFSPQGMNAEGYSYRNRFQSGVIPGMWSPGRLFAQSGHFTELDNGHDFYAPQSFVAKDGRRIVIGWMDMWESPMPSKREGWAGCMTLARELSESNGKLLQRPVHEAESLRQQHQSISPRTISNKYVLQENAQAVEIQLQWALKNSDAEHYGLQLGAGMRLYIDNQSERLVLWRYYPHENLDGYRSIPLPQGDMLALRIFIDTSSVEVFINDGEAVMSSRIYPQPEERELSLYASHGVAVLQHGALWQLG

[0047] CscB

[0048] MALNIPFRNAYYRFASSYSFLFFISWSLWWSLYAIWLKGHLGLTGTELGTLYSVNQFTSILFMMFYGIVQDKLGLKKPLIWCMSFILVLTGPFMIYVYEPLLQSNFSVGLILGALFFGLGYLAGCGLLDSFTEKMARNFHFEYGTARAWGSFGYAIGAFFAGIFFSISPHINFWLVSLFGAVFMMINMRFKDKDHQCIAADAGGVKKEDFIAVFKDRNFWVFVIFIVGTWSFYNIFDQQLFPVFYAGLFESHDVGTRLYGYLNSFQVVLEALCMAIIPFFVNRVGPKNALLIGVVIMALRILSCALFVNPWIISLVKLLHAIEVPLCVISVFKYSVANFDKRLSSTIFLIGFQIASSLGIVLLSTPTGILFDHAGYQTVFFAISGIVCLMLLFGIFFLSKKREQIVMETPVPSAI

[0049] CscK

[0050] MSAKVWVLGDAVVDLLPESDGRLLPCPGGAPANVAVGIARLGGTSGFIGRVGDDPFGALMQRTLLTEGVDITYLKQDEWHRTSTVLVDLNDQGERSFTFMVRPSADLFLETTDLPCWRHGEWLHLCSIALSAEPSRTSAFTAMTAIRHAGGFV SFDPNIREDLWQDEHLLRLCLRQALQLADVVKLSEEEWRLISGKTQNDRDICALAKEYEIAMLLVTKGAEGVVCYRGQVHHFAGMSVNCVDSTGAGDAFVAGLLTGLSSTGLSTDEREMRRIIDLAQRCGALAVTAKGAMTALPCRQELESEK

[0051] AlsE

[0052] MKISPSLMCMDLLKFKEQIEFIDSHADYFHIDIMDGHFVPNLTLSPFFVSQVKKLATKPLDCHLMVTRPQDYIAQLARAGADFITLHPETINGQAFRLIDEIRRHDMKVGLILNP ETPVEAMKYYIHKADKITVMTVDPGFAGQPFIPEMLDKLAELKAWREREGLEYEIEVVDGSCNQATYEKLMAAGADVFIVGTSGLFNHAENIDEAWRIMTAQILAAKSEVQPHAKTA

[0053] A6PP

[0054] MKYTVYLFDFDYTLADSSRGIVTCFRSVLERHGYTGITDDMIKRTIGKTLEESFSILTGITDADQLESFRQEYSKEADIYMNANTILFPDTLPTLTHLKKQGIRIGIISTK YRFRILSFLRNHMPDDWFDIIIGGEDVTHHKPDPEGLLLAIDRLKACPEEVLYIGDSTVDAGTAAAAGVSFTGVTSGMTTAQEFQAYPYDRIISTLGQLISVPEDKSGCPL

[0055] PtsG-F

[0056] MFKNAFANLQKFGKSLMLPVSVLPIAGILLGVGSANFSWLPAVVSHVMAEAGGSVFANMPLIFAIGVALGFTNNDGVSALAAVVAYGIMVKTMAVVAPLVLHLPAEEIASKHLADTGVLGGIISGAIAAYMFNRFYRIKLPEYLGFFAGKRFVPIISGLAAIFTGVVLSFIWPPIGSAIQTFSQWAAYQNPVVAFGIYGFIERCLVPFGLHHIWNVPFQMQIGEYTNAAGQVFHGDIP RYMAGDPTAGKLSGGFLFKMYGLPAAAIAIWHSAKPENRAKVGGIMISAALTSFLTGITEPIEFSFMFVAPILYIIHAILAGLAFPICILLGMRDGTSFSHGLIDFIVLSGNSSKLWLF PIVGIGYAIVYYTIFRVLIKALDLKTPGREDATEDAKATGTSEMAPALVAAFGGKENITNLDACITRLRVSVADVSKVDQAGLKKLGAAGVVVAGSGVQAIFGTKSDNLKTEMDEYIRNH

[0057] PTSD

[0058] MFKNAFANLQKVGKSLMLPVSVLPIAGILLGVGSANFSWLPAVVSHVMAEAGGSVFANMPLIFAIGVALGFTNNDGVSALAAVVAYGIMVKTMAVVAPLLVHLPAEEIASKHLADTGVLGGIISGAIAAYMFNRFYRIKLPEYLGFFFAGKRFVPIISGLAAIFTGVVLSFIWPPIGSAIQTFSQWAAYQNPVVAFGIYGFIERCLVPGLHHIWNVPFQMQIGEYTNAAGQVFHGDIP RYMAGDPTAGKLSGGFLFKMYGLPAAAAIAIWHSAKPENRAKVGGIMISAALTSFLTGITEPIEFSFMFVAPILYIIHAILAGLAFPICILLLGMRDGTSFSHGLIDFIVLSGNSSKLWLFPIVGIGYAIVYYTIFRVLIKALDLKTPGREDATEDAKATGTSEMAPALVAAFGGKENITNLDACITRLRVSVADVSKVDQAGLKKLGAAGVVVAGSGVQAIFGTKSDNLKTEMDEYIRNH

[0059] Early

[0060] MKTLLIIDANLGQARAYMAKTLLGAARKAKLEIIDNPNDAEMAIVLGDSIPNDSALNGKNVWLGDISRAVAHPELFLSEAKGHAKPYTAPVAATAPVAASGPKRVVAVTACPTGVAHTFMAAEAIETEAKKRGWWVKVETRGSVGAGNAITPEEVAADLVIVAADIEVDLAKFAGKPMYRTSTGLALKKTAQELDKAVAEATPYEPAGKAQTATTESKKESAGAYRHLLTGVSYMLPMVVAGGLCIALSFAFGIEAFKEPGTLAAALMQIGGGSAFALM VPVLAGYIAFSIADRPGLTPGLIGGMLAVSTGSGFIGGIIAGFLAGYAKLISTQLKLPQSMEALKPILIIPLISSLVVGLAMIYLIGKPVAGILEGLTHWLQTMGTANAVLLGAILGGMMCTDMGGPVNKAAYAFGVGLL STQTYGPMAAIMAAGMVPPLAMGLATMVARRKFDKAQQEGGKAALVLGLCFISEGAIPFAARDPMRVLPCCIVGGALTGAISMAIGAKLMAPHGGLFVLLIPGAITPVLGYLVAIIAGTLVAGLAYAFLKRPEVDAVAKAA

[0061] PtsI

[0062] MISGILASPGIAFGKALLLKEDEIVIDRKKISADQVDQEVERFLSGRAKASAQLETIKTKAGETFGEEKEAIFEGHIMLLEDEELEQEIIALIKDKHMTADAAAHEVIEGQASALEELDDEYLKERAADVRDIGKRLLRNILG LKIIDLSAIQDEVILVAADLTPSETAQLNLKKVLGFITDAGGRTSHTSIMARSLELPAIVGTGSVTSQVKNDDYLILDAVNNQVYVNPTNEVIDKMRAVQEQVASEKAELAKLKDLPAITLDGHQVEVCANIGTVRDVEGAERN GAEGVGLYRTEFLFMDRDALPTEEEQFAAYKAVAEACGSQAVIVRTMDIGGDKELPYNMNFPKEENPFLGWRAIRIAMDRREILDQLRAILRASAFGKLRIMFPMIISVEEVRALRKEIEIYKQELRDEGKAFDESIEIGVMVETPAATIARHLAKEVDFFSIGTNDLTQYTLAVDRGNDMISHLYQPMSPSVLNLIKQVIDASHAEGKWTGMCGELAGDERATLLLLGMGLDEFSMSAISIPRIKKIIRNTNFEDAKVLAEQALAQPTTDELMTLVNKFIEEKTIC

[0063] PtsH

[0064] MFQQEVTITAPNGLHTRPAAQFVKEAKGFTSEITVTSNGKSASAKSLFKLQTLGLTQGTVVTISAEGEDEQKAVEHLVKLMAELE

[0065] PfkA

[0066] MIKKIGVLTSGGDAPGMNAAIRGVVRSALTEGLEVMGIYDGYLGLYEDRMVQLDRYSVSDMINRGGTFLGSARFPEPFRDENIRAVAIENLKKRGIDALVVIGGDGSYMGAMRLTEMGFPCIGLPGTIDNDIKGTDYTIGFFTALSTVVEAIDRLRDTSSSHQRISVVEVMGRYCGDLTLAAAIAGGCEFVVVPEVEFSREDLVNEIKAGIAKGKKHAIVAITEHMCDVDELAHFIEKETGRETRATVLGHIQRGSPVPYDRILASRMGAYAIDLLLAGYGGRCVGIQNEQLVHHDIIDAIENMKRPFKGDWLDCAKKLY

[0067] Zwf

[0068] MAVTQTAQACDLVIFGAKGDLARRKLLPSLYQLEKAGQLNPDTRIIGVGRADWDKAAYTKVVREALETFMKETIDEGLWDTLSARLDFCNLDVNDTAAFSRLGAMLDQKNRITINYFAMPPSTFGAICKKGLGEAKNLAKPARVVMEKPLGTSLATSQEINDQVGEYFEECQVYRIDHYLGKETVLNLLALRFANSLFVNNWDNRTIDHVEITVAEEVGIEGRWGYFDKAGQMRDMIQNHLLQILCM IAMSPPSDLSADSIRDEKVVLKSLRRIDRSNVREKTVRGQYTAGFAQGKKVPGYLEEEGANKSSNTETFVAIRVDIDNWRWAGVFPYLRTGKRLPTKCSEVVVYFKTPELNLFKESWQDLPQNKLTIRLQPDEGVDIQVLNKVPGLDHKHNLQITKLDLSYSETFNQTHLADAYERLLLETMRGIQALFVRRDEVEEAWKWVDSITEAWAMNDAPKPYQAGTWGVASVAMITRDGRSWNEFE。

Claims

1. A method for the efficient synthesis of D-allulose by Escherichia coli using the nutritional sweetener sucrose, characterized in that: Using Escherichia coli JM109 (DE3) as the chassis host, the exogenous gene sucrose-6-phosphate hydrolase was introduced. cscA Sucrose permease cscB fructokinase cscK D-allulose-6-phosphate epimerase asE and D-allulose-6-phosphate phosphatase a6PP The preliminary synthetic pathway of D-allulose was constructed; by introducing... ptsG-F and knockout ptsG , fruA , ptsI and ptsH To recode the transport pathways of D-glucose and D-fructose produced by sucrose hydrolysis, thereby achieving the co-utilization of sucrose hydrolysis products; through pfkA and zwf Knockout was used to regulate carbon flux in the Embden-Meyerhof-Parnas (EMP) and pentose phosphate (PP) pathways, thereby enhancing the D-allulose synthesis pathway; the resulting recombinant strain E. coli (ΔPtsG, ΔFruA, ΔPtsI, ΔPtsH, ΔPfkA, ΔZwf, CscA, CscB, CscK, AlsE, A6PP, PtsG-F) were successfully constructed into a cell factory for the efficient synthesis of D-allulose using sucrose as a substrate by scaling up culture and optimizing cell growth. in cscA , cscB and cscK All were derived from Escherichia coli strain W, and their amino acid sequences are shown in SEQ ID NO 1, SEQ ID NO 2, and SEQ ID NO 3, respectively. asE Derived from Escherichia coli K12 strain, the amino acid sequence is shown in SEQ ID NO 4; a6PP Derived from Bacteroides fragilis NCTC 9343, the amino acid sequence is shown in SEQ ID NO 5; ptsG , fruA , ptsI , ptsH , pfkA and zwf The amino acid sequences are shown in SEQ ID NO 6, 7, 8, 9, 10, and 11, respectively; exogenous protein ptsG-F Derived from Escherichia coli strain K12 ptsG The mutant has its 12th amino acid, valine, replaced by phenylalanine. ptsG-F The amino acid sequence is shown in SEQ ID NO 12.

2. The method for efficient synthesis of D-allulose by Escherichia coli using the nutritional sweetener sucrose according to claim 1, characterized in that, Gene knockout in E. coli JM109 (DE3) based on the principle of λ Red homologous recombination pfkA and zwf .

3. The method for efficient synthesis of D-allulose by *Escherichia coli* using the nutritional sweetener sucrose according to claim 1, characterized in that, Recombinant strains E. coli (ΔPtsG, ΔFruA, ΔPtsI, ΔPtsH, ΔPfkA, ΔZwf,CscA, CscB, CscK, AlsE, A6PP, PtsG-F) were cultured in a 5 L microbioreactor. The culture medium contained 5-6 g / L glycerol, 24-25 g / L yeast extract, 12-14 g / L peptone, 2.31-2.35 g / L KH2PO4, 12.54-12.6 g / L K2HPO4, and sucrose.

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