CN115074376A

CN115074376A - Method for efficiently synthesizing D-psicose by fermentation of recombinant escherichia coli

Info

Publication number: CN115074376A
Application number: CN202210459067.6A
Authority: CN
Inventors: 范立海; 刘晨阳; 郭强; 郑辉东
Original assignee: Qingyuan Innovation Laboratory; Fuzhou University
Current assignee: Qingyuan Innovation Laboratory; Fuzhou University
Priority date: 2022-04-28
Filing date: 2022-04-28
Publication date: 2022-09-20
Anticipated expiration: 2042-04-28
Also published as: CN115074376B

Abstract

The invention provides a method for efficiently synthesizing D-psicose by utilizing recombinant escherichia coli fermentation. Uses colibacillus as chassis host bacteria, transfers protein gene by knocking out fructose specificity PTSFruASimultaneous overexpression of fructose non-phosphorylated transporter genepstG‑FFructokinase genemaKD-psicose-6-phosphate epimerase genealsEPsicose-6-phosphate phosphatase genea6PPEstablishing a synthetic pathway from D-fructose to D-psicose; further knock-out of D-fructose-6-phosphate kinase genepfkAAndpfkBregulating and controlling the carbon metabolism flux of the recombinant escherichia coli; overexpression of phosphoenolpyruvate carboxykinaseDue to the fact thatpckAAnd glycerol is used as a carbon source to improve the intracellular ATP concentration. The recombinant escherichia coli constructed by the invention is used for fermentation production, can effectively improve the substrate conversion rate, and provides an optimization scheme for the industrial development of biosynthesis of D-psicose.

Description

Method for efficiently synthesizing D-psicose by fermentation of recombinant escherichia coli

Technical Field

The invention belongs to the field of microbial metabolic engineering, and particularly relates to a method for efficiently synthesizing D-psicose by utilizing fermentation of recombinant escherichia coli.

Technical Field

D-psicose is a rare sugar with ultra-low calorie, and the sweetness is 70% of that of sucrose while the energy is only 0.3% of that of sucrose. A large number of researches report that the D-psicose has unique physiological characteristics in the aspects of preventing obesity, treating atherosclerosis, resisting hyperlipidemia, resisting inflammation, resisting oxidation, resisting hyperglycemia, protecting nerves and the like, can improve the gelation state of food, increase the flavor of the food, and even can relieve the oxidation effect in the food processing and storage processes. D-psicose has been approved as a generally recognized as safe product (GRAS) by the Food and Drug Administration (FDA) in the United states, and has great market prospects in the fields of food, beverages, health care and the like.

Since D-psicose is very scarce, development of a method for producing it has attracted considerable attention. The research on the chemical synthesis of D-psicose begins in 1960, but the research has not been broken through in industrial application due to the reasons of more side reactions, complicated purification, serious pollution and the like. In contrast, biosynthesis of D-psicose is highly specific and environmentally friendly. Although the enzyme method has advantages in terms of product conversion rate and product purification, the enzyme production and immobilization are high in cost and long in time, and especially the problem of thermal stability of key enzyme is not completely solved, so that the reusability of the biocatalyst is poor. In contrast, microbial fermentation for the production of D-psicose is an ideal alternative to enzymatic catalysis.

Disclosure of Invention

The present invention has been made in view of the above problems, and an object of the present invention is to provide a method for efficiently synthesizing D-psicose by fermentation using recombinant Escherichia coli. A synthesis path from D-fructose to D-psicose is established in escherichia coli through recombinant vector construction, carbon metabolic flux is reasonably regulated and controlled by means of gene knockout, an ATP intracellular synthesis path is introduced, and efficient synthesis of D-psicose is finally achieved.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for efficiently synthesizing D-psicose by fermentation of recombinant Escherichia coli inE. coliKnockout of fructose-specific PTS transfer protein Gene in JM109(DE3)fruASimultaneous overexpression of fructose non-phosphorylated transporter geneptsG-FFructokinase genemaKD-psicose-6-phosphate epimerase genealsEAnd psicose-6-phosphate phosphatase genea6PPFour genes are used for constructing a synthetic route of D-psicose; further by knocking out D-fructose-6-phosphate kinase genepfkAAndpfkBblocking the conversion of intracellular fructose-6-phosphate into fructose-1, 6-diphosphate, and leading the carbon flux of D-fructose to enter a product synthesis way to the maximum extent; reintroducing a heterologous phosphoenolpyruvate carboxykinase genepckAUsed for expressing PckA protein and realizing the intracellular circulation of ATP; and finally, performing oxygen-limited fermentation by using an M9 basal medium containing glycerol as a fermentation medium of the recombinant escherichia coli to realize the high-efficiency synthesis of the D-psicose.

The method for efficiently synthesizing the D-psicose by utilizing the recombinant escherichia coli fermentation specifically comprises the following steps:

(1) knock-outE. coliFructose-specific PTS transfer protein Gene of JM109(DE3)fruAAnd overexpressing a fructose non-phosphorylated transporter geneptsG-FFructokinase genemaKD-psicose-6-phosphate epimerase genealsEAnd psicose-6-phosphate phosphatase genea6PPFour genes to obtainE. coli(Δ FruA, PtsG-F, MaK, AlsE, A6 PP);

(2) in bacterial strainsE. coli(Δ FruA, PtsG-F, MaK, AlsE, A6PP) knock-out of D-fructose-6-phosphokinase GenepfkAAndpfkBto obtainE. coli (Δ FruA, Δ PfkA, Δ PfkB, PtsG-F, MaK, AlsE, A6 PP);

(3) in bacterial strainsE. coli (delta FruA, delta PfkA, delta PfkB, PtsG-F, MaK, AlsE, A6PP) based on the expression of phosphoenolpyruvate carboxykinase GenepckAObtaining recombinant Escherichia coliE. coli (ΔFruA, ΔPfkA, ΔPfkB, PtsG-F, MaK, AlsE, A6PP, PckA)；

(4) Fermentation culture using M9 minimal medium containing glycerolE. coli (Δ FruA, Δ PfkA, Δ PfkB, PtsG-F, MaK, AlsE, A6PP, PckA) strain.

The specific process of the step (1) is as follows: use of primersfruAF (SEQ ID number 1) andfruAPCR amplification of R (SEQ ID number 2) using pKD13 plasmid as template, followed by transfection of the PCR product into a plasmid containing pKD46E. coliJM109(DE3) was subjected to homologous recombination, single colony was picked for activation and competent cells were prepared, transfected with pep 20 plasmid, cultured overnight in a shaker at 30 ℃ and 220 rpm to eliminate resistance marker, and finally the pep 20 plasmid was lost under the culture conditions at 42 ℃ and 220 rpm to obtainE. coli(Δ FruA) strain; then artificially synthesized pETDuet-ptsG-F-maKAnd pRSFDuet-alsE-a6PPTransfection of recombinant plasmids intoE. coli(. DELTA.FruA) to giveE. coli(Δ FruA, PtsG-F, MaK, AlsE, A6 PP); wherein the content of the first and second substances,ptsG-F、maK、alsEanda6PPthe gene sequence of (A) is shown in SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO. 6.

The specific process of the step (2) is as follows: use of primerspfkA-F/pfkA-R andpfkB-F/pfkB-R is knocked out according to the method in step (1)pfkAAndpfkBgene, obtainingE. coli (Δ FruA, Δ PfkA, Δ PfkB, PtsG-F, MaK, AlsE, A6 PP); wherein the content of the first and second substances,pfkA-F andpfkAthe-R primer sequences are respectively shown as SEQ ID NO.7 and SEQ ID NO.8,pfkB-F andpfkBthe-R primer sequences are respectively shown as SEQ ID NO.9 and SEQ ID NO. 10.

The specific process of the step (3) is as follows: actinobacillus succinogenes: (Actinobacillus succinogenes) Of originpckAThe gene is artificially synthesized (SEQ ID NO. 11) after being optimized according to the codon preference of escherichia coli, and the gene is used as a templateUsing a primerpckAF (SEQ ID NO. 12) andpckAr (SEQ ID NO. 13) for PCR amplification using Xho I and Avr II for insertion of the gene fragment pRSFDuet-alsE-a6PPObtaining the recombinant vector pRSFDuet-alsE-a6PP- pckATransfection ofE. coli (Δ FruA, Δ PfkA, Δ PfkB, PtsG-F, MaK, AlsE, A6PP) to yield recombinant E.coliE. coli (ΔFruA, ΔPfkA, ΔPfkB, PtsG-F, MaK, AlsE, A6PP, PckA)。

The specific process of the step (4) is as follows: fermentation culture using M9 basal mediumE. coli (Δ FruA, Δ PfkA, Δ PfkB, PtsG-F, MaK, AlsE, A6PP, PckA) strain to which 8 g/L of glycerol was added as a carbon source for cell growth, 2.2 g/L D-fructose was added as a substrate for synthesis of D-psicose, protein expression was induced using 0.1 mM IPTG, and the bottle mouth was blocked with a rubber plug during fermentation.

The application of the method for efficiently synthesizing the D-psicose by utilizing the recombinant escherichia coli fermentation in the production of the D-psicose is disclosed.

Compared with the prior art, the invention has the following beneficial effects: compared with the existing enzyme catalysis method, the method has the advantages of simpler operation and larger reaction scale, and effectively solves the problem of high cost caused by enzyme reutilization and immobilization. Compared with the existing fermentation method based on the reversible epimerization synthesis path, the substrate conversion rate of the D-psicose synthesis path is obviously improved, and an optimization scheme is provided for the industrial development of the D-psicose synthesis method by the fermentation method.

Drawings

FIG. 1 shows recombinant Escherichia coliE. coli (Δ FruA, Δ PfkA, Δ PfkB, PtsG-F, MaK, AlsE, A6PP, PckA) metabolism scheme for the synthesis of D-psicose.

FIG. 2 is the recombinant plasmid pETDuet-ptsG-F-maKAnd pRSFDuet-alsE-a6PPA map of (a). A: pETDuet-ptsG-F-maK；B：pRSFDuet-alsE-a6PP。

FIG. 3 is a drawing showingE. coli (Δ FruA, PtsG-F, MaK, AlsE, A6 PP). A: adding 4 g/L of LB culture mediumD-fructose fermentation results; b: the fermentation result was obtained by adding 4 g/L D-fructose to LB medium and adding 50 mM potassium phosphate buffer.

FIG. 4 shows fermentation results after knocking out PfkA and PfkB. A:E. coli (Δ FruA, Δ PfkA, PtsG-F, MaK, AlsE, A6PP) strain; b:E. coli (Δ FruA, Δ PfkA, Δ PfkB, PtsG-F, MaK, AlsE, A6 PP).

FIG. 5 shows the recombinant plasmid pRSFDuet-alsE-a6PP-pckAA map of (a).

FIG. 6 shows recombinant Escherichia coliE. coli (Δ FruA, Δ PfkA, Δ PfkB, PtsG-F, MaK, AlsE, A6PP, PckA).

FIG. 7 shows recombinant Escherichia coliE. coli (Δ FruA, Δ PfkA, Δ PfkB, PtsG-F, MaK, AlsE, A6PP, PckA) fermentation results in M9 medium supplemented with 8 g/L of glycerol and 2.2 g/L D-fructose.

Detailed Description

The present invention is further illustrated by the following examples, which are intended to be illustrative only and not limiting.

Recombinant Escherichia coli in the present inventionE. coli The metabolic scheme for the synthesis of D-psicose (Δ FruA, Δ PfkA, Δ PfkB, PtsG-F, MaK, AlsE, A6PP, PckA) is shown in FIG. 1.

Example 1

Host bacteria in the underpanE. coliKnockout of fructose-specific PTS transfer protein gene based on JM109(DE3)fruAUsing a primerfruAF (SEQ ID NO. 1) andfruA-R (SEQ ID NO. 2) was PCR-amplified using pKD13 plasmid as a template, and the amplification product was transfected into a plasmid containing pKD46E. coliJM109(DE3) was subjected to homologous recombination, single colony activation and competent cell preparation were picked, transfected with pep 20 plasmid, cultured overnight at 30 ℃ and 220 rpm in a shaker to eliminate the resistance marker, and finally the pep 20 plasmid was lost under 42 ℃ and 220 rpm culture conditions to obtain a recombinant plasmidE. coli(Δ FruA) strain. The fructose non-phosphorylated transporter geneptsG-FFructokinase genemaKInsert pRSFDuet-1 to obtain pETDuet-ptsG-F-maKRecombinant plasmid(plasmid map see FIG. 2A). The D-psicose-6-phosphate epimerase genealsEAnd psicose-6-phosphate phosphatase genea6PPpRSFDuet-1 was inserted to obtain pRSFDuet-alsE-a6PPRecombinant plasmid (plasmid map see FIG. 2B). Wherein the fructose non-phosphorylated transporter geneptsG-FFructokinase genemaKD-psicose-6-phosphate epimerase genealsEAnd psicose-6-phosphate phosphatase genea6PPThe gene sequences of the gene are respectively shown in SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6,ptsG-Fthe gene is Escherichia coliE. coliJM109(DE3)ptsGThe 34 th nucleotide of the gene is mutated from G to T,maKandalsEthe gene is Escherichia coliE. coliOverexpression of JM109(DE3) own gene,a6PPthe gene is derived from bacteroides fragilis (B.), (Bacteroides fragilis) (ii) a In the present invention, a fructose non-phosphorylated transporter geneptsG-FFructokinase genemaKD-psicose-6-phosphate epimerase genealsEAnd psicose-6-phosphate phosphatase genea6PPAre all artificially synthesized. Artificially synthesized pETDuet-ptsG-F-maKAnd pRSFDuet-alsE-a6PPTransfection of recombinant plasmids intoE. coli(. DELTA.FruA) to giveE. coli(Δ FruA, PtsG-F, MaK, AlsE, A6 PP).

Will be provided withE. coli(Δ FruA, PtsG-F, MaK, AlsE, A6PP) Strain was activated overnight, inoculated to 50 mL of LB liquid medium containing 4 g/L D-fructose at an inoculum size of 1%, ampicillin was added thereto at a final concentration of 100 mg/mL and kanamycin was added thereto at a final concentration of 50 mg/mL, and fermentation-cultured in a shaker at 37 ℃ and 220 rpm for 72 hours, protein expression was induced by addition of 0.1 mM IPTG at a final concentration after 3 hours of fermentation culture, and sampling was performed at 12-hour intervals during fermentation culture using a preparation equipped with Sugar-Pak ^TM Measuring the content of D-fructose and the content of D-psicose in fermentation liquor by using a high performance liquid chromatograph (Chromaster HPLC, HITACHI) of an I (6.5 × 300 mm, 85, Waters) chromatographic column and a differential refraction detector (RID), wherein the mobile phase is deionized water, the flow rate is 0.5 mL/min, the column temperature is 85 ℃, the detector temperature is 55 ℃, and the sample injection amount is 20 muL. The results are shown in FIG. 3A, where the cells were in the course of fermentationMedium growth, weak ability and final OD of fermentation broth ₆₀₀ Only about 0.5, probably because the pH of the fermentation liquor is reduced due to the dephosphorylation reaction, the cell growth is influenced, and the pH of the final fermentation liquor is measured by a pH meter, and the measured pH value is about 3.9; however, D-psicose was apparently produced in the fermentation broth at a yield of 0.351 g/L and a yield of 0.159 g/g.

Will be provided withE. coli(Δ FruA, PtsG-F, MaK, AlsE, A6PP) strains were activated overnight, transferred to 50 mL of LB broth containing 4 g/L D-fructose in 1% inoculum size, added to the medium with a final concentration of 50 mM potassium phosphate buffer (purchased from a source leaf organism, cat # R26329) to adjust the pH, added with a final concentration of 100 mg/mL ampicillin and a final concentration of 50 mg/mL kanamycin, fermentatively cultured in a shaker at 37 ℃ and 220 rpm for 72 hours, added with a final concentration of 0.1 mM IPTG after 3 hours of fermentation culture to induce protein expression, sampled every 12 hours during fermentation culture, and prepared with Sugar-Pak ^TM Measuring the content of D-fructose and the content of D-psicose in fermentation liquor by using a high performance liquid chromatograph (Chromaster HPLC, HITACHI) of an I (6.5 × 300 mm, 85, Waters) chromatographic column and a differential refraction detector (RID), wherein the mobile phase is deionized water, the flow rate is 0.5 mL/min, the column temperature is 85 ℃, the detector temperature is 55 ℃, and the sample injection amount is 20 muL. The results are shown in FIG. 3B, where cell growth is alleviated and the final OD of the fermentation broth ₆₀₀ About 2.4 was achieved, and the D-psicose production was increased to 0.510 g/L.

Example 2

The strains obtained in example 1E. coli(Δ FruA, PtsG-F, MaK, AlsE, A6PP) based on the knockout method in example 1 using the corresponding knockout primerpfkA-F andpfkA-R knockout of 6-phosphofructokinase GenepfkATo obtainE. coli(Δ FruA, Δ PfkA, PtsG-F, MaK, AlsE, A6 PP). In bacterial strainsE. coli(Δ FruA, Δ PfkA, PtsG-F, MaK, AlsE, A6PP) based on the knockout method in example 1 using the corresponding knockout primerspfkB-F andpfkB-R knockout of 6-phosphofructokinase GenepfkBTo regulate the carbon metabolism flux of D-fructose to obtainE. coli (ΔFruA, ΔPfkA, ΔPfkB, PtsG-F, MaK, AlsE, A6PP)And (3) strain.pfkA-F andpfkAthe-R primer sequences are shown as SEQ ID NO.7 and SEQ ID NO. 8.pfkB-F andpfkBthe-R primer sequences are shown as SEQ ID NO.9 and SEQ ID NO. 10.

The strain was fermentatively cultured as in example 1. The strain was activated overnight, transferred to 50 mL of LB liquid medium containing 4 g/L D-fructose at an inoculum size of 1%, to the medium was added 50 mM potassium phosphate buffer (purchased from a source leaf organism, cat # R26329) to adjust pH, to which was added ampicillin 100 mg/mL to a final concentration and kanamycin 50 mg/mL to a final concentration, and fermentation-cultured for 72 hours at 37 ℃ and 220 rpm in a shaker, and after 3 hours of fermentation-culture, protein expression was induced by adding IPTG 0.1 mM to a final concentration, and sampling was performed every 12 hours during the fermentation-culture using a device equipped with Sugar-Pak ^TM Measuring the content of D-fructose and the content of D-psicose in fermentation liquor by using a high performance liquid chromatograph (Chromaster HPLC, HITACHI) of an I (6.5 × 300 mm, 85, Waters) chromatographic column and a differential refraction detector (RID), wherein the mobile phase is deionized water, the flow rate is 0.5 mL/min, the column temperature is 85 ℃, the detector temperature is 55 ℃, and the sample injection amount is 20 muL. The results are shown in FIG. 4, FIG. 4AE. coli (Δ FruA, Δ PfkA, PtsG-F, MaK, AlsE, A6PP) produced 0.721 g/L D-psicose in a yield of 0.180 g/g after 72 h of strain; in FIG. 4BE. coli The yield and yield of the (delta FruA, delta PfkA, delta PfkB, PtsG-F, MaK, AlsE and A6PP) strain are obviously improved and are respectively 0.816 g/L and 0.614 g/g, which shows that the reasonable limitation of the carbon metabolic flux of D-fructose is beneficial to the efficient conversion of synthetic reaction.

Example 3

Actinobacillus succinogenes: (Actinobacillus succinogenes) Phosphoenolpyruvate carboxykinase gene frompckAIs synthesized artificially after being optimized according to the codon preference of the escherichia coli, and the synthesized template sequence is SEQ ID NO. 11. Use of primerspckAF (SEQ ID NO. 12) andpckA-R (SEQ ID NO. 13) is artificially synthesizedpckAAfter PCR amplification of the template, the gene fragment was inserted into pRSFDuet-alsE-a6PPThen, pRSFDuet-alsE-a6PP-pckARecombinant plasmid (plasmid map is shown in FIG. 5), then transfected intoE. coli (Δ FruA, Δ PfkA, Δ PfkB, PtsG-F, MaK, AlsE, A6PP) to obtain recombinant Escherichia coliE. coli (ΔFruA, ΔPfkA, ΔPfkB, PtsG-F, MaK, AlsE, A6PP, PckA)。

The strain was fermentatively cultured as in example 1. The strain was activated overnight, transferred to 50 mL of LB liquid medium containing 4 g/L D-fructose at an inoculum size of 1%, to the medium was added 50 mM potassium phosphate buffer (purchased from a source leaf organism, cat # R26329) to adjust pH at a final concentration, to which was added ampicillin at a final concentration of 100 mg/mL and kanamycin at a final concentration of 50 mg/mL, and subjected to fermentation culture at 37 ℃ for 72 hours in a shaker at 220 rpm, and after 3 hours of fermentation culture, protein expression was induced by the addition of IPTG at a final concentration of 0.1 mM, and sampling was performed every 12 hours during the fermentation culture. During the fermentation culture, the bottle mouth is sealed by using a rubber plug, and an inducer is added or a sample is taken by an injector. Using a belt-Pak ^TM Measuring the content of D-fructose and the content of D-psicose in fermentation liquor by using a high performance liquid chromatograph (Chromaster HPLC, HITACHI) of an I (6.5 × 300 mm, 85, Waters) chromatographic column and a differential refraction detector (RID), wherein the mobile phase is deionized water, the flow rate is 0.5 mL/min, the column temperature is 85 ℃, the detector temperature is 55 ℃, and the sample injection amount is 20 muL. Fermentation experiments were carried out using the fermentation media referred to in example 1, the results are shown in FIG. 6, strainE. coli The yield of D-psicose in the fermentation broth (delta FruA, delta PfkA, delta PfkB, PtsG-F, MaK, AlsE, A6PP, PckA) was further increased to about 1.23 g/L, and the yield was about 0.683 g/g.

Example 4

In this example, the oxygen-limited fermentation experiments were performed on M9 minimal medium. Bacterial strainsE. coli (Δ FruA,. DELTA.PfkA,. DELTA.PfkB, PtsG-F, MaK, AlsE, A6PP, PckA) was activated overnight, transferred to M9 basal medium at an inoculum size of 1%, to which 8 g/L of glycerol and 2.2 g/L D-fructose were added together with ampicillin at a final concentration of 100 mg/mL and kanamycin at a final concentration of 50 mg/mL, and subjected to fermentation culture at 37 ℃ for 96 hours at 220 rpm in a shaker, and after 3 hours of fermentation culture, protein expression was induced by the addition of IPTG at a final concentration of 0.1 mM, and sampling was carried out at 20-hour intervals during the fermentation culture. During the fermentation culture, the bottle mouth of the culture bottle is sealed by using a rubber plug, and an inducer is added or a sample is taken by an injector. The results are shown in FIG. 7, 2.2 g/LD-fructose was completely converted into D-psicose (1.590 g/L) after 96 h, with a yield of about 0.722 g/g.

SEQUENCE LISTING

<110> Fuzhou university, Qingyuan innovation laboratory

<120> method for efficiently synthesizing D-psicose by utilizing recombinant escherichia coli fermentation

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gaaatccgcc gtcatgacat gaaagtgggg ctgatcctta acccggagac gccagttgag 360

gccatgaaat actatatcca taaggccgat aaaattacgg tcatgactgt cgatcccggc 420

tttgccggac aaccgttcat tcctgaaatg ctggataaac ttgccgaact gaaggcatgg 480

cgtgaacgag aaggtctgga gtacgaaatt gaggtggacg gttcctgcaa ccaggcaact 540

tacgaaaaac tgatggcggc aggggcggat gtctttatcg tcggcacttc cggcctgttt 600

aatcatgcgg aaaatatcga cgaagcatgg agaattatga ccgcgcagat tctggctgca 660

aaaagcgagg tacagcctca tgcaaaaaca gcataa 696

<210> 6

<211> 669

<212> DNA

<213> Artificial sequence

<400> 6

atgaaataca ctgtttattt attcgacttt gactatacac tggcagactc ttcgcgtggc 60

atcgtaacct gcttcagaag cgtactcgag agacatggct acaccggtat cactgatgat 120

atgatcaaac gcaccattgg gaaaacgctt gaagaatcgt tcagcatcct tacgggaatt 180

actgacgccg accaactgga atcattcaga caagagtatt ccaaagaggc agatatatac 240

atgaatgcaa ataccattct tttcccggat actctcccca cgcttacaca cctgaaaaaa 300

cagggaatcc gtatcggaat catttcaacc aaataccgat tccggatact gagtttcctg 360

agaaatcaca tgccggatga ttggtttgac atcattatcg gaggtgaaga tgtgacacat 420

cataaacccg atcctgaagg tttgctactt gccatcgacc gattgaaggc atgccccgaa 480

gaagtattat acatcggtga cagtacagtg gatgcaggaa cagccgccgc tgccggagtt 540

tcttttaccg gtgttaccag cggtatgact actgcccaag agtttcaggc ctatccctac 600

gacagaatca tcagtactct tggccagcta atctccgttc cggaagacaa atccggctgt 660

ccgctctga 669

<210> 7

<211> 71

<212> DNA

<213> Artificial sequence

<400> 7

atgattaaga aaatcggtgt gttgacaagc ggcggtgatg cgccaggcat gtgtaggctg 60

gagctgcttc g 71

<210> 8

<211> 70

<212> DNA

<213> Artificial sequence

<400> 8

ttaatacagt tttttcgcgc agtccagcca gtcacctttg aacggacgct attccgggga 60

tccgtcgacc 70

<210> 9

<211> 71

<212> DNA

<213> Artificial sequence

<400> 9

atggtacgta tctatacgtt gacacttgcg ccctctctcg atagcgcaac gtgtaggctg 60

gagctgcttc g 71

<210> 10

<211> 70

<212> DNA

<213> Artificial sequence

<400> 10

ttagcgggaa aggtaagcgt aaattttttg cgtatcgtca tgggagcaca attccgggga 60

tccgtcgacc 70

<210> 11

<211> 696

<212> DNA

<213> Artificial sequence

<400> 11

atgaaaatct ccccctcgtt aatgtgtatg gatctgctga aatttaaaga acagatcgaa 60

tttatcgaca gccatgccga ttacttccac atcgatatca tggacggtca ctttgtcccc 120

aatctgacac tctcaccgtt cttcgtaagt caggttaaaa aactggcaac taaaccgctc 180

gactgtcatc tgatggtgac gcggccgcag gattacattg ctcaactggc gcgtgcggga 240

gcagatttca tcactctgca tccggaaacc atcaacggcc aggcgttccg cctgattgat 300

gaaatccgcc gtcatgacat gaaagtgggg ctgatcctta acccggagac gccagttgag 360

gccatgaaat actatatcca taaggccgat aaaattacgg tcatgactgt cgatcccggc 420

tttgccggac aaccgttcat tcctgaaatg ctggataaac ttgccgaact gaaggcatgg 480

cgtgaacgag aaggtctgga gtacgaaatt gaggtggacg gttcctgcaa ccaggcaact 540

tacgaaaaac tgatggcggc aggggcggat gtctttatcg tcggcacttc cggcctgttt 600

aatcatgcgg aaaatatcga cgaagcatgg agaattatga ccgcgcagat tctggctgca 660

aaaagcgagg tacagcctca tgcaaaaaca gcataa 696

<210> 12

<211> 45

<212> DNA

<213> Artificial sequence

<400> 12

ccgctcgaga aggagatgaa aatctccccc tcgttaatgt gtatg 45

<210> 13

<211> 31

<212> DNA

<213> Artificial sequence

<400> 13

ccccatggtt agtggttacg gatgtactca t 31

Claims

1. High-efficiency fermentation agent by utilizing recombinant escherichia coliA process for the formation of D-psicose, characterized in that: to be provided withE.coliJM109(DE3) is a chassis host bacterium, and fructose specific PTS transfer protein gene is knocked outfruAAnd overexpresses the fructose non-phosphorylated transporter geneptsG-FFructokinase genemaKD-psicose-6-phosphate epimerase genealsEAnd psicose-6-phosphate phosphatase genea6PPFour genes establishing a biosynthetic pathway from D-fructose to D-psicose; further knocking out D-fructose-6-phosphokinase genepfkAAndpfkBthe reasonable regulation and control of the carbon flux of the D-fructose is realized; overexpression of phosphoenolpyruvate carboxykinase GenepckATo increase intracellular ATP concentration; and finally, supplying the D-fructose serving as a substrate raw material and glycerol serving as a carbon source for oxygen-limited fermentation, thereby ensuring the efficient conversion of the D-psicose.

2. The method for efficiently synthesizing D-psicose by fermentation of recombinant Escherichia coli according to claim 1, comprising the following steps:

(1) to be provided withE. coliJM109(DE3) is a chassis host bacterium, and homologous recombination technology is used for knocking out fructose specific PTS transfer protein genefruAAnd overexpressing a fructose non-phosphorylated transporter geneptsG-FFructokinase genemaKD-psicose-6-phosphate epimerase genealsEAnd psicose-6-phosphate phosphatase genea6PPFour genes to obtainE. coli(Δ FruA, PtsG-F, MaK, AlsE, A6 PP);

(2) in bacterial strainsE. coli(delta FruA, PtsG-F, MaK, AlsE, A6PP) and knocking out D-fructose-6-phosphokinase gene by using homologous recombination technologypfkAAndpfkBto block the conversion of D-fructose-6-phosphate to D-fructose-1, 6-diphosphate to obtainE. coli (Δ FruA, Δ PfkA, Δ PfkB, PtsG-F, MaK, AlsE, A6 PP);

(3) in bacterial strainsE. coli (delta FruA, delta PfkA, delta PfkB, PtsG-F, MaK, AlsE, A6PP) based on the expression of phosphoenolpyruvate carboxykinase genespckASo as to realize the intracellular cyclic regeneration of ATP and obtain the recombinant escherichia coliE. coli (ΔFruA, ΔPfkA, ΔPfkB, PtsG-F, MaK, AlsE, A6PP, PckA)；

(4) The strain is inoculated by using D-fructose as a substrate raw material, glycerol as a carbon source and an M9 basic culture mediumE. coli (Δ FruA, Δ PfkA, Δ PfkB, PtsG-F, MaK, AlsE, A6PP, PckA) to enhance oxygen-limited fermentationE. coli (Δ FruA,. DELTA.PfkA,. DELTA.PfkB, PtsG-F, MaK, AlsE, A6PP, PckA) and increased biomass in the fermentation broth.

3. The method for efficiently synthesizing D-psicose by fermentation using recombinant Escherichia coli according to claim 2, wherein: the specific process of the step (1) is as follows: using pKD13 plasmid as a template and primersfruA-F andfruAPCR amplification of-R, transfection of the amplification product into a plasmid containing pKD46 by electrochemical transformationE. coliJM109(DE3) was recombined and the resistance marker was knocked out using the pcp20 plasmid to obtainE. coli(Δ FruA) strain, wherein,fruA-F andfruA-R primer sequences are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2; artificial synthesis of pETDuet-ptsG-F-maKAnd pRSFDuet-alsE-a6PPRecombinant expression vector, transfected intoE. coli(. DELTA.FruA) to giveE. coli(Δ FruA, PtsG-F, MaK, AlsE, A6PP) strain, wherein,ptsG-F、maK、alsEanda6PPthe gene sequences of (A) are respectively shown in SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO. 6.

4. The method for efficiently synthesizing D-psicose by fermentation using recombinant Escherichia coli according to claim 2, wherein: the specific process of the step (2) is as follows: use of primerspfkA-F/pfkA-R andpfkB-F/pfkB-R is knocked out according to the method in step (1)pfkAAndpfkBgene, obtainingE. coli (Δ FruA,. DELTA.PfkA,. DELTA.PfkB, PtsG-F, MaK, AlsE, A6PP) strain, wherein,pfkA-F andpfkAthe-R primer sequences are respectively shown as SEQ ID number 7 and SEQ ID number 8,pfkB-F andpfkBthe-R primer sequences are respectively shown as SEQ ID NO.9 and SEQ ID NO.10。

5. The method for efficiently synthesizing D-psicose by fermentation using recombinant Escherichia coli according to claim 2, wherein: the specific process of the step (3) is as follows: actinobacillus succinogenes: (Actinobacillus succinogenes) Of originpckAThe gene is artificially synthesized after being optimized according to codon preference of escherichia coli, and primers are usedpckA-F andpckA-R is amplified by means of two enzymatic cleavage sites Xho I and Avr IIpckAGene insertion pRSFDuet-alsE-a6PPRecombinant expression vector to obtain the recombinant vector pRSFDuet-alsE-a6PP-pckAAnd then transfected intoE. coli (Δ FruA, Δ PfkA, Δ PfkB, PtsG-F, MaK, AlsE, A6PP) to obtain recombinant Escherichia coliE. coli (Δ FruA, Δ PfkA, Δ PfkB, PtsG-F, MaK, AlsE, A6PP, PckA), whereinpckAThe gene sequence is shown in SEQ ID NO.11,pckA-F andpckAthe-R primer sequences are respectively shown as SEQ ID NO.12 and SEQ ID NO. 13.

6. The method for efficiently synthesizing D-psicose by fermentation using recombinant Escherichia coli according to claim 2, wherein: the specific process of the step (4) is as follows: fermentation culture using M9 basal mediumE. coli (Δ FruA,. DELTA.PfkA,. DELTA.PfkB, PtsG-F, MaK, AlsE, A6PP, PckA) strain to which 8 g/L of glycerol was added as a carbon source for cell growth, 2.2 g/L D-fructose was added as a substrate for synthesis of D-psicose, protein expression was induced using 0.1 mM IPTG, and the mouth of the bottle was closed with a rubber plug during fermentation.

7. Use of the method for efficiently synthesizing D-psicose by fermentation of recombinant Escherichia coli according to claim 1 for producing D-psicose.