CN114426994A - D-psicose whole-cell synthesis method taking glycerol as substrate - Google Patents

Abstract

The invention provides a whole-cell synthesis method of D-psicose, which converts glycerol into D-psicose in one pot and relates to the field of biochemical engineering. In order to avoid thermodynamic equilibrium in the isomerase pathway and to reduce the production cost of D-psicose by using expensive dihydroxyacetone phosphate and D-glyceraldehyde, the present invention constructs a complete conversion pathway from glycerol to D-psicose. The recombinant strain is used as a whole-cell catalyst, so that the glycerol is converted into the D-psicose in one pot. Compared with other similar methods, the method has the advantages that the reaction system has no intermediate product fructose (isomer of D-psicose) residue, and the product is single (only D-psicose is produced without by-product D-sorbose), so that the separation and purification are convenient; compared with an enzyme catalysis reaction system, the method omits a complicated enzyme purification process, has a simple production process and lower cost, and is suitable for large-scale production.

Description

D-psicose whole-cell synthesis method taking glycerol as substrate

Technical Field

The invention belongs to the field of biochemical engineering, and particularly relates to a D-psicose whole-cell synthesis method using glycerol as a substrate.

Background

D-Psicose (D-Psicose or D-Allulose) is a rare functional sugar, a small molecular weight monosaccharide naturally present in various types of food products, first found in sugarcane and beet molasses, murinus and wheat. The sugar degree of the D-psicose is 70% of that of the sucrose, and the calorie is only 0.3% of that of the sucrose, so that the D-psicose is called non-caloric sugar and is an ideal sugar substitute with ultralow calorie. D-psicose is Generally Recognized As Safe (GRAS) as approved by the U.S. food and drug administration. It has tremendous physiological benefits including blood glucose inhibitory properties, neuroprotective effects, active oxygen scavenging activity, anti-obesity effects, and the like. The D-psicose can also inhibit fat accumulation in abdominal cavity, and has strong active oxygen (ROS) scavenging ability and glutathione reducing ability. In addition, the D-psicose also has a neuroprotective effect, and has good protection and treatment effects on diseases such as neurodegeneration and atherosclerosis. Based on the functions, the D-psicose has important application value in the aspects of food processing, medical care and the like.

The chemical synthesis method of D-psicose is complicated in process and has certain limitations, and the chemical production process is relatively serious in pollution, so that breakthrough progress is not made. At present, two routes are mainly used for synthesizing D-psicose by a biotransformation method: (1) the technical route based on the ketose 3-epimerase, i.e. the enzyme cascade consisting of glucose isomerase and ketose 3-epimerase, converts glucose into fructose and D-psicose in succession. However, this pathway is a reversible reaction, and there is a thermodynamic equilibrium such that the substrate (glucose), the intermediate (fructose) and the product (D-psicose) coexist in the reaction system, and after the thermodynamic equilibrium is reached, the reaction is in a dynamic equilibrium. Since glucose, fructose and D-psicose are isomers of each other, this phenomenon makes separation and purification of the product very difficult. (2) Based on the aldolase route, it is now common to catalyze the condensation of dihydroxyacetone phosphate (DHAP) and D-glycerol aldehyde to D-psicose and D-sorbose using L-rhamnose-1-phosphate aldolase and fructose-1-phosphorylase. However, the product selectivity of the process is poor, and the product contains both D-psicose and D-sorbose. The two are isomers, and the existence of the byproduct D-sorbose brings great troubles to the separation and purification of the product. In addition, dihydroxyacetone phosphate and D-glyceraldehyde are expensive and unstable, so that large-scale in vitro synthesis is difficult. The invention constructs a whole-cell catalyst, can directly synthesize cheap glycerol into D-psicose, only contains D-psicose in the product, and has no byproduct D-sorbose. The invention has larger application potential in the fields of food and medicine.

Disclosure of Invention

In view of the above, the present invention aims to provide a method for synthesizing D-psicose using glycerol in whole cells, thereby realizing direct synthesis of D-psicose using glycerol as an inexpensive substrate without production of D-sorbose as a byproduct.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a method for synthesizing D-psicose by using glycerol whole cells, wherein the D-psicose synthesis method takes escherichia coli as a basal cell to construct a whole-cell catalyst to convert the glycerol into the D-psicose.

Optionally, the escherichia coli is escherichia coli BL21(DE 3).

Optionally, the whole-cell catalyst construction method comprises the following steps: the glycerol kinase-encoding gene (glpK, SEQ ID NO:6), the glycerol-3-phosphate dehydrogenase-encoding gene (glpD, SEQ ID NO:7), the L-fucoidan-1-phosphate aldolase-encoding gene (FucA, SEQ ID NO:8) and the fructose-1-phosphorylase-encoding gene (YqaB, SEQ ID NO:9) derived from Escherichia coli MG1655 and the sugar alcohol oxidase-encoding gene (AldO, SEQ ID NO:10) derived from Streptomyces coelicolor M145 were cloned between the BamHI and HindIII sites of the vector pET28a (PB) using a one-step cloning kit (Nanjing Nodezae Biotech Co., Ltd.) (primers, see Table 1), respectively. The above pathway genes were assembled as monocistrons by multiple rounds of ePathBricks assembly (specific method references: Xu, P., et al.,2012, ACS Synthetic Biology,1(7):256-266), to obtain recombinant plasmid pET28a (PB) N-KDABO (plasmid map is shown in FIG. 2). Wherein the expression of all pathway genes is controlled by a T7 promoter and a T7 terminator. The recombinant plasmid is transformed into escherichia coli BL21(DE3), and the recombinant escherichia coli BL21/KDABO is obtained. The recombinant strain can be used for preparing a whole-cell catalyst and synthesizing glycerol into D-psicose.

Alternatively, the conditions for converting glycerol into D-psicose using the recombinant strain BL21/KDABO described above were: culturing the recombinant E.coli to OD₆₀₀0.6-0.8, IPTG was added to a final concentration of 0.05-0.5mM, and induction was carried out at 15-30 ℃ and 200rpm for 4-24 h. Centrifuging at 4 deg.C and 5000rpm to collect thallus, washing thallus with sterilized ultrapure water to remove culture medium, and resuspending thallus with 50mM phosphate buffer solution with pH of 6-8 to obtain resting cell which can be used as whole cell catalyst for subsequent reaction.

Optionally, the concentration of the substrate glycerol is 10-60 g/L.

Optionally, the conversion condition is 20-35 ℃ for 48 h.

Compared with the prior art, the invention has the following beneficial effects:

(1) the invention uses cheap raw material glycerol as a substrate instead of expensive dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde, greatly reduces the production cost of D-psicose, and is suitable for large-scale production.

(2) The product only contains D-psicose, and does not contain D-sorbose as a byproduct, thereby being beneficial to separation and purification of the product.

(3) Compared with a ketose 3-epimerase approach, the method avoids the problems of thermodynamic equilibrium and the like, is expected to obtain higher conversion rate, has single product, and is convenient for subsequent separation and purification.

(4) Compared with enzyme catalysis, the whole-cell transformation avoids a series of problems such as complicated enzyme purification steps, cofactors and the like, has mild reaction conditions, and is more suitable for industrial production.

Drawings

FIG. 1 is a schematic diagram of the synthetic pathway for the conversion of glycerol to D-psicose. This pathway consists of glycerol kinase (glpK), glycerol-3-phosphate dehydrogenase (glpD), sugar alcohol oxidase (AldO), L-fucoidan-1-phosphate aldolase (FucA), and fructose-1-phosphorylase (YqaB).

FIG. 2 is a map of recombinant plasmid pET28a (PB) N-KDABO. The recombinant plasmid contains a glycerol kinase coding gene (glpK), a glycerol-3-phosphate dehydrogenase coding gene (glpD), an L-fucoidan-1-phosphate aldolase coding gene (FucA), a fructose-1-phosphorylase coding gene (YqaB) and a sugar alcohol oxidase coding gene (AldO), and the expressions of the genes are controlled by a T7 promoter and a T7 terminator and are connected in series in the form of monocistrons to form a conversion pathway from glycerol to D-psicose.

FIG. 3. ion chromatography analysis of whole cell transformed products. Glycerol is used as a substrate, and after the recombinant strain BL21/KDABO whole cell transformation, D-psicose is synthesized, and a by-product D-sorbose is not synthesized. Wherein, a recombinant strain BL21/KDABO containing a synthetic pathway from glycerol to D-psicose is an experimental group, and a strain BL21/pET28a (PB) N containing an empty plasmid pET28a (PB) N is a blank control.

FIG. 4 Whole cell catalyst preparation and transformation conditions optimization. (A) Concentration optimization of inducer IPTG, (B) induction temperature optimization, (C) induction time optimization, (D) reaction temperature optimization of conversion of glycerol to D-psicose, (E) pH optimization of conversion of glycerol to D-psicose, (F) initial concentration optimization of substrate glycerol.

FIG. 5 shows the D-psicose production with time under the optimum conditions. The optimal conditions are as follows: the concentration of an inducer IPTG is 0.05mM, the induction temperature is 20 ℃, the induction time is 12h, the reaction temperature is 25 ℃, the reaction pH is 7.5, and the initial concentration of a substrate is 40 g/L.

FIG. 6 shows the experimental production verification of D-psicose synthesis method at the fermenter level.

Detailed description of the preferred embodiment

The invention provides a D-psicose whole-cell synthesis method using glycerol as a substrate, and the D-psicose synthesis method can directly convert the glycerol into the D-psicose by constructing a recombinant escherichia coli engineering strain.

The method for synthesizing D-psicose using glycerol whole cells and the use thereof according to the present invention will be described in detail with reference to the following examples, which should not be construed as limiting the scope of the present invention.

Detailed Description

Example 1: recombinant escherichia coli whole cell method for converting glycerol into D-psicose

The glycerol kinase-encoding gene (glpK, SEQ ID NO:6), the glycerol-3-phosphate dehydrogenase-encoding gene (glpD, SEQ ID NO:7), the L-fucoidan-1-phosphate aldolase-encoding gene (FucA, SEQ ID NO:8) and the fructose-1-phosphorylase-encoding gene (YqaB, SEQ ID NO:9) derived from Escherichia coli MG1655 and the sugar alcohol oxidase-encoding gene (AldO, SEQ ID NO:10) derived from Streptomyces coelicolor M145 were cloned between the BamHI and HindIII sites of the vector pET28a (PB) using a one-step cloning kit (Nanjing Nodezaki Biotech Co., Ltd.) (the primers used are shown in Table 1), respectively. The recipient plasmid was digested with NheI/SalI, the donor plasmid was digested with AvrII/SalI, and the genes of the above-mentioned pathways were assembled as monocistrons by multiple rounds of ePathBricks (see: Xu, P., et al, 2012, ACS Synthetic Biology,1(7):256-266) to obtain recombinant plasmid pET28a (PB) N-KDABO (plasmid map is shown in FIG. 2). Wherein the expression of all pathway genes is controlled by a T7 promoter and a T7 terminator. The recombinant plasmid is transformed into escherichia coli BL21(DE3), and the recombinant escherichia coli BL21/KDABO is obtained. The recombinant strain can be used for preparing a whole-cell catalyst and synthesizing glycerol into D-psicose.

TABLE 1 primers used in the present invention

Recombinant E.coli BL21/KDABO was cultured overnight in LB medium at 37 ℃ and 200rpm, transferred to a 250mL Erlenmeyer flask containing 25mL fresh LB medium at an inoculum size of 2% (v/v), and cultured at 37 ℃ and 200rpm to log phase. Then, IPTG was added to the cells at a final concentration of 500. mu.M, and the cells were further cultured at 25 ℃ and 200rpm for 6 hours to express the gene in the D-psicose-synthesizing pathway of glycerol. Centrifuging at 4 deg.C and 5000rpm for 5min, collecting thallus, washing thallus with sterilized ultrapure water to remove culture medium, and then resuspending thallus with 50mM Phosphate Buffer Solution (PBS) with pH of 7.0 to obtain resting cell which can be used as whole cell catalyst for converting glycerol into D-psicose. Coli BL21/pET28a (PB) N with empty plasmid pET28a (PB) N was used as a blank control, and the other operating conditions were the same.

Glycerol was added to the above reaction system to a final concentration of 40g/L, and the reaction was carried out at 30 ℃ for 48 hours. The reaction supernatant was detected by ion chromatography.

The method for ion chromatography analysis of D-psicose and D-sorbose is as follows: the ion chromatography model is a Saimeifei ICS-6000 high-pressure ion chromatograph, the detector is an electrochemical detector, the chromatographic column model is PA-20, the mobile phase A is ultrapure water, the mobile phase B is 200mM NaOH aqueous solution, and gradient elution is carried out to obtain (B%): 0min 10%, 15min 10%, 15.1min 100%, 25min 100%, 25.1min 10%, 35min 10%, constant flow rate 0.5mL/min, sample size 25 μ L, and column oven and detector temperature both 30 ℃.

The ion chromatographic analysis result shows that the peak-off time of D-sorbose and D-psicose is 8.267min and 9.475min respectively.

The blank (E.coli BL21/pET28a (PB) N) synthesized neither D-psicose nor D-sorbose; the experimental group (recombinant E.coli BL21/KDABO) synthesized only D-psicose, but not D-sorbose (FIG. 3). The results indicate that the constructed whole-cell catalyst can convert glycerol into D-psicose without D-sorbose as a byproduct.

Example 2: whole-cell catalyst preparation and optimization of D-psicose synthesis conditions

In order to improve the yield and the conversion rate of the D-psicose, the invention further optimizes the conditions for preparing the whole-cell catalyst and the reaction conditions for synthesizing the D-psicose by converting glycerol.

The invention firstly optimizes the preparation process of the whole-cell catalyst: the concentration range of the inducer IPTG concentration is 0.001-0.5mM, the temperature range of the pathway gene induction expression is 15-30 ℃, and the time range of the pathway gene induction expression is 4-20 h. The results showed that the optimal concentration of the inducer IPTG was 0.05mM (FIG. 4A), the optimal induction temperature was 20 deg.C (FIG. 4B) and the optimal induction time was 12h (FIG. 4C). The invention optimizes the reaction condition of the conversion of the glycerol to the D-psicose: the temperature range of the reaction is 20-35 ℃, the pH range of the reaction is 6.0-8.0, and the initial concentration range of the substrate glycerol is 10-60 g/L. As a result, the optimum reaction temperature was 25 ℃ C (FIG. 4D), the optimum reaction pH was 7.5 (FIG. 4E), and the optimum initial substrate concentration was 40g/L (FIG. 4F).

Under the above-described optimum conditions, the present invention analyzed the change in the production amount of D-psicose with time. Under the condition, the yield of D-psicose reaches the maximum at 48h, namely 3.55 g/L.

Example 3: preparation of D-psicose by using fermentation tank to amplify reaction process

In order to verify the feasibility of the D-psicose synthesis method in the fermenter level pilot production, the reaction system was scaled up using a 5L fermenter according to the invention based on example 2. Activating recombinant Escherichia coli BL21/KDABO overnight, transferring into a fermentation tank containing 3L LB medium with an inoculum size of 2% (v/v), and culturing at 37 deg.C and 200rpm with ventilation of 4NI/min to OD₆₀₀0.6-0.8. IPTG was added to a final concentration of 0.05mM,the induction was carried out at 20 ℃ and 200rpm for 12 hours at an aeration rate of 4NI/min to express the pathway gene. The cells were collected by centrifugation at 4 ℃ and 5000rpm for 15min, the cells were washed with sterilized ultrapure water to remove the medium, the cells were resuspended in a 50mM phosphate buffer solution having a pH of 7.5, and the medium was replaced with the phosphate buffer solution at a ratio of 1:1 to prepare a whole cell catalyst (resting cells). Glycerol was added to the reaction mixture to a final concentration of 50g/L, the reaction mixture was reacted at 25 ℃ and 200rpm with an aeration rate of 4NI/min, samples were taken at 12-hour intervals, and the D-psicose concentration was measured by ion chromatography. The results showed that the D-psicose yield reached 3.14g/L at 48h (FIG. 4). The result shows that the method constructed by the invention can be used for small-scale production on a fermentation tank and has larger application potential.

The foregoing is only an alternative embodiment of the present invention, and it should be noted that modifications and embellishments could be made by those skilled in the art without departing from the principle of the present invention, and these should be considered as the protection scope of the present invention.

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tcttccgaag tatacggtca gactaacatt ggcggcaaag gcggcacgcg tattccaatc 720

tccgggatcg ccggtgacca gcaggccgcg ctgtttggtc agttgtgcgt gaaagaaggg 780

atggcgaaga acacctatgg cactggctgc tttatgctga tgaacactgg cgagaaagcg 840

gtgaaatcag aaaacggcct gctgaccacc atcgcctgcg gcccgactgg cgaagtgaac 900

tatgcgttgg aaggtgcggt gtttatggca ggcgcatcca ttcagtggct gcgcgatgaa 960

atgaagttga ttaacgacgc ctacgattcc gaatatttcg ccaccaaagt gcaaaacacc 1020

aatggtgtgt atgtggttcc ggcatttacc gggctgggtg cgccgtactg ggacccgtat 1080

gcgcgcgggg cgattttcgg tctgactcgt ggggtgaacg ctaaccacat tatacgcgcg 1140

acgctggagt ctattgctta tcagacgcgt gacgtgctgg aagcgatgca ggccgactct 1200

ggtatccgtc tgcacgccct gcgcgtggat ggtggcgcag tagcaaacaa tttcctgatg 1260

cagttccagt ccgatattct cggcacccgc gttgagcgcc cggaagtgcg cgaagtcacc 1320

gcattgggtg cggcctatct cgcaggcctg gcggttggct tctggcagaa cctcgacgag 1380

ctgcaagaga aagcggtgat tgagcgcgag ttccgtccag gcatcgaaac cactgagcgt 1440

aattaccgtt acgcaggctg gaaaaaagcg gttaaacgcg cgatggcgtg ggaagaacac 1500

gacgaataa 1509

<210> 7

<211> 1506

<212> DNA

<213> Escherichia coli (Escherichia coli)

<400> 7

atggaaacca aagatctgat tgtgataggg ggcggcatca atggtgctgg tatcgcggca 60

gacgccgctg gacgcggttt atccgtgctg atgctggagg cgcaggatct cgcttgcgcg 120

acctcttccg ccagttcaaa actcattcac ggtggcctgc gctaccttga gcactatgaa 180

ttccgcctgg tcagcgaggc gctggctgaa cgtgaagtgc tgctgaaaat ggccccgcat 240

atcgccttcc cgatgcgttt tcgcctgcca catcgtccgc atctgcgccc ggcgtggatg 300

attcgcattg gtctgtttat gtacgatcat ctgggtaaac gcaccagctt gccgggatca 360

actggtttgc gttttggcgc aaattcagtg ttaaaaccgg aaattaagcg cggattcgaa 420

tattctgact gttgggtaga cgacgcccgt ctggtactcg ccaacgccca gatggtggtg 480

cgtaaaggcg gcgaagtgct tactcggact cgcgccacct ctgctcgccg cgaaaacggc 540

ctgtggattg tggaagcgga agatatcgat accggcaaaa aatatagctg gcaagcgcgc 600

ggcttggtta acgccaccgg cccgtgggtg aaacagttct tcgacgacgg gatgcatctg 660

ccttcgcctt atggcattcg cctgatcaaa ggcagccata ttgtggtgcc gcgcgtgcat 720

acccagaagc aagcctacat tctgcaaaac gaagataaac gtattgtgtt cgtgatcccg 780

tggatggacg agttttccat catcggcact accgatgtcg agtacaaagg cgatccgaaa 840

gcggtgaaga ttgaagagag tgaaatcaat tacctgctga atgtgtataa cacgcacttt 900

aaaaagcagt taagccgtga cgatatcgtc tggacctact ccggtgtgcg tccgctgtgt 960

gatgatgagt ccgactcgcc gcaggctatt acccgtgatt acacccttga tattcatgat 1020

gaaaatggca aagcaccgct gctgtcggta ttcggcggta agctgaccac ctaccgaaaa 1080

ctggcggaac atgcgctgga aaaactaacg ccgtattatc agggtattgg cccggcatgg 1140

acgaaagaga gtgtgctacc gggtggcgcc attgaaggcg accgcgacga ttatgccgct 1200

cgcctgcgcc gccgctatcc gttcctgact gaatcgctgg cgcgtcatta cgctcgcact 1260

tacggcagca acagcgagct gctgctcggc aatgcgggaa cggtaagcga tctcggggaa 1320

gatttcggtc atgagttcta cgaagcggag ctgaaatacc tggtggatca cgaatgggtc 1380

cgccgcgccg acgacgccct gtggcgtcgc acaaaacaag gcatgtggct aaatgcggat 1440

caacaatctc gtgtgagtca gtggctggtg gagtatacgc agcagaggtt atcgctggcg 1500

tcgtaa 1506

<210> 8

<211> 648

<212> DNA

<213> Escherichia coli (Escherichia coli)

<400> 8

atggaacgaa ataaacttgc tcgtcagatt attgacactt gcctggaaat gacccgcctg 60

ggactgaacc aggggacagc ggggaacgtc agtgtacgtt atcaggatgg gatgctgatt 120

acgcctacag gcattccata tgaaaaactg acggagtcgc atattgtctt tattgatggc 180

aacggtaaac atgaggaagg aaagctcccc tcaagcgaat ggcgtttcca tatggcagcc 240

tatcaaagca gaccggatgc caacgcggtt gttcacaatc atgccgttca ttgcacggca 300

gtttccattc ttaaccgatc gatccccgct attcactaca tgattgcggc ggctggcggt 360

aattctattc cttgcgcgcc ttatgcgacc tttggaacac gcgaactttc tgaacatgtt 420

gcgctggctc tcaaaaatcg taaggcaact ttgttacaac atcatgggct tatcgcttgt 480

gaggtgaatc tggaaaaagc gttatggctg gcgcatgaag ttgaagtgct ggcgcaactt 540

tacctgacga ccctggcgat tacggacccg gtgccagtgc tgagcgatga agagattgcc 600

gtagtgctgg agaaattcaa aacctatggg ttacgaattg aagagtaa 648

<210> 9

<211> 567

<212> DNA

<213> Escherichia coli (Escherichia coli)

<400> 9

atgtacgagc gttatgcagg tttaattttt gatatggatg gcacaatcct ggatacggag 60

cctacgcacc gtaaagcgtg gcgcgaagta ttagggcact acggtcttca gtacgatatt 120

caggcgatga ttgcgcttaa tggatcgccc acctggcgta ttgctcaggc aattattgag 180

ctgaatcagg ccgatctcga cccgcatgcg ttagcgcgtg aaaaaacaga agcagtaaga 240

agtatgctgc tggatagcgt cgaaccgctt cctcttgttg atgtggtgaa aagttggcat 300

ggtcgtcgcc caatggctgt aggaacgggg agtgaaagcg ccatcgctga ggcattgctg 360

gcgcacctgg gattacgcca ttattttgac gccgtcgtcg ctgccgatca cgtcaaacac 420

cataaacccg cgccagacac atttttgttg tgcgcgcagc gtatgggcgt gcaaccgacg 480

cagtgtgtgg tctttgaaga tgccgatttc ggtattcagg cggcccgtgc agcaggcatg 540

gacgccgtgg atgttcgctt gctgtga 567

<210> 10

<211> 1035

<212> DNA

<213> Streptomyces coelicolor

<400> 10

atggagtacc gcatcctcgg cctggccgcc gaggccgcct tcttcaccct gctctcggtg 60

ccgcccctgc tgctgagcct cctcggtctg ctcggctacg tcgactcctg gatcggcgcc 120

gacaccaccg agagcctgcg cgacaacatc ctggacgcct cccgcgccgt gctctccgag 180

aagggcgtcc ggcagatcac cgagccgatc ctggacgacg tgatgaaggg cggccggccc 240

gacgtcatct ccatcggctt cctcttcgcc ctgtggtccg gatcccgcgc ggtgaacgtc 300

ttcatcgaca ccatcaccgt gatgtacggc ctcgacggcg tccggggcat cgtcaggacc 360

cgcctgatgg ccttcctcct cttcatcgtg gccctgctga tcgggtcgat cgcgctgccg 420

ctgatggtgg ccggaccgga cgccgtggtg cgggtcgtgc cctggtcgac gacggtcgta 480

caggtgctgt actggccggt cgtcatcatc ctctccgtgg ccttcctgac cacgctgtac 540

cacgtgtcgg tgcccgtgcg ctcgccgtgg atcgaggacg tccccggcgc gctggtcgcc 600

ctcgccatgt gggtgctggg cagcttcctg ctgcgcatct acctcaccag caccgtcgag 660

ggccccacca tctacggctc cctcgccgcg cccgtcgccg tgctgctgtg gatcggggtg 720

tccgcgttcg cggtgctcgt cggcgccgcg gtcaacgcgg ccatcgaccg ggtgtggccg 780

gccgccgcga ccgccgccgc ccgcgaggcc aacgagcggc tgcgccaggc ccaggtcgcc 840

gagtacgtgg cccgcacgac cgcgaacggc gagggcgacc ccgacatgcc ctccgagttc 900

cccgagcgct ggtcccgctt cctgcccccc gaggacgtca cggcccggct gcgcacccaa 960

ccgaagagcg cgcctcccgc gaaccacacc caccatcaaa agcacgacca caaccaccgc 1020

gacgacgcct cctga 1035

<210> 11

<211> 5371

<212> DNA

<213> Artificial sequence (Synthetic DNA)

<400> 11

atccggagtc gactcctcct ttcgctagca aaaaacccct caagacccgt ttagaggccc 60

caaggggtta tgctagttat tgctcagcgg tggcagcagc caactcagct tcctttacta 120

gtttgttagc agccggatct cagtggtggt ggtggtggtg ctcgagtgcg gccgcaagct 180

tgtagacgga gctcgaattc ggatccgcga cccatttgct gtccaccagt catgcttgcc 240

atatggctgc cgcgcggcac caggccgctg ctgtgatgat gatgatgatg gctgctgccc 300

atggtatatc tccttcttaa agttaaacaa aattatttct agaggggaat tgttatccgc 360

tcacaattcc cctatagtga gtcgtattaa tttcgcggga tcgagatctc gatcctctac 420

gccggacgca tcgtggccgg catcaccggc gcctaggtgc ggttgctggc gcctatatcg 480

ccgacatcac cgatggggaa gatcgggctc gccacttcgg gctcatgagc gcttgtttcg 540

gcgtgggtat ggtggcaggc cccgtggccg ggggactgtt gggcgccatc tccttgcatg 600

caccattcct tgcggcggcg gtgctcaacg gcctcaacct actactgggc tgcttcctaa 660

tgcaggagtc gcataaggga gagcgtcgag atcccggaca ccatcgaatg gcgcaaaacc 720

tttcgcggta tggcatgata gcgcccggaa gagagtcaat tcagggtggt gaatgtgaaa 780

ccagtaacgt tatacgatgt cgcagagtat gccggtgtct cttatcagac cgtttcccgc 840

gtggtgaacc aggccagcca cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg 900

gcggagctga attacattcc caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg 960

ctgattggcg ttgccacctc cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg 1020

attaaatctc gcgccgatca actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc 1080

ggcgtcgaag cctgtaaagc ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg 1140

atcattaact atccgctgga tgaccaggat gccattgctg tggaagctgc ctgcactaat 1200

gttccggcgt tatttcttga tgtctctgac cagacaccca tcaacagtat tattttctcc 1260

catgaagacg gtacgcgact gggcgtggag catctggtcg cattgggtca ccagcaaatc 1320

gcgctgttag cgggcccatt aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat 1380

aaatatctca ctcgcaatca aattcagccg atagcggaac gggaaggcga ctggagtgcc 1440

atgtccggtt ttcaacaaac catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg 1500

ctggttgcca acgatcagat ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg 1560

cgcgttggtg cggatatctc ggtagtggga tacgacgata ccgaagacag ctcatgttat 1620

atcccgccgt taaccaccat caaacaggat tttcgcctgc tggggcaaac cagcgtggac 1680

cgcttgctgc aactctctca gggccaggcg gtgaagggca atcagctgtt gcccgtctca 1740

ctggtgaaaa gaaaaaccac cctggcgccc aatacgcaaa ccgcctctcc ccgcgcgttg 1800

gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg 1860

caacgcaatt aatgtaagtt agctcactca ttaggcaccg ggatctcgac cgatgccctt 1920

gagagccttc aacccagtca gctccttccg gtgggcgcgg ggcatgacta tcgtcgccgc 1980

acttatgact gtcttcttta tcatgcaact cgtaggacag gtgccggcag cgctctgggt 2040

cattttcggc gaggaccgct ttcgctggag cgcgacgatg atcggcctgt cgcttgcggt 2100

attcggaatc ttgcacgccc tcgctcaagc cttcgtcact ggtcccgcca ccaaacgttt 2160

cggcgagaag caggccatta tcgccggcat ggcggcccca cgggtgcgca tgatcgtgct 2220

cctgtcgttg aggacccggc taggctggcg gggttgcctt actggttagc agaatgaatc 2280

accgatacgc gagcgaacgt gaagcgactg ctgctgcaaa acgtctgcga cctgagcaac 2340

aacatgaatg gtcttcggtt tccgtgtttc gtaaagtctg gaaacgcgga agtcagcgcc 2400

ctgcaccatt atgttccgga tctgcatcgc aggatgctgc tggctaccct gtggaacacc 2460

tacatctgta ttaacgaagc gctggcattg accctgagtg atttttctct ggtcccgccg 2520

catccatacc gccagttgtt taccctcaca acgttccagt aaccgggcat gttcatcatc 2580

agtaacccgt atcgtgagca tcctctctcg tttcatcggt atcattaccc ccatgaacag 2640

aaatccccct tacacggagg catcagtgac caaacaggaa aaaaccgccc ttaacatggc 2700

ccgctttatc agaagccaga cattaacgct tctggagaaa ctcaacgagc tggacgcgga 2760

tgaacaggca gacatctgtg aatcgcttca cgaccacgct gatgagcttt accgcagctg 2820

cctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt 2880

cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg 2940

tgttggcggg tgtcggggcg cagccatgac ccagtcacgt agcgatagcg gagtgtatac 3000

tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatat atgcggtgtg 3060

aaataccgca cagatgcgta aggagaaaat accgcatcag gcgctcttcc gcttcctcgc 3120

tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg 3180

cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag 3240

gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc 3300

gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag 3360

gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga 3420

ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc 3480

atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg 3540

tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt 3600

ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca 3660

gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca 3720

ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag 3780

ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca 3840

agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg 3900

ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg aacaataaaa 3960

ctgtctgctt acataaacag taatacaagg ggtgttatga gccatattca acgggaaacg 4020

tcttgctcta ggccgcgatt aaattccaac atggatgctg atttatatgg gtataaatgg 4080

gctcgcgata atgtcgggca atcaggtgcg acaatctatc gattgtatgg gaagcccgat 4140

gcgccagagt tgtttctgaa acatggcaaa ggtagcgttg ccaatgatgt tacagatgag 4200

atggtcagac taaactggct gacggaattt atgcctcttc cgaccatcaa gcattttatc 4260

cgtactcctg atgatgcatg gttactcacc actgcgatcc ccgggaaaac agcattccag 4320

gtattagaag aatatcctga ttcaggtgaa aatattgttg atgcgctggc agtgttcctg 4380

cgccggttgc attcgattcc tgtttgtaat tgtcctttta acagcgatcg cgtatttcgt 4440

ctcgctcagg cgcaatcacg aatgaataac ggtttggttg atgcgagtga ttttgatgac 4500

gagcgtaatg gctggcctgt tgaacaagtc tggaaagaaa tgcataaact tttgccattc 4560

tcaccggatt cagtcgtcac tcatggtgat ttctcacttg ataaccttat ttttgacgag 4620

gggaaattaa taggttgtat tgatgttgga cgagtcggaa tcgcagaccg ataccaggat 4680

cttgccatcc tatggaactg cctcggtgag ttttctcctt cattacagaa acggcttttt 4740

caaaaatatg gtattgataa tcctgatatg aataaattgc agtttcattt gatgctcgat 4800

gagtttttct aagaattaat tcatgagcgg atacatattt gaatgtattt agaaaaataa 4860

acaaataggg gttccgcgca catttccccg aaaagtgcca cctgaaattg taaacgttaa 4920

tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc 4980

cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt 5040

tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa 5100

aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg 5160

gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg 5220

acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc 5280

tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa 5340

tgcgccgcta cagggcgcgt cccattcgcc a 5371

Claims (7)

1. A method for synthesizing D-psicose using glycerol whole cell is characterized in that glycerol is used as a substrate, and glycerol is converted into D-psicose using glycerol kinase (glpK, SEQ ID NO:1), glycerol-3-phosphate dehydrogenase (glpD, SEQ ID NO:2), sugar alcohol oxidase (AldO, SEQ ID NO:3), L-fucoidan-1-phosphate aldolase (FucA, SEQ ID NO:4) and fructose-1-phosphorylase (YqaB, SEQ ID NO:5) (the conversion pathway is shown in FIG. 1).

2. The transformation pathway of claim 1, wherein the glycerol kinase coding gene (glpK, SEQ ID NO:6), the glycerol-3-phosphate dehydrogenase coding gene (glpD, SEQ ID NO:7), the L-fucoidan-1-phosphate aldolase coding gene (FucA, SEQ ID NO:8) and the fructose-1-phosphorylase coding gene (YqaB, SEQ ID NO:9) are derived from Escherichia coli MG1655(Escherichia coli MG1655), and the sugar alcohol oxidase coding gene (AldO, SEQ ID NO:10) is derived from Streptomyces coelicolor M145(Streptomyces coelicolor M145).

3. The method for synthesizing D-psicose according to claim 1, wherein plasmid pET28a (PB) N (SEQ ID NO:11) is used as the expression vector of the transformation pathway, and recombinant plasmid pET28a (PB) N-KDABO (plasmid structure is shown in FIG. 2) containing all pathway genes is constructed.

4. According to the claim 1, claim 2 and claim 3, using Escherichia coli BL21(DE3) as expression host, and transforming the recombinant plasmid into Escherichia coli BL21(DE3) to obtain the engineering strain BL21/KDABO containing all pathway genes.

5. The method for the whole-cell synthesis of D-psicose according to claim 1 and the recombinant engineered strain according to claim 4, wherein the transformation conditions used are as follows: (1) culturing the recombinant engineering bacteria of the escherichia coli to OD in an LB culture medium₆₀₀Adding isopropyl-beta-D-thiogalactoside (IPTG) with final concentration of 0.05-0.5mM, and inducing gene expression at 15-30 deg.C and 200rpm for 4-24 h; (2) centrifuging at 4 deg.C and 5000rpm to collect thallus, washing thallus with sterilized ultrapure water to remove culture medium, and resuspending thallus with 50mM Phosphate Buffer Solution (PBS) with pH of 6-8 to obtain resting cell; (3) adding glycerol with the final concentration of 10-60g/L as a substrate, and reacting for 48h at 20-35 ℃; (4) the yield of D-psicose was analyzed using ion chromatography.

6. According to claim 1 and claim 5, the optimum conditions of the present invention are: inducer IPTG concentration: 0.05mM, induction temperature: 20 ℃, induction time 12h, reaction temperature: reaction pH at 25 ℃: 7.5, substrate glycerol concentration: 40 g/L.

7. According to claim 1 and claim 6, the D-psicose whole-cell synthesis method according to the present invention produces not less than 3.54g/L of D-psicose under optimal conditions.

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