CN115074376B - Method for efficiently synthesizing D-psicose by utilizing recombinant escherichia coli fermentation - Google Patents
Method for efficiently synthesizing D-psicose by utilizing recombinant escherichia coli fermentation Download PDFInfo
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- 238000000855 fermentation Methods 0.000 title claims abstract description 56
- 230000004151 fermentation Effects 0.000 title claims abstract description 56
- BJHIKXHVCXFQLS-PUFIMZNGSA-N D-psicose Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](O)C(=O)CO BJHIKXHVCXFQLS-PUFIMZNGSA-N 0.000 title claims abstract description 43
- 238000000034 method Methods 0.000 title claims abstract description 33
- 241000588724 Escherichia coli Species 0.000 title claims abstract description 24
- 230000002194 synthesizing effect Effects 0.000 title claims abstract description 12
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 claims abstract description 35
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 32
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims abstract description 25
- RFSUNEUAIZKAJO-VRPWFDPXSA-N D-Fructose Natural products OC[C@H]1OC(O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-VRPWFDPXSA-N 0.000 claims abstract description 22
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 15
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- 238000006243 chemical reaction Methods 0.000 claims abstract description 9
- GSXOAOHZAIYLCY-NGJCXOISSA-N D-psicose 6-phosphate Chemical compound OCC(=O)[C@H](O)[C@H](O)[C@H](O)COP(O)(O)=O GSXOAOHZAIYLCY-NGJCXOISSA-N 0.000 claims abstract description 8
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- RNBGYGVWRKECFJ-ZXXMMSQZSA-N alpha-D-fructofuranose 1,6-bisphosphate Chemical compound O[C@H]1[C@H](O)[C@](O)(COP(O)(O)=O)O[C@@H]1COP(O)(O)=O RNBGYGVWRKECFJ-ZXXMMSQZSA-N 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
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- BGWGXPAPYGQALX-ARQDHWQXSA-N beta-D-fructofuranose 6-phosphate Chemical compound OC[C@@]1(O)O[C@H](COP(O)(O)=O)[C@@H](O)[C@@H]1O BGWGXPAPYGQALX-ARQDHWQXSA-N 0.000 description 1
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Abstract
The invention provides a method for efficiently synthesizing D-psicose by utilizing recombinant escherichia coli fermentation. Coli is used as chassis host bacteria, and the fructose specificity PTS transfer protein gene is knocked outFruASimultaneous overexpression of fructose non-phosphorylated transporter genespstG‑FFructokinase genemaKD-psicose-6-phosphate epimerase genealsEPsicose-6-phosphate phosphatase genea6PPEstablishing a synthetic pathway from D-fructose to D-psicose; further knocking out D-fructose-6-phosphokinase genepfkAAndpfkBregulating and controlling carbon metabolism flux of recombinant escherichia coli; overexpression of phosphoenolpyruvate carboxykinase Gene againpckAAnd glycerol is used as a carbon source to improve 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 industrialized development of the biosynthesis of D-psicose.
Description
Technical Field
The invention belongs to the field of microbial metabolism engineering, and particularly relates to a method for efficiently synthesizing D-psicose by utilizing recombinant escherichia coli fermentation.
Technical Field
D-psicose is an ultra-low calorie rare sugar with sweetness of 70% and energy of only 0.3% 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 oxidization, 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 oxidization in the processes of food processing and storage. D-psicose has been approved by the united states Food and Drug Administration (FDA) as a generally recognized safety product (GRAS), and has a great market prospect in the fields of foods, beverages, health care, and the like.
Because D-psicose is very scarce, development of its production process has attracted great attention. The chemical synthesis research of D-psicose starts 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 enzymatic methods have advantages in terms of product conversion and product purification, the problems of high production and immobilization costs of enzymes and long time consumption, especially the thermal stability of key enzymes, have not been completely solved, resulting in poor reusability of biocatalysts. In contrast, microbial fermentation is an ideal alternative for the production of D-psicose for enzyme catalysis.
Disclosure of Invention
The invention aims to solve the problems and provide a method for efficiently synthesizing D-psicose by utilizing recombinant escherichia coli fermentation. The synthesis path from D-fructose to D-psicose is established in escherichia coli through recombinant vector construction, the carbon metabolism flow is reasonably regulated and controlled by means of gene knockout, and simultaneously, the ATP intracellular synthesis path is introduced, so that the efficient synthesis of D-psicose is finally realized.
In order to achieve the above purpose, the invention adopts the following technical scheme:
a method for efficiently synthesizing D-psicose by utilizing recombinant escherichia coli fermentation, wherein the recombinant escherichia coli is obtained byE. coliKnocking out fructose-specific PTS transfer protein gene in JM109 (DE 3)fruASimultaneous overexpression of fructose non-phosphorylated transporter genesptsG-FFructokinase genemaKD-psicose-6-phosphate epimerase genealsEAnd psicose-6-phosphate phosphatase genea6PPFour genes, constructing a synthesis path of D-psicose; further by knocking out the D-fructose-6-phosphate kinase genepfkAAndpfkBblocking the conversion of intracellular fructose-6-phosphate into fructose-1, 6-diphosphate, so that the carbon flux of D-fructose enters the product synthesis path to the maximum extent; reintroducing heterologous phosphoenolpyruvate carboxykinase genepckAFor expressing the PckA protein, realizing the intracellular circulation of ATP; and finally, using an M9 basic culture medium containing glycerol as a fermentation culture medium of recombinant escherichia coli to perform oxygen-limited fermentation, so as to realize the efficient synthesis of the D-psicose.
The method for efficiently synthesizing the D-psicose by utilizing recombinant escherichia coli fermentation comprises the following steps of:
(1) Knock-outE. coliFructose-specific PTS transfer protein gene of JM109 (DE 3)fruAAnd overexpress fructose non-phosphorylated transporter genesptsG-FFructokinase genemaKD-psicose-6-phosphate epimerase genealsEAnd psicose-6-phosphate phosphatase genea6PPFour genes, getE. coli(ΔFruA, ptsG-F, maK, alsE, A6 PP) strain;
(2) In the strainE. coliBased on (DeltaFruA, ptsG-F, maK, alsE, A6 PP), the D-fructose-6-phosphate kinase gene was knocked outpfkAAndpfkBobtainingE. coli (ΔFruA, ΔPfkA, ΔPfkB, ptsG-F, maK, alsE, A6 PP) strains;
(3) In the strainE. coli (DeltaFruA, deltaPfkA, deltaPfkB, ptsG-F, maK, alsE, A6 PP) based on the overexpression of the phosphoenolpyruvate carboxykinase genepckAObtaining recombinant escherichia coliE. coli (ΔFruA, ΔPfkA, ΔPfkB, PtsG-F, MaK, AlsE, A6PP, PckA)；
(4) Make the following stepsFermenting and culturing with M9 basal medium containing glycerolE. coli (ΔFruA, ΔPfkA, ΔPfkB, ptsG-F, maK, alsE, A6PP, pckA) strains.
The specific process of the step (1) is as follows: primer is usedfruA-F (SEQ ID NO. 1) andfruA-R (SEQ ID NO. 2) PCR amplification with pKD13 plasmid as template, followed by transfection of the PCR product into a plasmid containing pKD46E. coliHomologous recombination is carried out in JM109 (DE 3), competent cells are prepared by picking up single colony activation, after transfection of pep 20 plasmid, the cells are cultured overnight at 30℃in a shaker at 220 rpm to eliminate the resistance marker, and finally the pep 20 plasmid is lost under the culture conditions of 42℃and 220 rpm to obtainE. coli(Δfrua) strain; then artificially synthesized pETDuet-ptsG-F-maKAnd pRSFDuet-alsE-a6PPTransfection of recombinant plasmids intoE. coli(ΔFruA) to giveE. coli(ΔFruA, ptsG-F, maK, alsE, A6 PP) strain; wherein,ptsG-F、maK、alsEanda6PPthe gene sequences of (C) are shown as 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: primer is usedpfkA-F/pfkA-R andpfkB-F/pfkB-R knockdown according to the method in step (1)pfkAAndpfkBgenes, getE. coli (ΔFruA, ΔPfkA, ΔPfkB, ptsG-F, maK, alsE, A6 PP) strains; wherein,pfkA-F andpfkAthe sequences of the R primer are respectively shown as SEQ ID NO.7 and SEQ ID NO.8,pfkB-F andpfkBthe sequences of the R primers are shown as SEQ ID NO.9 and SEQ ID NO.10 respectively.
The specific process of the step (3) is as follows: actinobacillus succinogenes @ is preparedActinobacillus succinogenes) Of origin ofpckAThe gene was synthesized artificially (SEQ ID NO. 11) after optimization according to the codon preference of E.coli, and primers were used as templatespckA-F (SEQ ID NO. 12) andpckAPCR amplification of R (SEQ ID NO. 13) with insertion of the Gene fragment into pRSFDuet-alsE-a6PPObtaining recombinant vector pRSFDuet-alsE-a6PP- pckATransfection ofE. coli (ΔFruA, ΔPfkA, ΔPfkB, PtsG-F, MaK, AlsE, A6PP) to obtain recombinant Escherichia 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 (DeltaFruA, deltaPfkA, deltaPfkB, ptsG-F, maK, alsE, A6PP, pckA) strains, to which 8g/L glycerol was added as a carbon source for cell growth, 2.2g/L D-fructose was added as a synthetic substrate for D-psicose, protein expression was induced using 0.1mM IPTG, and the bottle mouth was blocked using a rubber plug during fermentation.
The method for efficiently synthesizing the D-psicose by utilizing recombinant escherichia coli fermentation is applied to the production of the D-psicose.
Compared with the prior art, the invention has the following beneficial effects: compared with the existing enzyme catalysis method, the method for preparing D-psicose by utilizing recombinant escherichia coli has the advantages of simpler and more convenient operation and larger reaction specification, and effectively solves the problems of high cost caused by enzyme reusability and immobilization. Compared with the existing fermentation method based on the reversible epimerization synthesis way, the substrate conversion rate of the D-psicose synthesis way is obviously improved, and an optimization scheme is provided for the industrialized development of the synthesis of the D-psicose by the fermentation method.
Drawings
FIG. 1 is a recombinant E.coliE. coli (ΔFruA, ΔPfkA, ΔPfkB, ptsG-F, maK, alsE, A6PP, pckA) A-psicose synthesis.
FIG. 2 is a recombinant plasmid pETDuet-ptsG-F-maKAnd pRSFDuet-alsE-a6PPIs a map of (3). A: pETDuet-ptsG-F-maK；B：pRSFDuet-alsE-a6PP。
FIG. 3 is a schematic view ofE. coli Fermentation results of (. DELTA.FruA, ptsG-F, maK, alsE, A6 PP). A: fermentation results of the addition of 4 g/L D-fructose using LB medium; b: fermentation results using LB medium to add 4 g/L D-fructose and 50 mM potassium phosphate buffer.
FIG. 4 shows the fermentation results after knocking out PfkA and PfkB. A:E. coli fermentation results of the (ΔFruA, ΔPfkA, ptsG-F, maK, alsE, A6 PP) strain; b:E. coli (ΔFruA, ΔPfkA, ΔPfkB, ptsG-F, maK, alsE, A6 PP).
FIG. 5 is a recombinant plasmid pRSFDuet-alsE-a6PP-pckAIs a map of (3).
FIG. 6 is a recombinant E.coliE. coli Fermentation results of (. DELTA.FruA,. DELTA.PfkA,. DELTA.PfkB,. PtsG-F, maK, alsE, A6PP, pckA).
FIG. 7 recombinant E.coliE. coli (ΔFruA, ΔPfkA, ΔPfkB, ptsG-F, maK, alsE, A6PP, pckA) fermentation results with addition of 8. 8g/L glycerol and 2.2. 2.2g/L D-fructose in M9 medium.
Detailed Description
The invention is further described below with reference to examples, which are provided to illustrate the invention and not to limit the invention.
Recombinant E.coli in the present inventionE. coli The metabolic scheme for the synthesis of D-psicose (ΔFruA, ΔPfkA, ΔPfkB, ptsG-F, maK, alsE, A6PP, pckA) is shown in FIG. 1.
Example 1
Host bacteria on chassisE. coliJM109 (DE 3) based deletion of fructose-specific PTS transfer protein genefruAPrimers were usedfruA-F (SEQ ID NO. 1) andfruAr (SEQ ID NO. 2) PCR amplification with pKD13 plasmid as template, transfection of the amplified product into a plasmid containing pKD46E. coliHomologous recombination is carried out in JM109 (DE 3), competent cells are prepared by picking up single colony activation, after transfection of pep 20 plasmid, the cells are cultured overnight at 30℃in a shaker at 220 rpm to eliminate the resistance marker, and finally the pep 20 plasmid is lost under the culture conditions of 42℃and 220 rpm to obtainE. coli(ΔFruA) strain. Fructose non-phosphorylated transporter genesptsG-FFructokinase genemaKInserting pRSFDuet-1 to obtain pETDuet-ptsG-F-maKRecombinant plasmid (plasmid map, see FIG. 2A). D-psicose-6-phosphate epimerase genealsEAnd psicose-6-phosphate phosphatase genea6PPInserting pRSFDuet-1 to obtain pRSFDuet-alsE-a6PPRecombinant plasmid (plasmid map, see FIG. 2B). Wherein the fructose non-phosphorylated transporter geneptsG-FFructoseKinase genemaKD-psicose-6-phosphate epimerase genealsEAnd psicose-6-phosphate phosphatase genea6PPThe gene sequences are respectively shown as SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6,ptsG-Fthe gene is Escherichia coliE. coliJM109 (DE 3)ptsGThe 34 th nucleotide of the gene is mutated from G to T,maKandalsEthe gene is Escherichia coliE. coliOverexpression of the JM109 (DE 3) self-gene,a6PPthe gene is derived from bacteroides fragilisBacteroides fragilis) The method comprises the steps of carrying out a first treatment on the surface of the In the present invention, fructose non-phosphorylated transporter geneptsG-FFructokinase genemaKD-psicose-6-phosphate epimerase genealsEAnd psicose-6-phosphate phosphatase genea6PPAre all synthesized artificially. Artificially synthesized pETDuet-ptsG-F-maKAnd pRSFDuet-alsE-a6PPTransfection of recombinant plasmids intoE. coli(ΔFruA) to giveE. coli(DeltaFruA, ptsG-F, maK, alsE, A6 PP) strain.
Will beE. coliThe (. DELTA.FruA, ptsG-F, maK, alsE, A6 PP) strain was activated overnight, transferred to 50 mL LB liquid medium containing 4 g/L D-fructose at an inoculum size of 1%, 100 mg/mL ampicillin and 50 mg/mL kanamycin were added thereto, and the mixture was fermented in a shaker at 37℃and 220 rpm for 72 h, protein expression was induced by adding 0.1mM IPTG at a final concentration after 3 hours of fermentation, and samples were taken every 12 hours at intervals during fermentation, using a shaker equipped with Sugar-Pak TM The D-fructose content and D-psicose content in the fermentation broth were determined by a high performance liquid chromatograph (Chromaster HPLC, HITACHI) of I (6.5X100X mm, 85, waters) chromatography column and a differential Refractive Index Detector (RID), the mobile phase was deionized water, the flow rate was 0.5 mL/min, the column temperature was 85 ℃, the detector temperature was 55 ℃, and the sample injection amount was 20. Mu.L. As shown in FIG. 3A, the cells were less able to grow during fermentation, and the final OD of the broth 600 Only about 0.5, probably due to the pH decrease of the broth resulting from the dephosphorylation reaction, affecting cell growth, and the pH of the final broth was further measured using a pH meter at a pH of about 3.9; however, D-psicose production was evident in the fermentation broth, and the yield was 0.351. 1 g/L, the yield is 0.159 g/g.
Will beE. coliThe (. DELTA.FruA, ptsG-F, maK, alsE, A6 PP) strain was activated overnight, transferred to 50 mL LB liquid medium containing 4 g/L D-fructose at an inoculum size of 1%, a final concentration of 50 mM potassium phosphate buffer (purchased from source leaf organisms, cat. No. R26329) was added to the medium to adjust pH, 100 mg/mL ampicillin and a final concentration of 50 mg/mL kanamycin were added thereto, and the mixture was fermented in a shaker at 37℃and 220 rpm for 72 h, protein expression was induced by adding 0.1mM IPTG at a final concentration after 3 hours of fermentation, and samples were taken every 12 hours during the fermentation culture using a shaker equipped with Sugar-Pak TM The D-fructose content and D-psicose content in the fermentation broth were determined by a high performance liquid chromatograph (Chromaster HPLC, HITACHI) of I (6.5X100X mm, 85, waters) chromatography column and a differential Refractive Index Detector (RID), the mobile phase was deionized water, the flow rate was 0.5 mL/min, the column temperature was 85 ℃, the detector temperature was 55 ℃, and the sample injection amount was 20. Mu.L. As shown in FIG. 3B, cell growth was alleviated and the final OD of the broth 600 Up to about 2.4, d-psicose yield increased to 0.510 g/L.
Example 2
The strain obtained in example 1E. coliBased on (DeltaFruA, ptsG-F, maK, alsE, A6 PP), the corresponding knockout primers were used according to the gene knockout method in example 1pfkA-F andpfkAr knockout of the phosphofructokinase 6 genepfkAObtainingE. coli(DeltaFruA,. DELTA.PfkA, ptsG-F, maK, alsE, A6 PP) strain. In the strainE. coliBased on (ΔFruA, ΔPfkA, ptsG-F, maK, alsE, A6 PP), the corresponding knockout primers were used according to the gene knockout method in example 1pfkB-F andpfkBr knockout of the phosphofructokinase 6 genepfkBTo regulate and control the carbon metabolism flux of D-fructose to obtainE. coli (ΔFruA, ΔPfkA, ΔPfkB, ptsG-F, maK, alsE, A6 PP) strains.pfkA-F andpfkAthe sequences of the R primers are shown as SEQ ID NO.7 and SEQ ID NO. 8.pfkB-F andpfkBthe sequences of the R primers are shown as SEQ ID NO.9 and SEQ ID NO. 10.
The strain was cultivated by fermentation according to the method in example 1. The strain was activated overnight and transferred to the strain containing 1% of the inoculum size4 g/L D-fructose in 50 mL LB liquid medium, adding final concentration 50 mM potassium phosphate buffer (purchased from source leaf organism, cat. No. R26329) to the medium to adjust pH, adding final concentration 100 mg/mL ampicillin and final concentration 50 mg/mL kanamycin thereto, fermenting and culturing 72 h in a shaker at 37℃and 220 rpm, adding final concentration 0.1mM IPTG to induce protein expression after 3 hours of fermentation culture, sampling every 12 hours during fermentation culture, and using a culture medium equipped with Sugar-Pak TM The D-fructose content and D-psicose content in the fermentation broth were determined by a high performance liquid chromatograph (Chromaster HPLC, HITACHI) of I (6.5X100X mm, 85, waters) chromatography column and a differential Refractive Index Detector (RID), the mobile phase was deionized water, the flow rate was 0.5 mL/min, the column temperature was 85 ℃, the detector temperature was 55 ℃, and the sample injection amount was 20. Mu.L. The results are shown in FIG. 4, FIG. 4AE. coli (ΔFruA, ΔPfkA, ptsG-F, maK, alsE, A6 PP) strain 72 h to yield 0.721 g/L D-psicose at a yield of 0.180 g/g; in FIG. 4BE. coli The yields and yields of the (. DELTA.FruA,. DELTA.PfkA,. DELTA.PfkB,. PtsG-F, maK, alsE, A6 PP) strains were significantly improved, respectively, at 0.816 g/L and 0.614 g/g, indicating that a reasonable limitation of the carbon metabolic flux of D-fructose was advantageous for efficient conversion of the synthesis reaction.
Example 3
Actinobacillus succinogenes @ is preparedActinobacillus succinogenes) Source phosphoenolpyruvate carboxykinase genepckAAnd (3) artificially synthesizing the recombinant DNA according to the codon preference of escherichia coli, wherein the synthesized template sequence is SEQ ID NO.11. Primer is usedpckA-F (SEQ ID NO. 12) andpckA-R (SEQ ID NO. 13) pair of syntheticpckAAfter PCR amplification of the template, the gene fragment was inserted into pRSFDuet-alsE-a6PPAfter that, pRSFDuet-alsE-a6PP-pckARecombinant plasmid (plasmid map shown in FIG. 5), and transfected againE. coli (DeltaFruA, deltaPfkA, deltaPfkB, ptsG-F, maK, alsE, A6 PP) to give recombinant E.coliE. coli (ΔFruA, ΔPfkA, ΔPfkB, PtsG-F, MaK, AlsE, A6PP, PckA)。
The strain was cultivated by fermentation according to the method in example 1. The strain was activated overnight and transferred to a strain containing 4 g/L D-fruits at 1% of the inoculum sizeTo 50 mL of LB liquid medium containing sugar, a final concentration of 50. 50 mM potassium phosphate buffer (purchased from source leaf organism, cat No. R26329) was added to adjust pH, and further 100. 100 mg/mL of ampicillin and 50. 50 mg/mL of kanamycin were added thereto, and the culture was fermented at 37℃and 220 rpm in a shaker for 72. 72 h, and protein expression was induced by adding 0.1mM IPTG to the culture medium after 3 hours of fermentation, and samples were taken every 12 hours during the fermentation. During fermentation culture, the bottle mouth is plugged by a rubber plug, and an inducer is added or sampled by a syringe. Using a Sugar-Pak equipped TM The D-fructose content and D-psicose content in the fermentation broth were determined by a high performance liquid chromatograph (Chromaster HPLC, HITACHI) of I (6.5X100X mm, 85, waters) chromatography column and a differential Refractive Index Detector (RID), the mobile phase was deionized water, the flow rate was 0.5 mL/min, the column temperature was 85 ℃, the detector temperature was 55 ℃, and the sample injection amount was 20. Mu.L. Fermentation experiments were performed using the fermentation medium referred to in example 1, the results of which 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 improved, about 1.23/g/L, and the yield was about 0.683/g/g.
Example 4
In this example, an oxygen limited fermentation experiment was performed on M9 basal medium. StrainE. coli (DeltaFruA, deltaPfkA, deltaPfkB, ptsG-F, maK, alsE, A6PP, pckA) were activated overnight, transferred to M9 basal medium at 1% inoculum size, 8. 8g/L glycerol and 2.2. 2.2g/L D-fructose were added thereto, 100 mg/mL ampicillin and 50 mg/mL kanamycin were added at a final concentration, 96 h was fermented in a shaker at 37℃and 220 rpm, protein expression was induced by adding 0.1mM IPTG at a final concentration after 3 hours of fermentation culture, and samples were taken every 20 hours during the fermentation culture. During fermentation culture, the bottle mouth of the culture bottle is plugged by a rubber plug, and an inducer is added or sampled by a syringe. As a result, as shown in FIG. 7, 2.2. 2.2g/L D-fructose was completely converted to D-psicose (1.590 g/L) after 96. 96 h, and the yield was about 0.722 g/g.
SEQUENCE LISTING
<110> university of Fuzhou, qingyuan Innovation laboratory
<120> a method for efficiently synthesizing D-psicose by fermentation of recombinant E.coli
<130>
<160> 13
<170> PatentIn version 3.3
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ccccatggtt agtggttacg gatgtactca t 31
Claims (3)
1. A method for efficiently synthesizing D-psicose by utilizing recombinant escherichia coli fermentation is characterized by comprising the following steps of: to be used forE.coli JM109 DE3 is chassis host bacterium, and specific PTS transfer of fructose is knocked outProtein genefruAAnd overexpress fructose non-phosphorylated transporter genesptsG-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 D-fructose carbon flux are realized; overexpression of phosphoenolpyruvate carboxykinase Gene againpckATo increase intracellular ATP concentration; finally, D-fructose is used as a substrate raw material, glycerol is used as a carbon source for oxygen-limited fermentation, and high-efficiency conversion of D-psicose is further ensured;
the method specifically comprises the following steps:
(1) To be used forE.coli JM109 DE3 is chassis host bacterium, and the fructose specific PTS transfer protein gene is knocked out by homologous recombination technologyfruAAnd overexpress fructose non-phosphorylated transporter genesptsG-FFructokinase genemaKD-psicose-6-phosphate epimerase genealsEAnd psicose-6-phosphate phosphatase genea6PPFour genes, getE.coli-ΔFruAPtsG-FMaKAlsEA6PP strain;
(2) In the strainE.coli-Based on DeltaFruAPtsG-FMaKAlsEA 6PP, the homologous recombination technology is utilized to knock out the D-fructose-6-phosphokinase genepfkAAndpfkBto block the conversion of D-fructose-6-phosphate to D-fructose-1, 6-biphosphoate to giveE.coli- ΔfraΔpfkaΔpfkbptsg-FMaKAlsEA6PP strain;
(3) In the strainE.coliOverexpression of the phosphoenolpyruvate carboxykinase Gene on the basis of ΔFruA ΔPfkA ΔPfkBPtsG-FMaKAlsEA6PPpckATo realize the intracellular circulation regeneration of ATP to obtain recombinant escherichia coliE.coli-ΔFruAΔPfkAΔPfkBPtsG-FMaKAlsEA6PPPckA；
(4) D-fructose is used as a substrate raw material, glycerol is used as carbon, and M9 basic culture medium is used for strainE.coli-Oxygen-limited fermentation of ΔFruA ΔPfkA ΔPfkBPtsG-FMaKAlsEA6PPPckA for fortificationE.coli-ATP producing ability of the ΔFruA ΔPfkA ΔPfkBPtsG-FMaKAlsEA6PPPckA strain and improving the production in fermentation brothA quantity of material;
the specific process of the step (1) is as follows: PCR amplification was performed using the pKD13 plasmid as a template and the primers fruA-F and fruA-R, and the amplified product was transfected into a plasmid containing pKD46 by electrochemical conversionE.coli JM109 DE3 was recombined and the resistance marker was knocked out using pep 20 plasmid after completion to obtainE.coli-Delta FruA strain, wherein the sequences of the fruA-F and fruA-R primers are shown as SEQ ID NO.1 and SEQ ID NO.2 respectively; artificially synthesizing pETDuet-ptsG-F-maK and pRSFDuet-alsE-a6PP recombinant expression vector, and transfecting toE.coli-ΔFruA to giveE.coli-The ΔFruAPtsG-FMaKAlsEA6PP strain, wherein,ptsG-F、maK、alsEanda6PPthe gene sequences of (a) are respectively shown as 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: knocking out the primers pfkA-F/pfkA-R and pfkB-F/pfkB-R according to the method in the step (1)E.coli-DeltaFruAPtsG-FMaKAlsEA 6PP StrainpfkAAndpfkBgenes, getE.coli-The ΔFruA ΔPfkA ΔPfkBPtsG-FMaKAlsEA6PP strain, wherein the primer sequences of pfkA-F and pfkA-R are shown in SEQ ID NO.7 and SEQ ID NO.8 respectively, and the primer sequences of pfkB-F and pfkB-R are shown in SEQ ID NO.9 and SEQ ID NO.10 respectively;
the specific process of the step (3) is as follows: actinobacillus succinogenesActinobacillus succinogenesOf origin ofpckAThe gene is synthesized artificially after optimization according to the codon preference of escherichia coli, amplified by using primers pckA-F and pckA-R, and digested with two restriction sites of Xho I and Avr IIpckAGene insertion pRSFDuet-alsE-a6PP recombinant expression vector to obtain recombinant vector pRSFDuet-alsE-a6PP-pckA, and then transfectionE.coli-DeltaFruA DeltaPfkA DeltaPfkBPtsG-FMaKAlsEA 6PP to obtain recombinant Escherichia coliE.coli-ΔFruA ΔPfkA ΔPfkBPtsG-FMaKAlsEA6PPPckA, wherein, artificially synthesizedpckAThe gene sequence is shown as SEQ ID NO.11, and the sequences of the pckA-F and pckA-R primers are shown as SEQ ID NO.12 and SEQ ID NO.13 respectively.
2. The efficient synthesis of D-alopecurone by recombinant E.coli fermentation according to claim 1A method of ketose, characterized by: the specific process of the step (4) is as follows: fermentation culture Using M9 basal MediumE.coli-The ΔFruA ΔPfkA ΔPfkBPtsG-FMaKAlsEA6PPPckA strain was added with 8g/L glycerol as a carbon source for cell growth, 2.2g/L D-fructose as a synthetic substrate for D-psicose, protein expression was induced using 0.1mM IPTG, and a plug was used to plug the bottle mouth during fermentation.
3. The use of a method for the efficient synthesis of D-psicose by recombinant escherichia coli fermentation according to claim 1 for the production of D-psicose.
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