CN117757707A - Genetically engineered bacterium, multi-bacterium mixed transformation system containing genetically engineered bacterium and application of multi-bacterium mixed transformation system - Google Patents
Genetically engineered bacterium, multi-bacterium mixed transformation system containing genetically engineered bacterium and application of multi-bacterium mixed transformation system Download PDFInfo
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Abstract
The invention provides a genetic engineering bacterium, a multi-bacterium mixed transformation system containing the genetic engineering bacterium and application thereof, belonging to the technical field of bioengineering; the invention adopts genetic engineering means to construct genetic engineering bacteria for jointly expressing D-psicose 3-epimerase (DAE) and L-rhamnose isomerase (L-RI) on the basis of bacillus subtilis WB800N, and then promotes the double-enzyme expression quantity of the genetic engineering bacteria by promoter engineering technology; the invention also prepares a multi-bacterium mixed conversion system based on the genetic engineering bacterium, and the genetic engineering bacterium and the multi-bacterium mixed conversion system can effectively improve the conversion efficiency of D-fructose, thereby realizing the efficient co-production of D-psicose and having good application prospect.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a genetic engineering bacterium, a multi-bacterium mixed transformation system containing the genetic engineering bacterium and application of the multi-bacterium mixed transformation system.
Background
D-psicose and D-psicose are natural rare sugar, have the characteristics of high sweetness and low calorie, are almost non-caloric sweeteners, have the effects of improving intestinal flora, reducing blood sugar, resisting oxidization, preventing obesity and the like, can be widely applied to the food fields of biscuits, fruit juice, beverages and the like instead of sucrose, have the physiological functions of resisting cancer, resisting tumor and the like, can be used as an intermediate for medical synthesis, and are applied to the clinical field of medicines.
At present, the preparation of D-psicose and D-psicose mainly comprises a chemical synthesis method and a biological synthesis method, wherein the chemical method has the defects of low efficiency, more byproducts, complicated purification, high cost, large pollution and the like, and is difficult to be used for industrially producing the D-psicose. The biosynthesis method uses biocatalyst and cheap D-fructose as substrate to catalyze the conversion of D-fructose into D-psicose and D-psicose. Specifically, D-fructose is isomerized to D-psicose using D-psicose 3-epimerase (DAE), and then D-psicose is isomerized to D-psicose using L-rhamnose isomerase. Although the biosynthesis method has the advantages of rapid reaction, environmental protection, low cost of raw materials and the like compared with the chemical synthesis method, the deficiency of low utilization rate of D-fructose exists in the multienzyme cascade biosynthesis, and the low yields of D-psicose and D-psicose are further caused.
In addition, the catalyst in the biosynthesis method is mostly in the form of pure enzyme, and the method of catalyzing the catalyst by using the pure enzyme has the advantages of high reaction speed, high efficiency and the like, but the pure enzyme has the defects of easy inactivation, instability, complicated purification process and the like, and although the stability and the recycling property of the catalyst can be enhanced by the method of immobilizing the enzyme, the method clearly increases the production cost. The whole cell catalysis method directly utilizes resting cells containing enzyme as a biocatalyst, and the enzyme has good stability and the preparation method is simple, so that the resting cells are used as the catalyst to meet the requirements of industrial development. The whole cell catalyst of D-psicose and D-psicose is mainly engineering escherichia coli, but endotoxin exists in the escherichia coli, the codon preference is unfavorable for the expression of exogenous genes, and the protein modification system after translation is imperfect, so that the protein expression forms inclusion bodies, and the activity of the protein is extremely low or inactive.
Therefore, it is necessary to construct a genetically engineered bacterium for efficiently co-producing D-psicose and a multi-bacterium mixed system containing the genetically engineered bacterium.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a genetic engineering bacterium, a multi-bacterium mixed transformation system containing the genetic engineering bacterium and application thereof; the invention adopts genetic engineering means to construct genetic engineering bacteria for jointly expressing D-psicose 3-epimerase (DAE) and L-rhamnose isomerase (L-RI) on the basis of bacillus subtilis WB800N, and then promotes the double-enzyme expression quantity of the genetic engineering bacteria by promoter engineering technology; the invention also prepares a multi-bacterium mixed conversion system based on the genetic engineering bacterium, and the genetic engineering bacterium and the multi-bacterium mixed conversion system can effectively improve the conversion efficiency of D-fructose, thereby realizing the efficient co-production of D-psicose and having good application prospect.
In order to achieve the technical purpose, the invention adopts the following technical means:
the invention firstly provides a genetic engineering bacterium for jointly expressing D-psicose 3-epimerase and L-rhamnose isomerase, wherein the genetic engineering bacterium comprises a recombinant vector for jointly expressing the D-psicose 3-epimerase and the L-rhamnose isomerase.
Preferably, the original strain of the genetically engineered bacterium is bacillus subtilis.
Preferably, the vector of the recombinant vector for the combined expression of D-psicose 3-epimerase and L-rhamnose isomerase comprises pP43NMK;
the promoter comprises P HpaⅡ 、P 43 、P spoVG Or P srfA One or two of them.
Preferably, the promoter is P HpaⅡ And P spoVG A promoter combination.
Preferably, the D-psicose 3-epimerase is derived from BlueTourette's bacillus (Blauthia product), designated BP-DAE, NCBI accession number: wp_148391986.1;
the amino acid sequence of the L-rhamnose isomerase is shown as SEQ ID No. 1.
The invention also provides a microbial agent, which comprises the genetically engineered bacterium.
The invention also provides a recombinant vector which expresses the D-psicose 3-epimerase and the L-rhamnose isomerase in a combined way.
The invention also provides a multi-bacterium mixed conversion system, which comprises:
the genetically engineered bacterium for jointly expressing the D-psicose 3-epimerase and the L-rhamnose isomerase; and/or
And (3) a mixed bacterial liquid of a genetically engineered bacterium which independently expresses D-psicose 3-epimerase and a genetically engineered bacterium which independently expresses L-rhamnose isomerase.
Preferably, in the mixed bacterial liquid, the content of the genetically engineered bacteria independently expressing the D-psicose 3-epimerase is 12.5-87.5%, and the content of the genetically engineered bacteria independently expressing the L-rhamnose isomerase is 87.5-12.5%.
The invention also provides application of the genetically engineered bacterium, microbial agent or multi-bacterium mixed conversion system in catalyzing D-fructose to prepare D-psicose and/or D-psicose.
The invention also provides a method for preparing the D-psicose and the D-psicose, which is to inoculate the genetic engineering bacteria or a mixed transformation system containing the genetic engineering bacteria, microbial agents or multiple bacteria into the D-fructose.
Compared with the prior art, the invention has the beneficial effects that:
the invention constructs the gene engineering bacteria for independently expressing DAE, the gene engineering bacteria for independently expressing L-RI and the gene engineering bacteria for jointly expressing D-psicose 3-epimerase and L-rhamnose isomerase by using probiotics bacillus subtilis as an expression host through a gene engineering technology. The invention designs two transformation systems for co-producing D-psicose and D-psicose by taking D-fructose as a substrate, wherein the first transformation system is a whole-cell mixed transformation system for independently expressing DAE and independently expressing L-RI according to a certain proportion, and the second transformation system is a whole-cell transformation system of genetically engineered bacteria for jointly expressing D-psicose 3-epimerase and L-rhamnose isomerase. The research shows that the two conversion systems have high capability of co-producing D-psicose and D-psicose. Wherein the yield of the whole cell transformation system of the genetically engineered bacterium for the combined expression of D-psicose 3-epimerase and L-rhamnose isomerase is relatively higher, probably because the combined expression of the double enzymes in the same strain is advantageous for the accumulation of intermediate (D-psicose). The invention also replaces single promoter P in the co-expression recombinant plasmid pP43NMK by tandem promoter combination 43 To increase the expression level of the double enzyme in the same strain, thereby further increasing the utilization rate of D-fructose and finally increasing the yield of D-psicose and D-psicose.
The promoter of the plasmid pP43NMK used in the invention is P 43 Compared with a T7 inducible promoter used in traditional escherichia coli, the constitutive self-inducible promoter does not need to add isopropyl thiogalactoside (IPTG) as an inducer, so that the operation steps are simplified, potential safety hazards caused by toxicity of the IPTG are avoided, and the production safety is further ensured and the production cost is controlled. The invention also optimizes the transformation of plasmid pP43NMK by amplifying other constitutive strong promoters derived from bacillus subtilis endogenous, and optimizes single promoter P 43 The expression of the target protein is enhanced by replacing other serial constitutive strong promoters to improve the transcription level, more rare sugar total amount is produced, and finally the D-fructose is effectively improvedCompared with other bacillus subtilis engineering bacteria expression systems, the transformation efficiency of the bacillus subtilis engineering bacteria has better application.
According to the invention, the expression plasmid is modified through promoter optimization, the B.subtilis WB800N is used as an expression host, the bacillus subtilis engineering bacteria capable of producing D-psicose and D-psicose with high yield are constructed, resting cells are used as catalysts for reaction, and the resting cells are different from other enzymes immobilized through engineering bacteria preparation, the preparation process of the resting cells is simple, the tolerance of the enzymes to acid and alkali and heat is improved to a certain extent, and the expression level of double enzymes is improved through serial combination of various promoters endogenous to bacillus subtilis, so that the utilization rate of D-fructose is improved, and finally the D-psicose and the D-psicose are efficiently co-produced.
Drawings
FIG. 1 shows agarose gel electrophoresis of a plasmid backbone pP43NMK, a bp-dae gene fragment (a), and a recombinant plasmid pP43NMK-bp-dae (b).
FIG. 2 shows agarose gel electrophoresis of plasmid backbone pP43NMK, rhaA gene fragment (a), recombinant plasmid pP43NMK-rhaA (b).
FIG. 3 shows agarose gel electrophoresis of the plasmid backbone pP43NMK (a) and the gene fragments of bp-dae-rhaA and rhaA-bp-dae (b), the recombinant plasmids pP43NMK-bp-dae-rhaA and pP43NMK-rhaA-bp-dae (c).
FIG. 4 shows SDS-PAGE protein gel electrophoresis of genetically engineered bacteria B.subtlis DAE (a), B.subtlis L-RI (b), B.subtlis DAE-L-RI and B.subtlis L-RI-DAE (c).
FIG. 5 shows the bioconversion of D-fructose by a bicistronic recombinant bacterium; the black histogram is B.subtitle DAE-L-RI, and the gray histogram is B.subtitle L-RI-DAE.
FIG. 6 (a) shows the D-fructose, D-psicose and D-psicose contents in different whole cell mixed transformation systems; FIG. 6 (b) shows the total content of D-psicose and D-psicose in different whole cell mixed transformation systems; FIG. 6 (c) shows the D-allose content in various whole cell mixed transformation systems.
FIG. 7 is a graph showing the effect of different transformation conditions on rare sugar production; wherein, (a) is a pH influence diagram; (b) Is warmA map of the influence of the degree; (c) is a graph showing the effect of D-fructose concentration; (d) is a graph of the effect of whole cell concentration; (e) Different Mn 2+ A graph of the influence of the concentration of metal ions; (f) The optimal system MS-8 bioconverts D-fructose into D-psicose and D-psicose.
FIG. 8 is a diagram showing the protein expression of genetically engineered bacteria with different combinations of tandem promoters.
FIG. 9 is a graph showing the production efficiency of rare sugars (D-psicose and D-psicose) of genetically engineered bacteria with different combinations of tandem promoters.
FIG. 10 is a graph showing the progress of bioconversion of D-fructose to D-psicose and D-psicose in the optimal system MS-8-D5.
Detailed Description
The invention will be further described with reference to the drawings and the specific embodiments, but the scope of the invention is not limited thereto. In the following examples, various processes and methods, which are not described in detail, are conventional methods well known in the art. The sources of the reagents used, the trade names and the components of the reagents are shown when the reagents appear for the first time, and the reagents which are the same as the sources shown for the first time are not specially indicated; the reagents, materials, etc. involved are commercially available without any particular explanation.
The preparation method of the culture medium used in the following examples includes:
(1) Liquid preparation of SPI-A: 0.4g of ammonium sulfate ((NH 4) is weighed out 2 SO 4 ) 1.2g of monopotassium phosphate (KH) 2 PO 4 ) 2.8g of anhydrous dipotassium hydrogen phosphate (K) 2 HPO 4 ) Sodium citrate trihydrate 0.2g (C 6 H 5 Na 3 O 7 ·3H 2 O), deionized water is added and the volume is fixed to 100mL. SPI-B liquid preparation: 0.04g of magnesium sulfate heptahydrate (MgSO 4 ·7H 2 O), deionized water is added and the volume is fixed to 100mL.100 XCAYE solution preparation: 0.2g casein hydrolysate, 1g yeast extract was weighed out, injected with deionized water and set to a volume of 10mL.
(2) Preparing SPI solution: 98mL of SPI-A solution, 98mL of SPI-B solution, 2mL of 50% glucose solution, 2mL of 100 XCAYE solution were taken and thoroughly mixed.
(3) Solution preparation of SPII: 98mL SPI,1mL 50mmol.L -1 Calcium chloride (CaCl) 2 ) 1mL 250 mmol.L solution -1 Magnesium chloride hexahydrate (MgCl) 2 ·6H 2 And O) fully and uniformly mixing the solution.
(4) Preparation of 100×egta: weighing 0.38g EGTA, dissolving in deionized water, adjusting pH to 8.0, and constant volume to 10mL
Example 1: construction of recombinant vector pP43NMK-bp-dae
(1) Based on the amino acid sequence encoding amino acid (NCBI accession number: WP_ 148391986.1) of D-psicose-3-epimerase produced in B.BlueTokida in NCBI database, the gene sequence bp-dae encoding D-psicose-3-epimerase and the gene element characteristics on vector pP43NMK (purchased from Hunan Fenghui Biotechnology Co., ltd.) were synthesized by the Suzhou Jin Wei intelligent company, and primers were designed using Oligo7.0 software, the sequences of which were shown as SEQ ID No. 2 to SEQ ID No. 5.
dae-F：5’-taactttaagaaggagatatacatatgtaccaagatctggcactgt-3’(SEQ ID No:2)；
dae-R：5’-tgcaggagctcccatggaggttacgcttgggtgatgaact-3’(SEQ ID No:3)；
pP43NMK-F:5’-atgtatatctccttcttaaagt-3’(SEQ ID No:4)；
pP43NMK-R:5’-cctccatgggagctcctg-3’(SEQ ID No:5)。
(2) Linearization of plasmid pP43NMK using pP43NMK-F and pP43NMK-R primers, PCR reaction parameters: pre-denaturation, 98 ℃ for 30s; denaturation, 10s at 98 ℃; annealing at 55 ℃ for 5s; extending at 72 ℃ for 35s; stopping extension at 72 ℃ for 1min; linearized plasmid pP43NMK was obtained after 32 cycles.
Amplification of bp-dae gene fragment containing homology arms using dae-F and dae-R primer pairs, PCR reaction parameters: pre-denaturation, 98 ℃ for 1min; denaturation, 10s at 98 ℃; annealing at 55 ℃ for 30s; extending at 72 ℃ for 15s; stopping extension at 72 ℃ for 5min; after 32 cycles, a bp-dae gene fragment containing the pP43NMK homology arm was obtained.
The obtained PCR amplification products were all detected by 1% agarose gel electrophoresis, and the detection results are shown in FIG. 1 (a), and it can be seen from the figure that the size of the band of the linearized plasmid pP43NMK is about 6700bp, and the size of the band of the bp-dae gene containing the pP43NMK homology arm is about 900bp, which all conform to the expected size. This indicates that the linearized plasmid pP43NMK and the bp-dae gene fragment containing the pP43NMK homology arm were successfully obtained.
(3) And (3) recombining and connecting the linearized plasmid pP43NMK obtained in the step (2) and the bp-dae gene fragment containing the pP43NMK homology arm according to a seamless cloning kit description method, mixing the linearized plasmid pP43NMK and the bp-dae gene fragment containing the pP43NMK homology arm according to a ratio of 1:3 (mol/mol), adding one volume of 2X MultiF Seamless Assembly Mix, and connecting at 50 ℃ for 30min to obtain a connecting product.
Then adding E.coli JM109 competent cells into the ligation product, mixing, standing on ice for 30min, carrying out heat shock at 42 ℃ for 60s, cooling on ice for 2min, adding 900 mu L of LB culture medium (yeast powder 5g/L, tryptone 10g/L and sodium chloride 10 g/L), incubating for 1h at 37 ℃ and then coating an ampicillin antibiotics LB plate (yeast powder 5g/L, tryptone 10g/L, sodium chloride 10g/L and agar powder 15 g/L) containing 50 mu g/mL for overnight culture, picking up positive transformants for overnight culture, extracting plasmids by using a plasmid miniextraction kit, obtaining recombinant plasmid pP43NMK-bp-dae, and verifying the size of the recombinant plasmid by agarose gel electrophoresis, and the electrophoresis results are shown in FIG. 1 (b).
As can be seen from FIG. 1 (b), the recombinant plasmid pP43NMK-bp-dae was about 7600bp in size, which corresponds to the theoretical value, in comparison with the plasmid standard band. This indicates that the recombinant plasmid pP43NMK-bp-dae was successfully obtained.
Example 2: construction of recombinant vector pP43NMK-rhaA
(1) According to the gene sequence rhaA of the L-rhamnose isomerase and the characteristics of gene elements on the vector pP43NMK, designing a primer by utilizing oligo7.0 software, wherein the primer sequences are shown as SEQ ID No. 6-SEQ ID No. 7.
rhaA-F：aggtaagagaggaatgtacacatgaccataaaagccaattatgac(SEQ ID No:6)；
rhaA-R：aggtaagagaggaatgtacacatgaccataaaagccaattatgac(SEQ ID No:7)。
(2) PCR amplification was performed using the Bacillus subtilis Bacillus subtilis 168 genome as template, using rhaA-F and rhaA-R primer pairs, PCR reaction parameters: pre-denaturation at 98℃for 1min; denaturation at 98℃for 10s; annealing at 55 ℃ for 30s; extending at 72 ℃ for 20s; after termination of the extension at 72℃for 5min,32 cycles, a rhaA gene fragment containing the homology arm of pP43NMK was obtained.
The PCR amplified products obtained were all detected by 1% agarose gel electrophoresis, and the detection results are shown in FIG. 2 (a). As can be seen from the figure, the band size of the rhaA gene containing pP43NMK homology arm, in which the linearized plasmid pP43NMK band size is about 6700bp, is about 1275bp, and is consistent with the expected size. This indicates that a rhaA gene fragment containing the pP43NMK homology arm was successfully obtained.
(3) And (3) carrying out recombination connection on the obtained linearization plasmid pP43NMK and rhaA gene fragments containing pP43NMK homology arms according to a seamless cloning kit description method, mixing the linearization plasmid pP43NMK and rhaA gene fragments containing pP43NMK homology arms according to a ratio of 1:3 (mol/mol), adding 2X MultiF Seamless Assembly Mix with a volume which is doubled, and connecting at 50 ℃ for 30min to obtain a connection product.
Adding the ligation product into E.coli JM109 competent cells, uniformly mixing, standing on ice for 30min, carrying out heat shock at 42 ℃ for 60s, cooling on ice for 2min, adding 900 mu L of LB culture medium, incubating at 37 ℃ for 1h, coating an ampicillin antibiotic LB plate containing 50 mu g/mL, culturing overnight, picking up positive transformants, culturing overnight, extracting plasmids by using a plasmid miniprep kit, and obtaining recombinant plasmids pP43NMK-rhaA.
The recombinant plasmid size was verified by agarose gel electrophoresis, and the electrophoresis results are shown in FIG. 2 (b). As can be seen from the figure, the recombinant plasmid size was about 7960bp compared with the plasmid standard band, which is consistent with the theoretical value, indicating that the recombinant plasmid pP43NMK-rhaA was successfully obtained.
EXAMPLE 3 construction of recombinant vectors pP43NMK-bp-dae-rhaA and pP43NMK-rhaA-bp-dae
(1) According to the gene sequence bp-dae of D-psicose 3-epimerase, the gene sequence rhaA of L-rhamnose isomerase and the gene element characteristics on the vector pP43NMK, designing a primer by utilizing oligo7.0 software, wherein the primer sequence is shown as SEQ ID No. 8-SEQ ID No. 15.
P1：aggtaagagaggaatgtacacatgaacaaaattggcatttattttg(SEQ ID No:8)；
P2：tttatggtcattaaatcgctcctttttaggtggcttagtttttgccatcttgatc(SEQ ID No:9)；
P3：gcaaaaactaagccacctaaaaaggagcgatttaatgaccataaaagccaattatgac(SEQ ID No:10)；
P4：gattacgccaagctttcatcattagacaatcggagaagatgc(SEQ ID No:11)；
P5：aggtaagagaggaatgtacacatgaccataaaagccaattatgac(SEQ ID No:12)；
P6：attttgttcattaaatcgctcctttttaggtggcttagacaatcggagaagatgc(SEQ ID No:13)；
P7：cgattgtctaagccacctaaaaaggagcgatttaatgaacaaaattggcatttatttt(SEQ ID No:14)；
P8：gattacgccaagctttcatcattagtttttgccatcttgatc(SEQ ID No:15)。
(2) Respectively using the pP43NMK-bp-dae obtained in the example 1 and the pP43NMK-rhaA obtained in the example 2 as templates, respectively amplifying to obtain bp-dae-rbs and rbs-rhaA gene fragments by using primer pairs P1/P2 and P3/P4, respectively amplifying to obtain rhaA-rbs and rbs-bp-dae by using primer pairs P5/P6 and P7/P8; reference is made to Table 1 for PCR reaction systems and procedures.
TABLE 1 PCR amplification System (50. Mu.L)
Note that: x is x 1 When the plasmid is used as a template<1.0ng, when genome is taken as a template, the genome is less than or equal to 100ng;
x 2 taking plasmid as a template for 1min and taking genome as a template for 3min;
x 3 3-5 ℃ lower than the Tm value of the primer;
x 4 the amplification efficiency of 2×Pfx Master Mix was 4kb/min, set according to the amplified fragment size;
wherein the denaturation, annealing and extension steps are cycled 32 times.
The PCR amplified products obtained were all detected by 1% agarose gel electrophoresis, and the detection results are shown in FIG. 3. As can be seen from the figure, the linearized plasmid pP43NMK band size is approximately 6700bp (FIG. 3 (a)), the bp-dae-rbs and rbs-rhaA gene band sizes are approximately 2300bp (FIG. 3 (b)), consistent with the expected size. This demonstrates the success of obtaining linearized plasmid pP43NMK with fragments containing bp-dae-rbs and rbs-rhaA genes.
(3) The obtained linearization plasmid pP43NMK and the gene fragments containing bp-dae-rbs and rbs-rhaA are respectively recombined and connected according to the description method of a seamless cloning kit, the linearization plasmid pP43NMK and the gene fragments containing bp-dae-rbs and rbs-rhaA are respectively mixed according to the ratio of 1:3 (mol/mol), and 2X MultiF Seamless Assembly Mix with the volume of one time is added, and the mixture is connected for 30 minutes at 50 ℃ to obtain a connection product.
The ligation product was added to E.coli JM109 competent cells and mixed well, placed on ice for 30min, heat-shocked at 42℃for 60s, cooled on ice for 2min, then added with 900. Mu.L of LB medium, incubated at 37℃for 1h, plated with LB plate containing 50. Mu.g/mL of ampicillin antibiotics, cultured overnight, then the positive transformants were picked up overnight for culturing, plasmids were extracted using a plasmid miniprep kit to obtain recombinant plasmids pP43NMK-bp-dae and pP43NMK-rhaA-bp-dae, and the recombinant plasmid sizes were verified by agarose gel electrophoresis, and the electrophoresis results are shown in FIG. 3 (c).
As can be seen from the figure, the recombinant plasmids pP43NMK-bp-dae-rhaA and pP43NMK-rhaA-bp-dae were approximately 8900bp in size, which is consistent with the theoretical value, in comparison with the plasmid standard band. This indicates that the recombinant plasmids pP43NMK-bp-dae-rhaA and pP43NMK-rhaA-bp-dae were obtained successfully.
EXAMPLE 4 construction and expression of genetically engineered bacteria
The embodiment respectively constructs the genetic engineering bacteria for independently expressing DAE, the genetic engineering bacteria for independently expressing L-RI and the genetic engineering bacteria for jointly expressing D-psicose 3-epimerase and L-rhamnose isomerase, and the specific construction method is as follows.
(1) Preparation and transformation of bacillus subtilis competence:
s1, taking out the B.subtilis WB800N glycerol tube storage strain from the temperature of minus 80 ℃, melting on ice, dipping a proper amount of bacterial liquid on a solid LB plate by using an inoculating needle in an ultra-clean workbench, streaking and separating, and culturing for 12h at the temperature of 37 ℃. And taking out the activated flat plate, picking single bacterial colony, transferring the single bacterial colony into 10mL of LB liquid medium, and carrying out shake culture at 37 ℃ for 10-12 h to obtain bacterial liquid.
S2, transferring 200 mu L of the bacterial liquid obtained in the step S1 into 10mL of SPI culture medium, shake culturing at 37 ℃ for 3 hours, and then starting to measure OD 600 When the culture grows to the end of logarithmic growth (about 4.5 h), 1mL is quickly inoculated into 10mL of SP II culture medium, and the culture is put into a shaking table (37 ℃ C., 100 rpm) for culturing for 1.5h, so that bacterial liquid is obtained.
S3, adding 100 mu L of 100 XEGTA solution into the bacterial liquid in the S2, culturing for 10min on a shaking table (37 ℃ C., 100 rpm), and sub-packaging into 500 mu L of each tube by using a 1.5mL sterile centrifuge tube.
S4, adding 5 mu L (about 500 ng) of recombinant plasmids pP43NMK-bp-dae, pP43NMK-rhaA, pP43NMK-bp-dae-rhaA and pP43NMK-rhaA-bp-dae into each tube of competent cells, gently mixing, and culturing for 30min at 100rpm at a temperature of below 37 ℃.
S5, transferring the centrifuge tube in S4 to 37 ℃, shaking at 250rpm for 1.5 hours, centrifuging at 4000rpm to collect thalli, discarding part of supernatant and re-suspending thalli, coating on LB plate (yeast powder 5g/L, tryptone 10g/L, sodium chloride 10g/L, agar powder 15 g/L) containing 50 mug/mL erythromycin, culturing overnight in a 37 ℃ incubator, and preserving positive transformant and naming as B.subtilis P 43 -DAE、B.subtilis P 43 -L-RI、B.subtilis P 43 DAE-L-RI and B.sub.ilis P 43 -L-RI-DAE。
(2) Taking B.subtilis WB800N protobacteria as blank control, and taking the different bacillus subtilis engineering bacteria B.subtilis P obtained in the step (1) 43 -DAE、B.subtilis P 43 -L-RI、B.subtilis P 43 DAE-L-RI and B.sub.ilis P 43 The L-RI-DAE was inoculated into an erythromycin LB liquid medium (yeast powder 5g/L, tryptone 10g/L and sodium chloride 10 g/L) containing 50. Mu.g/mL and cultured for 24 hours, and the protein expression was detected by SDS-PAGE protein gel electrophoresis.
FIGS. 4 (a) and 4 (b) are engineering bacteria B.subtilis P, respectively 43 DAE and B.sub.ulis P 43 The recombinant protein expression of the-L-RI is respectively 34.5 in engineering bacteria compared with the original bacteriaThe DAE and the L-RI were expressed correctly and efficiently, as shown by clear and bright protein bands at kDa and 49Da, which are consistent with the theoretical values.
FIG. 4 (c) shows engineering bacterium B.subtilis P 43 DAE-L-RI (FIG. 4c, lane 2) and B.subilis P 43 The recombinant protein expression of L-RI-DAE (FIG. 4 (c), lane 3) shows clear and bright protein bands at 34.5kDa and 49kDa for the engineering bacteria compared with the original bacteria, and accords with the theoretical value, and it is known that DAE and L-RI are successfully co-expressed in the same strain.
Example 5: one-step whole-cell mixed conversion of D-fructose to co-produce D-psicose and D-psicose
(1) Preparation of resting cells:
a single B.sub.lis-DAE positive clone was inoculated into erythromycin LB medium (yeast powder 5g/L, tryptone 10g/L and sodium chloride 10 g/L) containing 50. Mu.g/mL, cultured at 37℃and 200rpm for 12 hours as a seed solution, followed by 1% addition of 50. Mu.g/mL of TB medium (yeast powder 24g/L, tryptone 12g/L, glycerol 4mL, monopotassium phosphate 2.31g/L and monopotassium phosphate 16.42 g/L) for expansion culture, shaking culture at 37℃and 200rpm for 24 hours, centrifuging to discard the medium, collecting the cells, washing the collected cells with 50mM phosphate buffer of pH 7.0 for 2 times, and finally collecting the cells by centrifugation to obtain resting cells of B.sub.lis-DAE. And similarly, resting cells of other genetically engineered bacteria can be obtained.
(2) Selection of bicistronic:
preparation of a recombinant plasmid Gene engineering bacterium (B.subilis P) 43 -DAE-L-RI，B.subtilis P 43 -L-RI-DAE) and carrying out a transformation reaction with D-fructose as substrate, the initial transformation conditions being set as: the bacterial body amount is 20g/L; d-fructose concentration, 100g/L (50 mM phosphate solution pH 8.0); metal ion concentration, 1mM Mn 2+ The method comprises the steps of carrying out a first treatment on the surface of the The reaction temperature is 60 ℃; rotational speed, 220rpm; conversion time, 24h. The reaction conversion solution was obtained, and the total yield of D-psicose and D-psicose was measured, and the results are shown in FIG. 5.
As can be seen from FIG. 5, in the first step of D-fructose conversion reaction, B.subtilis P 43 DAE-L-RI (black histogram) convertibleMore D-fructose is D-psicose, which provides enough substrate for the conversion of D-psicose in the second conversion reaction, so that the reaction is finally converted to more D-psicose, and thus the sequence of BP-DAE co-expression with L-RI can be determined to be 5'-BP-DAE-rbs-rhaA-3' and engineering bacterium B.subilis P is used 43 -DAE-L-RI subsequent experiments were performed.
(3) Whole cell mixed transformation D-fructose:
recombinant strain B.subilis P to express BP-DAE, L-RI and Co-express BP-DAE and L-RI, respectively 43 -DAE、B.subtilis P 43 L-RI and B.subtilis P 43 Whole cells prepared from DAE-L-RI are designated as W1, W2 and W3. Based on different whole cell ratios, 8 whole cell mixed transformation systems are designed and named MS-1 to MS-8.
Wherein the systems MS-1, MS-7 and MS-8 only contain whole cells W2, W1 and W3 respectively; the actual mixing transformation systems (MS-2, MS-3, MS-4, MS-5 and MS-6) had the addition ratios of whole cells W1 and W2 of 1:7, 1:3, 1:1, 3:1 and 7:1, respectively.
The whole cell mixed transformation system is respectively resuspended by a proper amount of 50mM phosphate buffer solution with pH of 8.0 and sub-packaged in conical flasks, and the cell quantity is 20g/L; d-fructose concentration, 100g/L (50 mM phosphate solution pH 8.0); metal ion concentration, 1mM Mn 2+ The method comprises the steps of carrying out a first treatment on the surface of the The reaction temperature is 60 ℃; rotational speed, 220rpm; conversion time, 24h as initial conversion condition, and rare sugar production performance test was performed on the above 8 whole cell mixed systems.
The reaction solution was boiled for 10 minutes, centrifuged at high speed for 5 minutes, and then diluted with a corresponding multiple of 0.22 μm mixed fiber membrane, and analyzed by using a high performance liquid chromatograph RID detector under conditions of liquid chromatography column Hi-Plex Ca,300X7.7 mm, column temperature of 84℃and mobile phase ultra-pure water. The flow rate was 0.6mL/min, the single sample run time was 25min, and the detector temperature was 45 ℃. The contents of D-fructose, D-psicose and D-psicose in each mixed system are shown in FIG. 6 (a), the total amounts of D-psicose and D-psicose are shown in FIG. 6 (b), and the contents of D-psicose are shown in FIG. 6 (c).
As can be seen from FIGS. 6 (a) and 6 (b), D-psicose and D were mixed in MS-5The total amount of allose is 31.5g/L higher, but only contains engineering bacteria B.subtilis P 43 The total amount of D-psicose and D-psicose in the MS-8 system of DAE-L-RI is at most 33g/L, so the system is selected for the subsequent optimization of the conversion process.
(4) Condition optimization of whole cell catalytic reaction:
selecting pH, temperature, substrate concentration, thallus quantity and Mn 2+ The metal ion concentration is used as a key factor, the rare sugar yield is used as an index, and a single factor optimization experiment is carried out on the whole cell bioconversion process of the optimal system MS-8, and the result is shown in figure 7.
From the results shown in FIGS. 7 (a) - (e), it can be seen that the optimal conversion conditions for system MS-8 are: the bacterial body amount is 20g/L; d-fructose concentration, 100g/L (50 mM phosphate solution pH 8.5); metal ion concentration, 1mM Mn 2+ The method comprises the steps of carrying out a first treatment on the surface of the The reaction temperature was 65 ℃.
Under the optimal conditions, the transformation progress was monitored, and the results are shown in fig. 7 (f). The results show that under the optimal reaction conditions, the content of D-psicose gradually decreases, the content of D-psicose continuously increases, the balance is achieved in 18 hours, the production intensity of rare sugar reaches 1.94g/L/h, the total yield of D-psicose and D-psicose is 35g/L, and the yield is improved by 6.06% compared with 33g/L when the yield is not optimized.
(5) Tandem promoters optimize the amount of expression of the double enzymes:
s1, series connection of promoters:
PCR amplification of four strong constitutive promoters P from the B.subtilis 168 genome HpaⅡ (SEQ ID No:16)、P 43 (SEQ ID No:17)、P spoVG (SEQ ID No:18)、P srfA (SEQ ID No: 19), and performing tandem combination of the four strong promoters to obtain 14 tandem promoter combinations, replacing P in the co-expression recombinant plasmid pP43NMK-bp-dae-rhaA 43 The single promoter constructs 14 tandem promoter co-expression recombinant plasmids, and the recombinant plasmids are transformed into bacillus subtilis WB800N, and the obtained tandem promoter genetic engineering bacteria are named as B.subtilis Y (Y is D1-D14), and the expression is shown in Table 2; the expression of the recombinant protein of the genetically engineered bacterium is shown in figure 8.
SEQ ID No:16：
tactacctgtcccttgctgatttttaaacgagcacgagagcaaaacccccctttgctgaggtggcagagggcaggtttttttgtttcttttttctcgtaaaaaaaagaaaggtcttaaaggttttatggttttggtcggcactgccgacagcctcgcagagcacacactttatgaatataaagtatagtgtgttatactttacttggaagtggttgccggaaagagcgaaaatgcctcacatttgtgccacctaaaaaggagcgatttacat；
SEQ ID No:17：
tgataggtggtatgttttcgcttgaacttttaaatacagccattgaacatacggttgatttaataactgacaaacatcaccctcttgctaaagcggccaaggacgctgccgccggggctgtttgcgtttttgccgtgatttcgtgtatcattggtttacttatttttttgccaaagctgtaatggctgaaaattcttacatttattttacatttttagaaatgggcgtgaaaaaaagcgcgcgattatgtaaaatataaagtgatagcggtaccattataggtaagagaggaatgtacac；
SEQ ID No:18：
tgcggaagtaaacgaagtgtacggacaatattttgacactcacaaaccggcgagatcttgtgttgaagtcgcgagactcccgaaggatgcgttagtcgagatcgaagttattgcactggtgaaataataagaaaagtgattctgggagagccgggatcacttttttatttaccttatgcccgaaatgaaagctttatgacctaattgtgtaactatatcctattttttcaaaaaatattttaaaaacgagcaggatttcagaaaaaatcgtggaattgatacactaatgcttttatataggg；
SEQ ID No:19：
atatcgacaaaaatgtcatgaaagaatcgttgtaagacgctcttcgcaagggtgtctttttttgcctttttttcggtttttgcgcggtacacatagtcatgtaaagattgtaaattgcattcagcaataaaaaaagattgaacgcagcagtttggtttaaaaatttttatttttctgtaaataatgtttagtggaaatgattgcggcatcccgcaaaaaatattgctgtaaataaactggaatctttcggcatcccgcatgaaacttttcacccatttttcggtgataaaaacatttttttcatttaaactgaacggtagaaagataaaaaatattgaaaacaatgaataaatagccaaaattggtttcttattagggtggggtcttgcggtctttatccgcttatgttaaacgccgcaatgctgactgacggcagcctgctttaatagcggccatctgttttttgattggaagcactgctttttaagtgtagtactttgggctatttcggctgttagttcataagaattaaaagctgatatggataagaaagagaaaatgcgttgcacatgttcactgcttataaagattagg。
TABLE 2 tandem double promoter combinations
As can be seen from FIG. 8, it is associated with a single promoter P 43 (FIG. 8, lane 2) comparison, P, which is the combination of D5, D10 and D14 HpaⅡ -P spoVG (FIG. 8, lane 7), P spoVG -P 43 (FIG. 8, lane 12) and P srfA -P spoVG (FIG. 8, lane 16) the BP-DAE and L-RI protein bands were thicker under the tandem promoters, and it was initially determined that the co-expression amounts of the two enzymes under these combinations were more excellent. And replaced by a combination of D1, D3 and D4, i.e. P 43 -P 43 (FIG. 8, lane 3), P srfA -P srfA (FIG. 8, lane 5) and P 43 -P HpaⅡ (FIG. 5.4, lane 6) and the like, there is little band at the size position of the target protein; through preliminary analysis of SDS-PAGE, the expression condition of recombinant proteins of different tandem promoter genetic engineering bacteria can be known, and the optimal tandem promoter combination can be screened out by testing the rare sugar production capacity of the recombinant proteins.
S2, screening of tandem promoter combinations:
genetically engineered resting cells for the preparation of B.subtilis Y (wherein Y: D1 to D14) containing P according to the production efficiency of psicose and D psicose, i.e.the total amount of rare sugars 43 The single-promoter gene engineering bacteria system MS-8 is used as a control, and the system containing different tandem promoter gene engineering bacteria is screened, and the transformation conditions are as follows: the bacterial body amount is 20g/L; d-fructose concentration, 100g/L (50 mM phosphate solution pH 8.5); metal ion concentration, 1mM Mn 2+ The method comprises the steps of carrying out a first treatment on the surface of the The reaction temperature is 65 ℃; rotational speed, 220rpm; conversion time, 18h. The production efficiency of the rare sugars (D-psicose and D-psicose) of each system is shown in FIG. 9.
As can be seen from fig. 8 and 9, the rare sugar production is not found in the D1 and D4 promoter combinations, but the rare sugar production efficiency in the D3 promoter combinations is not much higher than that in the single promoter combinations, but rather is greatly reduced, which may be related to gene silencing or co-suppression caused by the repeated use of the same constitutive promoter to express multiple genes;the detection result is consistent with the SDS-PAGE analysis result, D5 (P HpaⅡ -P spoVG ) The BP-DAE and L-RI co-expression level under the combination of the promoters is optimal, and the system is named as MS-8-D5, the total yield of rare sugar is 39g/L, and the production efficiency is as high as 2.17g/L/h.
(6) Reaction progress of the optimal system MS-8-D5 for converting D-fructose:
the reaction progress of the system MS-8-D5 bioconversion D-fructose was measured under the optimum conditions, and the consumption of D-fructose and the production of rare sugar at each time period were recorded, and the results are shown in FIG. 10.
As can be seen from FIG. 10, D-fructose was largely converted into D-psicose by BP-DAE 4 hours ago, at which stage the D-psicose content was low; as the reaction proceeds, the content of D-psicose starts to decrease from 8h, the L-RI continuously catalyzes the D-psicose to be D-psicose, the content of D-psicose gradually increases, the content of D-psicose, D-psicose and D-fructose in the system tends to be stable when the reaction proceeds for about 16h, the balance ratio is 15:24:61, and the D-fructose conversion rate under the system is as high as 39%.
The reaction of converting the D-fructose by the MS-8-D5 system reaches reaction balance after 16 hours, the actual production efficiency of the D-psicose and the D-psicose is 2.44g/L/h (16 hours), the total yield of rare sugar is 39g/L, and compared with 35g/L produced by the MS-8 system, the actual production efficiency of the D-psicose and the D-psicose is improved by 11.42 percent; in other studies, the double enzyme cascade reaction using 100g/L of D-fructose as a substrate and pure enzyme forms of DAE (from Agrobacterium sp.ATCC 31749) and L-RI (from Thermoanaerobacterium saccharolyticum NTOU 1) as catalysts was carried out, but the balance ratio of D-allose, D-allose and D-fructose was 12:22:66, the yield of D-allose was 12g/L, the total yield of rare sugars was 34g/L, and the whole cell yield of the genetically engineered bacterium involved in the patent was relatively higher, pure enzyme was not required and the whole cell preparation method was simple. Therefore, the whole cell transformation system of the double enzyme cascade constructed by optimizing the promoter can effectively utilize cheap sugar to be transformed into rare sugar with higher added value.
Example 6: construction and application of a multi-bacterium mixed transformation system:
preparation of recombinant Strain B.subilis P 43 -DAE、B.subtilis P 43 L-RI and B.subtilis P 43 The whole cells of DAE-L-RI are then inoculated into TB medium for culture, and the cells are collected by centrifugation and washed twice with buffer solution to obtain the corresponding resting cells, which are designated as W1, W2 and W3, respectively.
The W1 and W2 were combined in different ratios, and 8 multi-fungus mixed transformation systems were designed, and specific combination ratios are shown in Table 3:
TABLE 3 combination ratio Table of Multi-fungus Mixed conversion System
And the initial transformation conditions were set to: the bacterial body amount is 20g/L; d-fructose concentration, 100g/L (50 mM phosphate solution pH 8.0); metal ion concentration, 1mM Mn 2+ The method comprises the steps of carrying out a first treatment on the surface of the The reaction temperature is 60 ℃; rotational speed, 220rpm; conversion time, 24h. The total yield of D-psicose and D-psicose was examined using the above conditions.
In the system MS-1, since only whole cells W2 expressing L-RI are contained, but whole cells W1 expressing BP-DAE are not contained, the system MS-1 does not have the capability of converting D-fructose into D-psicose, and D-psicose cannot be produced; d-allose can be produced by a mixed conversion system (MS-2 to MS-7) containing different ratios of whole cells W1 and W2, the content of D-allose is gradually increased along with the continuous increase of the ratio of the whole cells W1, when the addition ratio of the whole cells W1 to W2 is 3:1 (namely, the system MS-5) reaches the highest yield, the ratio of the whole cells W1 is continuously increased afterwards, the yield of D-allose is reduced (as shown in a figure 6 (c)), and in the system MS-7, although the system MS-7 only contains the whole cells W1, the host bacillus subtilis WB800N has the encoding genes rhaA of L-RI and L-RI which are expressed in the bacterial culture process, so the D-allose can be still converted into the D-allose; in the system MS-8, although only whole cell W3 was contained, the yield of D-allose was comparable to that of the mixed system MS-5.
As shown in FIG. 6 (b) and FIG. 6 (c), wherein the optimal transformation system MS-8 produced D-psicose at 22g/L and D-psicose at 11g/L; the yield of the D-psicose in the mixed conversion system MS-5 is 21g/L, the yield of the D-psicose is 10.5g/L, the total yield of the D-psicose and the D-psicose is slightly lower than 33g/L of the system MS-8, wherein the balance ratio of the D-psicose, the D-psicose and the D-fructose in the system MS-5 and the system MS-8 is 10.5:21:68.5 and 11:22:67 respectively; this may be related to the efficiency of the transport of the metabolite within the cell, with a single whole cell with both enzymes as a catalyst effectively increasing the local concentration of the intermediate product facilitating the next conversion reaction; the metabolites, in turn, reduce their transformation efficiency and, to some extent, yield, among cells.
In summary, the invention constructs the bacillus subtilis engineering bacteria capable of producing D-psicose and D-psicose with high yield by optimizing the promoter, transforming the expression plasmid and using B.subtilis WB800N as the expression host, and uses resting cells of the bacillus subtilis engineering bacteria as the catalyst for reaction, and the resting cells have simple preparation process, improve the tolerance of the enzyme to acid and alkali and heat to a certain extent, and improve the expression level of double enzymes by serially combining various promoters endogenous to the bacillus subtilis, thereby improving the utilization rate of D-fructose and finally efficiently co-producing the D-psicose and the D-psicose. The examples are preferred embodiments of the present invention, but the present invention is not limited to the above-described embodiments, and any obvious modifications, substitutions or variations that can be made by one skilled in the art without departing from the spirit of the present invention are within the scope of the present invention.
Claims (10)
1. A genetically engineered bacterium for the combined expression of D-psicose 3-epimerase and L-rhamnose isomerase, characterized in that the genetically engineered bacterium comprises a recombinant vector for the combined expression of D-psicose 3-epimerase and L-rhamnose isomerase.
2. The genetically engineered bacterium that expresses D-psicose 3-epimerase and L-rhamnose isomerase in combination of claim 1, wherein the starting strain of the genetically engineered bacterium is bacillus subtilis.
3. The genetically engineered bacterium that expresses D-psicose 3-epimerase and L-rhamnose isomerase in combination of claim 1, wherein the vector of the recombinant vector that expresses D-psicose 3-epimerase and L-rhamnose isomerase in combination comprises pP43NMK;
the promoter comprises P HpaⅡ 、P 43 、P spoVG Or P srfA One or two of them.
4. The genetically engineered bacterium for the combined expression of D-psicose 3-epimerase and L-rhamnose isomerase according to claim 3, wherein the promoter is P HpaⅡ And P spoVG A promoter combination.
5. A microbial agent comprising the genetically engineered bacterium of any one of claims 1 to 4.
6. A recombinant vector, characterized in that the recombinant vector expresses D-psicose 3-epimerase and L-rhamnose isomerase in combination.
7. A multi-bacterial hybrid transformation system, characterized in that the multi-bacterial hybrid transformation system comprises:
the genetically engineered bacterium of any one of claims 1 to 4 that expresses D-psicose 3-epimerase and L-rhamnose isomerase in combination; and/or
And (3) a mixed bacterial liquid of a genetically engineered bacterium which independently expresses D-psicose 3-epimerase and a genetically engineered bacterium which independently expresses L-rhamnose isomerase.
8. The multi-bacterium mixed transformation system according to claim 7, wherein the content of the genetically engineered bacterium which expresses D-psicose 3-epimerase alone in the mixed bacterial liquid is 12.5 to 87.5%, and the content of the genetically engineered bacterium which expresses L-rhamnose isomerase alone is 87.5 to 12.5%.
9. Use of the genetically engineered bacterium of any one of claims 1 to 4, or the microbial agent of claim 5, or the multi-bacterial mixed conversion system of any one of claims 7 to 8 for catalyzing D-fructose to prepare D-psicose and/or D-psicose.
10. A process for producing D-psicose and D-psicose, which comprises inoculating the genetically engineered bacterium according to any one of claims 1 to 4, the microbial agent according to claim 5, or the multi-bacterial mixed transformation system according to any one of claims 7 to 8 to D-fructose.
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