CN118064346A - Recombinant genetic engineering strain, construction method thereof and fermentation production method of PQQ - Google Patents

Recombinant genetic engineering strain, construction method thereof and fermentation production method of PQQ Download PDF

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CN118064346A
CN118064346A CN202410465220.5A CN202410465220A CN118064346A CN 118064346 A CN118064346 A CN 118064346A CN 202410465220 A CN202410465220 A CN 202410465220A CN 118064346 A CN118064346 A CN 118064346A
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CN118064346B (en
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袁强
郭帅印
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Shandong Juntai Pharmaceutical Co ltd
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Abstract

The invention belongs to the technical field of PQQ production, relates to a technology for producing PQQ by fermentation, and in particular relates to a recombinant genetic engineering strain, a construction method thereof and a fermentation production method of PQQ. Coli BL21 (DE 3) was used as an original strain, and the proB gene, argA gene, tyrR gene, trpE gene, pheA gene were knocked out, and PK gene, gdhA gene, fpK gene, PQQ synthesis gene cluster pqqABCDE/F and tdD gene were transferred to overexpress PK gene, gdhA gene, fpK gene, PQQ synthesis gene cluster pqqABCDE/F and tdD gene. Experiments show that the highest fermentation yield of the engineering strain can reach 2.6 g/L.

Description

Recombinant genetic engineering strain, construction method thereof and fermentation production method of PQQ
Technical Field
The invention belongs to the technical field of PQQ production, relates to a technology for producing PQQ by fermentation, and in particular relates to a recombinant genetic engineering strain, a construction method thereof and a fermentation production method of PQQ.
Background
The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.
PQQ and pyrroloquinoline quinone are important redox prosthetic groups, have various physiochemical functions, and have wide application in the fields of food, medicine and health, agriculture and animal husbandry and the like. With the development of medicine, cosmetics, health care products and food industry, the market demand of PQQ is gradually increased year by year, at present, the global main supplier is Mitsubishi gas chemistry in Japan, and in addition, PQQ is prepared by plant extraction, but the plant extraction has the problems of low content, high extraction difficulty, long production period and the like, and the chemical synthesis process is quite complex, the yield is low and the like, so that the method is not easy to popularize. In contrast, the biological fermentation preparation of PQQ has the advantages of mild conditions, easy control of the synthesis process, no environmental pollution and the like, and is the development direction of the future industrialized production of the PQQ.
The inventor researches and learns that the main problem of preparing the PQQ by biological fermentation is that the yield is low, in order to improve the yield of producing the PQQ by biological fermentation, the gene of the strain is recombined by adopting biological engineering at present, and the metabolic pathway of the strain is changed, so that the yield of the PQQ is changed, for example Yang Xuepeng and the like, and the yield of 1.98g/L is obtained by expressing the PQQ synthesis gene cluster pqqABCDEF from G.oxydans 621H in escherichia coli ESCHERICHIA COLI. Sun Jiguo et al expressed the PQQ synthetic gene cluster pqqABCDEF from K.pneumoniae in E.coli ESCHERICHIA COLI with a PQQ yield of 1700 nmol/L. Meulenberg et al cloned expression in E.coli of the 6.7 kb PQQ gene cluster derived from K.pneumoniae with a PQQ yield of only 280 nmol/L. Although the above-described method can improve the yield of PQQ to some extent, the yield thereof still remains to be further improved.
Disclosure of Invention
In order to solve the defects of the prior art, the invention aims to provide a recombinant genetic engineering strain, a construction method thereof and a fermentation production method of PQQ, and the invention enhances the synthesis force of Glu and Tyr through a CRISPR/Cas9 gene editing technology, overexpresses a PQQ synthesis gene cluster and promotes synthesis of a related gene tldD, so as to construct an escherichia coli engineering strain with high PQQ yield, and experiments show that the highest fermentation yield of the engineering strain can reach 2.6 g/L.
In order to achieve the above object, the present invention provides the following technical solutions:
In the first aspect, a recombinant genetically engineered strain is prepared by taking escherichia coli BL21 (DE 3) as a starting strain, knocking out a proB gene, an argA gene, a tyrR gene, a trpE gene and a pheA gene, transferring PK genes, gdhA genes, fpK genes and PQQ synthetic gene clusters pqqABCDE/F and tldD genes to enable the recombinant genetically engineered strain to overexpress the PK genes, gdhA genes, fpK genes and PQQ synthetic gene clusters pqABCDE/F and tldD genes.
In some embodiments, the target PAM sequence of the proB gene is shown in SEQ ID No.2, the target PAM sequence of the argA gene is shown in SEQ ID No.1, the target PAM sequence of the tyrR gene is shown in SEQ ID No.7, the target PAM sequence of the trpE gene is shown in SEQ ID No.8, the target PAM sequence of the pheA gene is shown in SEQ ID No.9, the base sequence of the PK gene is shown in SEQ ID No.44, the base sequence of the gdhA gene is shown in SEQ ID No.45, the base sequence of the fpK gene is shown in SEQ ID No.46, the base sequence of the pqqABCDE/F gene cluster is shown in SEQ ID No.47, and the base sequence of the tdd gene is shown in SEQ ID No. 48.
In a second aspect, a method for constructing the recombinant genetic engineering strain comprises the following steps:
Knocking out proB gene, argA gene, tyrR gene, trpE gene and pheA gene in escherichia coli genome to obtain mutant strain;
And transferring PK genes, gdhA genes, fpK genes and PQQ synthesis gene clusters pqqABCDE/F and tldD genes into the mutant strain to obtain the escherichia coli genetic engineering strain.
In some embodiments, the knockout is performed using CRISPR/Cas9 gene editing techniques.
Specifically, the process of knocking out by using CRISPR/Cas9 gene editing technology comprises the construction process of sgRNA vector and the construction process of donor DNA.
More specifically, in the construction process of the sgRNA vector, the primer sequences of the proB gene are respectively shown as SEQ ID NO.5 and SEQ ID NO.6, the primer sequences of the argA gene are respectively shown as SEQ ID NO.3 and SEQ ID NO.4, the primer sequences of the tyrR gene are respectively shown as SEQ ID NO.10 and SEQ ID NO.11, the primer sequences of the trpE gene are respectively shown as SEQ ID NO.12 and SEQ ID NO.13, and the primer sequences of the pheA gene are respectively shown as SEQ ID NO.14 and SEQ ID NO. 15.
More specifically, in the construction process of the donor DNA, the primer sequences for knocking out the proB gene are shown as SEQ ID NO. 20-23 respectively, the primer sequences for knocking out the argA gene are shown as SEQ ID NO. 16-19 respectively, the primer sequences for knocking out the tyrR gene are shown as SEQ ID NO. 24-27 respectively, the primer sequences for knocking out the trpE gene are shown as SEQ ID NO. 28-31 respectively, and the primer sequences for knocking out the pheA gene are shown as SEQ ID NO. 32-35 respectively.
In some embodiments, the PK gene, the gdhA gene, the fpK gene, the PQQ synthesis gene cluster pqqABCDE/F, and the tdD gene are transferred to the mutant strain by electrotransformation.
In the third aspect, the fermentation production method of the PQQ is realized by adopting the recombinant genetic engineering strain to ferment.
In some embodiments, the recombinant genetically engineered strain is seed cultured prior to performing the fermentation. Specifically, the culture medium for seed culture is LB culture medium.
In some embodiments, the conditions of the fermentation are: the temperature is 28-32 ℃ and the time is 48-72 h.
In some embodiments, the fermentation medium comprises ：0.9~1.1 g/L Glu、2.9~3.1 g/L L-Tyr、5.9~6.1 g/L KH2PO4、16.3~16.5 g/L K2HPO4、4.9~5.1 g/L (NH4)2SO4、1.0~1.2 g/L citric acid, 0.9-1.1 g/L MgSO 4, 9.9-10.1 g/L yeast extract, 6.9-7.1 g/L glucose, 0.09-0.11 g/L vitamin B1, and 1.9-2.1 mL/L trace elements.
In particular, the trace elements include ：9.9~10.1 g/L FeSO4·7H2O、1.52~1.53 g/L CaCl2、2.1~2.2 g/L ZnSO4·7H2O、0.9~1.1g MnSO4·4H2O、0.9~1.1 g/L CuSO4·5H2O、0.09~0.11 g/L (NH4)6Mo7O24·4H2O、0.19~0.21 g/L Na2B4O7·10H2O、0.9~1.1 g/L NiCl2、0.9~1.1 g/L H3BO3、9.9~10.1 mL/L HCl.
In some embodiments, the fermented feed medium contains: 490-510 g/L glucose, 6.9-7.1 g/L MgSO 4, 9.9-10.1 g/L Glu, 29-30 g/L Tyr, 0.9-1.1 mL/L trace element.
The beneficial effects of the invention are as follows:
The invention utilizes metabolic engineering means to increase the accumulation of glutamic acid by knocking out genes proB, argA, over-expressed genes PK and gdhA; the tyrR gene is knocked out to remove feedback regulation inhibition of tyrosine synthesis, the trpE and pheA are knocked out to simultaneously overexpress genes fpK to enhance tyrosine synthesis, and finally the PQQ synthesis gene cluster pqqABCDE/F and the gene tldD related to PQQ synthesis are overexpressed, so that the recombinant genetic engineering strain is constructed. The recombinant genetic engineering strain can accumulate Glu and Tyr with high efficiency, thereby effectively improving the fermentation efficiency of PQQ and achieving the purpose of high-yield PQQ. Experimental researches show that the highest yield of continuous fermentation of the recombinant genetic engineering strain provided by the invention can reach 2.6g/L, and a new scheme is provided for industrialized mass production of PQQ.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
FIG. 1 shows the PQQ synthesis pathway constructed in E.coli BL21 (DE 3) according to the present invention;
FIG. 2 is a schematic diagram showing the principle of the preparation of the donor DNA in the example of the present invention;
FIG. 3 is a construction map of pACYC-T7p-PK-RBS-gdhA-T7p-fpK-T7T overexpression vector in the example of the invention;
FIG. 4 is a diagram showing construction of pRSF-T7p-pqqABCDE/F-T7p-tldD-T7T overexpression vector in an embodiment of the present invention;
FIG. 5 is a bar graph of liquid phase analysis of PQQ yields in an embodiment of the invention.
Detailed Description
In order to enable those skilled in the art to more clearly understand the technical scheme of the present invention, the technical scheme of the present invention will be described in detail with reference to specific embodiments.
Examples
In E.coli BL21 (DE 3) (obtained by purchase), the PQQ synthesis pathway was constructed DE novo, the synthesis pathway of which is shown in FIG. 1, and Glu and Tyr synthesis forces were enhanced by CRISPR/Cas9 gene editing technique according to the synthesis pathway.
Preparing a corresponding sgRNA vector for gene knockout:
1. Knockout of the gene argA and proB blocks glutamate metabolism, promoting glutamate accumulation. Target PAM sequences of the genes argA and proB are shown in the following table:
Constructing an sgRNA vector by designing primers according to a PAM sequence, wherein the designed corresponding primers are shown in the following table:
The pTarget-sgRNA vector was used as a template, and the primers sg-argA-F/sg-argA-R, sg-proB-F/sg-proB-R were used for whole plasmid PCR, the polymerase used was KOD-Plus-Neo, the PCR procedure was 95℃for 3min,95℃for 10s,58℃for 10s,68℃for 1min,68℃for 5min,95℃for 10s,58℃for 10s,68℃for 1min, and 25 cycles were performed by digestion with nuclease DpnI to remove the template, and the product was recovered by the purification kit for use. The escherichia coli DH5a competence is transformed by a chemical method, positive clones are obtained by identification and sequencing, and the transformation method comprises the following steps: 50ng of whole plasmid PCR recovery product is added into 100ul competent cells, gently mixed, then kept stand on ice for 20min, subjected to water bath heat shock at 42 ℃ for 90s, kept stand on ice for 5min, 800ul of LB culture medium is added, 37 ℃ and 220r shaking table is incubated for 45min, finally a resistance plate is coated, the culture is carried out overnight at 37 ℃, and the monoclonal is selected for sequencing to obtain positive clones.
2. The knockdown gene tyrR releases upstream tyrosine synthesis feedback regulation inhibition, promotes tyrosine synthesis, and the knockdown genes trpE and pheA block synthesis tyrosine branch metabolism, and promotes tyrosine accumulation. The PAM sequences of the gene tyrR, trpE and pheA targets are shown in the following table:
Constructing an sgRNA vector by designing primers according to a PAM sequence, wherein the designed corresponding primers are shown in the following table:
Preparing corresponding donor DNA for gene knockout:
1. the donor DNA is designed for knocking out gene argA primer:
GGGTTACTCAACGCGAACTGCTTATA up-argA-F, shown as SEQ ID NO. 16;
ATCGCGGCACACCTCTTTGCATGATTATTCGAAATTAGTGT up-argA-R, shown as SEQ ID NO. 17;
dw-argA-F GCAAAGAGGTGTGCCGCGATGAAAATCGTCGGATGCGACATGCGTAAC, shown as SEQ ID NO. 18;
dw-argA-R GTACGAAGGTCGACCGGTGATGATTGC, shown in SEQ ID NO. 19.
The reference genome GenBank: CP001509 was used for primer design, E.coli BL21 (DE 3) genome was used as a template, and primers up-iclR-F/up-iclR-R and dw-iclR-R were used for PCR, respectively, the polymerase used was KOD-Plus-Neo, the PCR procedure was 3min pre-denatured at 95 ℃, 10s annealed at 58 ℃, 1min extended at 68 ℃, 5min extended at 68 ℃, 10s denatured at 95 ℃, 10s annealed at 58 ℃, 1min extended at 68 ℃, and 25 cycles were performed. Then, the product recovery was performed by the purification kit to perform the next overlap extension PCR, 100ng of each purified product was mixed and added to the polymerase, and the PCR procedure was performed for 10 cycles of 95℃pre-denaturation for 3min,95℃denaturation for 10s, 58℃annealing for 10s,68℃extension for 1min, 68℃final extension for 5min,95℃denaturation for 10s, 58℃annealing for 10s,68℃extension for 1min, and then the product recovery was performed by the purification kit as a Donor DNA for use.
2. The donor DNA is designed for knocking out a gene proB primer:
TCAGTTGATCGTTAATTTGTGTTTC of up-proB-F, shown as SEQ ID NO. 20;
CTGCTCCGATTCTCTGCCATTCAATTTTAGGAAAAATGATATC of up-proB-R, shown as SEQ ID NO. 21;
dw-proB-F GAATGGCAGAGAATCGGAGCAGGCTGATGCTGGAACAAATG, shown as SEQ ID NO. 22;
dw-proB-R GGATCACCGCATTACCGGTTTTCAGGC, shown in SEQ ID NO. 23.
And respectively carrying out PCR (polymerase chain reaction) on the two pairs of primers and the E.coli BL21 (DE 3) genome as a template to prepare an upstream homology arm and a downstream homology arm, and then carrying out overlap extension PCR on the upstream homology arm and the downstream homology arm to finally obtain the donor DNA.
3. The design of the donor DNA is that the tyrR primer of the knockout gene is designed:
GTCGATATGGCGTTTATCGCC of up-tyrR-F, shown as SEQ ID No. 24;
CATATTCGCGCGGGAACCTTCACCTGAAAAAAGAACAATTAATATG of up-tyrR-R, shown as SEQ ID NO. 25;
dw-tyrR-F GGTGAAGGTTCCCGCGCGAATATGCCTGATGGTGCAACACCATCAG, shown as SEQ ID No. 26;
dw-tyrR-R GCGCAGACTTTTACTCTCGTGGCAAAAG, shown as SEQ ID NO. 27.
And respectively carrying out PCR (polymerase chain reaction) on the two pairs of primers and the E.coli BL21 (DE 3) genome as a template to prepare an upstream homology arm and a downstream homology arm, and then carrying out overlap extension PCR on the upstream homology arm and the downstream homology arm to finally obtain the donor DNA.
4. The donor DNA is designed for knocking out gene trpE primer:
up-trpE-F GTGGTGTCATGGTCGGTGATC as shown in SEQ ID No. 28;
GTCAGCCATGTTATTCTCTAATTTTGTTCAAAAAAAAGCCCGC of up-trpE-R, shown as SEQ ID NO. 29;
dw-trpE-F GAGAATAACATGGCTGACATTCTGCTGCTCGA, shown as SEQ ID NO. 30;
dw-trpE-R CCATTAAAATGGGCGTTGATGGTTAAAC as shown in SEQ ID NO. 31.
And respectively carrying out PCR (polymerase chain reaction) on the two pairs of primers and the E.coli BL21 (DE 3) genome as a template to prepare an upstream homology arm and a downstream homology arm, and then carrying out overlap extension PCR on the upstream homology arm and the downstream homology arm to finally obtain the donor DNA.
5. Design of donor DNA (deoxyribonucleic acid) knockout gene pheA primer
CACAAGGGTTTGTTGCTGACGCCACAATC of up-pheA-F, shown as SEQ ID NO. 32;
CCGGCACCTTTTCAAGTGTTGCCTTTTTGTTATCAATAAAAAAGGC of up-pheA-R, shown as SEQ ID NO. 33;
dw-pheA-F GCAACACTTGAAAAGGTGCCGGATGATGTGAATCATCCGGCACTG, shown as SEQ ID NO. 34;
dw-pheA-R GCGTTTATTCAGGCTCTGCGCCACTTTG, shown as SEQ ID NO. 35.
And respectively carrying out PCR (polymerase chain reaction) on the two pairs of primers and the E.coli BL21 (DE 3) genome as a template to prepare an upstream homology arm and a downstream homology arm, and then carrying out overlap extension PCR on the upstream homology arm and the downstream homology arm to finally obtain the donor DNA.
The preparation principle of the donor DNA is shown in FIG. 2.
Constructing an over-expression vector:
The overexpression of the genes PK and gdhA promotes the synthesis of glutamic acid, the overexpression of the gene fpK promotes the synthesis of tyrosine, and the overexpression of the gene cluster pqqABCDE/F and the gene tldD promote the conversion of glutamic acid and tyrosine to synthesize PQQ. The two plasmid system was introduced to overexpress gene PK, gdhA, fpK, gene cluster pqqABCDE/F and tldD. Wherein the PK and gdhA genes are expressed in polycistronic form and the fpK gene is expressed in monocistronic form on the vector pACYCduent-1. The gene clusters pqqABCDE/F and tldD were expressed as monocistronic sequences on vector pRSFduent-1, respectively. Besides vectors pACYCduent-1 and pRSFduent-1, vectors having both expression cassettes, pETduent-1, pCDFduent-1, etc. may be used in this experiment. The carrier patterns are shown in figures 3-4.
Gene PK, gdhA, fpK and pACYC-T7p-PK-RBS-gdhA-T7p-fpK-T7T overexpression vector are synthesized by Beijing Hua big genes.
Construction of pRSF-T7p-pqqABCDE/F-T7p-tldD-T7T overexpression vector:
The gene cluster pqqABCDE/F and the gene tldD are amplified, wherein the gene cluster pqqABCDE/F and the gene tldD are all from G.oxydans 621H, and the genome of G.oxydans 621H is taken as a template for PCR amplification, and the primer design is carried out by referring to genome information of genome Accession number: CP000009, and the designed related primers are shown in the following table.
The genome of G, oxydans 621H is used as a template, a primer Pqq-F/Pqq-R is used for amplifying a 3.7kb PQQ synthetase gene cluster, a primer TldD-F/TldD-R is used for amplifying a tldD gene, a primer Bacb-1-F/Bacb-1-R is used for amplifying a carrier skeleton, polymerase is KOD-Plus-Neo, a PCR program is 95 ℃ pre-denatured for 3min, 10s is denatured at 95 ℃,10 s is annealed at 58 ℃, 3min is extended at 68 ℃, 5min is extended at 68 ℃,10 s is denatured at 95 ℃,10 s is annealed at 58 ℃, 1min is extended at 68 ℃, 25 cycles are performed, the PCR product is purified and recovered by a purification kit, firstly, a seamless cloning is performed on the amplification product of Pqq-F/Pqq-R and Bacb-1-F/Bacb-1-R, a positive carrier pRSF-T7 p-pqqABCDEF-T7T is obtained by sequencing, the carrier 3535-F/3792-F/Pqq-R is used as a template, a positive carrier pRSF-T7 p-35-T7T is obtained by sequencing, and a PCR kit is used for carrying out a seamless cloning with the amplification product of the step of carrying out the amplification kit, and the step of carrying out the PCR method is performed on the amplification product of the amplification kit of the step of carrying out the amplification kit for carrying out the seamless cloning.
Constructing a synthetic PQQ recombinant engineering strain:
The plasmid pACYC-T7p-PK-RBS-gdhA-T7p-fpK-T7T was co-transformed with the plasmid pRSF-T7p-pqqABCDE/F-T7p-tldD-T7T into the strains BL21 (DE 3) ΔargA, ΔproB, ΔtyrR, ΔtrpE, ΔpheA by means of electrotransformation. The process of the electrotransformation method is as follows:
(1) Electrotransformation competent preparation:
(1-1) picking single colony and shaking the colony in LB culture medium at 37 ℃ overnight;
(1-2) 2ml of overnight culture was transferred to 200ml of LB medium and vigorously shake-cultured on a shaking table at 37℃until OD600 = 0.6 (about 2.5-3 hours);
(1-3) rapidly placing the bacterial liquid on ice, and cooling the bacterial liquid on the ice for 10 minutes;
(1-4) centrifuging 3000g at 4 ℃ for 5 minutes, and collecting thalli;
(1-5) adding 30ml of 10% sterile glycerol solution, and re-suspending the cells;
(1-6) centrifuging 3000g at 4 ℃ for 5 minutes, and collecting thalli;
(1-7) 3ml of 10% sterile glycerol solution, re-suspending the thallus, and sub-packaging at-80 ℃ according to actual conditions for standby.
(2) Electric conversion:
respectively mixing 50ng of plasmid vector with 100ul competent cells, adding into 0.2cm electric shock cup after cooling, setting electric shock parameters to 2.5KV,200Ω, preferably electric shock constant of about 5ms, rapidly adding SOC recovery medium 37 ℃, recovering for 1h at 220r, and coating double-antibody plate with kanamycin and chloramphenicol resistance.
Shake flask fermentation test:
And (3) culturing and activating the engineering strain on an LB solid medium in a constant temperature oven with the marking temperature of 37 ℃ overnight, picking up the monoclonal strain into a shaking flask with the volume of 20mL of LB medium with the temperature of 37 ℃ and culturing the monoclonal strain in a shaking table with 220r overnight to obtain seed liquid, transferring the seed liquid into a shaking flask with the volume of 100mL of TB medium with the temperature of 37 ℃ according to the inoculation amount of 1%, culturing the strain with 220r until the OD600 = 0.6-0.8, adding an IPTG inducer to obtain the final concentration of 0.2Mm, and culturing the strain with 220r for 24 hours at the temperature of 37 ℃. 10ml of bacterial liquid is taken and crushed by ultrasonic waves, and liquid phase analysis is carried out, so that the result shows that PQQ synthesis products exist, and the yield reaches 472mg/L.
5L-fermenter test:
Inoculating the recombinant bacteria into a 500ml medicine bottle with 100ml LB culture medium, culturing overnight at 37 ℃ with a 220r shaking table to obtain seed liquid, transferring the seed liquid into a 5L fermentation tank containing 2.5L fermentation culture medium according to 2% of inoculation amount, and fermenting culture medium ：1g/L Glu、3g/L L-Tyr、6 g/L KH2PO4、16.4 g/L K2HPO4、5 g/L (NH4)2SO4、1.1 g/L citric acid, 1 g/L MgSO 4, 10g/L yeast extract, 7 g/L glucose, 0.1 g/L vitamin B1 and 2 mL/L microelements; the microelements comprise 10 g/L FeSO4·7H2O、1.53 g/L CaCl2、2.2 g/L ZnSO4·7H2O、1g MnSO4·4H2O、1 g/L CuSO4·5H2O、0.1 g/L (NH4)6Mo7O24·4H2O、0.2 g/L Na2B4O7·10H2O、1 g/L NiCl2、1 g/L H3BO3、10 mL/L HCl, , firstly, the microelements are dissolved in hydrochloric acid, and then deionized water is added to fix the volume to adjust the pH value to 7.0; feed medium: 500g/L glucose, 7 g/L MgSO 4, 10g/L Glu, 30g/L Tyr, 1 mL/L trace elements. And (3) continuously feeding, culturing and fermenting for 48-72 h at the temperature of 30 ℃, taking a proper amount of bacterial liquid, carrying out ultrasonic crushing, and carrying out liquid phase analysis, wherein the yield of the PQQ reaches 1.5 g/L-2.6 g/L, as shown in a figure 5, wherein the yield of the PQQ reaches 2.6g/L after the fermentation is carried out for 72 h.
PK base sequence:
ATGAGAAAAACTAAAATTGTTTGTACCATCGGTCCGGCAAGTGAAAGTATTGAAATGCTTACGAAATTAATGGAGTCAGGAATGAACGTGGCTCGATTAAACTTTTCTCACGGAGATTTTGAGGAGCACGGTGCAAGAATTAAAAATATCCGCGAAGCAAGTAAAAAACTTGGCAAGAACGTTGGAATTCTGCTTGATACAAAAGGTCCTGAAATCCGCACACATACAATGGAAAACGGCGGTATTGAGCTTGAAACAGGCAAAGAGCTCATTATTTCAATGGACGAGGTTGTAGGAACAAAAGATAAAATTTCAGTGACATATGAAGGTTTAGTCCATGACGTTGAACAAGGTTCAACGATTCTGTTAGATGACGGCCTTATCGGTCTTGAGGTACTTGATGTAGATGCCGCTAAACGCGAAATCAAAACAAAAGTATTAAACAACGGAACACTCAAAAATAAAAAAGGTGTTAACGTACCGGGCGTAAGTGTCAATCTTCCGGGGATTACTGAAAAGGATGCGCGAGACATCGTTTTCGGTATTGAGCAAGGAGTAGACTTCATCGCACCATCTTTCATTCGACGTTCTACGGATGTGCTCGAAATCCGTGAGCTTCTTGAAGAGCACAACGCTCAGGATATTCAAATCATCCCTAAAATCGAAAACCAAGAGGGCGTTGACAACATCGATGCGATTCTCGAAGTGTCTGACGGCTTAATGGTTGCACGCGGAGACTTAGGTGTGGAAATTCCAGCTGAAGAAGTGCCGCTTGTGCAAAAAGAACTGATCAAAAAATGCAACGCGCTGGGCAAACCTGTTATTACAGCGACACAAATGCTTGACAGCATGCAGCGCAACCCGCGTCCGACTCGTGCGGAAGCAAGTGACGTTGCAAACGCGATCTTCGACGGAACAGATGCGATCATGCTTTCTGGTGAAACTGCTGCCGGAAGTTACCCGGTTGAAGCAGTTCAAACAATGCATAACATCGCGTCCCGTTCTGAAGAAGCATTAAATTATAAAGAAATTCTCTCAAAACGCAGAGACCAAGTGGGCATGACAATTACAGACGCAATTGGACAATCTGTCGCACATACGGCGATTAACCTGAATGCTGCTGCGATCGTAACGCCGACAGAAAGCGGCCATACAGCACGTATGATTGCAAAATACCGTCCGCAGGCTCCGATTGTTGCGGTTACTGTAAATGACTCTATTTCCAGAAAGCTTGCCCTCGTATCTGGCGTATTCGCGGAAAGCGGCCAAAATGCGAGCTCAACAGATGAGATGCTTGAGGATGCTGTCCAAAAATCATTGAACAGCGGAATTGTAAAACACGGCGATCTTATCGTTATTACAGCTGGCACTGTCGGTGAGTCCGGCACTACGAACTTAATGAAGGTTCATACTGTCGGCGATATCATCGCTAAAGGCCAAGGCATTGGACGCAAATCAGCTTACGGTCCGGTTGTCGTTGCACAAAATGCAAAAGAAGCTGAGCAAAAAATGACTGACGGTGCGGTACTTGTTACCAAAAGCACTGACCGTGATATGATTGCATCCCTTGAAAAAGCGTCTGCTCTTATTACAGAAGAAGGCGGTTTGACTAGCCATGCTGCGGTAGTCGGATTAAGCCTTGGCATCCCGGTTATCGTGGGTCTGGAAAATGCGACATCTATTTTGACAGATGGCCAGGATATTACAGTTGACGCTTCCAGAGGCGCAGTCTATCAAGGCCGTGCGAGCGTTCTTTAA, As shown in SEQ ID NO. 44.
GdhA base sequence:
ATGTCCACGGTCCTGGAATACGTTGACCCTCTGGAAGGCTTTCGCGGCTGGCTGGTGTACGATGGCGGCTCTTGCCGTATCGCTGCAGGTGGTTGTCGCGTACAGCAGGGCCTGACTCGTGATACCCTGGTGTCTCTGGCATCCCGTATGACGCTGAAAGAACGTCTGCTGGGCATCAACGTTGACGGCGCAAAATGCGGCATCGACTACGATCCGCGTAGCCCTGGCAAAGCCGCGGCCGTTCGTCGCTTCCTGGCGTTCCTGCGCGAAGAACTGGTCCACCGTTTCTCCATGGGTAGCGACATGGGCACCCGTTGGGATGAACTGGAACGTCTGGCCGCGGCAGAGGGTATCCCATCCATCAAATACGCGGTACGCCGTGCGCAGGAATTTACCGACGATGAATTTTTTGCGCGCCTGCGCACCCTGCAGGCTCCAGTTGGTGCTCTGACTCTGGCACAGCGTCGTGCGGGTCATGCCCTGGCTGAAGCAGCAGTTGGTGCAGCACGTGCGGCAGGCCTGGGTCCGCGTGCAACTTGTGCGCTGCAGGGCTTTGGTAATCTGGGTCGTGCCGCGGCGTGTACCCTGGCTGAAGTGGGTATGACGGTGGTGGCGGTTTCCGACGAATTCGGTTGTGTTGCTGACCACCATGGCCTGGATATCCCGGGCATGCTGGCGGCCCCGTTCGGCACTCCAGTCCAGGCGTCCGTCCGTCGTGGTGAACAGCTGCCTTCCACCGCCCTGTTCAAAATTCCGGCTGACATCAGCATTCTGGCTGCCACCGAAAACGCTATCAGCCTGCCGCAAGTAGGCGAACTGCCGACCCCTGTGGTCGTGGTTGGCGCCAACTGCGGTCTGACCCCGGATGTTGAAGCAGCGCTGGACCGTGCGGGCGTACTGGTTATTCCGGATTTTGTTGGTGGCATCGGCGGCTCTGCCTCCGTGGAGGCTCTGTTCGGTCCGGAACACCGTCCGACTGAAGTTCACGTGCTGGGTGGCGTGACCCAGATCATGCGTCAGCTGGTTGACCACCTGATTTCCGAAGCTCGTCGTCGTGGCACGTCTGCTCGTTCCGTTGCGCTGCATCTGGCGCAGACGACCATCCCGGTGCCGGACCGCCGCCCGTATGGCCGTAGCCGTTTCCTGGCGTAA, As shown in SEQ ID NO. 45.
FpK base sequence:
ATGACCTCCCCTGTTATCGGTACCCCATGGAAAAAACTGAACGCGCCAGTTAGCGAAGAAGCCATTGAAGGCGTAGACAAGTACTGGCGCGCTGCTAACTACCTGAGCATCGGCCAGATTTACCTGCGCTCTAACCCGCTGATGAAGGAACCGTTCACCCGTGAAGATGTCAAACACCGTCTGGTTGGTCATTGGGGTACTACCCCGGGCCTGAACTTCCTGATCGGTCACATCAATCGTCTGATCGCTGATCACCAGCAGAACACCGTGATCATTATGGGCCCGGGTCACGGTGGTCCGGCGGGTACCGCACAGTCTTATCTGGACGGCACCTATACCGAATACTTCCCAAACATTACCAAAGACGAAGCCGGTCTGCAGAAATTTTTTCGTCAGTTCTCTTACCCGGGTGGCATCCCAAGCCATTACGCTCCGGAAACTCCTGGCTCTATCCACGAAGGTGGTGAGCTGGGCTACGCACTGTCTCATGCGTATGGTGCTGTTATGAACAACCCATCCCTGTTCGTGCCGGCCATTGTTGGCGACGGTGAAGCCGAAACTGGTCCGCTGGCTACCGGCTGGCAGAGCAACAAACTGATTAACCCACGTACTGACGGCATCGTTCTGCCGATCCTGCACCTGAATGGCTACAAAATTGCGAACCCGACCATTCTGAGCCGCATCAGCGACGAGGAACTGCATGAATTCTTCCACGGTATGGGCTACGAACCGTACGAATTTGTTGCAGGCTTCGATAACGAAGATCATCTGTCTATCCACCGTCGCTTCGCCGAACTGTTTGAGACCGTATTCGATGAGATCTGTGATATCAAAGCCGCTGCTCAGACCGACGATATGACGCGTCCGTTCTACCCGATGATCATCTTTCGTACTCCGAAAGGTTGGACCTGTCCGAAATTCATTGACGGTAAGAAAACCGAAGGTTCTTGGCGTTCTCATCAAGTTCCGCTGGCATCCGCTCGTGATACTGAGGCGCACTTCGAAGTTCTGAAAAACTGGCTGGAATCTTATAAACCGGAAGAACTGTTCGATGAGAACGGCGCTGTGAAACCAGAGGTCACGGCATTCATGCCGACGGGCGAACTGCGTATCGGTGAGAACCCGAACGCCAACGGTGGCCGTATTCGTGAAGAACTGAAACTGCCGAAACTGGAAGATTACGAAGTTAAGGAAGTCGCCGAGTACGGTCATGGCTGGGGCCAACTGGAAGCAACTCGCCGCCTGGGCGTGTACACTCGCGACATCATTAAAAACAACCCGGATTCTTTCCGTATCTTTGGTCCGGACGAAACCGCATCTAACCGTCTGCAGGCCGCGTATGATGTTACGAACAAACAGTGGGACGCTGGTTACCTGTCTGCGCAGGTCGATGAACACATGGCAGTGACCGGCCAGGTTACGGAACAACTGTCCGAACATCAGATGGAAGGTTTCCTGGAGGGCTACCTGCTGACCGGCCGCCACGGCATCTGGAGCAGCTATGAGTCCTTCGTTCACGTAATCGACTCTATGCTGAACCAGCATGCGAAGTGGCTGGAAGCTACTGTTCGTGAAATCCCGTGGCGTAAACCTATCTCCTCTATGAACCTGCTGGTGTCTTCTCATGTATGGCGTCAGGACCATAACGGTTTCAGCCATCAGGACCCGGGTGTAACTTCTGTTCTGCTGAACAAGTGCTTCAACAACGACCACGTTATTGGCATCTACTTCCCGGTCGACTCCAACATGCTGCTGGCGGTGGCGGAGAAATGTTACAAAAGCACCAACAAAATCAACGCGATCATCGCTGGTAAACAACCAGCTGCGACCTGGCTGACCCTGGATGAAGCGCGTGCGGAGCTGGAGAAAGGTGCCGCAGAATGGAAATGGGCGTCTAATGTGAAATCCAACGACGAAGCGCAAATCGTTCTGGCTGCTACTGGTGATGTTCCGACTCAAGAAATCATGGCGGCCGCAGATAAACTGGACGCAATGGGTATCAAATTCAAAGTGGTGAACGTTGTTGACCTGGTTAAACTGCAGTCTGCTAAAGAGAACAACGAGGCACTGTCCGACGAAGAGTTCGCCGAACTGTTCACCGAAGACAAACCGGTCCTGTTTGCGTACCATTCCTACGCTCGTGATGTGCGCGGTCTGATCTACGATCGTCCAAACCATGACAACTTCAACGTTCACGGTTACGAAGAACAAGGTAGCACCACTACTCCTTACGACATGGTGCGTGTTAACAATATCGACCGTTATGAACTGCAGGCTGAAGCACTGCGCATGATTGATGCCGACAAATACGCGGACAAGATCAACGAACTGGAAGCGTTTCGCCAAGAAGCCTTCCAGTTCGCAGTTGACAACGGTTACGACCACCCGGATTACACCGATTGGGTTTACTCTGGTGTAAACACTAATAAACAAGGCGCCATTTCTGCCACCGCAGCGACCGCGGGTGACAACGAATAA, As shown in SEQ ID NO. 46.
PqqABCDE/F base sequence:
ATGGCCTGGAACACACCGAAAGTTACCGAAATCCCGCTGGGCGCAGAAATCAACTCGTATGTCTGCGGCGAGAAGAAATAAGCCGCTTTCCCGGGGACCCGTCCTTGAGGAATAATGGCACGGCCGCTCCCCCATGGAGCGGCCGTTTTCGTTCATGGGTGCTCTGTGGTGCCCCAGTCAGACGGTTTGTGAAAAAATGATTGATGTCATCGTGCTTGGCGCGGCGGCAGGGGGCGGTTTTCCGCAGTGGAACTCCGCAGCACCCGGCTGTGTGGCCGCCCGCACGCGACAGGGCGCGAAAGCCCGGACCCAGGCCTCCCTTGCCGTCAGTGCCGACGGAAAGCGCTGGTTCATTCTCAACGCCTCGCCCGATCTGCGGCAGCAGATCATCGATACGCCGGCCCTGCATCATCAGGGCAGCCTGCGTGGAACGCCCATTCAGGGCGTCGTCCTGACCTGCGGCGAGATCGACGCCATAACCGGGCTTCTGACCCTGCGTGAGCGTGAGCCTTTTACCCTGATGGGCAGCGACTCGACCCTTCAGCAGCTTGCGGACAATCCGATCTTCGGTGCGCTCGATCCGGAAATCGTCCCACGTGTTCCGCTCATTCTCGATGAAGCCACGTCCCTGATGAACAAGGACGGGATTCCGTCCGGTCTTTTGCTCACGGCCTTCGCCGTTCCGGGCAAGGCGCCGCTTTACGCGGAAGCCGCAGGGTCACGCCCGGACGAGACGCTGGGCCTTTCCATTACGGATGGATGCAAGACGATGCTCTTCATTCCCGGCTGTGCGCAGATCACGTCGGAAATCGTGGAACGGGTAGCGGCAGCCGATCTCGTGTTCTTTGACGGGACACTGTGGCGGGATGACGAAATGATCCGCGCCGGGTTGAGCCCGAAGAGCGGACAGCGGATGGGACATGTGTCCGTGAATGATGCCGGGGGACCGGTCGAATGTTTCACGACATGCGAAAAACCCCGTAAAGTGTTGATTCATATCAACAACTCCAATCCAATTCTGTTCGAAGACAGCCCCGAACGCAAAGACGTCGAACGCGCCGGATGGACGGTTGCGGAAGACGGCATGACTTTCAGACTGGACACACCATGACGCTCCTCACACCTGACCAGCTTGAAGCACAGCTTCGCCAGATCGGGGCCGAGCGGTATCACAACCGGCACCCGTTCCATCGCAAGCTGCATGACGGCAAGCTGGACAAGGCACAGGTTCAGGCTTGGGCGCTGAACCGCTATTATTATCAGGCCCGCATCCCGGCGAAGGATGCGACGCTTCTCGCACGTCTGCCGACGGCCGAACTGCGCCGCGAATGGCGTCGCCGGATCGAGGACCATGACGGCACGGAGCCCGGAACGGGCGGTGTTGCGCGCTGGCTGATGCTGACGGATGGTCTGGGGCTGGACCGGGATTATGTGGAAAGCCTCGATGGTCTGCTTCCAGCCACGCGCTTCTCGGTCGATGCCTATGTGAACTTCGTGCGGGACCAGTCGATTCTGGCGGCCATTGCGTCGTCGCTGACGGAACTGTTTTCGCCCACGATCATCAGCGAGCGCGTCTCGGGGATGCTGCGGCACTACGACTTTGTGTCGGAAAAGACGCTGGCCTATTTCACGCCGCGCCTGACGCAGGCCCCGCGGGATTCCGATTTCGCGCTGGCCTATGTCCGCGAAAAGGCCCGCACGCCGGAGCAGCAGAAAGAAGTCCTGGGAGCGCTGGAGTTCAAGTGCTCCGTGCTGTGGACGATGCTGGATGCGCTCGACTACGCCTATGTGGAAGGCCACATTCCGCCGGGGGCTTTCGTTCCATGACGGAGGCCCCGCATGTCGTGGCGGAGGGGACGGTTCTCTCCTTTGCCCGGGGGCATCGTCTCCAGCACGATCGTGTGCGGGACGTGTGGATCGTGCAGGCGCCTGAAAAAGCATTTGTAGTTGAGGGCGCCGCGCCGCATATTCTGCGGCTGCTGGATGGGAAGCGCAGCGTCGGCGAGATCATCCAGCAGCTTGCAATCGAGTTTTCCGCCCCGCGTGAGGTCATTGCGAAAGATGTCCTCGCGCTTCTTTCTGAACTGACAGAAAAGAACGTCCTGCACACATGACACTCCCTTCGCCGCCGATGAGCCTTCTGGCTGAACTGACGCATCGATGCCCGCTTTCCTGCCCCTACTGCTCCAATCCGCTTGAACTCGAACGCAAGGCGGCAGAACTCGACACGGCCACCTGGACTGCCGTACTGGAGCAGGCGGCCGAGCTTGGGGTGCTCCAGGTTCATTTCTCTGGCGGCGAGCCTATGGCGCGGCCTGATCTGGTCGAACTGGTCTCCGTCGCACGGAGACTCAACCTGTATTCCAACTTGATCACGTCCGGCGTGTTGCTGGACGAACCGAAACTGGAAGCTCTCGACAGGGCGGGGCTGGATCACATCCAGCTCTCTTTCCAAGACGTGACGGAGGCGGGAGCCGAGCGTATCGGCGGTCTCAAGGGAGCGCAGGCCCGCAAGGTTGCGGCGGCGCGGCTCATCCGCGCGTCCGGCATTCCGATGACGCTCAATTTTGTGGTGCACAGGGAAAATGTCGCCCGTATCCCCGAGATGTTCGCCCTGGCGCGGGAACTCGGAGCGGGGCGGGTGGAGATCGCGCATACCCAGTATTATGGCTGGGGGCTGAAAAACCGTGAGGCGCTTCTTCCCAGCCGGGATCAGCTGGAGGAATCCACACGCGCCGTGGAAGCGGAGCGCGCTAAGGGTGGTTTGTCCGTTGATTATGTGACGCCGGACTATCATGCAGACCGGCCCAAGCCCTGCATGGGGGGATGGGGCCAGCGTTTCGTGAATGTCACACCTTCGGGCCGGGTCCTGCCGTGTCATGCAGCCGAAATCATTCCGGATGTCGCATTCCCGAATGTGCAGGATGTGACCCTGTCCGAAATCTGGAACATCTCACCGCTGTTCAACATGTTCCGCGGGACGGACTGGATGCCGGAGCCCTGCCGCTCCTGCGAGCGCAAGGAGCGTGACTGGGGCGGGTGTCGCTGTCAGGCGATGGCGCTGACGGGGAATGCCGCGAATACCGATCCCGTATGCAGTCTCTCCCCCTATCACGATCGGGTGGAGCAGGCCGTCGAGAACAACATGCAGCCAGAAAGCACGTTGTTCTACAGGCGTTATACGTAA, As shown in SEQ ID NO. 47.
TldD base sequence:
ATGTCTGTTGCTGCTGATGCTCTGGGCGCTCTTGCGACGACCGATGCCCTGTTTTTCGGGCGGCCTGATTCAAAGCTGACCCGTGACGATGCCCGGGCGCTCGTCAATCAGGGGCTTGATGGCGTGGATGACGGCGAACTGTTCCTGGAATACCGGGAGAACGAAAGCATCAGCCTGGATGACGGGACGATCCGTTCGGCGAGCTTCAATACGTCTTCGGGGTTCGGGCTGCGGGCTGTTCTGGGGACGGAGACGGCTTTTGCGCATGCGGACGATATCAGCCGGGATGCTCTGGAGCGGGCTGTCAGTACGGTGGGTGCCGTGCGGCAGGGACGCAGCGGCATCATGGCGCCGGGACCGCAGGCGACGAACCGGCGGCTTTATGGCGACTCCCGCCCGCTTGAAGGAACGGATTTTGCAGCACGTGCGGCGGTTCTGAGCGAGATCGATGCCTATGCGCGGGGTCTGGATTCCCGGGTGGTGCAGGTCAGTGCCGTACTGAGTTCCGAATGGCAGGCCGTGCAGATCATGCGGCGCGCGGACTCCGGCGGCGATGTGGCGGACCTGCGGCCGCTGGTGCGCATGAACGTGTCTGTCGTCGTGGAAAAGGACGGTCAGCGTGAGAGCGGAAGCTGTGGTCTGGGAGGGCGCTATGAACTGGACCGGCTGCTGGCGCCGGAAACGTGGCGCGATGCCGTCGACAAGGCCCTGAAACAGGCTCTGATCACGCTGGAAGCCGCACCGGCGCCCGCAGGGGAAATGGATGTGGTGCTTGGTCCGGGATGGCCGGGCATTTTGCTGCATGAGGCTGTCGGGCACGGGCTGGAAGGCGATTTCAACCGCAAGGGGACCTCGTCCTTCTCGGGGATGATCGGCAAGCGTGTGGCGTCTCCAGGCGTGACTGTGGTCGATGATGGAACGCTTCCGGAGCGTCGTGGCTCGCTGAGTGTTGATGATGAAGGAACGCCGACGTCCCGGACCGTTCTGATCGAGGACGGAATTCTGACCGGATACCTTCAGGACCGGCTGAATGCCCGTCTGATGGGCACGAAATCCACCGGGAACGGACGCCGGGAATCCTATGCACATGCTCCGATGCCGCGCATGACCAATACGCTCATGCTGGAAGGAAGCGCGACCACCGATGAGATGATCCGCTCGATGAAGCGCGGGCTTTATGCCGTGAACTTCGGCGGCGGCCAGGTGGATATCACGTCGGGCAAGTTCGTGTTTGCGGCTTCGGAAGCCTATCTGGTCGAGGACGGGAAAATCATCCGTCCGGTCAAGGGGGCTACGCTGATCGGCAATGGGGCGGACGCGATGAACCAGATTTCCATGATCGGATCCGATGTGGCGCTGGATCCTGGCATCGGAACCTGCGGCAAGGCCGGTCAGGGCGTGCCGGTAGGCGTCGGGCAGCCGACCCTGAAGATTTCGGGTCTGACGGTGGGTGGTACGGCCTAA, As shown in SEQ ID No. 48.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (10)

1. A recombinant genetic engineering strain is characterized in that escherichia coli BL21 (DE 3) is taken as an original strain, and a proB gene, an argA gene, a tyrR gene, a trpE gene and a pheA gene are knocked out, and are transferred into PK genes, gdhA genes, fpK genes and PQQ synthetic gene clusters pqqABCDE/F and tldD genes to enable the recombinant genetic engineering strain to overexpress the PK genes, the gdhA genes, fpK genes and the PQQ synthetic gene clusters pqqABCDE/F and tldD genes.
2. The recombinant genetic engineering strain of claim 1, wherein the target PAM sequence of the proB gene is shown in SEQ ID No.2, the target PAM sequence of the argA gene is shown in SEQ ID No.1, the target PAM sequence of the tyrR gene is shown in SEQ ID No.7, the target PAM sequence of the trpE gene is shown in SEQ ID No.8, the target PAM sequence of the pheA gene is shown in SEQ ID No.9, the base sequence of the PK gene is shown in SEQ ID No.44, the base sequence of the gdhA gene is shown in SEQ ID No.45, the base sequence of the fpK gene is shown in SEQ ID No.46, the base sequence of the pqqABCDE/F of the PQQ synthetic gene cluster is shown in SEQ ID No.47, and the base sequence of the tdd gene is shown in SEQ ID No. 48.
3. A method of constructing a recombinant genetically engineered strain of claim 1, comprising the steps of:
Knocking out proB gene, argA gene, tyrR gene, trpE gene and pheA gene in escherichia coli genome to obtain mutant strain;
And transferring PK genes, gdhA genes, fpK genes and PQQ synthesis gene clusters pqqABCDE/F and tldD genes into the mutant strain to obtain the escherichia coli genetic engineering strain.
4. The method of claim 3, wherein the CRISPR/Cas9 gene editing technique is used for knockout.
5. The method of claim 4, wherein the process of knocking out by CRISPR/Cas9 gene editing technology comprises the process of constructing sgRNA vector and the process of constructing donor DNA.
6. The method of constructing a recombinant genetic engineering strain according to claim 3, wherein the PK gene, the gdhA gene, the fpK gene, and the PQQ synthesis gene cluster pqqABCDE/F and the tdD gene are transferred into the mutant strain by an electrotransformation method.
7. A method for producing PQQ by fermentation, which is characterized by comprising the step of fermenting the recombinant genetic engineering strain according to claim 1 or 2.
8. The method for producing PQQ according to claim 7, wherein the recombinant genetic engineering strain is seed-cultured before the fermentation.
9. The method for the fermentative production of PQQ according to claim 7, wherein the conditions of the fermentation are: the temperature is 28-32 ℃ and the time is 48-72 h.
10. The method for producing PQQ by fermentation according to claim 7, wherein the fermentation medium comprises ：0.9~1.1 g Glu、2.9~3.1 g L-Tyr、5.9~6.1 g/L KH2PO4、16.3~16.5 g/L K2HPO4、4.9~5.1 g/L (NH4)2SO4、1.0~1.2 g/L citric acid, 0.9-1.1 g/L MgSO 4, 9.9-10.1 g/L yeast extract, 6.9-7.1 g/L glucose, 0.09-0.11 g/L vitamin B1, and 1.9-2.1 mL/L trace elements;
The fermented feed medium contains: 490-510 g/L glucose, 6.9-7.1 g/L MgSO 4 , 9.9-10.1 g/L Glu, 29-30 g/L Tyr, 0.9-1.1 mL/L trace element.
CN202410465220.5A 2024-04-18 2024-04-18 Recombinant genetic engineering strain, construction method thereof and fermentation production method of PQQ Active CN118064346B (en)

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