CN117866868B - L-high proline production strain and construction method and application thereof - Google Patents
L-high proline production strain and construction method and application thereof Download PDFInfo
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- 238000010276 construction Methods 0.000 title abstract description 7
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- HXEACLLIILLPRG-YFKPBYRVSA-N L-pipecolic acid Chemical compound [O-]C(=O)[C@@H]1CCCC[NH2+]1 HXEACLLIILLPRG-YFKPBYRVSA-N 0.000 claims abstract description 70
- 238000000855 fermentation Methods 0.000 claims abstract description 37
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- 239000008103 glucose Substances 0.000 claims abstract description 24
- 238000012269 metabolic engineering Methods 0.000 claims abstract description 7
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides an L-homoproline production strain, a construction method and application thereof, wherein the production strain is obtained by further modifying an original strain E.coli W3110 by utilizing a metabolic engineering means, the E.coli W3110 is taken as a chassis, L-lysine is transformed into alpha-aminoadipic semialdehyde by introducing exogenous L-lysine 6-aminotransferase, D-1-piperidine-6-carboxylic acid is produced by spontaneous reaction, L-homoproline is produced by reducing the L-homoproline into L-homoproline through delta 1 -pyrroline-5-carboxylic acid reductase, glucose is taken as a substrate by utilizing a fermentation method, L-homoproline is directly synthesized from glucose in a fermentation tank, and the L-homoproline production strain has the advantages of low production cost, no toxic metabolic byproducts, high conversion rate, short fermentation period and the like, and the fermentation yield of 50h L-homoproline can reach 40.5 g/L, and has very good industrial application value.
Description
Technical Field
The invention relates to the technical production field of genetic engineering and fermentation engineering, in particular to an L-high proline production strain, a construction method and application thereof.
Background
L-homoproline (L-Pipecolic acid), also known as L-pipecolic acid, is a commercially valuable natural cyclic non-proteinogenic alpha-amino acid found in a variety of animals, plants and microorganisms. L-homoproline is a key component of many natural and synthetic bioactive molecules, such as the immunosuppressant rapamycin, the antineoplastic agent swainsonine, the peptide antibiotic virginiamycin, and the like, and therefore has high market value.
At present, the preparation of the L-homoproline mainly comprises a chemical synthesis method and a biological synthesis method. Traditional chemical synthesis methods have large limitations in production due to the disadvantages of using noble metal catalysts, higher pressures and temperatures, complicated separation and purification steps, lower yields, and unsustainable raw materials.
The microorganism direct fermentation method for synthesizing the L-homoproline has the advantages of safe production raw materials, low cost, simple process, easy industrialization and the like, and becomes the mode for producing the L-homoproline first choice at the present stage. The method of synthesizing L-homoproline by microorganisms mainly catalyzes the decomposition of L-lysine by lysine ring deaminase and lysine-6-dehydrogenase, but researches show that the turnover number of the L-lysine ring deaminase is low (about 0.6-1 s), so that the biocatalytic method requires very high enzyme load; lysine-6-dehydrogenase has a large restriction in its expression due to incompatibility with the intracellular environment.
Disclosure of Invention
The invention aims to provide an L-high proline producing strain.
Another technical problem to be solved by the present invention is to provide a method for constructing the strain for producing L-homoproline.
Another technical problem to be solved by the present invention is to provide an application of the strain for producing L-homoproline.
In order to solve the technical problems, the technical scheme of the invention is as follows:
An L-homoproline producing strain, namely strain PIP11, is obtained by further modifying an original strain E.coli W3110 by utilizing a metabolic engineering means, and comprises the steps of adjusting the expression intensity of cadA, ldcC, ppc, lysC, asd, dapA, lysA, pntAB genes, and carrying out heterologous expression on lat and proC genes, wherein the method comprises the following specific steps:
knocking out cadA gene on genome to make it not express,
Knocking out the ldcC gene on the genome so that the gene is not expressed,
The ppc gene overexpression was controlled at ycdN pseudogene locus using the P trc promoter,
The P trc promoter was used to control overexpression of lysC gene at the rph pseudogene locus,
The P trc promoter was used to control the overexpression of asd gene at fhia pseudogene locus,
The P trc promoter was used to control the overexpression of the dapA gene at the yjiP pseudogene locus,
The lysA gene overexpression was controlled at yciQ pseudogene locus using the P trc promoter,
The P trc promoter was used to control the overexpression of the exogenous lat gene at yeeL pseudogene locus,
The P trc promoter was used to control the overexpression of the exogenous lat gene at mbhA pseudogene locus,
The P trc promoter is used to control the over-expression of the exogenous proC gene at the ilvG pseudogene locus,
The P trc promoter was used to control pntAB gene overexpression at ygaY pseudogene locus.
Preferably, the above-mentioned L-homoproline producing strain, the metabolic engineering means is CRISPR-Cas9 gene editing technology.
Preferably, the L-homoproline producing strain described above, the starting strain E.coli W3110, has accession number ATCC 273250.
Preferably, the nucleotide sequence of the P trc promoter of the L-homoproline producing strain is shown in a sequence table SEQ ID NO. 1.
Preferably, the nucleotide sequence of the cadA gene of the L-homoproline producing strain is shown in a sequence table SEQ ID NO. 2.
Preferably, the nucleotide sequence of the ldcC gene of the L-homoproline producing strain is shown in a sequence table SEQ ID NO. 3.
Preferably, the nucleotide sequence of the ppc gene of the L-homoproline producing strain is shown in a sequence table SEQ ID NO. 4.
Preferably, the nucleotide sequence of the lysC gene of the L-homoproline producing strain is shown in a sequence table SEQ ID NO. 5.
Preferably, the nucleotide sequence of the asd gene of the L-homoproline production strain is shown in a sequence table SEQ ID NO. 6.
Preferably, the nucleotide sequence of the dapA gene of the L-homoproline producing strain is shown in a sequence table SEQ ID NO. 7.
Preferably, the nucleotide sequence of the lysA gene of the L-homoproline producing strain is shown in a sequence table SEQ ID NO. 8.
Preferably, the L-homoproline production strain is obtained by deriving Flavobacterium lutescens from the lat gene and performing codon optimization, and the nucleotide sequence of the lat gene is shown as a sequence table SEQ ID NO. 9.
Preferably, the L-homoproline producing strain is characterized in that the proC gene is derived from corynebacterium glutamicum ATCC 13032, and the nucleotide sequence of the proC gene is shown in a sequence table SEQ ID NO. 10.
Preferably, the nucleotide sequence of the pntAB gene of the L-homoproline production strain is shown in a sequence table SEQ ID NO. 11.
The construction method of the L-homoproline production strain is based on the original strain E.coli W3110, and comprises the following specific steps:
(1) Knocking out cadA gene on the genome of an original strain E.coli W3110 to obtain a strain PIP01;
(2) Knocking out the ldcC gene on the genome based on the strain PIP01 to obtain a strain PIP02;
(3) Based on the strain PIP02, using a P trc promoter to control the overexpression of the ppc gene at ycdN pseudogene sites to obtain the strain PIP03;
(4) Based on the strain IP03, controlling lysC gene overexpression at an rph pseudogene locus by using a P trc promoter to obtain a strain PIP04;
(5) Based on the strain PIP04, using a P trc promoter to control the overexpression of asd genes at fhia pseudogene loci to obtain a strain PIP05;
(6) Based on the strain PIP05, using a P trc promoter to control the overexpression of the dapA gene at yjiP pseudogene sites to obtain a strain PIP06;
(7) Based on the strain PIP06, using a P trc promoter to control lysA gene over-expression at yciQ pseudogene sites to obtain a strain PIP07;
(8) Based on the strain PIP07, using a P trc promoter to control the over-expression of the exogenous lat gene at yeeL pseudogene sites to obtain a strain PIP08;
(9) Taking the strain PIP08 as an original strain, and controlling the overexpression of an exogenous lat gene by using a P trc promoter at mbhA pseudogene locus to obtain a strain PIP09;
(10) Taking the strain PIP09 as an original strain, and controlling the overexpression of an exogenous proC gene by using a P trc promoter at an ilvG pseudogene locus to obtain a strain PIP10;
(11) The strain PIP10 is taken as a starting strain, and the P trc promoter is used for controlling the overexpression of the pntAB gene at ygaY pseudogene sites to obtain the strain PIP11.
The L-homoproline producing strain is applied to the fermentation production of L-homoproline.
Preferably, the L-homoproline producing strain is used for producing L-homoproline by fermentation in a mechanically stirred tank, and is synthesized by seed culture and fermentation culture with glucose as a substrate.
Preferably, the application of the strain for producing L-homoproline comprises the following specific steps:
(1) Seed culture: the culture temperature is 35 ℃, the pH value of the culture is maintained at 7.0+/-0.2 by automatically feeding 25% ammonia water solution, the dissolved oxygen value of the culture is maintained at 40% by adjusting the stirring rotation speed or ventilation quantity, and the inoculation requirement is met when the OD 600nm is 20;
(2) Fermentation culture: the inoculation amount is 30%, the culture temperature is 35 ℃, the pH is controlled to be 7.0+/-0.2 by automatically feeding 25% ammonia water solution, the dissolved oxygen value of the culture is maintained to be 40% by adjusting the stirring rotation speed or ventilation amount, the glucose concentration in the tank is controlled to be less than or equal to 0.5g/L by feeding 80% (mass volume fraction) glucose solution, and the fermentation period is less than or equal to 50h.
Preferably, the application of the L-homoproline production strain comprises inoculating a storage strain at-80deg.C on an activating inclined plane without resistance, culturing at 37deg.C for 12 hr, and passaging for 2 times; eluting the activated thalli on the inclined plane by sterilized distilled water, and transferring the thalli into a mechanical stirring type fermentation tank to start seed culture.
Preferably, the use of the above-mentioned L-homoproline producing strain, the seed culture medium used in the seed culture: 30g/L of glucose, 5g/L of yeast powder, 1g/L of peptone 2g/L,(NH4)2SO41g/L,KH2PO43g/L,MgSO4·7H2O 0.8g/L, monosodium glutamate and the balance of water.
Preferably, the use of the above-mentioned L-homoproline producing strain, the fermentation medium used in the fermentation culture: 30g/L of glucose, 5g/L of yeast powder, 0.3g/L of peptone 2g/L,(NH4)2SO41g/L,KH2PO44g/L,MgSO4·7H2O 1.5g/L, methionine, 0.5g/L of isoleucine, 20mg/L of FeSO 4·7H2 O and the balance of water.
The above culture medium can be prepared by standard method.
Preferably, when the L-homoproline producing strain is used for fermentation culture, 9mg of pyridoxal phosphate (PLP) and 2.25g of choline chloride (namely, pyridoxal phosphate (PLP) 6mg/L of sugar solution and 1.5g/L of choline chloride) are added to 1.5L of 80% (mass volume fraction) glucose solution.
The beneficial effects are that:
the L-homoproline producing strain is a strain PIP11 obtained by further modifying an original strain E.coli W3110 by utilizing a metabolic engineering means, takes E.coli W3110 as a chassis, converts L-lysine into alpha-aminoadipic semialdehyde by introducing exogenous L-lysine 6-aminotransferase lat genes, then spontaneously reacts to produce D-1-piperidine-6-carboxylic acid, reduces the D-1-piperidine-6-carboxylic acid into L-homoproline by delta 1 -pyrroline-5-carboxylic acid reductase proC genes, produces L-homoproline by utilizing a strain PIP11 fermentation method, takes glucose as a substrate, and directly synthesizes L-homoproline from glucose in a fermentation tank.
Drawings
FIG. 1 is a diagram showing the process of the genetic engineering of L-homoproline producing strain from the head synthesis pathway.
Detailed Description
In order to enable those skilled in the art to better understand the technical scheme of the present invention, the technical scheme of the present invention will be further described in detail below with reference to the specific embodiments.
The percentage "%" referred to in the examples is the mass percentage, the percentage of the solution is the gram of the solute contained in 100mL, and the percentage between the liquids is the volume ratio of the solution at 25 ℃.
The starting strain used in the examples was wild-type E.coli W3110, accession number ATCC 273250, see sequence listing for the corresponding promoters and genes, etc.
As shown in FIG. 1, an L-homoproline producing strain was constructed by the following method: based on E.coli W3110 (ATCC 273250), the lysine decarboxylase cadA and ldcC genes were knocked out so as not to be expressed, the phosphoenolpyruvate carboxylase ppc gene was overexpressed using the P trc promoter at ycdN pseudogene site, the aspartokinase lysC gene was overexpressed using the P trc promoter at rph pseudogene site, the aspartate semialdehyde dehydrogenase asd gene was overexpressed using the P trc promoter at fhia pseudogene site, and the dihydropyridine dicarboxylic acid synthase dapA gene was overexpressed using the P trc promoter at yjiP pseudogene site. The P trc promoter was used to control the overexpression of the diaminopimelate decarboxylase lysA gene at the yciQ pseudogene locus, the P trc promoter was used to control the overexpression of the exogenous L-lysine-6-aminotransferase lat gene at the yeeL pseudogene locus, the P trc promoter was used to control the overexpression of the exogenous L-lysine-6-aminotransferase lat gene at the mbhA pseudogene locus, the P trc promoter was used to control the overexpression of the exogenous delta 1 -pyrroline-5-carboxylate reductase proC gene at the ilvG pseudogene locus, and the P trc promoter was used to control the membrane border-inducible enzyme pntAB gene at the ygaY pseudogene locus.
Example 1
1. Method for gene editing
The adopted gene editing method refers to literature （Li Y,Lin Z,Huang C,et al. Metabolic engineering of Escherichia coli using CRISPR-Cas9 meditated genome editing. Metabolic Engineering,2015,31：13-21.）., and the method relates to engineering plasmids pREDCas and pGRB, wherein pREDCas carries an elimination system of a gRNA expression plasmid pGRB, a Red recombination system of lambda phage, a Cas9 protein expression system and the resistance of Qamycin (working concentration: 100 mg/L); pGRB pUC18 was used as a backbone, comprising the promoter J23100, the gRNA-Cas9 binding domain sequence and the terminator sequence, and ampicillin resistance (working concentration: 100 mg/L). The terminology involved in the following embodiments is explained in this article.
2. The primers used in the strain construction are shown in Table 1.
TABLE 1 primers involved in the construction of strains
| Primer name | Primer sequence (5 '-3') | Sequence number |
| cadA-pGRB-S | AGTCCTAGGTATAATACTAGTAAGAAACACCAAACGCAACCGTTTTAGAGCTAGAA | SEQ ID NO.12 |
| cadA-pGRB-A | TTCTAGCTCTAAAACGGTTGCGTTTGGTGTTTCTTACTAGTATTATACCTAGGACT | SEQ ID NO.13 |
| cadA-U-S | ACTGGGTTGCGTGTTCTGC | SEQ ID NO.14 |
| cadA-U-A | GTACGGAAGGATCATATTGGCGTCAGTCAAAAATAACGCCGCACA | SEQ ID NO.15 |
| cadA-D-S | TGTGCGGCGTTATTTTTGACTGACGCCAATATGATCCTTCCGTAC | SEQ ID NO.16 |
| cadA-D-A | AAACCAGAGAAGCATATGCGCT | SEQ ID NO.17 |
| ldcC-pGRB-S | AGTCCTAGGTATAATACTAGTTGTACGCCGGGGCATATGGGGTTTTAGAGCTAGAA | SEQ ID NO.18 |
| ldcC-pGRB-A | TTCTAGCTCTAAAACCCCATATGCCCCGGCGTACAACTAGTATTATACCTAGGACT | SEQ ID NO.19 |
| ldcC-U-S | TGGGTATCATTGCTCCGCG | SEQ ID NO.20 |
| ldcC-U-A | GATAAGGCAGGATCATATTTGCCGCAAAAATCACGCCGCAAATTCG | SEQ ID NO.21 |
| ldcC-D-S | CGAATTTGCGGCGTGATTTTTGCGGCAAATATGATCCTGCCTTATC | SEQ ID NO.22 |
| ldcC-D-A | CGGCGGGAACGGAAATGAGAA | SEQ ID NO.23 |
| ycdN-pGRB-U | AGTCCTAGGTATAATACTAGTGCGTGGAAATCATCATGGCTGTTTTAGAGCTAGAA | SEQ ID NO.24 |
| ycdN-pGRB-D | TTCTAGCTCTAAAACAGCCATGATGATTTCCACGCACTAGTATTATACCTAGGACT | SEQ ID NO.25 |
| ycdN-U-S | GATTTTGACGCCACCAACACC | SEQ ID NO.26 |
| ycdN-U-A | GTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAACCAATCCACATCACACAATCCATC | SEQ ID NO.27 |
| ycdN-D-S | CTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATGAAGGGATTTTTGGCTATCAGG | SEQ ID NO.28 |
| ycdN-D-A | GTATTCGCCAGGCTGTAAATTC | SEQ ID NO.29 |
| ycdN-ppc-S | CGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGAACGAACAATATTCCGCATTGC | SEQ ID NO.30 |
| ycdN-ppc-A | TCACCGACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTAGCCGGTATTACGCATACCTGC | SEQ ID NO.31 |
| rph-pGRB-S | AGTCCTAGGTATAATACTAGTTGCGACGTGCTTCAGGCTGAGTTTTAGAGCTAGAA | SEQ ID NO.32 |
| rph-pGRB-A | TTCTAGCTCTAAAACTCAGCCTGAAGCACGTCGCAACTAGTATTATACCTAGGACT | SEQ ID NO.33 |
| rph-U-S | ATAGCGCAGGGTACATTCCACT | SEQ ID NO.34 |
| rph-U-A | GAAATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAACCTTCTTCAATAGAGGCGGTACA | SEQ ID NO.35 |
| rph-D-S | TGCCGCAGAGACCGACATCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAAT | SEQ ID NO.36 |
| rph-D-A | ACAGCGGTTGTGGTGGCA | SEQ ID NO.37 |
| rph-lysC-S | CGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGTCTGAAATTGTTGTCTCCAAATTT | SEQ ID NO.38 |
| rph-lysC-A | GACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTACTCAAACAAATTACTATGCAGTT | SEQ ID NO.39 |
| fhia-pGRB-S | AGTCCTAGGTATAATACTAGTTGACGTGCGTAACCAGCTGCGTTTTAGAGCTAGAA | SEQ ID NO.40 |
| fhia-pGRB-A | TTCTAGCTCTAAAACGCAGCTGGTTACGCACGTCAACTAGTATTATACCTAGGACT | SEQ ID NO.41 |
| fhia-U-S | GGGCAATGGTGTTGATACTGG | SEQ ID NO.42 |
| fhia-U-A | AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAATCGCCAGAATCATCATCCC | SEQ ID NO.43 |
| fhia-D-S | AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAAT CAAGCAGGAGCTGACGGTGT | SEQ ID NO.44 |
| fhia-D-A | TGCACCAATGCTGGATACTTACA | SEQ ID NO.45 |
| fhia-asd-S | ATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGAAAAATGTTGGTTTTATCGGCTG | SEQ ID NO.46 |
| fhia-asd-A | CGACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTACGCCAGTTGACGAAGCATC | SEQ ID NO.47 |
| yjiP-pGRB-S | AGTCCTAGGTATAATACTAGTTGGAAAGCGCCTCGGGGAATGTTTTAGAGCTAGAA | SEQ ID NO.48 |
| yjiP-pGRB-A | TTCTAGCTCTAAAACATTCCCCGAGGCGCTTTCCAACTAGTATTATACCTAGGACT | SEQ ID NO.49 |
| yjiP-U-S | GCCATACCGCCAGCAAGAT | SEQ ID NO.50 |
| yjiP-U-A | AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAGCAGATATTCCCCTTTCCACC | SEQ ID NO.51 |
| yjiP-D-S | AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATGACGGATGACAAACGCAAAGC | SEQ ID NO.52 |
| yjiP-D-A | AAAGGCGGATTTTTACTGTGGA | SEQ ID NO.53 |
| yjiP-dapA-S | CTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGTTCACGGGAAGTATTGTCGC | SEQ ID NO.54 |
| yjiP-dapA-A | ACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTACAGCAAACCGGCATGCTTAA | SEQ ID NO.55 |
| yciQ-pGRB-S | AGTCCTAGGTATAATACTAGTAAACAACGTTTCTTGCCTCAGTTTTAGAGCTAGAA | SEQ ID NO.56 |
| yciQ-pGRB-A | TTCTAGCTCTAAAACTGAGGCAAGAAACGTTGTTTACTAGTATTATACCTAGGACT | SEQ ID NO.57 |
| yciQ-U-S | TTACTTGAAGCATTGGGCGAAC | SEQ ID NO.58 |
| yciQ-U-A | AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAACCAGTCAAGATGCCAGGGTTC | SEQ ID NO.59 |
| yciQ-D-S | AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATGTCTGACAAGAACCAGCAAATCCT | SEQ ID NO.60 |
| yciQ-D-A | ATAGCTTCACCGTGGGCATAAC | SEQ ID NO.61 |
| yciQ-lysA-S | CGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGCCACATTCACTGTTCAGCA | SEQ ID NO.62 |
| yciQ-lysA-A | GACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTAAAGCAATTCCAGCGCCAGT | SEQ ID NO.63 |
| yeeL-pGRB-S | AGTCCTAGGTATAATACTAGTAACACAGCAATACGGTACGCGTTTTAGAGCTAGAA | SEQ ID NO.64 |
| yeeL-pGRB-A | TTCTAGCTCTAAAACGCGTACCGTATTGCTGTGTTACTAGTATTATACCTAGGACT | SEQ ID NO.65 |
| yeeL-U-S | TTCATCGGGACGAGTGGAGA | SEQ ID NO.66 |
| yeeL-U-A | TCCACACATTATACGAGCCGGATGATTAATTGTCAACCATAGCATCGCCAATCTGATCGGG | SEQ ID NO.67 |
| yeeL-D-S | AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATACCCAAAGGTGAAGATA | SEQ ID NO.68 |
| yeeL-D-A | CATTCCCTCTACAGAACTAG | SEQ ID NO.69 |
| yeeL-lat-S | CGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGAGCTTATTAGCGCCACTGG | SEQ ID NO.70 |
| yeeL-lat-A | TCACCGACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTACGCGCGGCGCGGG | SEQ ID NO.71 |
| mbhA-pGRB-S | TGTGTGAAATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAACACGGTGGCAGGTTTTGG | SEQ ID NO.72 |
| mbhA-pGRB-A | AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATGACCAAAAGTGCGTCCGATAC | SEQ ID NO.73 |
| mbhA-U-S | GCCAGCACGAACATAATCCC | SEQ ID NO.74 |
| mbhA-U-A | GGTCTGTTTCCTGCTAGCACTATACCTAGGACTGAGCTAGCCGTAAACACGGTGGCAGGTTTTGG | SEQ ID NO.75 |
| mbhA-D-S | AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATGACCAAAAGTGCGTCCGATAC | SEQ ID NO.76 |
| mbhA-D-A | CGGCGTAATCACAAACTGGC | SEQ ID NO.77 |
| ilvG-pGRB-S | AGTCCTAGGTATAATACTAGTTATCGGCACTGACGCATTTCGTTTTAGAGCTAGAA | SEQ ID NO.78 |
| ilvG-pGRB-A | AGTCCTAGGTATAATACTAGTGGAAGAGTTGCCGCGCATCAGTTTTAGAGCTAGAA | SEQ ID NO.79 |
| ilvG-U-S | ACCGAGGAGCAGACAATGAATAA | SEQ ID NO.80 |
| ilvG-U-A | AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAGGTGATGGCAACAACAGGGA | SEQ ID NO.81 |
| ilvG-D-S | CTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCTATCTACGCGCCGTTGTTGTT | SEQ ID NO.82 |
| ilvG-D-A | GCGCTGGCTAACATGAGGAA | SEQ ID NO.83 |
| ilvG-proC-S | CGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGACAACAATTGCTGTAATCGGC | SEQ ID NO.84 |
| ilvG-proC-A | CAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGCTAGCGCTTTCCGAGTTCTTCAG | SEQ ID NO.85 |
| ygaY-pGRB-S | AGTCCTAGGTATAATACTAGTCACTGATGGCGCTGGCATTAGTTTTAGAGCTAGAA | SEQ ID NO.86 |
| ygaY-pGRB-A | TTCTAGCTCTAAAACTAATGCCAGCGCCATCAGTGACTAGTATTATACCTAGGACT | SEQ ID NO.87 |
| ygaY-U-S | CCTACAAACCACATCGCACATT | SEQ ID NO.88 |
| ygaY-U-A | TCCACACATTATACGAGCCGGATGATTAATTGTCAAACACCGAAGCAACCCAAAAGACGGT | SEQ ID NO.89 |
| ygaY-D-S | AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATTTGCTTGCCGCTCCACC | SEQ ID NO.90 |
| ygaY-D-A | GGAGTAGGGCTTTCCATAGAGTGT | SEQ ID NO.91 |
| ygaY-pntAB-S | CCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGCGAATTGGCATACCAAGAG | SEQ ID NO.92 |
| ygaY-pntAB-A | CAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTACAGAGCTTTCAGGATTGCATCCAC | SEQ ID NO.93 |
Example 2
This example is intended to illustrate the procedure for knocking out the genomic cadA gene, which is specifically as follows:
① The escherichia coli W3110 genome is used as a template, cadA-U-S, cadA-U-A and cadA-D-S, cadA-D-A are respectively used as primers, an upstream homology arm and a downstream homology arm are obtained through PCR amplification, the upstream homology arm and the downstream homology arm are used as templates, a delta cadA gene knockout fragment is obtained through overlap PCR, and the gene integration fragment consists of the cadA upstream homology arm and the cadA downstream homology arm.
② Constructing a DNA fragment containing a target sequence for pGRB-cadA by using cadA-pGRB-S and cadA-pGRB-A as primers through a PCR annealing program, carrying out transformation and transformation on the DNA fragment into Top10 transformation competent cells, screening to obtain positive transformants, and extracting plasmid pGRB-cadA;
③ The delta cadA gene knockout fragment obtained in the step ①、② and pGRB-cadA plasmid are electrotransferred into E.coli W3110 strain, positive transformant is obtained through screening, pGRB-cadA plasmid is eliminated and named PIP01.
Example 3
This example is intended to illustrate the procedure for knocking out the genomic ldcC gene, and is described as follows:
① The E.coli W3110 genome is used as a template, the upstream homology arm and the downstream homology arm are obtained through PCR amplification by respectively using the ldcC-U-S, ldcC-U-A and the ldcC-D-S, ldcC-D-A as primers, the delta ldcC gene knockout fragment is obtained through overlapping PCR by using the same as the template, and the gene integration fragment consists of the ldcC upstream homology arm and the ldcC downstream homology arm.
② Constructing a DNA fragment containing a target sequence for pGRB-ldcC by using the ldcC-pGRB-S and the ldcC-pGRB-A as primers through a PCR annealing procedure, carrying out transformation and transformation on the DNA fragment into Top10 transformation competent cells, screening to obtain positive transformants, and extracting plasmid pGRB-ldcC;
③ The ΔldcC knockout fragment obtained in step ①、② was electrotransferred into PIPO1 strain together with pGRB-ldcC plasmid, and positive transformants were obtained by selection, and pGRB-ldcC plasmid was deleted and designated PIP02.
Example 4
This example is intended to illustrate the steps for controlling ppc gene overexpression at ycdN pseudogene locus using the P trc promoter, as follows:
① The E.coli W3110 genome is used as a template, ycdN-U-S, ycdN-U-A, ycdN-D-S, ycdN-D-A and ycdN-ppc-S, ycdN-ppc-A are respectively used as primers, an upstream homology arm, a downstream homology arm and a target gene fragment are obtained through PCR amplification, and then the P trc -ppc (ycdN) gene integration fragment is obtained through overlap PCR by using the same as a template, wherein the gene integration fragment consists of ycdN upstream homology arm, P trc -ppc target gene and ycdN downstream homology arm.
② DNA fragments containing target sequences used in pGRB-ycdN were constructed by PCR annealing procedure using ycdN-pGRB-S and ycdN-pGRB-A as primers, transformed into Top10 transformed competent cells, screened to obtain positive transformants, and plasmids pGRB-ycdN were extracted.
③ The P trc -ppc (ycdN) gene integrated fragment obtained in the step ①、② was electrotransferred into the PIP02 strain together with the pGRB-ycdN plasmid, and positive transformants were obtained by selection, and the pGRB-ycdN plasmid was deleted and designated PIP03.
Example 5
This example is intended to illustrate the procedure for controlling overexpression of lysC gene at the rph pseudogene locus using the P trc promoter, and is specifically as follows:
① The escherichise:Sub>A coli W3110 genome is used as se:Sub>A template, rph-U-S, rph-U-A, rph-D-S, rph-D-A and rph-lysC-S, rph-lysC-A are respectively used as primers, an upstream homology arm, se:Sub>A downstream homology arm and se:Sub>A target gene fragment are obtained through PCR amplification, and then the P trc -lysC (rph) gene integration fragment is obtained through overlap PCR by using the same as se:Sub>A template, wherein the gene integration fragment consists of an rph upstream homology arm, se:Sub>A P trc -lysC target gene and an rph downstream homology arm.
② Constructing a DNA fragment containing a target sequence for pGRB-rph by using rph-pGRB-S and rph-pGRB-A as primers through a PCR annealing procedure, transforming the DNA fragment into Top10 transformed competent cells, screening to obtain positive transformants, and extracting plasmid pGRB-rph.
③ The P trc -lysC (rph) gene-integrated fragment obtained in step ①、② was electroporated with pGRB-rph plasmid into PIP03 strain, and positive transformants were obtained by selection, and pGRB-rph plasmid was deleted and designated PIP04.
Example 6
This example is intended to illustrate the steps for controlling the overexpression of asd gene using the P trc promoter at fhia pseudogene locus, as follows:
① The E.coli W3110 genome is used as a template, fhia-U-S, fhia-U-A, fhia-D-S, fhia-D-A and fhia-asd-S, fhia-asd-A are respectively used as primers, an upstream homology arm, a downstream homology arm and a target gene fragment are obtained through PCR amplification, and then the P trc -asd (fhia) gene integration fragment is obtained through overlapping PCR by using the same as the template, wherein the gene integration fragment consists of fhia upstream homology arm, P trc -asd target gene and fhia downstream homology arm.
② DNA fragments containing target sequences used in pGRB-fhia were constructed by PCR annealing procedure using fhia-pGRB-S and fhia-pGRB-A as primers, transformed into Top10 transformed competent cells, screened to obtain positive transformants, and plasmids pGRB-fhia were extracted.
③ The P trc -asd (fhia) gene integrated fragment obtained in the step ①、② was electrotransformed with the pGRB-fhia plasmid into PIP04 strain, and positive transformants were obtained by selection, and the pGRB-fhia plasmid was deleted and designated PIP05.
Example 7
This example is intended to illustrate the procedure for controlling overexpression of dapA gene using the P trc promoter at yjiP pseudogene locus, as follows:
① The E.coli W3110 genome is used as a template, yjiP-U-S, yjiP-U-A, yjiP-D-S, yjiP-D-A and yjiP-dapA-S, yjiP-dapA-a are respectively used as primers, an upstream homology arm, a downstream homology arm and a target gene fragment are obtained through PCR amplification, and then the P trc -dapA (rph) gene integration fragment is obtained through overlapping PCR by using the same as the template, wherein the gene integration fragment consists of a yjiP upstream homology arm, a P trc -dapA target gene and a yjiP downstream homology arm.
② DNA fragments containing target sequences used in pGRB-yjiP were constructed by PCR annealing procedure using yjiP-pGRB-S and yjiP-pGRB-A as primers, transformed into Top10 transformed competent cells, screened to obtain positive transformants, and plasmids pGRB-yjiP were extracted.
③ The P trc -dapA (yjiP) gene integrated fragment obtained in the step ①、② and a pGRB-yjiP plasmid were electrotransferred into the PIP05 strain, and positive transformants were obtained by screening, and the pGRB-yjiP plasmid was deleted and named PIP06.
Example 8
This example is intended to illustrate the steps for controlling overexpression of lysA gene using the P trc promoter at the yciQ pseudogene locus, as follows:
① The E.coli W3110 genome is used as a template, yciQ-U-S, yciQ-U-A, yciQ-D-S, yciQ-D-A and yciQ-lysA-S, yciQ-lysA-a are respectively used as primers, an upstream homology arm, a downstream homology arm and a target gene fragment are obtained through PCR amplification, and then the P trc -lysA (yciQ) gene integration fragment is obtained through overlap PCR by using the same as a template, wherein the gene integration fragment consists of a yciQ upstream homology arm, a P trc -lysA target gene and a yciQ downstream homology arm.
② DNA fragments containing target sequences used in pGRB-yciQ were constructed by PCR annealing procedure using yciQ-pGRB-S and yciQ-pGRB-A as primers, transformed into Top10 transformed competent cells, screened to obtain positive transformants, and plasmids pGRB-yciQ were extracted.
③ The P trc -lysA (yciQ) gene integrated fragment obtained in the step ①、② was electrotransferred into PIP06 strain together with pGRB-yciQ plasmid, and positive transformants were obtained by selection, and the pGRB-yciQ plasmid was deleted and designated PIP07.
Example 9
This example is intended to illustrate the steps of using the P trc promoter to control exogenous overexpression of the lat gene at the yeeL pseudogene locus, as follows:
① The method comprises the steps of taking an escherichia coli W3110 genome as a template, respectively taking yeeL-U-S, yeeL-U-A, yeeL-D-S, yeeL-D-A as a primer, obtaining an upstream homology arm and a downstream homology arm through PCR amplification, taking Flavobacterium lutescens genome as the template, taking yeeL-lat-S and yeeL-lat-A as primers, obtaining a target gene fragment through PCR amplification, carrying out codon optimization on the target gene fragment, taking the upstream homology arm, the downstream homology arm and the target gene fragment subjected to the codon optimization as templates, and obtaining a P trc -lat (yeeL) gene integration fragment through overlap PCR, wherein the gene integration fragment consists of the yeeL upstream homology arm, the P trc -lat target gene and the yeeL downstream homology arm.
② DNA fragments containing target sequences used in pGRB-yeeL were constructed by PCR annealing procedure using yeeL-pGRB-S and yeeL-pGRB-A as primers, transformed into Top10 transformed competent cells, screened to obtain positive transformants, and plasmids pGRB-yeeL were extracted.
③ The P trc -lat (yeeL) gene integrated fragment obtained in the step ①、② and a pGRB-yeeL plasmid are electrically transferred into the PIP07 strain, positive transformants are obtained through screening, and the pGRB-yeeL plasmid is eliminated and named PIP08.
Example 10
This example is intended to illustrate the steps of using the P trc promoter to control exogenous overexpression of the lat gene at the mbhA pseudogene locus, as follows:
① The method comprises the steps of taking an escherichia coli W3110 genome as a template, respectively taking mbhA-U-S, mbhA-U-A, mbhA-D-S, mbhA-D-A as a primer, obtaining an upstream homology arm and a downstream homology arm through PCR amplification, taking Flavobacterium lutescens genome as the template, taking yeeL-lat-S and yeeL-lat-A as primers, obtaining a target gene fragment through PCR amplification, carrying out codon optimization on the target gene fragment, taking the upstream homology arm, the downstream homology arm and the target gene fragment subjected to the codon optimization as templates, and obtaining a P trc -lat (mbhA) gene integration fragment through overlap PCR, wherein the gene integration fragment consists of the mbhA upstream homology arm, the P trc -lat target gene and the mbhA downstream homology arm.
② DNA fragments containing target sequences used in pGRB-mbhA were constructed by PCR annealing procedure using mbhA-pGRB-S and mbhA-pGRB-A as primers, transformed into Top10 transformed competent cells, screened to obtain positive transformants, and plasmids pGRB-mbhA were extracted.
③ The P trc -lat (mbhA) gene integrated fragment obtained in the step ①、② and the pGRB-mbhA plasmid are electrically transferred into the PIP08 strain, positive transformants are obtained through screening, and the pGRB-mbhA plasmid is eliminated and named PIP09.
Example 11
This example is intended to illustrate the procedure for controlling exogenous overexpression of the proC gene at the ilvG pseudogene locus using the P trc promoter, as follows:
① The method comprises the steps of taking an escherichise:Sub>A coli W3110 genome as se:Sub>A template, respectively taking ilvG-U-S, ilvG-U-A, ilvG-D-S, ilvG-D-A as se:Sub>A primer, obtaining an upstream homology arm and se:Sub>A downstream homology arm through PCR amplification, taking se:Sub>A corynebacterium glutamicum ATCC 13032 genome as the template, taking ilvG-proC-S and ilvG-proC-A as primers, obtaining se:Sub>A target gene fragment through PCR amplification, taking the upstream homology arm, the downstream homology arm and the target gene fragment as templates, and obtaining se:Sub>A P trc -proC (ilvG) gene integration fragment through overlap PCR, wherein the gene integration fragment consists of an ilvG upstream homology arm, se:Sub>A P trc -proC target gene and an ilvG downstream homology arm.
② Ext> byext> usingext> pGRBext> -ext> ilvGext> -ext> Sext> andext> pGRBext> -ext> ilvGext> -ext> Aext> asext> primersext>,ext> constructingext> aext> DNAext> fragmentext> containingext> aext> targetext> sequenceext> forext> pGRBext> -ext> ilvGext> throughext> aext> PCRext> annealingext> programext>,ext> transformingext> theext> DNAext> fragmentext> intoext> Topext> 10ext> transformedext> competentext> cellsext>,ext> screeningext> toext> obtainext> positiveext> transformantsext>,ext> andext> extractingext> plasmidext> pGRBext> -ext> ilvGext>.ext>
③ The P trc -proC (ilvG) gene integration fragment obtained in step ①、② was electrotransferred into PIP09 strain together with pGRB-ilvG plasmid, and positive transformants were obtained by selection, the PGRB plasmid was deleted and designated PIP10.
Example 12
This example is intended to illustrate the steps for controlling the overexpression of the pntAB gene at the ygaY pseudogene locus using the P trc promoter, as follows:
① The E.coli W3110 genome is used as ase:Sub>A template, ygaY-U-S, ygaY-U-A, ygaY-D-S, ygaY-D-A and ygaY-pntAB-S, ygaY-pntAB-A are respectively used as primers, an upstream homology arm, ase:Sub>A downstream homology arm and ase:Sub>A target gene fragment are obtained through PCR amplification, and then the P trc -pntAB (ygaY) gene integration fragment is obtained through overlap PCR by using the same as ase:Sub>A template, wherein the gene integration fragment consists of ygaY upstream homology arm, P trc -pntAB target gene and ygaY downstream homology arm.
② DNA fragments containing target sequences used in pGRB-ygaY were constructed by PCR annealing procedure using ygaY-pGRB-S and ygaY-pGRB-A as primers, transformed into Top10 transformed competent cells, screened to obtain positive transformants, and plasmids pGRB-ygaY were extracted.
③ The P trc -pntAB (ygaY) gene integrated fragment obtained in the step ①、② was electrotransferred into PIP010 strain together with pGRB-ygaY plasmid, and positive transformants were obtained by selection, and the pGRB-ygaY and pREDCas plasmids were deleted and named PIP11.
Example 13
The strain PIP11 is used as an L-homoproline production strain, and the method for producing L-homoproline by using the production strain is described in the embodiment, wherein a 5L mechanical stirring type fermentation tank is used, and the specific culture mode is as follows:
Seed activation: inoculating the preserved strain at-80deg.C on non-activated slant, culturing at 37deg.C for 12 hr, and passaging for 2 times; eluting the activated thallus on the inclined plane by sterilized distilled water, and transferring the thallus to a 5L mechanical stirring type fermentation tank to start seed culture.
Seed culture: the culture temperature is 35 ℃, the pH value of the culture is maintained at 7.0+/-0.2 by automatically feeding 25% ammonia water solution, the dissolved oxygen value of the culture is maintained at 40% by adjusting the stirring rotation speed or ventilation quantity, and the inoculation requirement is met when the OD 600nm is 20; the seed culture medium adopted is as follows: 30g/L of glucose, 5g/L of yeast powder, 1g/L of peptone 2g/L,(NH4)2SO41g/L,KH2PO43g/L,MgSO4·7H2O 0.8g/L, monosodium glutamate and the balance of water.
Fermentation culture: the fermentation inoculation amount is 30%, the culture temperature is 35 ℃, the culture pH is maintained at 7.0+/-0.2 by automatically feeding 25% ammonia water solution, the culture dissolved oxygen value is maintained at 40% by adjusting the stirring rotation speed or ventilation amount, the glucose concentration in a tank is controlled to be less than or equal to 0.5g/L by feeding 80% (mass volume fraction) glucose solution, meanwhile, 9 mgPLP and 2.25 g choline chloride (namely PLP 6mg/L sugar solution and choline chloride 1.5g/L sugar solution) are added into 1.5L 80% (mass volume fraction) glucose solution along with the glucose solution fed, and the fermentation period is 50h; the fermentation medium adopted is: 30g/L of glucose, 5g/L of yeast powder, 0.3g/L of peptone 2g/L, (NH4)2SO41g/L,KH2PO44g/L,MgSO4·7H2O 1.5g/L, methionine, 0.5g/L of isoleucine, 20mg/L of FeSO 4·7H2 O and the balance of water.
Example 14
The effect of PLP and choline chloride in L-homoproline fermentation applications is illustrated in this example using PIP11 as a producer strain. The fermentation culture was conducted in accordance with example 13, except that four control groups were provided with the addition or non-addition of PLP and choline chloride to the 80% glucose solution, and the results of the fermentation in the four fermenter groups for 50 hours were shown in Table 2.
TABLE 2 influence of PLP and Choline chloride on biomass of cells and L-homoproline production
| Group 1 | Group 2 | Group 3 | Group 4 | |
| PLP addition (mg/L sugar solution) | 0 | 0 | 6 | 6 |
| Choline chloride addition (g/L sugar solution) | 0 | 1.5 | 0 | 1.5 |
| Bacterial biomass OD 600nm | 151.7 | 159.2 | 154.9 | 157.5 |
| L-high proline yield g/L | 29.8 | 33.4 | 36.1 | 40.5 |
In conclusion, the L-homoproline production strain is obtained by directionally modifying the metabolic synthesis path of L-homoproline, and in the constructed L-homoproline production strain, lysine decarboxylase genes cadA and ldcC are derived from escherichia coli, so that the L-lysine can be catalyzed to be catabolized in cells to generate pentanediamine and carbon dioxide, and the accumulation of L-lysine serving as a precursor of L-homoproline can be effectively improved by reducing the expression level of cadA and ldcC genes. The phosphoenolpyruvate carboxylase ppc is derived from escherichia coli, and the coded enzyme can catalyze the conversion of phosphoenolpyruvic acid into oxaloacetic acid and promote the accumulation of L-lysine which is a precursor of L-homoproline. The aspartokinase gene lysC, the aspartyl semialdehyde dehydrogenase gene asd, the dihydropyridine dicarboxylic acid synthase gene dapA and the diaminopimelate decarboxylase gene lysA are all derived from escherichia coli, and the coded enzyme can effectively improve the accumulation of L-lysine as a precursor of L-homoproline. L-lysine-6-aminotransferase gene lat, derived from a Flavobacterium lutescens codon-optimized lat gene, encodes a key enzyme catalyzing the production of L-homoproline, and L-lysine can be transamidated into alpha-aminoadipic semialdehyde by using cofactor alpha-ketoglutarate. The delta 1 -pyrroline-5-carboxylic acid reductase gene proC is derived from escherichia coli, and the coded enzyme catalyzes the production of L-homoproline. The membrane boundary induction enzyme pntAB gene can improve the intracellular NADPH conversion rate and the L-high proline yield.
The L-homoproline production strain takes glucose as a carbon source, L-homoproline is directly synthesized from the head in a fermentation tank, pyridoxal phosphate (PLP) and choline chloride are added in the fermentation process of the strain, so that on one hand, the catalytic requirement of L-lysine 6-aminotransferase can be ensured, on the other hand, the activity of the strain can be ensured, the production benefit is improved, the production cost in the fermentation process is low, the heredity is stable, and the high economic benefit is realized, thereby laying a foundation for realizing the large-scale production of L-homoproline.
The foregoing is merely illustrative of the preferred embodiments of this invention, and it will be appreciated by those skilled in the art that variations and modifications of the invention and strain changes, which are carried out by or based on the methods of this invention, may be made without departing from the spirit of this invention.
Claims (9)
1. An L-homoproline producing strain, characterized in that: is obtained by further modifying an original strain E.coli W3110 ATCC 273250 by using a metabolic engineering means, and is specifically as follows:
knocking out cadA gene on genome to make it not express,
Knocking out the ldcC gene on the genome so that the gene is not expressed,
The ppc gene overexpression was controlled at ycdN pseudogene locus using the P trc promoter,
The P trc promoter was used to control overexpression of lysC gene at the rph pseudogene locus,
The P trc promoter was used to control the overexpression of asd gene at fhia pseudogene locus,
The P trc promoter was used to control the overexpression of the dapA gene at the yjiP pseudogene locus,
The lysA gene overexpression was controlled at yciQ pseudogene locus using the P trc promoter,
The P trc promoter was used to control the overexpression of the exogenous lat gene at yeeL pseudogene locus,
The P trc promoter was used to control the overexpression of the exogenous lat gene at mbhA pseudogene locus,
The P trc promoter is used to control the over-expression of the exogenous proC gene at the ilvG pseudogene locus,
The P trc promoter was used to control pntAB gene overexpression at ygaY pseudogene locus; wherein,
The nucleotide sequence of the P trc promoter is shown in a sequence table SEQ ID NO. 1; the nucleotide sequence of the ppc gene is shown in a sequence table SEQ ID NO. 4; the nucleotide sequence of the lysC gene is shown in a sequence table SEQ ID NO. 5; the nucleotide sequence of the asd gene is shown in a sequence table SEQ ID NO. 6; the nucleotide sequence of the dapA gene is shown in a sequence table SEQ ID NO. 7; the nucleotide sequence of the lysA gene is shown in a sequence table SEQ ID NO. 8; the nucleotide sequence of the lat gene is shown in a sequence table SEQ ID NO. 9; the nucleotide sequence of the proC gene is shown in a sequence table SEQ ID NO. 10; the nucleotide sequence of the pntAB gene is shown in a sequence table SEQ ID NO. 11.
2. The L-homoproline producing strain of claim 1, which is characterized in that: the nucleotide sequence of the cadA gene is shown in a sequence table SEQ ID NO. 2; the nucleotide sequence of the ldcC gene is shown in a sequence table SEQ ID NO. 3.
3. The method for constructing an L-homoproline producing strain according to claim 1 or 2, characterized in that: the specific steps are as follows, based on the original strain E.coli W3110:
(1) Knocking out cadA genes on the genome of an original strain E.coli W3110 to obtain a strain PIP01;
(2) Knocking out the ldcC gene on the genome based on the strain PIP01 to obtain a strain PIP02;
(3) Based on the strain PIP02, using a P trc promoter to control the overexpression of the ppc gene at ycdN pseudogene sites to obtain the strain PIP03;
(4) Based on the strain IP03, controlling lysC gene overexpression at an rph pseudogene locus by using a P trc promoter to obtain a strain PIP04;
(5) Based on the strain PIP04, using a P trc promoter to control the overexpression of asd genes at fhia pseudogene loci to obtain a strain PIP05;
(6) Based on the strain PIP05, using a P trc promoter to control the overexpression of the dapA gene at yjiP pseudogene sites to obtain a strain PIP06;
(7) Based on the strain PIP06, using a P trc promoter to control lysA gene over-expression at yciQ pseudogene sites to obtain a strain PIP07;
(8) Based on the strain PIP07, using a P trc promoter to control the over-expression of the exogenous lat gene at yeeL pseudogene sites to obtain a strain PIP08;
(9) Taking the strain PIP08 as an original strain, and controlling the overexpression of an exogenous lat gene by using a P trc promoter at mbhA pseudogene locus to obtain a strain PIP09;
(10) Taking the strain PIP09 as an original strain, and controlling the overexpression of an exogenous proC gene by using a P trc promoter at an ilvG pseudogene locus to obtain a strain PIP10;
(11) The strain PIP10 is taken as a starting strain, and the P trc promoter is used for controlling the overexpression of the pntAB gene at ygaY pseudogene sites to obtain the strain PIP11.
4. Use of the L-homoproline producing strain of claim 1 or 2 for the fermentative production of L-homoproline.
5. The use of the strain for producing L-homoproline according to claim 4, wherein: the L-homoproline is produced by fermentation in a mechanically stirring type fermentation tank, and is synthesized by seed culture and fermentation culture and glucose as a substrate.
6. The use of the strain for producing L-homoproline according to claim 5, wherein: the method comprises the following specific steps:
(1) Seed culture: the culture temperature is 35 ℃, the pH value of the culture is maintained at 7.0+/-0.2 by automatically feeding 25% ammonia water solution, the dissolved oxygen value of the culture is maintained at 40% by adjusting the stirring rotation speed or ventilation quantity, and the inoculation requirement is met when the OD 600nm is 20;
(2) Fermentation culture: the inoculation amount is 30%, the culture temperature is 35 ℃, the pH is controlled to be 7.0+/-0.2 by automatically feeding 25% ammonia water solution, the dissolved oxygen value of the culture is maintained to be 40% by adjusting the stirring speed or ventilation, and the glucose concentration in the tank is controlled to be less than or equal to 0.5g/L by feeding 80% glucose solution.
7. The use of the strain for producing L-homoproline according to claim 6, characterized in that: inoculating the preserved strain at-80deg.C on non-activated slant, culturing at 37deg.C for 12 hr, and passaging for 2 times; eluting the activated thalli on the inclined plane by sterilized distilled water, and transferring the thalli into a mechanical stirring type fermentation tank to start seed culture.
8. The use of the strain for producing L-homoproline according to claim 6, characterized in that: the seed culture medium adopted in the seed culture comprises: 30g/L of glucose, 5g/L of yeast powder, 1g/L of peptone 2g/L,(NH4)2SO4 1g/L,KH2PO43g/L,MgSO4·7H2O 0.8g/L, monosodium glutamate and the balance of water; the fermentation culture medium adopted in the fermentation culture comprises the following components: 30g/L of glucose, 5g/L of yeast powder, 0.3g/L of peptone 2g/L,(NH4)2SO41g/L,KH2PO44g/L,MgSO4·7H2O 1.5g/L, methionine, 0.5g/L of isoleucine, 20mg/L of FeSO 4·7H2 O and the balance of water.
9. The use of the strain for producing L-homoproline according to claim 6, characterized in that: during fermentation culture, pyridoxal phosphate and choline chloride are fed along with the glucose solution.
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| CN112522223A (en) * | 2021-02-18 | 2021-03-19 | 天津科技大学 | Genetically engineered bacterium for producing L-sarcosine and construction method and application thereof |
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