CN116656587A - Construction method of metabolic engineering escherichia coli for producing tetrahydropyrimidine - Google Patents

Construction method of metabolic engineering escherichia coli for producing tetrahydropyrimidine Download PDF

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CN116656587A
CN116656587A CN202310552067.5A CN202310552067A CN116656587A CN 116656587 A CN116656587 A CN 116656587A CN 202310552067 A CN202310552067 A CN 202310552067A CN 116656587 A CN116656587 A CN 116656587A
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tetrahydropyrimidine
seq
escherichia coli
ptac
amino acid
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魏哲
吕厚臣
陈鑫鑫
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Shanghai Huamao Pharmaceutical Co ltd
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Shanghai Huamao Pharmaceutical Co ltd
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Abstract

The invention discloses a metabolic engineering escherichia coli construction method for producing tetrahydropyrimidine, and belongs to the technical field of bioengineering. The invention provides a method for producing engineering bacteria ECT3-10 by using tetrahydropyrimidine under low salt condition without antibiotics and inducer, wherein the method uses a protein scaffold strategy to carry out multienzyme assembly on the tetrahydropyrimidine synthetase for the first time, accelerates the transfer of intermediate substrates, improves the expression intensity of the tetrahydropyrimidine and the synthesis rate, optimizes the expression intensity of the precursor pathway enzyme aspartate semialdehyde dehydrogenase and aspartokinase by promoter engineering for the first time, and the constructed recombinant escherichia coli takes glucose as a substrate, and after fed-batch fermentation for 48 hours, the yield of the tetrahydropyrimidine can reach 19.5g/L.

Description

Construction method of metabolic engineering escherichia coli for producing tetrahydropyrimidine
Technical Field
The invention relates to a metabolic engineering escherichia coli construction method for producing tetrahydropyrimidine, and belongs to the technical field of bioengineering.
Background
Tetrahydropyrimidine (1, 4,5, 6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) is a cyclic amino acid derivative of L-aspartic acid, which protects living cells by alleviating the toxic effects of extreme temperatures, high osmotic pressure, dehydration and radiation on proteins, nucleic acids and cell membranes. Tetrahydropyrimidines have been developed for a variety of applications including inhibition of neurodegenerative diseases, skin protection against cell damage and aging, anti-inflammatory treatments, organ transplant maintenance, and other biomedical applications.
Commercial tetrahydropyrimidine is produced mainly by the halophilic bacteria "bacterial milking" technique, in which cells are first cultured under high salinity conditions, extracellular induction biosynthesis and intracellular accumulation are performed, and then the cells are rapidly transferred to low permeability conditions to trigger release of the tetrahydropyrimidine (CN 102286564 a), but the process has long cycle and low yield. Chen et al (CN 111607551B) constructed metabolic pathways for producing aspartic acid and its derivatives and glutamic acid in antibacterial halophiles, enhanced anabolic pathway flow of target products, weakened or blocked branches and catabolism, and realized recombinant halophiles to produce aspartic acid and its derivatives tetrahydropyrimidine, etc. The king et al (CN 112501102B) knocked out crr gene encoding glucose specific enzyme domain A of phosphotransferase system, iclR gene encoding glyoxylate branch transcription inhibitor and thrA gene encoding bifunctional aspartokinase/homoserine dehydrogenase with E.coli MG1655 as starting strain, and overexpressed gene clusters ectABC, phosphoenolpyruvate carboxylase PPC, aspartdh and anti-feedback inhibition gene eclysC encoding aspartokinase III, and the recombinant strain could reach 25.34g/L under batch fermentation by condition optimization.
However, the production process of the recombinant industrial strain of the tetrahydropyrimidine at the present stage is not separated from the addition of antibiotics or inducers, the stability of the strain is poor, the production cost is high, and the development of the related research and the promotion of commercial application of the tetrahydropyrimidine are affected.
Disclosure of Invention
According to the invention, by utilizing a means of synthetic biology technology and genetic engineering, escherichia coli is taken as an initial strain, the protein scaffold GBD-SH3-PDZ is applied to a tetrahydropyrimidine synthesis system, the ectoine synthesis gene cluster ectoABC from the halomonas elongata is subjected to multienzyme assembly, and meanwhile, the copy number ratio of the synthetase is optimized through a system, so that the conversion efficiency of a precursor L-aspartic acid-beta-semialdehyde to tetrahydropyrimidine is greatly improved. Then, the expression intensities of aspartokinase gene lysC from corynebacterium glutamicum and aspartyl semialdehyde dehydrogenase gene asd from halophila are regulated and combined through promoter engineering, and finally, all optimized gene clusters are integrated into the genome of the escherichia coli by using a CRISPR technology in one step, so that the recombinant escherichia coli without antibiotics and inducers is obtained for producing tetrahydropyrimidine.
The invention relates to an escherichia coli engineering bacterium for producing tetrahydropyrimidine, which contains (GBD) a -(SH3) b -(PDZ) c A protein scaffold; wherein a=1 or 2, b=1 or 2, c=1 or 2; simultaneously expressing the gene clusters EctA, ectB and EctC of the tetrahydropyrimidine synthase, and aspartokinase lysC from corynebacterium glutamicum and aspartyl semialdehyde dehydrogenase asd from halomonas elongata; the amino acid sequence of GBD is shown as SEQ ID NO.1, the amino acid sequence of SH3 is shown as SEQ ID NO.2, and the amino acid sequence of PDZ is shown as SEQ ID NO. 3.
In one embodiment of the invention, the tetrahydropyrimidine synthase gene clusters EctA, ectB, and EctC each comprise any one of the following binding ligands: PDZ binding ligand, GBD binding ligand, SH3 binding ligand; the tetrahydropyrimidine synthase gene clusters EctA, ectB, and EctC comprise binding ligands that are different; the tetrahydropyrimidine synthase gene clusters EctA, ectB, and EctC are directly linked to a ligand.
In one embodiment of the present invention, the C-terminal ends of the tetrahydropyrimidine synthetases EctA, ectB, and EctC are randomly combined and fused with the GBD protein ligand GBDlig, SH3 protein ligand SH3lig, and PDZ protein ligand PDZlig, respectively, to form 6 combinations.
In one embodiment of the invention, the escherichia coli engineering bacteria express protein scaffold GBD-SH3-PDZ genes, including carrier proteins GBD, SH3 and PDZ, and can specifically recognize and bind to ligand GBDlig, SH3lig and PDZlig sequences respectively.
In one embodiment of the invention, the PDZ binding ligand PDZlig has an amino acid sequence shown in SEQ ID No.4, the GBD binding ligand GBDlig has an amino acid sequence shown in SEQ ID No.5, and the SH3 binding ligand SH3lig has an amino acid sequence shown in SEQ ID No. 6.
In one embodiment of the invention, the fusion is that the ectoA with the amino acid sequence shown as SEQ ID NO.7 is directly connected with the SH3lig shown as SEQ ID NO.6 to obtain ectoA-SH 3lig; directly connecting the ectoB with the amino acid sequence shown as SEQ ID NO.8 with the GDBlig with the amino acid sequence shown as SEQ ID NO.5 to obtain ectoB-GDBlig; direct connection of the ecto with the amino acid sequence shown as SEQ ID NO.9 and the PDZlig shown as SEQ ID NO.4 is carried out to obtain the ecto-PDZlig.
In one embodiment of the invention, the protein scaffold is obtained by GSlinker ligation (GBD) a -(GS) n -(SH3) b -(GS) n -(PDZ) c A protein scaffold; where n=any positive integer, a=any natural number. .
In one embodiment of the present invention, the amino acid sequence of the tetrahydropyrimidine synthase EctA is shown as SEQ ID NO.7, the amino acid sequence of the tetrahydropyrimidine synthase EctB is shown as SEQ ID NO.8, the amino acid sequence of the tetrahydropyrimidine synthase EctC is shown as SEQ ID NO.9, the amino acid sequence of the aspartokinase lysC is shown as SEQ ID NO.10, and the amino acid sequence of the aspartyl semialdehyde dehydrogenase asd is shown as SEQ ID NO. 11.
In one embodiment of the invention, the protein scaffold (GBD) a -(SH3) b -(PDZ) c Regulating and controlling by adopting a strong constitutive promoter Ptac with a sequence shown as SEQ ID NO. 12; the gene clusters EctA, ectB and EctC of the tetrahydropyrimidine synthase are regulated by a strong constitutive promoter Ptac with a sequence shown as SEQ ID NO. 12.
In one embodiment of the present invention, the expression levels of aspartokinase gene lysC and aspartyl semialdehyde dehydrogenase gene asd are regulated using constitutive promoters Ptac, PJ23119, pcspA, PJ 23100.
In one embodiment of the invention, the sequences of the constitutive promoters Ptac, PJ23119, pcspA and PJ23100 are shown in SEQ ID NO. 12-15 respectively.
In one embodiment of the present invention, the protein scaffold GBD-SH3-PDZ gene, ligand protein gene cluster, aspartate semialdehyde dehydrogenase gene asd and aspartokinase gene lysC are integrated into the E.coli genome by CRISPR technology.
The invention also provides a method for preparing the tetrahydropyrimidine, which comprises the step of preparing the tetrahydropyrimidine by adopting the escherichia coli genetic engineering bacteria for fermentation.
In one embodiment of the invention, the escherichia coli engineering bacteria are fermented in a fermentation medium for at least 48 hours, the pH is controlled to be 7.0 in the fermentation process, and the concentration of glucose control residual sugar is controlled to be 5g/L.
In one embodiment of the invention, the escherichia coli engineering bacteria are cultivated by fed-batch fermentation for at least 48 hours, and the specific steps are as follows:
(1) Seed culture, namely inoculating the escherichia coli engineering bacteria into a 250mL round bottom triangular flask filled with fed-batch fermentation seed culture medium after plate activation, wherein 50mL of seed culture medium is used for each scraping loop of an inoculating loop, and culturing for 8 hours at 37 ℃ at 220rpm/min to obtain seed liquid;
(2) Fermenting and culturing, namely inoculating the seed solution obtained in the step (1) into a 5-L fermentation tank containing 2L fermentation medium at an inoculum size of 10% (v/v), culturing at 37 ℃, controlling the stirring speed and aeration rate to maintain dissolved oxygen at 20%, controlling pH at 7.0 by adding ammonia water, and controlling the concentration of residual sugar at 5g/L by adding glucose.
In one embodiment of the invention, the seed medium for fed-batch fermentation in step (1) consists of: yeast extract 10.0g, peptone 5.0g, KH 2 PO 4 2.0g, citric acid2.0g，NaCl 1.0g，MgSO 4 ·7H 2 O1.0 g, biotin 0.1mg, d-glucose 25.0g, trace element L ml, adjust pH to 7.0, and fix volume to 1L with deionized water.
In one embodiment of the invention, the fermentation medium for fed-batch fermentation in step (2) consists of: yeast extract 6.0g, peptone 4.0g, na 2 HPO 4 ·12H 2 O 25.0g，KH 2 PO 4 6.0g, 2.0g of citric acid and MgSO 4 ·7H 2 O1.0 g, biotin 0.1mg, d-glucose 20.0g, trace element solution L ml, adjust pH to 7.0, and fix volume to 1L with deionized water.
In one embodiment of the invention, the trace element solution composition is (g/L): feCl 3 ·6H 2 O 2.4，CoCl 2 ·6H 2 O 0.8，CuCl 2 ·2H 2 O 0.15，ZnCl 2 0.3，Na 2 MoO 4 ·2H 2 O 0.3，H 3 BO 3 0.075，MnSO 4 1.2，CaCl 2 ·2H 2 O10, dissolved in 120mM HCl, was fixed in volume with deionized water.
The invention also claims the application of the escherichia coli engineering bacteria or the method in the aspect of producing foods, medicines, health-care products or cosmetics containing tetrahydropyrimidine.
Advantageous effects
The invention uses a protein scaffold strategy to carry out multienzyme assembly on the tetrahydropyrimidine synthase for the first time, accelerates the transfer of intermediate substrates, improves the tetrahydropyrimidine and the synthesis rate, optimizes the expression strength of the precursor pathway enzymes aspartate semialdehyde dehydrogenase and aspartate kinase for the first time through promoter engineering, and achieves the following effects:
(1) According to the invention, the protein scaffold GBD-SH3-PDZ is applied to multi-enzyme assembly of the tetrahydropyrimidine synthetase derived from the halophila, and the number and distance of the synthetases are optimized and the reaction rate is accelerated by randomly combining the C ends of the synthetases EctA, ectB and EctC with the ligand GBDlig of GBD protein, the ligand SH3lig of SH3 protein and the ligand PDZlig of PDZ protein respectively, and screening the number of the scaffold proteins GBD, SH3 and PDZ and the length of the connecting peptide.
(2) The invention regulates and combines the expression intensity of aspartokinase gene lysC from corynebacterium glutamicum and aspartyl semialdehyde dehydrogenase gene asd from halophila by promoter engineering, optimizes the precursor synthesis path by utilizing the strategy of combining path enzymes of promoters Ptac, PJ23119, pcspA and PJ23100 with different intensities, realizes that the yield of batch fed-batch fermentation tetrahydropyrimidine can reach 19.5g/L under the conventional fermentation condition of non-high salt concentration, has no addition of antibiotics and inducers compared with the existing process for producing tetrahydropyrimidine by escherichia coli, and has the advantages of lower raw material cost, simple culture process, small equipment loss and low production cost of the tetrahydropyrimidine.
Drawings
FIG. 1 is a schematic representation of a protein scaffold strategy.
FIG. 2 is a graph of the yield of tetrahydropyrimidine from pathway optimized recombinant strains.
FIG. 3 is a graph of tetrahydropyrimidine yield for 48h of fed-batch fermentation of recombinant strains.
Fig. 4 is a high performance liquid chromatogram of a tetrahydropyrimidine standard sample.
FIG. 5 is a high performance liquid chromatogram of a fermentation broth of a tetrahydropyrimidine-producing strain.
Detailed Description
Coli e.coli W3110 as referred to in the examples below is a commercially available host, enzyme reagents such as PrimeSTAR DNA polymerase, phosphorylase, DNAMarker, solution I, avril and the like as referred to were purchased from TaKaRa (da), clonExpress one-step directed cloning kit as referred to was purchased from Vazyme Biotech (south kyo), glue recovery kit as referred to was purchased from Thermo fisher Scientific, plasmid extraction kit as referred to was purchased from bioengineering (Shanghai) limited, various analytically pure reagents as referred to were purchased from the national drug group, and tetrahydropyrimidine standard samples as referred to were purchased from Sigma-Aldrich (Shanghai).
The following examples relate to the following media:
LB solid medium (g/L): peptone 10, yeast powder 5, sodium chloride 10, agar powder 20.
LB liquid medium (g/L): peptone 10, yeast powder 5 and sodium chloride 10.
Batch fermentation medium (g/L): yeast extract 10.0g, peptone 5.0g, KH 2 PO 4 2.0g, citric acid 2.0g,NaCl 1.0g,MgSO 4 ·7H 2 O1.0 g, biotin 0.1mg, d-glucose 25.0g, trace element L ml, adjust pH to 7.0, and fix volume to 1L with deionized water.
The fermentation medium composition of fed-batch fermentation is: yeast extract 6.0g, peptone 4.0g, na 2 HPO 4 ·12H 2 O25.0g，KH 2 PO 4 6.0g, 2.0g of citric acid and MgSO 4 ·7H 2 O1.0 g, biotin 0.1mg, d-glucose 20.0g, trace element solution L ml, adjust pH to 7.0, and fix volume to 1L with deionized water.
The trace element solution comprises the following components (g/L): feCl 3 ·6H 2 O 2.4，CoCl 2 ·6H 2 O 0.8，CuCl 2 ·2H 2 O 0.15，ZnCl 2 0.3，Na 2 MoO 4 ·2H 2 O 0.3，H 3 BO 3 0.075，MnSO 4 1.2，CaCl 2 ·2H 2 O10, dissolved in 120mM HCl, was fixed in volume with deionized water.
The detection method involved in the following examples is as follows:
detection of tetrahydropyrimidine:
(1) Preparing a detection sample: 1mL of the fermentation broth was centrifuged (13000 rpm,2 min), and the obtained supernatant was diluted with ultrapure water to an appropriate multiple, filtered through a 0.22 μm aqueous microporous membrane, and poured into a liquid bottle.
(2) HPLC detection: tetrahydropyrimidine was measured using an Agilent 1260 high performance liquid chromatograph. The sample injection amount is set to be 5 mu l, the chromatographic column is 250*4.6mm 5um Hypersil GOLD aQ, the column temperature is 30 ℃, the mobile phase is 2% acetonitrile/98% water, the flow rate is 0.6mL/min, and the ultraviolet detection wavelength is 210nm.
The preparation method of competent cells involved in the following examples:
1. coli was prepared as competent cells, transformed with plasmid pCas9, plated on kanamycin plates and cultured at 30 ℃ as in example 1.
2. Single colonies on the plates were picked up and inoculated into SOB liquid medium containing kanamycin, and cultured overnight at 30℃and 220r/min for 12 hours.
3. Transferring into SOB liquid culture medium containing kanamycin at 2% inoculum size, continuously culturing at 30deg.C under 220r/min, and adding L-arabinose with final concentration of 10mmol/L to induce lambda-RED recombinase expression when OD600 is about 0.1-0.2.
4. And continuing to culture until the OD600 is about 0.6, centrifuging the ice bath bacterial liquid for 5min at 4 ℃ and 4000r/min for about 10-15min, and collecting bacterial cells.
5. The cells were washed 3 times with pre-chilled 10% glycerol and concentrated 100-fold to prepare electrotransformation competent cells.
Example 1: construction of recombinant plasmid of tetrahydropyrimidine synthase gene
The method comprises the following specific steps:
(1) Directly connecting the ectoA with the amino acid sequence shown as SEQ ID NO.7 with SH3lig with the amino acid sequence shown as SEQ ID NO.6 to obtain ectoA-SH 3lig; directly connecting the ectoB with the amino acid sequence shown as SEQ ID NO.8 with the GDBlig with the amino acid sequence shown as SEQ ID NO.5 to obtain ectoB-GDBlig; directly connecting the ecto with the amino acid sequence shown as SEQ ID NO.9 with the PDZlig shown as SEQ ID NO.4 to obtain ecto-PDZlig; the ectoA-SH 3lig, ectoB-GDBlig and ectoC-PDZlig are all synthesized by Jin Wei intelligent company.
(2) The primer pRSF-F/R is designed, and the plasmid pRSFDuet-1 is used as a template for PCR amplification to obtain a linearized vector.
pRSF-F：taaggtaccctcgagtctggtaaaga；
pRSF-R：atttcctaatgcaggagtcgcataaggg。
(3) And (3) correspondingly connecting the fragments obtained in the steps (1) and (2) by utilizing one-step cloning enzyme, uniformly mixing the obtained recombinant vector with E.coli JM109 competent cells, and then placing in ice for 30min.
(4) The above ice-bath mixed system was rapidly heat-shocked (42 ℃ C., 90 s) and then rapidly placed on ice, allowed to stand for 2min, then 900. Mu.L of LB liquid medium without antibiotics was added to the sterile chamber, and the culture was resumed (40-60 min,37 ℃ C., 220 rpm).
(5) Centrifuging the culture solution (4000-5000 rpm,2 min), discarding 800 mu L of supernatant, uniformly mixing and concentrating to 200 mu L, coating a kanamycin resistance plate, culturing at a constant temperature of 37 ℃ for 12-16h, selecting positive clones for colony PCR verification, and obtaining plasmids with recombinant tetrahydropyrimidine synthetic gene clusters after sequencing is correct:
pRSFDuet-1-ectoB-GDBlig-ectoA-SH 3 lig-ectoC-PDZlig, designated pR-ECT.
(6) Designing a primer Ptac-F/R, carrying out plasmid circular PCR by taking a plasmid pR-ECT as a template, transferring into E.coli JM109 competent cells, and screening according to the method to obtain a recombinant plasmid with a promoter correctly replaced by Ptac, namely: the ectoine synthase gene clusters ectoine A-SH3lig, ectoine B-GDBlig and ectoine C-PDZlig are regulated and controlled by a strong constitutive promoter Ptac with a sequence shown as SEQ ID NO. 12; designated pR-Ptac-ECT.
Ptac-F：atcagacctttgtttaactttaagaaggagatataccgagatatacataatgcagaccca；
Ptac-R：taaacaaaggtctgatcacattatacgagccgatgattaattgtcaaatttcctaatgcaggagtcgcataa。
Example 2: construction of protein scaffold GBD-SH3-PDZ recombinant plasmid
The method comprises the following specific steps:
(1) The scaffold protein GBD fragment (SEQ ID NO. 1), SH3 fragment (SEQ ID NO. 2) and PDZ fragment (SEQ ID NO. 3) were synthesized separately, and all were submitted to Jin Wei Intelligence company.
(2) The primer pRSF-Ptac-F/R was designed and PCR amplification was performed using the plasmid pR-Ptac-ECT prepared in example 1 as a template to obtain a linearized vector.
pRSF-Ptac-F：taaggtaccctcgagtctggtaaaga；
pRSF-Ptac-R：tccttcttaaagttaaacaaaggtctgatcacattatacgagccgatgattaattgtcaaatttcgcgggatcgagatct。
(3) Ligating the fragments obtained in step (1) and (2) by using one-step cloning enzyme, transferring into E.coli JM109 competent cells according to the method of example 1, and screening according to the method of example 1 to obtain recombinant protein scaffold with correctly replaced promoter PtacPlasmid of GBD-SH3-PDZ, wherein the protein scaffold GBD-SH3-PDZ is GBD- (GS) obtained by GSlinker ligation 9 -SH3-(GS) 9 PDZ protein scaffold, designated plasmid pR-Ptac-ECT-Ptac-G 1 S 1 P 1 。
(4) Designing a primer GBD-F/R, pR-GBD-F/R; SH3-F/R, pR-SH3-F/R and PDZ-F/R, pR-PDZ-F/R, pR-Ptac-ECT-Ptac-G obtained in step (3) 1 S 1 P 1 As vector templates, fragments GBD-1, GBD-2, SH3-1, SH3-2, PDZ-1, PDZ-2 were obtained.
GBD-F：accaaagcggatatcggtaccccg；
GBD-R：agaaccagaaccagaaccagaacctctagaagaaccagaaccagaacccggcgcctgacgacgcagttcg；
pR-GBD-F：gctgaatacgttcgtgcgctgtt；
pR-GBD-R：agaaccagaaccagaaccagaacctctagaagaaccagaaccagaacccggcgcctgacgacgcagttcg；
SH3-F：gctgaatacgttcgtgcgctgttc；
SH3-R：agaaccagaaccagaaccagaaccggatccagaaccagaaccagaaccacggtatttttcaacgtacg；
pR-SH3-F：ttacagcgtcgtcgtgttaccg；
pR-SH3-R：agaaccagaaccagaaccagaaccggatccagaaccagaaccagaaccacggtatttttcaacgtacg；
PDZ-F：ggttctggttctggttctggatccggttctggttctggttctggttctttacagcgtcgtcgtgttac；
PDZ-R：tttgaagtacgggctaacttc；
pR-PDZ-F：tagtgccaccgctgagcaataact；
pR-PDZ-R：tttgaagtacgggctaacttctt；
(5) Fragments GBD-1 and GBD-2, SH3-1 and SH3-2, PDZ-1 and PDZ-2 are respectively connected by utilizing one-step cloning enzyme, E.coli JM109 competent cells are transferred according to the method of the example 1, and plasmids of recombinant protein scaffolds GBD-SH3-PDZ containing different protein data, the promoters of which are correctly replaced by Ptac, are obtained by screening according to the method of the example 1, so that the following vectors are respectively obtained: pR-Ptac-ECT-Ptac-G 2 S 1 P 1 (the protein scaffold is (GBD) 2 -(GS) 9 -SH3-(GS) 9 -PDZ)、pR-Ptac-ECT-Ptac-G 1 S 2 P 1 (the protein scaffold is GBD- (GS) 9 -(SH3) 2 -(GS) 9 -PDZ)、pR-Ptac-ECT-Ptac-G 1 S 1 P 2 (the protein scaffold is GBD- (GS) 9 -SH3-(GS) 9 -(PDZ) 2 )。
(6) With pR-Ptac-ECT-Ptac-G 2 S 1 P 1 As a carrier template, the fragments SH3-3 and SH3-4 are obtained by PCR through primers SH3-F/R, pR-SH 3-F/R; plasmid pR-Ptac-ECT-Ptac-G of recombinant protein scaffold obtained by the method of step (5) 2 S 2 P 1 (the protein scaffold is (GBD) 2 -(GS) 9 -(SH3) 2 -(GS) 9 -PDZ)。
Example 3: construction of recombinant strain of gene integration genome of tetrahydropyrimidine synthase
The genome of E.coli was edited using the pCas9-pTargetF system (ref: jiang Y, chen B, duan C, et al Multigene editing in the Escherichia coli genome using the CRISPR-Cas9 system. Appl Environ Microbiol,2015,81 (7): 2506-2514.) as follows:
1. the primer Ptac-ECT-F/R is designed to pR-Ptac-ECT, pR-Ptac-ECT-Ptac-G 1 S 1 P 1 、pR-Ptac-ECT-Ptac-G 2 S 1 P 1 、pR-Ptac-ECT-Ptac-G 1 S 2 P 1 、pR-Ptac-ECT-Ptac-G 1 S 1 P 2 、pR-Ptac-ECT-Ptac-G 2 S 2 P 1 As a vector template, PCR was performed to obtain fragments Ptac-ECT-Ptac-G, respectively 1 S 1 P 1 、Ptac-ECT-Ptac-G 2 S 1 P 1 、Ptac-ECT-Ptac-G 1 S 2 P 1 、Ptac-ECT-Ptac-G 1 S 1 P 2 、Ptac-ECT-Ptac-G 2 S 2 P 1 The method comprises the steps of carrying out a first treatment on the surface of the The primers FlgG-UP-F/R, flgG-DN-F/R were designed, and PCR was performed using the E.coli genome (NCBI No.: NC-000913.3) as a template to obtain fragments FlgG-UP and FlgG-DN, which were fused to the fragments Ptac-ECT-Ptac-G, respectively, using a one-step cloning enzyme 1 S 1 P 1 、Ptac-ECT-Ptac-G 2 S 1 P 1 、Ptac-ECT-Ptac-G 1 S 2 P 1 、Ptac-ECT-Ptac-G 1 S 1 P 2 、Ptac-ECT-Ptac-G 2 S 2 P 1 To obtain a certain concentration of homologous recombination fragments.
Ptac-ECT-F：ttgacaattaatcatcggctcgtataatgtgatc；
Ptac-ECT-R：caaaaaacccctcaagacccgtttagag；
FlgG-UP-F：atcactattgctgccgatggca；
FlgG-UP-R：cgttttggcgatccataatgaactgat；
FlgG-DN-F：atgcgttaagtatcaccatcggtcg；
FlgG-DN-R：gccatagttaatcggctgagcaga。
FlgG-UP：
atcactattgctgccgatggcacaatctcggcgctcaatccgggcgatccggcaaatacggttgcgccagtagggcgtcttaaactggtg
aaagccacgggcagcgaagtgcagcgcggtgacgacggcatttttcgtttaagcgcagaaacccaggccacgcgtgggccggtactg
caggcagatccaaccttgcgtgtgatgtcgggggttctggaaggcagtaacgtcaatgccgttgcggcaatgagcgacatgattgccag
cgcgcggcgttttgaaatgcagatgaaggtgatcagcagcgtcgatgataacgcaggccgtgccaaccaactgctgtcgatgagttaatt
gaaaggatacatgacaagtataagttgcccgatgcgcaagtttatcgggtctatgggggcaatcgcaatttatcgattttgcgagcacttgt
aggccggataaggcgtttacgccgcatccggcaagaagacatatgcactttgtcactaatccactacaggacattttatgatcagttcattatggatcgccaaaacg；
FlgG-DN：
atgcgttaagtatcaccatcggtcgtgatggcgtggtcagcgtaacccaacaaggccaggcagctccggttcaggttgggcagctcaat
ctcaccacctttatgaatgacaccgggctggagagcattggcgaaaacctctacaccgaaacgcaatcctctggtgcaccgaacgaaag
cacgccgggcctgaacggcgcgggactgctgtatcaagggtatgttgaaacgtctaacgtcaacgtggcggaagaactggtcaatatg
attcaggtgcaacgcgcttacgaaatcaacagtaaagcggtgtccaccaccgatcagatgctgcaaaaactgacgcaactctaaggctt
aaccggtggcaggttcaccggtttactgatttttgaagatgatagccatgcaaaaaaacgctgcgcatacttatgccatttccagcttgttgg
tgctttcactaaccggctgcgcctggataccctccacgccgctggtgcagggggcgaccagtgcacaaccggttcccggtccgacgcccgtcgccaacggttctattttccagtctgctcagccgattaactatggc。
2. The homologous recombination fragment obtained in the step 1 is electrically transformed into escherichia coli BL21 (DE 3), not less than 100ng of pTargetF plasmid with the constructed expression target point of FlgG locus sgRNA (the nucleotide sequence is cggattacaaatcggcacgg) and 400ng of the homologous recombination fragment are added into 50 mu L competent cells before the electric transformation, the electric transformation is carried out in a 2.5kV and 2mm electric rotating cup, and 1mL of precooled SOC culture medium is quickly added.
3. Cells were thawed by culturing at 30℃for 1-2h at 220r/min, then plated on SOC plates containing kanamycin and spectinomycin, cultured overnight at 30℃and PCR and DNA sequencing performed on transformants to confirm that the genome modification was correct.
4. Elimination of plasmid pTargetF:
the correct single colony containing plasmid pCas9 and pTargetF was inoculated into 2mL LB liquid medium containing kanamycin and IPTG, cultured at 30℃for 8-16h, diluted and spread on LB plates containing kanamycin, the elimination of plasmid pTargetF was further confirmed by verifying the resistance of single colony to spectinomycin, and cells successfully eliminated pTargetF were used for the next round of genome engineering by transferring plasmid pTargetF expressing sgRNA.
5. Elimination of plasmid pCas:
single colonies, from which pTargetF was successfully eliminated, were inoculated into LB liquid medium, cultured overnight at 37℃and the elimination of plasmid pCas was further confirmed by plating on LB plates without resistance and containing kanamycin, and verifying the resistance of single colonies to kanamycin.
6. After the pCas9 and pTargetF plasmids are eliminated, recombinant strains integrated with protein scaffolds GBD-SH3-PDZ and tetrahydropyrimidine synthetic genes are obtained, and the recombinant strains are respectively arranged on the large intestinal rodsThe FlgG site of the bacterium has incorporated therein fragments Ptac-ECT, ptac-ECT-Ptac-G 1 S 1 P 1 、Ptac-ECT-Ptac-G 2 S 1 P 1 、Ptac-ECT-Ptac-G 1 S 2 P 1 、Ptac-ECT-Ptac-G 1 S 1 P 2 、Ptac-ECT-Ptac-G 2 S 2 P 1 Is named ECT1, ECT2, ECT3, ECT4, ECT5, ECT6, respectively.
Example 4: tetrahydropyrimidine synthase gene integration genome recombinant strain batch fermentation
The method comprises the following specific steps:
(1) Recombinant strains ECT1, ECT2, ECT3, ECT4, ECT5 and ECT6 obtained in example 3 were taken from glycerol tubes stored at-80℃respectively, streaked onto a non-resistant LB solid medium using a sterile inoculating loop, and cultured at 37℃for 12-16 hours under constant temperature in a sealed manner until single colonies were grown.
(2) Single colonies are respectively picked from the activated LB plates and inoculated into 50mL centrifuge tubes containing LB culture medium (the liquid loading amount is 5 mL), and the culture is carried out for 8 hours at 37 ℃ and 220r/min, so as to obtain seed liquid.
(3) Transferring the cultured seed liquid into a batch fermentation culture medium with a conical bottle sample amount of 30mL and a rate of 250mL according to an inoculation amount of 2% (v/v), wherein the culture temperature is 37 ℃, the rotating speed is 220r/min, and fermenting for 48h.
The content of the tetrahydropyrimidine in the fermentation broths of the recombinant strains ECT1, ECT2, ECT3, ECT4, ECT5 and ECT6 is detected respectively, as shown in figures 4-5, wherein figure 4 shows the detection result of the liquid chromatography of the tetrahydropyrimidine standard sample, and figure 5 shows the detection result of the liquid chromatography of the tetrahydropyrimidine fermentation sample.
The results show that heterologous synthesis of tetrahydropyrimidine is realized in escherichia coli, and as shown in fig. 1, recombinant strains ECT1, ECT2, ECT3, ECT4, ECT5 and ECT6 have the highest yield of recombinant strain ECT3 in fermentation for 48h, the yield is 0.37mg/L, and no inducer or antibiotic is required to be added in the fermentation process, so that recombinant strain ECT3 is further modified as a starting strain.
Example 5: construction of pathway gene optimized recombinant plasmid
The method comprises the following specific steps:
(1) Chemical Synthesis of aspartokinase lysC (SEQ ID NO. 10) derived from Corynebacterium glutamicum and aspartyl semialdehyde dehydrogenase asd (SEQ ID NO. 11) derived from Salmonella elongata were synthesized by Jin Wei Intelligence company.
(2) PCR amplification was performed using the primer pRSF-F/R and the plasmid pR-Ptac-ECT prepared in example 1 as a template to obtain a linearized vector.
(3) The lysC and asd gene fragments were ligated to the linearized vector of step (2) by using one-step cloning enzyme, respectively, and the correct substitution of the promoter with Ptac-containing plasmid containing two recombinant foreign genes, designated pR-Ptac-Asd-Ptac-LysC, was confirmed as in example 1.
(4) Designing a primer Ptac-Asd-F/R, PJ23119-Asd-F/R, pcspA-Asd-F/R, PJ23100-Asd-F/R, ptac-LysC-F/R, PJ23119-LysC-F/R, pcspA-LysC-F/R, PJ23100-LysC-F/R, pR-Asd-LysC-F/R, and carrying out PCR amplification by using a plasmid pR-Ptac-Asd-Ptac-LysC as a template to obtain fragments:
①Ptac-Asd、②PJ23119-Asd、③PcspA-Asd、④PJ23100-Asd、⑤Ptac-LysC、⑥
PJ23119-LysC、⑦PcspA-LysC、⑧PJ23100-LysC、⑨pR-Asd-LysC；
using one-step cloning enzyme to connect (1) (6) (9), (1) (7) (9), (1) (8) (9), (2) (5) (9), (2) (6) (9), (2) (7) (9), (2) (8) (9), (3) (5) (9), (3) (6) (9), (3) (7) (9), (3) (8) (9), (4) (5) (9), (4) (6) (9), (4) (7) (9), (4) (8) (9) correspondingly; recombinant plasmids containing different promoters were obtained by verification as in example 1:
pR-Ptac-Asd-PJ23119-LysC, pR-Ptac-Asd-PcspA-LysC, pR-Ptac-Asd-PJ23100-LysC, pR-PJ23119-Asd-Ptac-LysC, pR-PJ23119-Asd-PJ23119-LysC, pR-PJ23119-Asd-PcspA-LysC, pR-PJ23119-Asd-PJ23100-LysC, pR-PcspA-Asd-Ptac-LysC pR-PcspA-Asd-PJ23119-LysC, pR-PcspA-Asd-PcspA-LysC, pR-PcspA-Asd-PJ23100-LysC, pR-PJ23100-Asd-Ptac-LysC, pR-PJ23100-Asd-PJ23119-LysC, pR-PJ23100-Asd-PcspA-LysC, pR-PJ23100-Asd-PJ 23100-LysC.
The primer sequences involved are as follows:
Ptac-Asd-F：
ttgacaattaatcatcggctcgtataatgtgatcagacctttgtttaactttaagaaggagatatacccctgtagaaataattttgtttaactttaa taaggagatataccat；
Ptac-Asd-R：atttcgattatgcggccgtgtacaat；
PJ23119-Asd-F：ttgacagctagctcagtcctaggtataatcctgtagaaataattttgtttaacttt；
pj23119-asd-r：atttcgattatgcggccgtgtacaat；
PcspA-Asd-F：
ccgattaatcataaatatgaaaaataattgttgcatcacccgccaatgcgtggcttaatgcacatcacctgtagaaataattttgtttaacttta
at；
PcspA-Asd-R：atttcgattatgcggccgtgtacaat；
PJ23100-Asd-F：ttgacggctagctcagtcctaggtacagtgctagccctgtagaaataattttgtttaactttaataag；
PJ23100-Asd-R：atttcgattatgcggccgtgtacaat；
Ptac-LysC-F：
ttgacaattaatcatcggctcgtataatgtgatcagacctttgtttaactttaagaaggagatataccccatcttagtatattagttaagtataaa
ggagatatacc；
Ptac-LysC-R：caaaaaacccctcaagacccgtttaga；
PJ23119-LysC-F：ttgacagctagctcagtcctaggtataatccatcttagtatattagttaagtataaaggagatatacc；
PJ23119-LysC-R：caaaaaacccctcaagacccgtttaga；
PcspA-LysC-F：
ccgattaatcataaatatgaaaaataattgttgcatcacccgccaatgcgtggcttaatgcacatcaccatcttagtatattagttaagtataaaggagatatacc；
PcspA-LysC-R：caaaaaacccctcaagacccgtttaga；
PJ23100-LysC-F：ttgacggctagctcagtcctaggtacagtgctagcccatcttagtatattagttaagtataaaggagatatacc；
PJ23100-LysC-R：caaaaaacccctcaagacccgtttaga；
pR-Asd-LysC-F：ctctaaacgggtcttgaggggttttttg；
pR-Asd-LysC-R：atttcctaatgcaggagtcgcata。
example 6: pathway optimized strain construction and batch fermentation
In the same manner as in example 4, the exogenous aspartokinase gene lysC and aspartyl semialdehyde dehydrogenase gene Asd replaced with the promoter obtained in example 5 were integrated into the motA site (nucleotide sequence tatctgcgcctgattatcag) of recombinant strain ECT3, recombinant strains were obtained in which the fragment Ptac-LysC-Ptac-Asd, PJ23119-LysC-Ptac-Asd, pcspA-LysC-Ptac-Asd, PJ23100-LysC-Ptac-Asd, ptac-LysC-PJ23119-Asd, PJ23119-LysC-PJ23119-Asd, pcspA-LysC-PJ23119-Asd, PJ23100-LysC-PJ23119-Asd, ptac-LysC-PcspA-Asd, PJ23119-LysC-PcspA-Asd, pcspA-LysC-PcspA-Asd, PJ23100-LysC-PcspA-Asd, ptac-LysC-PJ23110-Asd, PJ 23119-LysC-23110-PcspA-P3756-P23110-PJ-LysC-P3735-PJ-P37-Asd were integrated at the motA site of the genome of ECT3, respectively, ECT3-1, ECT3-2, ECT3-3, ECT3-4, ECT3-5, ECT3-6, ECT3-7, ECT3-8, ECT3-9, ECT3-10, ECT3-11, ECT3-12, ECT3-13, ECT3-14, ECT3-15, ECT3-16, respectively.
Batch fermentation was performed as in example 4, and the recombinant strain ECT3-10 was detected to have a yield of up to 1.49g/L (as shown in FIG. 2) after 48 hours of batch fermentation, and no inducer or antibiotic was added at all during the fermentation.
Example 7: fed-batch fermentation of engineering bacteria ECT3-10 for producing tetrahydropyrimidine
The method comprises the following specific steps:
(1) Plate activation: the tetrahydropyrimidine producing strain ECT3-10 constructed in example 6 was inoculated onto a non-resistant LB solid medium from a glycerol tube preserved at-80℃and inoculated with a sterile loop, and cultured at 37℃for 12 hours under constant temperature in a sealed manner until single colonies were grown
(2) Seed culture: a loop of the single colonies cultured in the step (1) was scraped off by using an inoculating loop, and cultured in a 250mL round bottom triangular flask with a liquid loading amount of 50mL batch fermentation medium at 37℃and 220rpm for 8 hours to obtain a seed liquid with an OD of 6-8.
(3) Fed-batch fermentation in a 5-L fermentation tank: inoculating the seed solution obtained in the step (2) into a 5-L fermentation tank containing 2L of batch fermentation medium according to 10% (v/v), culturing at 37 ℃, controlling the rotation speed of a stirring paddle and ventilation to maintain dissolved oxygen at 20%, controlling pH at 7.0 by adding ammonia water, feeding glucose to control the concentration of residual sugar at 5g/L, and fermenting for 48h (figure 3), wherein the yield of tetrahydropyrimidine reaches 19.5g/L, and no inducer or antibiotics are required to be added in the fermentation process.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims (10)

1. An escherichia coli engineering bacterium for producing tetrahydropyrimidine, which is characterized by comprising (GBD) a -(SH3) b -(PDZ) c A protein scaffold; wherein a=1 or 2, b=1 or 2, c=1 or 2; the amino acid sequence of GBD is shown as SEQ ID NO.1, the amino acid sequence of SH3 is shown as SEQ ID NO.2, and the amino acid sequence of PDZ is shown as SEQ ID NO. 3;
meanwhile, the escherichia coli engineering bacteria express tetrahydropyrimidine synthase gene clusters EctA, ectB and EctC from the halophila elongata, and aspartokinase lysC from corynebacterium glutamicum and aspartyl semialdehyde dehydrogenase asd from the halophila elongata.
2. The engineered escherichia coli of claim 1, wherein the tetrahydropyrimidine synthase gene clusters EctA, ectB, and EctC each comprise any one of the following binding ligands: PDZ binding ligand, GBD binding ligand, SH3 binding ligand; the tetrahydropyrimidine synthase gene clusters EctA, ectB, and EctC comprise binding ligands that are different; the tetrahydropyrimidine synthase gene clusters EctA, ectB, and EctC are directly connected with the ligand.
3. The escherichia coli engineering bacterium according to claim 2, wherein the amino acid sequence of the PDZ binding ligand is shown as SEQ ID NO.4, the amino acid sequence of the GBD binding ligand is shown as SEQ ID NO.5, and the amino acid sequence of the SH3 binding ligand is shown as SEQ ID NO. 6.
4. The genetically engineered escherichia coli of claim 3, wherein the protein scaffold is obtained by GSlinker ligation (GBD) a -(GS) n -(SH3) b -(GS) n -(PDZ) c A protein scaffold; where n=any positive integer.
5. The genetically engineered escherichia coli of claim 4, wherein the amino acid sequence of the tetrahydropyrimidine synthase EctA is shown as SEQ ID NO.7, the amino acid sequence of the tetrahydropyrimidine synthase EctB is shown as SEQ ID NO.8, the amino acid sequence of the tetrahydropyrimidine synthase EctC is shown as SEQ ID NO.9, the amino acid sequence of the aspartokinase lysC is shown as SEQ ID NO.10, and the amino acid sequence of the aspartyl semialdehyde dehydrogenase asd is shown as SEQ ID NO. 11.
6. The genetically engineered escherichia coli of claim 5, wherein the protein scaffold (GBD) a -(SH3) b -(PDZ) c Regulating and controlling by adopting a strong constitutive promoter Ptac with a sequence shown as SEQ ID NO. 12; the gene clusters EctA, ectB and EctC of the tetrahydropyrimidine synthase are regulated and controlled by a strong constitutive promoter Ptac with a nucleotide sequence shown as SEQ ID NO. 12.
7. The genetically engineered escherichia coli of claim 6, wherein the expression levels of aspartokinase lysC and aspartyl semialdehyde dehydrogenase asd are regulated by a constitutive promoter with a nucleotide sequence shown as any one of SEQ ID No. 13-15.
8. A method for preparing tetrahydropyrimidine is characterized in that the method adopts the escherichia coli genetic engineering bacteria as defined in any one of claims 1 to 7 to ferment and prepare the tetrahydropyrimidine.
9. The method according to claim 8, wherein the escherichia coli engineering bacteria are fermented in a fermentation medium for at least 48 hours, the pH is controlled to be 7.0 during the fermentation, and glucose is added to control the concentration of residual sugar to be 5g/L.
10. Use of the genetically engineered escherichia coli of any one of claims 1-7 in preparing foods, medicines, health-care products or cosmetics containing tetrahydropyrimidine.
CN202310552067.5A 2023-05-16 2023-05-16 Construction method of metabolic engineering escherichia coli for producing tetrahydropyrimidine Pending CN116656587A (en)

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