CN110157750B - Improved lysine decarboxylase, production method and application thereof - Google Patents
Improved lysine decarboxylase, production method and application thereof Download PDFInfo
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- C12Y401/01—Carboxy-lyases (4.1.1)
- C12Y401/01018—Lysine decarboxylase (4.1.1.18)
Abstract
The present invention relates to an improved lysine decarboxylase, a method for producing the same, and a method for decarboxylating lysine using the same.
Description
Technical Field
The present invention relates to an improved lysine decarboxylase, a method for producing the same, and a method for decarboxylating lysine using the same.
Background
Lysine decarboxylase (lysine decarboxylase, abbreviated as LDC, EC 4.1.1.18) is widely found in microorganisms, animals such as insects and higher plants, and can strip L-lysine to remove one carboxyl group to produce 1, 5-pentanediamine (cadaverine) and CO 2 . The 1, 5-pentanediamine has quite wide application, for example, the 1, 5-pentanediamine can be polymerized with dibasic acid to synthesize novel nylon, and has high application value in industrial production. At present, the microbial method for producing the pentanediamine mainly adopts the following two methods, namely microbial fermentation production and microbial in-vitro enzyme catalysis production.
The lysine decarboxylase CadA (or called inducible LdcI) derived from escherichia coli is a double-layered, five-flap complex composed of 10 subunits (each of about 80kDa in size) (Kanjee, et al, the EMBO Journal 30:931-944,2011;Kanjee et al, biochemistry 50,9388-9398,2011). Under lower pH conditions (5.0), cadA will appear under microscope to be a stack of 10-mers, which progressively decrease as pH increases (around 6.5), corresponding toThe number of individual 10 mers in (a) increases, and the number of dimers increases; at a pH of about 8.0, most of the reaction product exists as a dimer; the corresponding lysine-converting activity develops a phenotype of increasing pH and decreasing activity (Kanjee, et al, the EMBO Journal 30:931-944,2011;Kanjee et al, biochemistry 50,9388-9398,2011). Decarboxylating lysine in the medium to CO with expression of lysine decarboxylase and exertion of lysine decarboxylation activity 2 And pentamethylenediamine, which is transported extracellular by the CadB transporter, this reaction necessarily occurs with a gradual increase in the pH of the culture conditions (Meng et al, journal of Bacteriology 174:2659-2669,1992;Snider et al, journal of Biological Chemistry 281:1532-1546,2006).
Disclosure of Invention
The present inventors have found that, in addition to the effect of the environmental pH on lysine decarboxylase activity, the change in the environmental temperature during fermentation also has a great effect on lysine decarboxylase-expressing strains, and that the temperature can be regulated by the temperature control system of the fermenter, but this causes energy consumption and an increase in cost. To this end, the present inventors have creatively made an improved lysine decarboxylase which is economical and efficient, a method for producing the same, and a method for decarboxylating lysine using the same.
In particular, in a first aspect, there is provided a method for decarboxylating lysine using a host cell expressing an improved lysine decarboxylase having the amino acid sequence of SEQ ID NO:8 or a sequence having at least 70% sequence identity thereto, e.g. at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70% identical, or a sequence consisting of SEQ ID NO:7 or with SEQ ID NO:7, e.g., a sequence encoding at least 70% identity, e.g., at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70% identity, comprising the step of contacting the expressed modified lysine decarboxylase with lysine.
In a second aspect, there is provided a method for producing lysine decarboxylase comprising culturing a polypeptide comprising the nucleotide sequence of SEQ ID NO:7 or with SEQ ID NO:7, e.g., a host cell having a sequence at least 70% identical, e.g., at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70% identical.
In some embodiments, the host cells are cultured at a temperature of 30 to 42 ℃, e.g., 30 ℃,42 ℃.
In some embodiments, the sequence SEQ ID NO. 17 upstream of the cadA gene start codon in the host cell is replaced with SEQ ID NO. 18.
In some embodiments, the host cell is transformed with plasmid pCIB71-M. In some embodiments, lysine decarboxylation reaction is performed at a temperature of 35-50 ℃, e.g., 35 ℃, 37 ℃, 45 ℃, 50 ℃.
In some embodiments, lysine decarboxylation is performed at a pH of 6.0 to 8.0, e.g., at a pH of 6.0 or 8.0.
In a third aspect, there is provided a nucleotide sequence of SEQ ID NO:7 or with SEQ ID NO:7 has at least 70% identity, e.g., a sequence at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70% identity.
In a fourth aspect, there is provided a lysine decarboxylase having the amino acid sequence of SEQ ID NO:8 or a sequence having at least 70% sequence identity thereto, e.g., at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70% identity.
In the modified lysine decarboxylase producing strain, the modified lysine decarboxylase is fused with a prion protein peptide at the 3' end of a lysine decarboxylase gene, and compared with the wild type lysine decarboxylase, the modified lysine decarboxylase producing strain has the advantages that the lysine conversion rate is not influenced by the pH of the reaction condition basically, and the modified lysine decarboxylase has better pH tolerance.
In some embodiments, the invention firstly takes the genome of escherichia coli (Escherichia coli MG1165K 12) as a template to clone lysine decarboxylase gene cadA (SEQ ID: no. 1), constructs the lysine decarboxylase gene cadA into a proper expression vector, and optimizes the 5' end of the lysine decarboxylase gene cadA by a PCR method so that the lysine decarboxylase gene cadA can be successfully expressed in escherichia coli E.coli BL21 or honeycomb Ha Funi bacteria (Hafnia alvei). Then, a sequence is synthesized by utilizing a gene assembly method, the amino acid sequence encoded by the sequence is the shortest peptide fragment sequence (SEQ ID: no 3) of New1 protein prion derived from Saccharomyces cerevisiae (Saccharomyces cerevisiae) which retains the activity of the protein prion, the nucleotide sequence (SEQ ID: no 4) is obtained after codon optimization, and the operations of codon optimization and DNA assembly are referred to Hoover DM & Lubkowski J, nucleic Acids Research30:10,2002. The optimized New1 gene sequence is fused to the 3' end of cadA gene, and GSGSG connecting sequence is added to obtain modified cadA-M gene sequence (SEQ ID: no 7) which codes for cadA-M modified protein (SEQ ID: no 8).
In one embodiment of the invention, a plasmid capable of expressing a wild-type CadA protein or a CadA-M capable of expressing a modified protein is transformed into a hive Ha Funi (h.alvei) strain to obtain a wild-type CIB strain or a modified CIB-M strain. The lysine conversion rates of the cells and the cell lysates of the wild-type CIB71 strain and the modified CIB71-M strain are respectively detected under different pH conditions, and the modified CIB71-M is basically not influenced by the pH of the reaction conditions no matter the cells and the cell lysates, so that the modified CIB71-M has better pH tolerance.
In some embodiments, the encoding gene cadA/ldcI (SEQ ID No: 1) of lysine decarboxylase cadA/LdcI (SEQ ID No: 2) is an inducible lysine decarboxylase derived from E.coli; the lysine decarboxylase gene may also be a cell from other microorganisms, animals or plants, including but not limited to bacillus subtilis (Bacillus subtilis), bacillus alcalophilus (Bacillus halodurans), streptomyces coelicolor (Streptomyces coelicolor), hafnia alvei (h.alvei) or corynebacterium glutamicum (Corynebacterium glutamicum), akkerr-gracile bacillus (Klebsiella oxytoca), and the like. The lysine decarboxylase may be derived from a strain or a genetically engineered bacterium obtained by mutagenesis or random modification of the above strain. Preferably, the inducible lysine decarboxylase gene from E.coli is selected. Lysine decarboxylase may also be an improved version of the above-described source of lysine decarboxylase (including natural and artificial recombinant modifications) or an active fragment (a truncated version of a protein fragment that retains lysine decarboxylase activity).
In some embodiments, the amino acid sequence of the lysine decarboxylase is the sequence of SEQ ID NO. 2 or a variant thereof having lysine decarboxylase activity. In some embodiments, the amino acid sequence of the lysine decarboxylase is a sequence having at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70% homology to SEQ ID No. 2 and which retains lysine decarboxylase activity.
In some embodiments, the amino acid sequence of the modified lysine decarboxylase is the sequence of SEQ ID NO. 8 or a variant thereof having lysine decarboxylase activity. In some embodiments, the amino acid sequence of the modified lysine decarboxylase is a sequence having at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70% homology to SEQ ID No. 8 and which retains lysine decarboxylase activity.
In some embodiments, the sequence of the lysine decarboxylase gene is the sequence of SEQ ID NO.1 or a variant or homologous sequence thereof encoding a peptide having lysine decarboxylase activity. In some embodiments, the sequence of the lysine decarboxylase gene is a sequence having at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70% homology to SEQ ID NO. 1.
In some embodiments, the sequence of the modified lysine decarboxylase gene is the sequence of SEQ ID NO.7 or a variant or homologous sequence thereof encoding a peptide having lysine decarboxylase activity. In some embodiments, the sequence of the modified lysine decarboxylase gene is a sequence having at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70% homology to SEQ ID NO. 7.
In some embodiments, the constructed recombinant expression plasmid vector containing the gene encoding lysine decarboxylase is derived from a replicable backbone plasmid in a host cell.
The replicable backbone plasmid may be any plasmid capable of replication in a host bacterium, and in one embodiment, the stable recombinant expression plasmid may be derived from a backbone plasmid capable of replication in E.coli (E.coli) and Hafnia alvei (Hafnia alvei), including, but not limited to, pUC (pUC 18, pUC 19), pBR322 plasmids and their derived plasmids.
In some embodiments, the host bacteria used to construct the wild-type or modified lysine decarboxylase expression plasmids are, for example, E.coli BL21 (DE 3), E.coli Top10, E.coli JM109 strains.
In some embodiments, the microorganism selected as a host for expression of the wild-type or modified lysine decarboxylase may be E.coli (B.subtilis), streptomyces coelicolor (S.coelicolor), hafnia alvei (H.alvei) or Corynebacterium glutamicum (C.glutamicum), preferably Hafnia alvei (H.alvei).
In some embodiments, the cell Ha Funi (H.alvei) selected as a host for expression of the wild-type or modified lysine decarboxylase is a Hafnia alvei (H.alvei) cell that does not contain an endogenous plasmid, either a strain that does not naturally contain an endogenous plasmid or a strain that has been deleted from an endogenous plasmid as selected by a means. In one embodiment, the hafnia alvei is CGMCC1.1009.
In some embodiments, the present invention studies the effect of culture temperature on the growth of wild-type CIB71 strain and modified CIB71-M strain, cell lysine conversion rate by varying the culture temperature (20 ℃,30 ℃,42 ℃) and found that the cell lysine conversion rate of the modified CIB71-M strain did not significantly differ at the culture temperatures of 30 ℃ and 42 ℃. OD of wild-type CIB71 and modified strain CIB71-M under culture conditions of 42℃as compared with other culture temperatures 560nm The values were significantly reduced, but at 42℃the OD units were measured 560nm The lysine conversion rate of the modified CIB71-M strain is obviously higher than that of the wild CIB71 strainPotential, and is significantly higher than unit OD under culture conditions of 20deg.C and 30deg.C 560nm Lysine conversion of (c). Similarly, wild-type CIB71 strain has a unit OD under culture conditions of 42 ℃ 560nm The lysine conversion rate of (C) is obviously higher than the unit OD under the culture condition of 20 ℃ and 30 DEG C 560nm Lysine conversion of (c). It was demonstrated that a incubation temperature of 42℃may significantly increase the activity of intracellular CadA or CadA-M.
In some embodiments, the temperature at which the cells are cultured may be any temperature that will allow growth of the cells, and suitable temperatures include 30-42 ℃, such as 30 ℃ 42 ℃.
In some embodiments, the time of culturing may be about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days.
In some embodiments, the medium of the transformant contains polypeptides, peptones, vitamins, trace elements and minerals, and such medium may include, but is not limited to, conventional LB medium (prepared from tryptone, yeast extract and sodium chloride dissolved in water).
In the present invention, the person skilled in the art can determine the range of pH of the medium used. For example, in some embodiments, the pH of the medium ranges from 5.0 to 9.0, with pH conditions of 6.0 to 8.0 being preferred.
In some embodiments, cellular lysine conversion is calculated using nuclear magnetic resonance methods to detect the amount of pentylene diamine produced by lysine-catalyzed lysine conversion.
Drawings
FIG. 1 OD after overnight culture of wild-type strain CIB71 and modified strain CIB71-M under culture conditions of different temperatures 560nm And (5) measuring a value.
FIG. 2 measurement of lysine conversion after the wild-type strain CIB71 and the modified strain CIB71-M were cultured overnight under different temperature conditions.
FIG. 3 Unit OD after overnight culture of wild-type strain CIB71 and modified strain CIB71-M under culture conditions of different temperatures 560nm Lysine conversion calculations of (2).
FIG. 4 inoculation of the same OD 560nm Cell lysine conversion rate after overnight culture of the wild-type strain CIB71 and the modified strain CIB71-M at 42 ℃.
The invention will be further illustrated with reference to specific examples, which are given solely for the purpose of illustration and are not intended to limit the scope of the invention. Any alterations, modifications, or examples of implementations that come within the spirit of the invention, using equivalents of the examples, are to be understood as being within the scope of the invention as claimed.
Detailed Description
Example 1
Cloning of lysine decarboxylase Gene cadA
The lysine decarboxylase (SEQ ID No: 2) encoding gene cadA (SEQ ID No: 1) was amplified from the genome of E.coli MG1655K12 (E.coli MG1655K12, purchased from Beijing Tian-zen Biotechnology Co., ltd.) using primers cadA-SacI-F (SEQ ID No: 9) and cadA-XbaI-R (SEQ ID No: 10), and after double cleavage by SacI and XbaI, was ligated into the same double digested pUC18 plasmid (purchased from Takara Bio Inc.). By CaCl 2 Competent preparation is carried out by the method, the connection product is transformed into E.coli BL21 (purchased from Takara biological engineering (Dalian) Co.) cells by a heat shock method, ampicillin antibiotics (100 mug/ml final concentration) are added into LB culture medium for screening, and plasmid pCIB60 is obtained after cloning, PCR and sequencing verification are correct.
The plasmid pCIB60 is used as a template, and primers cadA-F2 (SEQ ID No: 11) and cadA-R2 (SEQ ID No: 12) are further used for optimizing the sequence of the pCIB60 plasmid upstream of the start codon ATG of the cadA gene, so that the 26bp sequence of the cadA gene upstream of the start codon ATG is deleted, and the cadA can be successfully translated into proteins in E.coli BL21 and Hafnia alvei. Specifically, a 46bp sequence upstream of the ATG of the original cadA gene start codon was taken for illustration, and the sequence 5'-ATTTCACACAGGAAACAGCTATGACCATGATTACGAATTCGAGCTC-3' (SEQ ID No: 17) was replaced with 5'-ATTTCACACAGGAAACAGCT-3' (SEQ ID No: 18). After PCR amplification, the PCR product was digested with DpnI restriction enzyme and transformed into E.coli BL21 by heat shock method, and the plasmid pCIB71 containing the cadA gene start codon ATG upstream sequences SEQ ID No.18 and SEQ ID No.1 was obtained after sequencing verification.
TABLE 1 primers used in the present invention.
Example 2
Cloning of the improved lysine decarboxylase Gene cadA-M
According to the report of Osherovichet al, PLOS Biology 2:4,2004, the shortest peptide fragment sequence capable of determining the activity of New1 protein prion from Saccharomyces cerevisiae (S.cerevisiae) was obtained, and amino acids 2-101 (SEQ ID NO: 3) thereof were subjected to codon optimization to facilitate the expression thereof in E.coli (E.coli) and Hafnia alvei, the optimized nucleotide sequence being SEQ ID NO:4. In addition, a connecting sequence GSGSG of a short peptide is added to the N end of a New1 protein SEQ ID NO. 3 to form SEQ ID NO. 5, and the corresponding DNA sequence is shown as SEQ ID NO. 6. The manipulation of codon optimisation and DNA assembly is described in Hoover DM & Lubkowski J Nucleic Acids Research30:10,2002.
The terminator codon of the cadA gene in plasmid pCIB71 was deleted using primers cadAT-XbaI-R (SEQ ID NO: 13) and cadAT-XbaI-F (SEQ ID NO: 14) to give plasmid pCIB138, which was constructed in the same manner as the plasmid pCIB 71. PCR amplification of SEQ ID NO. 6 was performed using primers New1-XbaI-F (SEQ ID NO. 15) and New1-HindIII-R (SEQ ID NO. 16), with XbaI and HindIII restriction sites at both ends, respectively, digestion of the PCR product and plasmid pCIB71 using XbaI and HindIII restriction enzymes, respectively, ligation of the ligation product, transformation of the ligation product into E.coli BL21 using a heat shock method, sequencing verification of the ligation product to obtain modified plasmid pCIB71-M, which contains cadA (SEQ ID NO. 1) -ligation sequence-New 1 prion protein domain gene (SEQ ID NO. 6) modification gene, the corresponding nucleotide sequence of which is SEQ ID NO.7, and the encoded amino acid sequence of which is SEQ ID NO. 8. Plasmid pCIB71 and plasmid pCIB71-M were transformed into competent cells of Hafnia alvei (H.alvei, specifically, hafnia alvei CGMCC1.1009, available from China general microbiological culture Collection center) respectively, and screened in LB solid plates containing ampicillin at a final concentration of 100. Mu.g/ml to obtain strains CIB71 and CIB71-M containing the two plasmids respectively.
Example 3
Effect of different culture temperatures on growth of wild-type and modified lysine decarboxylase expressing strains
5 individual clones of wild-type CIB71 and modified CIB71-M strains were individually picked up in 5ml LB liquid tubes containing ampicillin at a final concentration of 100. Mu.g/. Mu.l, and after shaking in a shaking table (Shanghai's world Wide laboratory Co., ltd.) of extraordinary small capacity thermostatted culture table, SPH-200B; set at 200rpm, the same applies hereinafter) at 37℃for about 24 hours, the respective strains were cultured in a 1:: 100 (also containing ampicillin at a final concentration of 100. Mu.g/ml, three replicates were set) was transferred to fresh LB liquid tubes and shaken in a shaker at different temperatures (20 ℃,30 ℃,42 ℃) for the same period of time, about 24 hours. Determination of OD of each Strain Using visible Spectrophotometer 560nm The effect of different culture temperatures on the growth of each strain was examined and the results are shown in FIG. 1.
The culture temperature of the strain was 20 ℃,30 ℃ and 42 ℃ respectively, and the OD of the wild-type CIB71-M 560nm OD of the modified strain CIB71-M was 4.0, 4.2 and 3.25, respectively 560nm The values were 3.7, 4.5 and 1.36, respectively.
Example 4
Effect of different culture temperatures on lysine conversion of wild-type and modified lysine decarboxylase-expressing strains
5 individual clones of wild-type CIB71 and modified CIB71-M strains were individually picked up in 5ml LB liquid tubes containing ampicillin at a final concentration of 100. Mu.g/. Mu.l, and after shaking in a shaking table (Shanghai's world Wide laboratory Co., ltd.) of extraordinary small capacity thermostatted culture table, SPH-200B; set at 200rpm, the same applies hereinafter) at 37℃for about 24 hours, the respective strains were cultured in a 1:: 100 (also containing 100. Mu.g/. Mu.l ampicillin in final concentration, three replicates) were transferred to fresh LB liquid tubes and shaken in a shaker at different temperatures (20 ℃,30 ℃,42 ℃) for the same period of time, about 24 hours. And taking the bacterial cells with the same volume to carry out lysine transformation experiments. The conversion of each strain to 1, 5-pentanediamine was analyzed by measuring the percentage of L-lysine converted during the same period of time.
The preparation of the reaction system (pH 6.0) and the nuclear magnetic resonance measurement method were as follows:
the reaction system was formulated as follows:
| bacterial liquid cultivated overnight | 700μl |
| Lys-HCl(400g/L,pH6.0) | 300μl |
| Pyridoxal phosphate coenzyme (PLP, 20 mM) | 5μl |
Specifically, 700. Mu.l of each strain was cultured overnight in a 1.5ml EP tube, 300. Mu.l (400 g/L, pH 6.0) of lysine hydrochloric acid solution and coenzyme PLP (0.1 mM final concentration) were added, the reaction was carried out at 37℃and 250rpm for 2hrs, the reaction mixture was centrifuged (12000 rpm,3 min) after the completion of the reaction, 500. Mu.l of the reaction mixture supernatant was placed in a nuclear magnetic tube, and 100. Mu. L D was added 2 O: DMSO = 30:1 as internal standard, nuclear magnetic resonance (BRUKER ULTRASHIED) TM400 PLUS, beckman NMR) to scan the hydrogen spectrum of the mixture and integrate to obtain L-lysine remaining in the reaction systemThe peak area of 1, 5-pentanediamine produced was calculated using the formula (A-2 XC)/4, and the L-lysine conversion was calculated by dividing the peak area of 1, 5-pentanediamine produced (A-2 XC)/4 by the total peak area of L-lysine remaining and 1, 5-pentanediamine produced C+ (A-2 XC)/4.
As shown in FIG. 2, the lysine conversion rate of the modified strain CIB71-M was 3.3% and 28.1% when the culture temperatures of the strain were 20℃and 30℃respectively, and the lysine conversion rate of the wild-type CIB71 was 2.43% and 9.89% respectively, which were significantly higher than those of the wild-type strain; the method comprises the steps of carrying out a first treatment on the surface of the When the culture temperature of the strain was 42 ℃, the lysine conversion rate of the wild-type CIB71 was 61.0%, and the lysine conversion rate of the modified CIB71-M strain was 36.2%; the lysine conversion rate of the modified CIB71-M strain is not significantly different under the conditions that the culture temperature is 30 ℃ and 42 ℃; and under the conditions of 30 ℃ and 42 ℃, the lysine conversion rate of the wild-type CIB71 strain is obviously different. The lysine conversion rate of the modified strain is obviously smaller than that of the wild strain along with the change of the culture temperature.
OD of wild-type CIB71 and modified strain CIB71-M determined in example 3 were combined 560nm The value data can calculate the lysine conversion rate and OD of two strains at each culture temperature 560nm Ratio of (1), i.e. the same OD 560nm In theory the percentage of lysine that can be converted in a reaction time of 2 hrs. As a result, the unit OD under the culture conditions of 42℃is shown in FIG. 3 560nm The lysine conversion rate of the modified CIB71-M strain of (C) is obviously higher than that of the wild CIB71 strain, and is also obviously higher than that of the modified CIB71-M strain per se in unit OD under the culture condition of 20 ℃ and 30 DEG C 560nm Lysine conversion of (c). Similarly, wild-type CIB71 strain has a unit OD under culture conditions of 42 ℃ 560nm The lysine conversion rate of (C) is obviously higher than the unit OD under the culture condition of 20 ℃ and 30 DEG C 560nm Lysine conversion of (c). It was demonstrated that a incubation temperature of 42℃may significantly increase the activity of intracellular CadA or CadA-M.
As a further verification we firstThree individual clones of the wild-type CIB71 strain and the modified CIB71-M strain were first selected and cultured in LB tubes at 42℃overnight, and the OD of each individual clone of the two strains was measured 560nm According to the measured OD 560nm Value, concentrate the bacterial liquid of the modified bacterial strain CIB71-M to OD 560nm OD with wild CIB71 Strain 560nm The same OD is then taken 560nm The monoclonal bacterial solutions of the two strains are respectively transferred into fresh LB test tubes according to the proportion of 1:100, are placed at 42 ℃ for shaking culture for about 24hrs, and are taken to carry out lysine transformation experiments, and the preparation of a reaction system (pH 6.0) and a nuclear magnetism measurement method are the same as above. The results of measuring lysine conversion of cells are shown in FIG. 4, when initial OD of two strains are inoculated 560nm At the same time, after the same time of culture, the OD of the end of both strains 560nm Also approaching the same, it can be seen from FIG. 4 that the lysine conversion of the modified strain CIB71-M (87.54%) was significantly higher than that of the wild-type strain CIB71 (66.4%) by measuring the lysine conversion of the same volume of cells over the same time period.
Sequence information:
SEQ ID NO.1 Escherichia coli cadA nucleic acid sequence
ATGAACGTTATTGCAATATTGAATCACATGGGGGTTTATTTTAAAGAAGAACCCATCCGTGAACTTCATCGCGCGCTTGAACGTCTGAACTTCCAGATTGTTTACCCGAACGACCGTGACGACTTATTAAAACTGATCGAAAACAATGCGCGTCTGTGCGGCGTTATTTTTGACTGGGATAAATATAATCTCGAGCTGTGCGAAGAAATTAGCAAAATGAACGAGAACCTGCCGTTGTACGCGTTCGCTAATACGTATTCCACTCTCGATGTAAGCCTGAATGACCTGCGTTTACAGATTAGCTTCTTTGAATATGCGCTGGGTGCTGCTGAAGATATTGCTAATAAGATCAAGCAGACCACTGACGAATATATCAACACTATTCTGCCTCCGCTGACTAAAGCACTGTTTAAATATGTTCGTGAAGGTAAATATACTTTCTGTACTCCTGGTCACATGGGCGGTACTGCATTCCAGAAAAGCCCGGTAGGTAGCCTGTTCTATGATTTCTTTGGTCCGAATACCATGAAATCTGATATTTCCATTTCAGTATCTGAACTGGGTTCTCTGCTGGATCACAGTGGTCCACACAAAGAAGCAGAACAGTATATCGCTCGCGTCTTTAACGCAGACCGCAGCTACATGGTGACCAACGGTACTTCCACTGCGAACAAAATTGTTGGTATGTACTCTGCTCCAGCAGGCAGCACCATTCTGATTGACCGTAACTGCCACAAATCGCTGACCCACCTGATGATGATGAGCGATGTTACGCCAATCTATTTCCGCCCGACCCGTAACGCTTACGGTATTCTTGGTGGTATCCCACAGAGTGAATTCCAGCACGCTACCATTGCTAAGCGCGTGAAAGAAACACCAAACGCAACCTGGCCGGTACATGCTGTAATTACCAACTCTACCTATGATGGTCTGCTGTACAACACCGACTTCATCAAGAAAACACTGGATGTGAAATCCATCCACTTTGACTCCGCGTGGGTGCCTTACACCAACTTCTCACCGATTTACGAAGGTAAATGCGGTATGAGCGGTGGCCGTGTAGAAGGGAAAGTGATTTACGAAACCCAGTCCACTCACAAACTGCTGGCGGCGTTCTCTCAGGCTTCCATGATCCACGTTAAAGGTGACGTAAACGAAGAAACCTTTAACGAAGCCTACATGATGCACACCACCACTTCTCCGCACTACGGTATCGTGGCGTCCACTGAAACCGCTGCGGCGATGATGAAAGGCAATGCAGGTAAGCGTCTGATCAACGGTTCTATTGAACGTGCGATCAAATTCCGTAAAGAGATCAAACGTCTGAGAACGGAATCTGATGGCTGGTTCTTTGATGTATGGCAGCCGGATCATATCGATACGACTGAATGCTGGCCGCTGCGTTCTGACAGCACCTGGCACGGCTTCAAAAACATCGATAACGAGCACATGTATCTTGACCCGATCAAAGTCACCCTGCTGACTCCGGGGATGGAAAAAGACGGCACCATGAGCGACTTTGGTATTCCGGCCAGCATCGTGGCGAAATACCTCGACGAACATGGCATCGTTGTTGAGAAAACCGGTCCGTATAACCTGCTGTTCCTGTTCAGCATCGGTATCGATAAGACCAAAGCACTGAGCCTGCTGCGTGCTCTGACTGACTTTAAACGTGCGTTCGACCTGAACCTGCGTGTGAAAAACATGCTGCCGTCTCTGTATCGTGAAGATCCTGAATTCTATGAAAACATGCGTATTCAGGAACTGGCTCAGAATATCCACAAACTGATTGTTCACCACAATCTGCCGGATCTGATGTATCGCGCATTTGAAGTGCTGCCGACGATGGTAATGACTCCGTATGCTGCATTCCAGAAAGAGCTGCACGGTATGACCGAAGAAGTTTACCTCGACGAAATGGTAGGTCGTATTAACGCCAATATGATCCTTCCGTACCCGCCGGGAGTTCCTCTGGTAATGCCGGGTGAAATGATCACCGAAGAAAGCCGTCCGGTTCTGGAGTTCCTGCAGATGCTGTGTGAAATCGGCGCTCACTATCCGGGCTTTGAAACCGATATTCACGGTGCATACCGTCAGGCTGATGGCCGCTATACCGTTAAGGTATTGAAAGAAGAAAGCAAAAAATAA
SEQ ID NO. 2CadA polypeptide sequence
MNVIAILNHMGVYFKEEPIRELHRALERLNFQIVYPNDRDDLLKLIENNARLCGVIFDWDKYNLELCEEISKMNENLPLYAFANTYSTLDVSLNDLRLQISFFEYALGAAEDIANKIKQTTDEYINTILPPLTKALFKYVREGKYTFCTPGHMGGTAFQKSPVGSLFYDFFGPNTMKSDISISVSELGSLLDHSGPHKEAEQYIARVFNADRSYMVTNGTSTANKIVGMYSAPAGSTILIDRNCHKSLTHLMMMSDVTPIYFRPTRNAYGILGGIPQSEFQHATIAKRVKETPNATWPVHAVITNSTYDGLLYNTDFIKKTLDVKSIHFDSAWVPYTNFSPIYEGKCGMSGGRVEGKVIYETQSTHKLLAAFSQASMIHVKGDVNEETFNEAYMMHTTTSPHYGIVASTETAAAMMKGNAGKRLINGSIERAIKFRKEIKRLRTESDGWFFDVWQPDHIDTTECWPLRSDSTWHGFKNIDNEHMYLDPIKVTLLTPGMEKDGTMSDFGIPASIVAKYLDEHGIVVEKTGPYNLLFLFSIGIDKTKALSLLRALTDFKRAFDLNLRVKNMLPSLYREDPEFYENMRIQELAQNIHKLIVHHNLPDLMYRAFEVLPTMVMTPYAAFQKELHGMTEEVYLDEMVGRINANMILPYPPGVPLVMPGEMITEESRPVLEFLQMLCEIGAHYPGFETDIHGAYRQADGRYTVKVLKEESKK
SEQ ID NO. 3 New1 protein (position 2-101) amino acid sequence from Saccharomyces cerevisiae
FPPKKFKDLNSFLDDQPKDPNLVASPFGGYFKNPAADAGSNNASKKSSYQQQRNWKQGGNYQQGGYQSYDSNYNNYNNYNNYNNYNNYNNYNKYNGQGYQ
New1 protein nucleotide sequence with optimized SEQ ID NO 4 codon and artificial synthesis
TTTCCGCCGAAAAAGTTCAAAGACCTGAACTCTTTCCTGGACGACCAGCCGAAAGACCCGAACCTGGTTGCGTCTCCGTTCGGTGGCTACTTCAAAAACCCAGCGGCGGACGCGGGTTCTAACAACGCGTCTAAGAAATCTTCTTACCAGCAGCAGCGTAACTGGAAACAGGGTGGCAACTATCAGCAAGGTGGTTACCAGTCTTACGACTCTAATTACAACAACTACAACAACTACAATAACTATAATAACTACAACAACTACAACAATTATAACAAATACAACGGTCAGGGCTACCAG
SEQ ID NO. 5 New1 protein amino acid sequence with GSGSG linker, wherein the underlined sequence is the linker sequence. And (5) manually synthesizing.
GSGSGFPPKKFKDLNSFLDDQPKDPNLVASPFGGYFKNPAADAGSNNASKKSSYQQQRNWKQGGNYQQGGYQSYDSNYNNYNNYNNYNNYNNYNNYNKYNGQGYQ
The codon-optimized New1 nucleotide sequence and the linker nucleotide sequence corresponding to SEQ ID NO. 6, wherein the underlined sequence is the linker sequence.
GGTTCTGGCTCTGGTTTTCCGCCGAAAAAGTTCAAAGACCTGAACTCTTTCCTGGACGACCAGCCGAAAGACCCGAACCTGGTTGCGTCTCCGTTCGGTGGCTACTTCAAAAACCCAGCGGCGGACGCGGGTTCTAACAACGCGTCTAAGAAATCTTCTTACCAGCAGCAGCGTAACTGGAAACAGGGTGGCAACTATCAGCAAGGTGGTTACCAGTCTTACGACTCTAATTACAACAACTACAACAACTACAATAACTATAATAACTACAACAACTACAACAATTATAACAAATACAACGGTCAGGGCTACCAG
SEQ ID NO. 7cadA-M nucleotide sequence
ATGAACGTTATTGCAATATTGAATCACATGGGGGTTTATTTTAAAGAAGAACCCATCCGTGAACTTCATCGCGCGCTTGAACGTCTGAACTTCCAGATTGTTTACCCGAACGACCGTGACGACTTATTAAAACTGATCGAAAACAATGCGCGTCTGTGCGGCGTTATTTTTGACTGGGATAAATATAATCTCGAGCTGTGCGAAGAAATTAGCAAAATGAACGAGAACCTGCCGTTGTACGCGTTCGCTAATACGTATTCCACTCTCGATGTAAGCCTGAATGACCTGCGTTTACAGATTAGCTTCTTTGAATATGCGCTGGGTGCTGCTGAAGATATTGCTAATAAGATCAAGCAGACCACTGACGAATATATCAACACTATTCTGCCTCCGCTGACTAAAGCACTGTTTAAATATGTTCGTGAAGGTAAATATACTTTCTGTACTCCTGGTCACATGGGCGGTACTGCATTCCAGAAAAGCCCGGTAGGTAGCCTGTTCTATGATTTCTTTGGTCCGAATACCATGAAATCTGATATTTCCATTTCAGTATCTGAACTGGGTTCTCTGCTGGATCACAGTGGTCCACACAAAGAAGCAGAACAGTATATCGCTCGCGTCTTTAACGCAGACCGCAGCTACATGGTGACCAACGGTACTTCCACTGCGAACAAAATTGTTGGTATGTACTCTGCTCCAGCAGGCAGCACCATTCTGATTGACCGTAACTGCCACAAATCGCTGACCCACCTGATGATGATGAGCGATGTTACGCCAATCTATTTCCGCCCGACCCGTAACGCTTACGGTATTCTTGGTGGTATCCCACAGAGTGAATTCCAGCACGCTACCATTGCTAAGCGCGTGAAAGAAACACCAAACGCAACCTGGCCGGTACATGCTGTAATTACCAACTCTACCTATGATGGTCTGCTGTACAACACCGACTTCATCAAGAAAACACTGGATGTGAAATCCATCCACTTTGACTCCGCGTGGGTGCCTTACACCAACTTCTCACCGATTTACGAAGGTAAATGCGGTATGAGCGGTGGCCGTGTAGAAGGGAAAGTGATTTACGAAACCCAGTCCACTCACAAACTGCTGGCGGCGTTCTCTCAGGCTTCCATGATCCACGTTAAAGGTGACGTAAACGAAGAAACCTTTAACGAAGCCTACATGATGCACACCACCACTTCTCCGCACTACGGTATCGTGGCGTCCACTGAAACCGCTGCGGCGATGATGAAAGGCAATGCAGGTAAGCGTCTGATCAACGGTTCTATTGAACGTGCGATCAAATTCCGTAAAGAGATCAAACGTCTGAGAACGGAATCTGATGGCTGGTTCTTTGATGTATGGCAGCCGGATCATATCGATACGACTGAATGCTGGCCGCTGCGTTCTGACAGCACCTGGCACGGCTTCAAAAACATCGATAACGAGCACATGTATCTTGACCCGATCAAAGTCACCCTGCTGACTCCGGGGATGGAAAAAGACGGCACCATGAGCGACTTTGGTATTCCGGCCAGCATCGTGGCGAAATACCTCGACGAACATGGCATCGTTGTTGAGAAAACCGGTCCGTATAACCTGCTGTTCCTGTTCAGCATCGGTATCGATAAGACCAAAGCACTGAGCCTGCTGCGTGCTCTGACTGACTTTAAACGTGCGTTCGACCTGAACCTGCGTGTGAAAAACATGCTGCCGTCTCTGTATCGTGAAGATCCTGAATTCTATGAAAACATGCGTATTCAGGAACTGGCTCAGAATATCCACAAACTGATTGTTCACCACAATCTGCCGGATCTGATGTATCGCGCATTTGAAGTGCTGCCGACGATGGTAATGACTCCGTATGCTGCATTCCAGAAAGAGCTGCACGGTATGACCGAAGAAGTTTACCTCGACGAAATGGTAGGTCGTATTAACGCCAATATGATCCTTCCGTACCCGCCGGGAGTTCCTCTGGTAATGCCGGGTGAAATGATCACCGAAGAAAGCCGTCCGGTTCTGGAGTTCCTGCAGATGCTGTGTGAAATCGGCGCTCACTATCCGGGCTTTGAAACCGATATTCACGGTGCATACCGTCAGGCTGATGGCCGCTATACCGTTAAGGTATTGAAAGAAGAAAGCAAATCTAGAGGTTCTGGCTCTGGTTTTCCGCCGAAAAAGTTCAAAGACCTGAACTCTTTCCTGGACGACCAGCCGAAAGACCCGAACCTGGTTGCGTCTCCGTTCGGTGGCTACTTCAAAAACCCAGCGGCGGACGCGGGTTCTAACAACGCGTCTAAGAAATCTTCTTACCAGCAGCAGCGTAACTGGAAACAGGGTGGCAACTATCAGCAAGGTGGTTACCAGTCTTACGACTCTAATTACAACAACTACAACAACTACAATAACTATAATAACTACAACAACTACAACAATTATAACAAATACAACGGTCAGGGCTACCAGTAA
SEQ ID NO. 8CadA-M polypeptide sequence
MNVIAILNHMGVYFKEEPIRELHRALERLNFQIVYPNDRDDLLKLIENNARLCGVIFDWDKYNLELCEEISKMNENLPLYAFANTYSTLDVSLNDLRLQISFFEYALGAAEDIANKIKQTTDEYINTILPPLTKALFKYVREGKYTFCTPGHMGGTAFQKSPVGSLFYDFFGPNTMKSDISISVSELGSLLDHSGPHKEAEQYIARVFNADRSYMVTNGTSTANKIVGMYSAPAGSTILIDRNCHKSLTHLMMMSDVTPIYFRPTRNAYGILGGIPQSEFQHATIAKRVKETPNATWPVHAVITNSTYDGLLYNTDFIKKTLDVKSIHFDSAWVPYTNFSPIYEGKCGMSGGRVEGKVIYETQSTHKLLAAFSQASMIHVKGDVNEETFNEAYMMHTTTSPHYGIVASTETAAAMMKGNAGKRLINGSIERAIKFRKEIKRLRTESDGWFFDVWQPDHIDTTECWPLRSDSTWHGFKNIDNEHMYLDPIKVTLLTPGMEKDGTMSDFGIPASIVAKYLDEHGIVVEKTGPYNLLFLFSIGIDKTKALSLLRALTDFKRAFDLNLRVKNMLPSLYREDPEFYENMRIQELAQNIHKLIVHHNLPDLMYRAFEVLPTMVMTPYAAFQKELHGMTEEVYLDEMVGRINANMILPYPPGVPLVMPGEMITEESRPVLEFLQMLCEIGAHYPGFETDIHGAYRQADGRYTVKVLKEESKSRGSGSGFPPKKFKDLNSFLDDQPKDPNLVASPFGGYFKNPAADAGSNNASKKSSYQQQRNWKQGGNYQQGGYQSYDSNYNNYNNYNNYNNYNNYNNYNKYNGQGYQ
SEQ ID NO. 17 original cadA Gene A46 bp sequence upstream of the start codon
ATTTCACACAGGAAACAGCTATGACCATGATTACGAATTCGAGCTC
SEQ ID NO.18 sequence upstream of the start codon of the corresponding cadA gene after optimization
ATTTCACACAGGAAACAGCT
Sequence listing
<110> Shanghai Kaiser Biotechnology research and development center Co., ltd
Kaisai Biological Industry Co.,Ltd.
<120> an improved lysine decarboxylase, process for producing the same and use thereof
<130> LZ1705996CN01
<160> 18
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2148
<212> DNA
<213> Escherichia coli (Escherichia coli)
<400> 1
atgaacgtta ttgcaatatt gaatcacatg ggggtttatt ttaaagaaga acccatccgt 60
gaacttcatc gcgcgcttga acgtctgaac ttccagattg tttacccgaa cgaccgtgac 120
gacttattaa aactgatcga aaacaatgcg cgtctgtgcg gcgttatttt tgactgggat 180
aaatataatc tcgagctgtg cgaagaaatt agcaaaatga acgagaacct gccgttgtac 240
gcgttcgcta atacgtattc cactctcgat gtaagcctga atgacctgcg tttacagatt 300
agcttctttg aatatgcgct gggtgctgct gaagatattg ctaataagat caagcagacc 360
actgacgaat atatcaacac tattctgcct ccgctgacta aagcactgtt taaatatgtt 420
cgtgaaggta aatatacttt ctgtactcct ggtcacatgg gcggtactgc attccagaaa 480
agcccggtag gtagcctgtt ctatgatttc tttggtccga ataccatgaa atctgatatt 540
tccatttcag tatctgaact gggttctctg ctggatcaca gtggtccaca caaagaagca 600
gaacagtata tcgctcgcgt ctttaacgca gaccgcagct acatggtgac caacggtact 660
tccactgcga acaaaattgt tggtatgtac tctgctccag caggcagcac cattctgatt 720
gaccgtaact gccacaaatc gctgacccac ctgatgatga tgagcgatgt tacgccaatc 780
tatttccgcc cgacccgtaa cgcttacggt attcttggtg gtatcccaca gagtgaattc 840
cagcacgcta ccattgctaa gcgcgtgaaa gaaacaccaa acgcaacctg gccggtacat 900
gctgtaatta ccaactctac ctatgatggt ctgctgtaca acaccgactt catcaagaaa 960
acactggatg tgaaatccat ccactttgac tccgcgtggg tgccttacac caacttctca 1020
ccgatttacg aaggtaaatg cggtatgagc ggtggccgtg tagaagggaa agtgatttac 1080
gaaacccagt ccactcacaa actgctggcg gcgttctctc aggcttccat gatccacgtt 1140
aaaggtgacg taaacgaaga aacctttaac gaagcctaca tgatgcacac caccacttct 1200
ccgcactacg gtatcgtggc gtccactgaa accgctgcgg cgatgatgaa aggcaatgca 1260
ggtaagcgtc tgatcaacgg ttctattgaa cgtgcgatca aattccgtaa agagatcaaa 1320
cgtctgagaa cggaatctga tggctggttc tttgatgtat ggcagccgga tcatatcgat 1380
acgactgaat gctggccgct gcgttctgac agcacctggc acggcttcaa aaacatcgat 1440
aacgagcaca tgtatcttga cccgatcaaa gtcaccctgc tgactccggg gatggaaaaa 1500
gacggcacca tgagcgactt tggtattccg gccagcatcg tggcgaaata cctcgacgaa 1560
catggcatcg ttgttgagaa aaccggtccg tataacctgc tgttcctgtt cagcatcggt 1620
atcgataaga ccaaagcact gagcctgctg cgtgctctga ctgactttaa acgtgcgttc 1680
gacctgaacc tgcgtgtgaa aaacatgctg ccgtctctgt atcgtgaaga tcctgaattc 1740
tatgaaaaca tgcgtattca ggaactggct cagaatatcc acaaactgat tgttcaccac 1800
aatctgccgg atctgatgta tcgcgcattt gaagtgctgc cgacgatggt aatgactccg 1860
tatgctgcat tccagaaaga gctgcacggt atgaccgaag aagtttacct cgacgaaatg 1920
gtaggtcgta ttaacgccaa tatgatcctt ccgtacccgc cgggagttcc tctggtaatg 1980
ccgggtgaaa tgatcaccga agaaagccgt ccggttctgg agttcctgca gatgctgtgt 2040
gaaatcggcg ctcactatcc gggctttgaa accgatattc acggtgcata ccgtcaggct 2100
gatggccgct ataccgttaa ggtattgaaa gaagaaagca aaaaataa 2148
<210> 2
<211> 715
<212> PRT
<213> Escherichia coli (Escherichia coli)
<400> 2
Met Asn Val Ile Ala Ile Leu Asn His Met Gly Val Tyr Phe Lys Glu
1 5 10 15
Glu Pro Ile Arg Glu Leu His Arg Ala Leu Glu Arg Leu Asn Phe Gln
20 25 30
Ile Val Tyr Pro Asn Asp Arg Asp Asp Leu Leu Lys Leu Ile Glu Asn
35 40 45
Asn Ala Arg Leu Cys Gly Val Ile Phe Asp Trp Asp Lys Tyr Asn Leu
50 55 60
Glu Leu Cys Glu Glu Ile Ser Lys Met Asn Glu Asn Leu Pro Leu Tyr
65 70 75 80
Ala Phe Ala Asn Thr Tyr Ser Thr Leu Asp Val Ser Leu Asn Asp Leu
85 90 95
Arg Leu Gln Ile Ser Phe Phe Glu Tyr Ala Leu Gly Ala Ala Glu Asp
100 105 110
Ile Ala Asn Lys Ile Lys Gln Thr Thr Asp Glu Tyr Ile Asn Thr Ile
115 120 125
Leu Pro Pro Leu Thr Lys Ala Leu Phe Lys Tyr Val Arg Glu Gly Lys
130 135 140
Tyr Thr Phe Cys Thr Pro Gly His Met Gly Gly Thr Ala Phe Gln Lys
145 150 155 160
Ser Pro Val Gly Ser Leu Phe Tyr Asp Phe Phe Gly Pro Asn Thr Met
165 170 175
Lys Ser Asp Ile Ser Ile Ser Val Ser Glu Leu Gly Ser Leu Leu Asp
180 185 190
His Ser Gly Pro His Lys Glu Ala Glu Gln Tyr Ile Ala Arg Val Phe
195 200 205
Asn Ala Asp Arg Ser Tyr Met Val Thr Asn Gly Thr Ser Thr Ala Asn
210 215 220
Lys Ile Val Gly Met Tyr Ser Ala Pro Ala Gly Ser Thr Ile Leu Ile
225 230 235 240
Asp Arg Asn Cys His Lys Ser Leu Thr His Leu Met Met Met Ser Asp
245 250 255
Val Thr Pro Ile Tyr Phe Arg Pro Thr Arg Asn Ala Tyr Gly Ile Leu
260 265 270
Gly Gly Ile Pro Gln Ser Glu Phe Gln His Ala Thr Ile Ala Lys Arg
275 280 285
Val Lys Glu Thr Pro Asn Ala Thr Trp Pro Val His Ala Val Ile Thr
290 295 300
Asn Ser Thr Tyr Asp Gly Leu Leu Tyr Asn Thr Asp Phe Ile Lys Lys
305 310 315 320
Thr Leu Asp Val Lys Ser Ile His Phe Asp Ser Ala Trp Val Pro Tyr
325 330 335
Thr Asn Phe Ser Pro Ile Tyr Glu Gly Lys Cys Gly Met Ser Gly Gly
340 345 350
Arg Val Glu Gly Lys Val Ile Tyr Glu Thr Gln Ser Thr His Lys Leu
355 360 365
Leu Ala Ala Phe Ser Gln Ala Ser Met Ile His Val Lys Gly Asp Val
370 375 380
Asn Glu Glu Thr Phe Asn Glu Ala Tyr Met Met His Thr Thr Thr Ser
385 390 395 400
Pro His Tyr Gly Ile Val Ala Ser Thr Glu Thr Ala Ala Ala Met Met
405 410 415
Lys Gly Asn Ala Gly Lys Arg Leu Ile Asn Gly Ser Ile Glu Arg Ala
420 425 430
Ile Lys Phe Arg Lys Glu Ile Lys Arg Leu Arg Thr Glu Ser Asp Gly
435 440 445
Trp Phe Phe Asp Val Trp Gln Pro Asp His Ile Asp Thr Thr Glu Cys
450 455 460
Trp Pro Leu Arg Ser Asp Ser Thr Trp His Gly Phe Lys Asn Ile Asp
465 470 475 480
Asn Glu His Met Tyr Leu Asp Pro Ile Lys Val Thr Leu Leu Thr Pro
485 490 495
Gly Met Glu Lys Asp Gly Thr Met Ser Asp Phe Gly Ile Pro Ala Ser
500 505 510
Ile Val Ala Lys Tyr Leu Asp Glu His Gly Ile Val Val Glu Lys Thr
515 520 525
Gly Pro Tyr Asn Leu Leu Phe Leu Phe Ser Ile Gly Ile Asp Lys Thr
530 535 540
Lys Ala Leu Ser Leu Leu Arg Ala Leu Thr Asp Phe Lys Arg Ala Phe
545 550 555 560
Asp Leu Asn Leu Arg Val Lys Asn Met Leu Pro Ser Leu Tyr Arg Glu
565 570 575
Asp Pro Glu Phe Tyr Glu Asn Met Arg Ile Gln Glu Leu Ala Gln Asn
580 585 590
Ile His Lys Leu Ile Val His His Asn Leu Pro Asp Leu Met Tyr Arg
595 600 605
Ala Phe Glu Val Leu Pro Thr Met Val Met Thr Pro Tyr Ala Ala Phe
610 615 620
Gln Lys Glu Leu His Gly Met Thr Glu Glu Val Tyr Leu Asp Glu Met
625 630 635 640
Val Gly Arg Ile Asn Ala Asn Met Ile Leu Pro Tyr Pro Pro Gly Val
645 650 655
Pro Leu Val Met Pro Gly Glu Met Ile Thr Glu Glu Ser Arg Pro Val
660 665 670
Leu Glu Phe Leu Gln Met Leu Cys Glu Ile Gly Ala His Tyr Pro Gly
675 680 685
Phe Glu Thr Asp Ile His Gly Ala Tyr Arg Gln Ala Asp Gly Arg Tyr
690 695 700
Thr Val Lys Val Leu Lys Glu Glu Ser Lys Lys
705 710 715
<210> 3
<211> 100
<212> PRT
<213> Saccharomyces cerevisiae (Saccharomyces cerevisiae)
<400> 3
Phe Pro Pro Lys Lys Phe Lys Asp Leu Asn Ser Phe Leu Asp Asp Gln
1 5 10 15
Pro Lys Asp Pro Asn Leu Val Ala Ser Pro Phe Gly Gly Tyr Phe Lys
20 25 30
Asn Pro Ala Ala Asp Ala Gly Ser Asn Asn Ala Ser Lys Lys Ser Ser
35 40 45
Tyr Gln Gln Gln Arg Asn Trp Lys Gln Gly Gly Asn Tyr Gln Gln Gly
50 55 60
Gly Tyr Gln Ser Tyr Asp Ser Asn Tyr Asn Asn Tyr Asn Asn Tyr Asn
65 70 75 80
Asn Tyr Asn Asn Tyr Asn Asn Tyr Asn Asn Tyr Asn Lys Tyr Asn Gly
85 90 95
Gln Gly Tyr Gln
100
<210> 4
<211> 300
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> codon-optimized New1 protein nucleotide sequence
<400> 4
tttccgccga aaaagttcaa agacctgaac tctttcctgg acgaccagcc gaaagacccg 60
aacctggttg cgtctccgtt cggtggctac ttcaaaaacc cagcggcgga cgcgggttct 120
aacaacgcgt ctaagaaatc ttcttaccag cagcagcgta actggaaaca gggtggcaac 180
tatcagcaag gtggttacca gtcttacgac tctaattaca acaactacaa caactacaat 240
aactataata actacaacaa ctacaacaat tataacaaat acaacggtca gggctaccag 300
<210> 5
<211> 105
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> New1 protein amino acid sequence with GSGSG linker, wherein GSGSG is linker sequence
<400> 5
Gly Ser Gly Ser Gly Phe Pro Pro Lys Lys Phe Lys Asp Leu Asn Ser
1 5 10 15
Phe Leu Asp Asp Gln Pro Lys Asp Pro Asn Leu Val Ala Ser Pro Phe
20 25 30
Gly Gly Tyr Phe Lys Asn Pro Ala Ala Asp Ala Gly Ser Asn Asn Ala
35 40 45
Ser Lys Lys Ser Ser Tyr Gln Gln Gln Arg Asn Trp Lys Gln Gly Gly
50 55 60
Asn Tyr Gln Gln Gly Gly Tyr Gln Ser Tyr Asp Ser Asn Tyr Asn Asn
65 70 75 80
Tyr Asn Asn Tyr Asn Asn Tyr Asn Asn Tyr Asn Asn Tyr Asn Asn Tyr
85 90 95
Asn Lys Tyr Asn Gly Gln Gly Tyr Gln
100 105
<210> 6
<211> 315
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> nucleotide sequence of New1 nucleotide sequence after codon optimization and nucleotide sequence of linker corresponding thereto, wherein GGTTC TGGCTCTGGT is linker sequence
<400> 6
ggttctggct ctggttttcc gccgaaaaag ttcaaagacc tgaactcttt cctggacgac 60
cagccgaaag acccgaacct ggttgcgtct ccgttcggtg gctacttcaa aaacccagcg 120
gcggacgcgg gttctaacaa cgcgtctaag aaatcttctt accagcagca gcgtaactgg 180
aaacagggtg gcaactatca gcaaggtggt taccagtctt acgactctaa ttacaacaac 240
tacaacaact acaataacta taataactac aacaactaca acaattataa caaatacaac 300
ggtcagggct accag 315
<210> 7
<211> 2466
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> cadA-M nucleotide sequence
<400> 7
atgaacgtta ttgcaatatt gaatcacatg ggggtttatt ttaaagaaga acccatccgt 60
gaacttcatc gcgcgcttga acgtctgaac ttccagattg tttacccgaa cgaccgtgac 120
gacttattaa aactgatcga aaacaatgcg cgtctgtgcg gcgttatttt tgactgggat 180
aaatataatc tcgagctgtg cgaagaaatt agcaaaatga acgagaacct gccgttgtac 240
gcgttcgcta atacgtattc cactctcgat gtaagcctga atgacctgcg tttacagatt 300
agcttctttg aatatgcgct gggtgctgct gaagatattg ctaataagat caagcagacc 360
actgacgaat atatcaacac tattctgcct ccgctgacta aagcactgtt taaatatgtt 420
cgtgaaggta aatatacttt ctgtactcct ggtcacatgg gcggtactgc attccagaaa 480
agcccggtag gtagcctgtt ctatgatttc tttggtccga ataccatgaa atctgatatt 540
tccatttcag tatctgaact gggttctctg ctggatcaca gtggtccaca caaagaagca 600
gaacagtata tcgctcgcgt ctttaacgca gaccgcagct acatggtgac caacggtact 660
tccactgcga acaaaattgt tggtatgtac tctgctccag caggcagcac cattctgatt 720
gaccgtaact gccacaaatc gctgacccac ctgatgatga tgagcgatgt tacgccaatc 780
tatttccgcc cgacccgtaa cgcttacggt attcttggtg gtatcccaca gagtgaattc 840
cagcacgcta ccattgctaa gcgcgtgaaa gaaacaccaa acgcaacctg gccggtacat 900
gctgtaatta ccaactctac ctatgatggt ctgctgtaca acaccgactt catcaagaaa 960
acactggatg tgaaatccat ccactttgac tccgcgtggg tgccttacac caacttctca 1020
ccgatttacg aaggtaaatg cggtatgagc ggtggccgtg tagaagggaa agtgatttac 1080
gaaacccagt ccactcacaa actgctggcg gcgttctctc aggcttccat gatccacgtt 1140
aaaggtgacg taaacgaaga aacctttaac gaagcctaca tgatgcacac caccacttct 1200
ccgcactacg gtatcgtggc gtccactgaa accgctgcgg cgatgatgaa aggcaatgca 1260
ggtaagcgtc tgatcaacgg ttctattgaa cgtgcgatca aattccgtaa agagatcaaa 1320
cgtctgagaa cggaatctga tggctggttc tttgatgtat ggcagccgga tcatatcgat 1380
acgactgaat gctggccgct gcgttctgac agcacctggc acggcttcaa aaacatcgat 1440
aacgagcaca tgtatcttga cccgatcaaa gtcaccctgc tgactccggg gatggaaaaa 1500
gacggcacca tgagcgactt tggtattccg gccagcatcg tggcgaaata cctcgacgaa 1560
catggcatcg ttgttgagaa aaccggtccg tataacctgc tgttcctgtt cagcatcggt 1620
atcgataaga ccaaagcact gagcctgctg cgtgctctga ctgactttaa acgtgcgttc 1680
gacctgaacc tgcgtgtgaa aaacatgctg ccgtctctgt atcgtgaaga tcctgaattc 1740
tatgaaaaca tgcgtattca ggaactggct cagaatatcc acaaactgat tgttcaccac 1800
aatctgccgg atctgatgta tcgcgcattt gaagtgctgc cgacgatggt aatgactccg 1860
tatgctgcat tccagaaaga gctgcacggt atgaccgaag aagtttacct cgacgaaatg 1920
gtaggtcgta ttaacgccaa tatgatcctt ccgtacccgc cgggagttcc tctggtaatg 1980
ccgggtgaaa tgatcaccga agaaagccgt ccggttctgg agttcctgca gatgctgtgt 2040
gaaatcggcg ctcactatcc gggctttgaa accgatattc acggtgcata ccgtcaggct 2100
gatggccgct ataccgttaa ggtattgaaa gaagaaagca aatctagagg ttctggctct 2160
ggttttccgc cgaaaaagtt caaagacctg aactctttcc tggacgacca gccgaaagac 2220
ccgaacctgg ttgcgtctcc gttcggtggc tacttcaaaa acccagcggc ggacgcgggt 2280
tctaacaacg cgtctaagaa atcttcttac cagcagcagc gtaactggaa acagggtggc 2340
aactatcagc aaggtggtta ccagtcttac gactctaatt acaacaacta caacaactac 2400
aataactata ataactacaa caactacaac aattataaca aatacaacgg tcagggctac 2460
cagtaa 2466
<210> 8
<211> 821
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> CadA-M modified polypeptide sequence
<400> 8
Met Asn Val Ile Ala Ile Leu Asn His Met Gly Val Tyr Phe Lys Glu
1 5 10 15
Glu Pro Ile Arg Glu Leu His Arg Ala Leu Glu Arg Leu Asn Phe Gln
20 25 30
Ile Val Tyr Pro Asn Asp Arg Asp Asp Leu Leu Lys Leu Ile Glu Asn
35 40 45
Asn Ala Arg Leu Cys Gly Val Ile Phe Asp Trp Asp Lys Tyr Asn Leu
50 55 60
Glu Leu Cys Glu Glu Ile Ser Lys Met Asn Glu Asn Leu Pro Leu Tyr
65 70 75 80
Ala Phe Ala Asn Thr Tyr Ser Thr Leu Asp Val Ser Leu Asn Asp Leu
85 90 95
Arg Leu Gln Ile Ser Phe Phe Glu Tyr Ala Leu Gly Ala Ala Glu Asp
100 105 110
Ile Ala Asn Lys Ile Lys Gln Thr Thr Asp Glu Tyr Ile Asn Thr Ile
115 120 125
Leu Pro Pro Leu Thr Lys Ala Leu Phe Lys Tyr Val Arg Glu Gly Lys
130 135 140
Tyr Thr Phe Cys Thr Pro Gly His Met Gly Gly Thr Ala Phe Gln Lys
145 150 155 160
Ser Pro Val Gly Ser Leu Phe Tyr Asp Phe Phe Gly Pro Asn Thr Met
165 170 175
Lys Ser Asp Ile Ser Ile Ser Val Ser Glu Leu Gly Ser Leu Leu Asp
180 185 190
His Ser Gly Pro His Lys Glu Ala Glu Gln Tyr Ile Ala Arg Val Phe
195 200 205
Asn Ala Asp Arg Ser Tyr Met Val Thr Asn Gly Thr Ser Thr Ala Asn
210 215 220
Lys Ile Val Gly Met Tyr Ser Ala Pro Ala Gly Ser Thr Ile Leu Ile
225 230 235 240
Asp Arg Asn Cys His Lys Ser Leu Thr His Leu Met Met Met Ser Asp
245 250 255
Val Thr Pro Ile Tyr Phe Arg Pro Thr Arg Asn Ala Tyr Gly Ile Leu
260 265 270
Gly Gly Ile Pro Gln Ser Glu Phe Gln His Ala Thr Ile Ala Lys Arg
275 280 285
Val Lys Glu Thr Pro Asn Ala Thr Trp Pro Val His Ala Val Ile Thr
290 295 300
Asn Ser Thr Tyr Asp Gly Leu Leu Tyr Asn Thr Asp Phe Ile Lys Lys
305 310 315 320
Thr Leu Asp Val Lys Ser Ile His Phe Asp Ser Ala Trp Val Pro Tyr
325 330 335
Thr Asn Phe Ser Pro Ile Tyr Glu Gly Lys Cys Gly Met Ser Gly Gly
340 345 350
Arg Val Glu Gly Lys Val Ile Tyr Glu Thr Gln Ser Thr His Lys Leu
355 360 365
Leu Ala Ala Phe Ser Gln Ala Ser Met Ile His Val Lys Gly Asp Val
370 375 380
Asn Glu Glu Thr Phe Asn Glu Ala Tyr Met Met His Thr Thr Thr Ser
385 390 395 400
Pro His Tyr Gly Ile Val Ala Ser Thr Glu Thr Ala Ala Ala Met Met
405 410 415
Lys Gly Asn Ala Gly Lys Arg Leu Ile Asn Gly Ser Ile Glu Arg Ala
420 425 430
Ile Lys Phe Arg Lys Glu Ile Lys Arg Leu Arg Thr Glu Ser Asp Gly
435 440 445
Trp Phe Phe Asp Val Trp Gln Pro Asp His Ile Asp Thr Thr Glu Cys
450 455 460
Trp Pro Leu Arg Ser Asp Ser Thr Trp His Gly Phe Lys Asn Ile Asp
465 470 475 480
Asn Glu His Met Tyr Leu Asp Pro Ile Lys Val Thr Leu Leu Thr Pro
485 490 495
Gly Met Glu Lys Asp Gly Thr Met Ser Asp Phe Gly Ile Pro Ala Ser
500 505 510
Ile Val Ala Lys Tyr Leu Asp Glu His Gly Ile Val Val Glu Lys Thr
515 520 525
Gly Pro Tyr Asn Leu Leu Phe Leu Phe Ser Ile Gly Ile Asp Lys Thr
530 535 540
Lys Ala Leu Ser Leu Leu Arg Ala Leu Thr Asp Phe Lys Arg Ala Phe
545 550 555 560
Asp Leu Asn Leu Arg Val Lys Asn Met Leu Pro Ser Leu Tyr Arg Glu
565 570 575
Asp Pro Glu Phe Tyr Glu Asn Met Arg Ile Gln Glu Leu Ala Gln Asn
580 585 590
Ile His Lys Leu Ile Val His His Asn Leu Pro Asp Leu Met Tyr Arg
595 600 605
Ala Phe Glu Val Leu Pro Thr Met Val Met Thr Pro Tyr Ala Ala Phe
610 615 620
Gln Lys Glu Leu His Gly Met Thr Glu Glu Val Tyr Leu Asp Glu Met
625 630 635 640
Val Gly Arg Ile Asn Ala Asn Met Ile Leu Pro Tyr Pro Pro Gly Val
645 650 655
Pro Leu Val Met Pro Gly Glu Met Ile Thr Glu Glu Ser Arg Pro Val
660 665 670
Leu Glu Phe Leu Gln Met Leu Cys Glu Ile Gly Ala His Tyr Pro Gly
675 680 685
Phe Glu Thr Asp Ile His Gly Ala Tyr Arg Gln Ala Asp Gly Arg Tyr
690 695 700
Thr Val Lys Val Leu Lys Glu Glu Ser Lys Ser Arg Gly Ser Gly Ser
705 710 715 720
Gly Phe Pro Pro Lys Lys Phe Lys Asp Leu Asn Ser Phe Leu Asp Asp
725 730 735
Gln Pro Lys Asp Pro Asn Leu Val Ala Ser Pro Phe Gly Gly Tyr Phe
740 745 750
Lys Asn Pro Ala Ala Asp Ala Gly Ser Asn Asn Ala Ser Lys Lys Ser
755 760 765
Ser Tyr Gln Gln Gln Arg Asn Trp Lys Gln Gly Gly Asn Tyr Gln Gln
770 775 780
Gly Gly Tyr Gln Ser Tyr Asp Ser Asn Tyr Asn Asn Tyr Asn Asn Tyr
785 790 795 800
Asn Asn Tyr Asn Asn Tyr Asn Asn Tyr Asn Asn Tyr Asn Lys Tyr Asn
805 810 815
Gly Gln Gly Tyr Gln
820
<210> 9
<211> 30
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer cadA-SacI-F
<400> 9
tccgagctca tgaacgttat tgcaatattg 30
<210> 10
<211> 28
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer cadA-XbaI-R
<400> 10
gcctctagac cacttccctt gtacgagc 28
<210> 11
<211> 44
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer cadA-F2
<400> 11
atttcacaca ggaaacagct atgaacgtta ttgcaatatt gaat 44
<210> 12
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer cadA-R2
<400> 12
agctgtttcc tgtgtgaaat 20
<210> 13
<211> 34
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer cadAt-XbaI-R
<400> 13
gtcgactcta gatttgcttt cttctttcaa tacc 34
<210> 14
<211> 35
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer cadAt-XbaI-F
<400> 14
gaagaaagca aatctagagt cgacctgcag gcatg 35
<210> 15
<211> 30
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer New1-XbaI-F
<400> 15
ggctctagag gttctggctc tggttctccg 30
<210> 16
<211> 31
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer New1-HindIII-R
<400> 16
ggcaagcttt tactggtagc cctgaccgtt g 31
<210> 17
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> 46bp sequence upstream of the start codon of the original cadA Gene
<400> 17
atttcacaca ggaaacagct atgaccatga ttacgaattc gagctc 46
<210> 18
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> sequence upstream of the start codon of the corresponding cadA Gene after optimization
<400> 18
atttcacaca ggaaacagct 20
Claims (12)
1. A method for decarboxylating lysine using a host cell expressing an improved lysine decarboxylase consisting of SEQ ID NO:8 or consists of the sequence set forth in SEQ ID NO:7, wherein the sequence SEQ ID NO:17 is replaced with SEQ ID NO:18 upstream of the initiation codon of the cadA gene in said host cell, said method comprising the step of contacting the expressed modified lysine decarboxylase with lysine.
2. A method for producing lysine decarboxylase, the method comprising culturing a polypeptide comprising the nucleotide sequence of SEQ ID NO:7, and a host cell having the sequence shown in seq id no.
3. The method of claim 1 or 2, wherein the host cell is cultured at a temperature of 30 ℃ to 42 ℃.
4. The method of claim 1 or 2, wherein the host cell is cultured at a temperature of 30 ℃ or 42 ℃.
5. The method of claim 1 or 2, wherein the host cell is selected from the group consisting of escherichia coli (e.coli), bacillus subtilis (b.subilis), streptomyces coelicolor (s.coelicolor), hafnia alvei (h.alvei) and corynebacterium glutamicum (c.glutamicum).
6. The method of claim 5, wherein the host cell is hafnia alvei (h.alvei).
7. The method of claim 5, wherein the host cell is hafnia alvei that does not contain an endogenous plasmid.
8. The method of claim 5, wherein the host cell is transformed with a plasmid comprising the sequences set forth in SEQ ID NO.7 and SEQ ID NO. 18.
9. The method of claim 5, wherein lysine decarboxylation is performed at a pH of 6.0 to 8.0.
10. The method of claim 9, wherein lysine decarboxylation reaction is performed at a pH of 6.0 or 8.0.
11. A polynucleotide having the sequence of SEQ ID NO:7.
12. a lysine decarboxylase consisting of SEQ ID NO:8, and a sequence shown in SEQ ID NO.
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Citations (4)
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|---|---|---|---|---|
| WO2013151139A1 (en) * | 2012-04-05 | 2013-10-10 | 三井化学株式会社 | Method for producing 1,5-pentamethylenediamine, method for producing 1,5-pentamethylenediisocyanate, method for producing polyisocyanate composition, and method for storing catalyst cell |
| KR20160085602A (en) * | 2015-01-08 | 2016-07-18 | 광운대학교 산학협력단 | Method for preparing lysine decarboxylase using recombinant microorganism |
| CN107164352A (en) * | 2017-05-16 | 2017-09-15 | 中国科学院天津工业生物技术研究所 | New lysine decarboxylase mutant and its application |
| CN110300801A (en) * | 2016-11-24 | 2019-10-01 | 上海凯赛生物技术研发中心有限公司 | The control of the protein-protein interaction of acid decarboxylase |
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2018
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| WO2013151139A1 (en) * | 2012-04-05 | 2013-10-10 | 三井化学株式会社 | Method for producing 1,5-pentamethylenediamine, method for producing 1,5-pentamethylenediisocyanate, method for producing polyisocyanate composition, and method for storing catalyst cell |
| KR20160085602A (en) * | 2015-01-08 | 2016-07-18 | 광운대학교 산학협력단 | Method for preparing lysine decarboxylase using recombinant microorganism |
| CN110300801A (en) * | 2016-11-24 | 2019-10-01 | 上海凯赛生物技术研发中心有限公司 | The control of the protein-protein interaction of acid decarboxylase |
| CN107164352A (en) * | 2017-05-16 | 2017-09-15 | 中国科学院天津工业生物技术研究所 | New lysine decarboxylase mutant and its application |
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