WO2022001121A1 - 一种用于合成戊二胺的赖氨酸脱羧酶及其应用 - Google Patents

一种用于合成戊二胺的赖氨酸脱羧酶及其应用 Download PDF

Info

Publication number
WO2022001121A1
WO2022001121A1 PCT/CN2021/076305 CN2021076305W WO2022001121A1 WO 2022001121 A1 WO2022001121 A1 WO 2022001121A1 CN 2021076305 W CN2021076305 W CN 2021076305W WO 2022001121 A1 WO2022001121 A1 WO 2022001121A1
Authority
WO
WIPO (PCT)
Prior art keywords
lysine
lysine decarboxylase
buffer
amino acid
expression vector
Prior art date
Application number
PCT/CN2021/076305
Other languages
English (en)
French (fr)
Inventor
黄玉红
薛雅鞠
张锁江
Original Assignee
中国科学院过程工程研究所
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 中国科学院过程工程研究所 filed Critical 中国科学院过程工程研究所
Publication of WO2022001121A1 publication Critical patent/WO2022001121A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/001—Amines; Imines
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y401/00—Carbon-carbon lyases (4.1)
    • C12Y401/01—Carboxy-lyases (4.1.1)
    • C12Y401/01018—Lysine decarboxylase (4.1.1.18)

Definitions

  • the application belongs to the technical field of genetic engineering, in particular to a lysine decarboxylase for synthesizing pentamethylene diamine and its application, in particular to a lysine decarboxylase gene sequence, protein sequence and The constructed expression vector and recombinant engineering bacteria.
  • Nylon is widely used in many fields such as fibers and engineering plastics due to its excellent mechanical properties, heat resistance, corrosion resistance and other properties.
  • China's nylon production, production capacity and demand have all shown an increasing trend.
  • the production and consumption of nylon 66 is huge, but because the synthesis technology of adiponitrile, the precursor of its core monomer hexamethylenediamine, has been monopolized by foreign companies, resources are tight, and the cost fluctuates greatly, and it is mainly derived from petroleum.
  • Pentamethylenediamine is the product of lysine decarboxylation, and it is a homologue with hexamethylenediamine.
  • a variety of nylon 5X products can be synthesized from pentamethylenediamine and dibasic acid, such as nylon 52, 5T, 54, 56, 510, 516, 518, etc., have better characteristics such as light weight loss, moisture absorption and perspiration, temperature resistance, abrasion resistance, dyeability and intrinsic flame retardancy, and have broad development prospects.
  • the synthesis of bio-nylon 5X can not only reduce the dependence on petroleum resources, but also break the monopoly of the output and technology of hexamethylenediamine products by multinational enterprises, and has broad application prospects in the fields of national defense and aerospace.
  • lysine decarboxylase The key to bio-based nylon 5X is the efficient synthesis of its core monomer, pentamethylenediamine. Efficient and stable lysine decarboxylase is the core of bio-based pentamethylene diamine synthesis. Lysine decarboxylase has a wide range of sources. Currently, the microorganisms reported to have lysine decarboxylase are mainly Escherichia coli (E.coli), Hafnia alvei, Bacillus halodurans, and Bacillus cereus.
  • Bacillus cereus Bacterium cadaveris, Burkholderia vietnamensia, Chromobacterium violaceum, Vibrio cholerae, Streptomyces polosus, rumination Bacteria such as Selenomonas ruminantium, Salmonella typhimurium, etc., but only a few sources of lysine decarboxylase have been studied in depth, such as lysine from Escherichia coli and Hafnia albicans acid decarboxylase.
  • CN105316270B, CN105368766A and CN104498519A disclose that different genetic engineering strains are constructed by using the inducible lysine decarboxylase CadA of Escherichia coli to catalyze the synthesis of pentamethylenediamine in whole cells.
  • Tianjin University of Science and Technology used the temperature-regulated promoter pR-pL and the signal peptide pelBs to transform the overexpression vector, and the Institute of Microbiology, Chinese Academy of Sciences integrated T7CadB into the chassis cell genome to increase the production of pentamethylenediamine.
  • CN106148373A, EP3118312B1 and US7189543 carried out sequence modification of the inducible lysine decarboxylase CadA of Escherichia coli.
  • Ajinomoto Company of Japan screened mutants with higher thermostability through the directed evolution of CadA, and Mitsui Chemicals Co., Ltd. Mutants with 10-20% increased activity are disclosed in the patent.
  • mutant enzyme lines are limited to the transformation of Escherichia coli inducible lysine decarboxylase CadA, and the source is very single.
  • the mutant strains have low activity and catalytic intensity in the process of catalytic conversion of high concentration of lysine, and the cell cycle utilization rate is poor. , increasing the operating time, production cost, reducing the yield of pentamethylenediamine and restricting the development of industrialization.
  • the purpose of this application is to provide a lysine decarboxylase for synthesizing pentamethylene diamine and its application, including the amino acid sequence of the lysine decarboxylase, the nucleotide sequence encoding the same , gene expression vectors and recombinant engineering bacteria.
  • the present application provides a lysine decarboxylase for synthesizing pentamethylenediamine, which can catalyze L-lysine to generate pentamethylenediamine, the lysine decarboxylase having (I), (II) or Amino acid sequence shown in any one of (III):
  • the application provides a novel high-efficiency lysine decarboxylase, the lysine decarboxylase can catalyze L-lysine to generate pentamethylene diamine, and the lysine decarboxylase can be induced by constructing an expression vector and genetically engineered bacteria The expression is obtained, and the whole cell catalyzes the synthesis of pentamethylene diamine from lysine hydrochloride. Meanwhile, the lysine decarboxylase provided by the present application is still stable at higher temperature and pH, and has higher yield.
  • the lysine decarboxylase represented by SEQ ID NO.1 is denoted as LdcEdw, and the similarity between the amino acid sequence and Escherichia coli CadA is 86.7%;
  • the lysine decarboxylase represented by SEQ ID NO.2 is named It is LdcAer, and its similarity with Escherichia coli CadA is 75.77%;
  • the lysine decarboxylase represented by SEQ ID NO.3 is named LdcSal, and the similarity between the amino acid sequence and Escherichia coli CadA is 92.3%; SEQ ID NO.
  • the lysine decarboxylase represented by 4 was named LdcKle, and the amino acid sequence was 94.4% similar to Escherichia coli CadA.
  • the amino acid sequence has a great influence on the three-dimensional structure and enzymatic properties of the enzyme, and sometimes a small amount of amino acid difference may lead to a large difference in properties between the two enzymes, and the impact is unpredictable. Therefore, it is the differences between the two that endow LdcEdw with different enzymatic properties from Escherichia coli CadA, and it is these differences that enable LdcEdw to achieve efficient conversion of high concentrations of lysine hydrochloride.
  • amino acid sequence shown in any one of SEQ ID NO. 88%, 90%, 92%, 94%, 96%, 98% or 99%, etc. homology of amino acid sequences can also achieve the conversion of lysine hydrochloride.
  • the lysine decarboxylase described in this application is derived from Hafnia alvei, Bacillus halodurans, Bacillus cereus, Bacterium cadaveris, Burke Burkholderia vietnamensia, Chromobacterium violaceum, Edwardsiella tarda, Vibrio cholerae, Streptomyces polosus, Lunamonas ruminants (Selenomonas ruminantium), Salmonella typhimurium, Salmonella bongori, Serratia, Bordetella, Vibrio cholerae, Aeromonas Lysine decarboxylase genes and mutants of bacteria of the genus Aeromonas and Klebsiella.
  • the lysine decarboxylase is derived from Edwardsiella tarda, Klebsiella, Aeromonas or Salmonella bongori.
  • a second aspect a nucleotide encoding the lysine decarboxylase of the first aspect, the nucleotide having the nucleotide shown in any one of (i), (ii) or (iii) sequence:
  • LdcEdw its nucleic acid sequence is the sequence shown in SEQ ID NO.5.
  • the nucleic acid sequence is codon-optimized, the GC content is 43%, and the similarity with the nucleic acid sequence of Escherichia coli CadA is 74.5%.
  • the nucleic acid sequence of described LdcAer is the sequence shown in SEQ ID NO.6.
  • the nucleic acid sequence is codon-optimized, the GC content is 45%, and the similarity with the nucleic acid sequence of Escherichia coli CadA is 69.2%.
  • the nucleic acid sequence of described LdcSal is the sequence shown in SEQ ID NO.7.
  • the nucleic acid sequence is codon-optimized, the GC content is 43%, and the similarity with the nucleic acid sequence of Escherichia coli CadA is 78.1%.
  • the nucleic acid sequence of described LdcKle is the sequence shown in SEQ ID NO.8.
  • the nucleic acid sequence is codon-optimized, the GC content is 43%, and the similarity with the nucleic acid sequence of Escherichia coli CadA is 78.6%.
  • the present application also provides a gene expression vector.
  • the gene expression vector comprises: a nucleotide sequence encoding the amino acid sequence as described in the first aspect or the nucleotide as described in the second aspect.
  • the gene expression vector is a pET plasmid, preferably a pETDuet plasmid.
  • the expression vector may be pETDuet, or may be various expression vectors commonly used in the art for expressing target genes in Escherichia coli.
  • the gene expression vector further comprises a nucleotide sequence encoding a lysine pentamethylene diamine antiporter.
  • the application also provides a construction method of the gene expression vector as described in the third aspect, the construction method comprising the following steps:
  • the gene expression vector is obtained by inserting the nucleotide sequence encoding the amino acid sequence described in the first aspect or the nucleotide sequence described in the second aspect between the restriction enzyme cleavage sites of the plasmid.
  • the construction method also includes the manipulation of inserting the lysine pentamethylene diamine antiporter gene.
  • the lysine pentamethylene diamine antiporter gene further includes a signal peptide.
  • the signal peptide comprises an E. coli periplasmic secretory signal peptide.
  • the expression vector takes pelB signal peptide as an example, but it is not limited to this signal peptide, which can be common in E. coli, such as dsbA, hlyA, lamB, malE, ompA, ompF, ompT, phoA, etc.
  • the construction method specifically includes the following steps:
  • the lysine decarboxylase gene ldc and the lysine pentanediamine antiporter gene cadB were inserted between the NcoI/SacI and Bgl II/Pac I restriction sites of the pETDuet plasmid, respectively, to construct the plasmid pETDuet- ldc-cadB, and then introduce pelB before the cadB sequence, and connect through the NdeI/Bgl II restriction enzyme site. Finally, the constructed expression vector is pETDuet-ldc-pelB-cadB.
  • a fifth aspect a recombinant engineering bacterium for synthesizing pentamethylenediamine, comprising the gene expression vector described in the third aspect and/or the nucleotide encoding the lysine decarboxylase described in the first aspect.
  • the engineered bacteria can be E. coli BL21 (DE3).
  • the present application also provides a method for preparing pentamethylenediamine using the host cell as described in the fifth aspect, the method comprising the steps of:
  • the bacterial liquid obtained after culturing and inducing the recombinant engineered bacteria is centrifuged and resuspended to obtain a bacterial suspension, which is mixed and reacted with a buffer containing lysine hydrochloride and pyridoxal phosphate (PLP). and centrifugation to obtain the pentamethylenediamine.
  • a buffer containing lysine hydrochloride and pyridoxal phosphate (PLP). pyridoxal phosphate
  • the temperature of the reaction is 35-65°C, for example, it can be 35°C, 40°C, 45°C, 50°C, 55°C, 60°C or 65°C, etc.
  • the time is 0.5-24h, For example, it can be 0.5h, 1h, 1.5h, 2h, 2.5h, 3h, 5h, 6h, 8h, 10h, 12h, 15h, 16h, 18h, 20h or 24h, etc., preferably 1-4h.
  • the shaking rate during the reaction is 400-800 rpm, such as 400 rpm, 500 rpm, 550 rpm, 600 rpm, 650 rpm, 700 rpm or 800 rpm, and the like.
  • the rotation speed during the centrifugation is 8000-12000rpm, for example, it can be 8000rpm, 8500rpm, 9000rpm, 10000rpm, 10500rpm, 11000rpm or 12000rpm, etc.
  • the time is 1-3min, for example, it can be 1min, 1.5min, 2min, 2.5min or 3min, etc.
  • the molar concentration of lysine hydrochloride in the buffer is 0.1-3M, such as 0.1M, 0.5M, 0.8M, 1M, 1.2M, 1.5M, 1.8M, 2M, 2.5M or 3M et al.
  • the molar concentration of PLP in the buffer is 0.1-0.5 mM, such as 0.1 mM, 0.15 mM, 0.2 mM, 0.25 mM, 0.3 mM, 0.35 mM, 0.4 mM, 0.45 mM or 0.5 mM, etc.
  • the buffer includes any one of sodium acetate buffer, phosphate buffer, Tris-HCl buffer or sodium carbonate buffer, preferably phosphate buffer.
  • the pH of the buffer solution is 5-11, for example, it can be 5, 6, 6.4, 7, 7.2, 7.5, 8, 9, 10 or 11, etc., preferably 6-10; The same solution was used for suspension.
  • cryopreservation operation is also included, and the cryopreservation is: cryopreservation at -80°C for more than 1 hour.
  • the method for preparing pentamethylene diamine comprises the steps:
  • the method for detecting lysine and pentamethylenediamine is:
  • the application also provides a bio-based pentamethylene diamine synthesis using the lysine decarboxylase as described in the first aspect, the gene expression vector as described in the third aspect or the recombinant host cell as described in the fifth aspect. applications in .
  • the application provides a novel lysine decarboxylase capable of efficiently synthesizing pentamethylene diamine, and the novel and efficient lysine decarboxylase can be induced by constructing an expression vector and genetically engineered bacteria at the same time. Expression obtained, when whole cell catalyzes lysine hydrochloride to synthesize pentamethylenediamine, it remains stable at pH 5-9 and temperature 40-60 °C, and the catalytic efficiency is high, and the catalytic strength can reach 136-204g/L /h;
  • the novel lysine decarboxylase provided by this application can realize near-complete conversion of high-concentration lysine hydrochloride.
  • the pH is 6.5 and the temperature is 50°C
  • the conversion rate of pentamethylenediamine is the largest, which can reach 100%.
  • the catalytic strength can reach 204g/L/h, and its activity and catalytic strength are significantly higher than those of the existing reported Escherichia coli CadA and its mutants, which is conducive to the efficient synthesis of high-concentration pentamethylenediamine, and has extremely high industrial application prospects.
  • Figure 1 is a schematic diagram of the pETDuet-ldcEdw-pelB-cadB expression vector constructed in Example 1.
  • FIG. 2 is a graph showing the variation of LdcEdw whole-cell catalysis with reaction time in Example 5.
  • FIG. 3 is a graph showing the variation of LdcAer whole-cell catalysis with reaction time in Example 5.
  • the type of the expression vector there is no special requirement for the type of the expression vector, and it can be various expression vectors commonly used in the art that can express the target gene in Escherichia coli, such as plasmids and the like.
  • the construction method of the expression vector can adopt various methods commonly used in the art, such as ligating the target gene into the vector after enzyme digestion.
  • the HPLC detector used is: SPD-20A diode array detector; the detection column is: C18 column (Shim-pack GIST-HP-C18 column, 2.1 ⁇ 100 mm, 3 ⁇ m particle size).
  • HPLC detector SPD-20A diode array detector; detection column: C18 column; detection temperature: 35°C; injection volume: 5 ⁇ L, wavelength: 284 nm.
  • mobile phase A is acetonitrile
  • mobile phase B is 25mM pH 4.8 sodium acetate buffer solution
  • flow rate 0.5mL/min
  • time program proportion of mobile phase B: 0min 80%; 2min 75%; 22min 51.7%; 22.01min 80%; 27min 80%.
  • This example provides a gene expression vector containing lysine decarboxylase and an engineered strain expressing the same.
  • the lysine decarboxylase is named LdcEdw, its amino acid sequence is SEQ ID NO.1, and the similarity with Escherichia coli CadA is 86.7%; after codon optimization, the nucleotide sequence of synthesizing LdcEdw is SEQ ID NO.5 , the GC content of the nucleotide sequence is 43%, and the similarity with the nucleic acid sequence of Escherichia coli CadA is 74.5%.
  • the lysine decarboxylase is named LdcAer, its amino acid sequence is SEQ ID NO.2, and the similarity with Escherichia coli CadA is 75.77%; after codon optimization, the nucleotide sequence of synthetic LdcAer is SEQ ID NO.6 , the GC content of the nucleotide sequence is 45%, and the similarity with the nucleic acid sequence of Escherichia coli CadA is 69.2%.
  • the lysine decarboxylase is named LdcSal, its amino acid sequence is SEQ ID NO.3, and the similarity with Escherichia coli CadA is 92.3%; after codon optimization, the nucleotide sequence of synthetic LdcSal is SEQ ID NO.7 , the GC content of the nucleotide sequence is 43%, and the similarity with the nucleic acid sequence of Escherichia coli CadA is 78.1%.
  • the lysine decarboxylase is named LdcKle, its amino acid sequence is SEQ ID NO.4, and the similarity with Escherichia coli CadA is 94.4%; after codon optimization, the nucleotide sequence of synthesizing LdcKle is SEQ ID NO.8 , the GC content of the nucleotide sequence is 43%, and the similarity with the nucleic acid sequence of Escherichia coli CadA is 78.6%.
  • this application also uses EcCadA (GenBank: WP_001295383.1) derived from Escherichia coli as a comparison; and LdcEdw, LdcAer, LdcSal, LdcKle and CadA are respectively constructed in pETDuet between NcoI/SacI of the plasmid; at the same time, the CadB (GenBank: WP_000092909.1) gene was constructed between BglII/PacI of the pETDuet plasmid; His tag was inserted before the nucleotide sequence encoding the protein;
  • the signal peptide pelB was introduced before the cadB sequence to construct pETDuet-ldcEdw-pelB-cadB (as shown in Figure 1), pETDuet-ldcAer-pelB-cadB, pETDuet-ldcSal-pelB-cadB, pETDuet-ldcKle-pelB-cadB and pETDuet-EccadA-pelB-cadB plasmids were transferred into E.coli BL21 (DE3) chassis cells respectively to construct genetically engineered strains that catalyze the synthesis of pentanediamine in whole cells, named EDW, AER, SAL, KLE and WT. Store at -80°C.
  • This example is a whole-cell catalysis comparison of gene expression vectors containing lysine decarboxylase LdcEdw, LdcAer and EcCadA, respectively.
  • the engineering strains EDW, AER and WT obtained in Example 1 were cultured in 5 mL of LB medium supplemented with 100 mg/L ampicillin at 37° C. overnight to obtain seed liquid.
  • the seed solution was transferred into 50 mL of LB medium with 100 mg/L ampicillin at a transfer volume of 1%, and cultured at 37 °C.
  • IPTG iso-isopropylamine
  • propyl- ⁇ -D-thiogalactopyranoside was induced, and cultured at 20°C for 20h. Centrifuge at 4000 rpm, collect the cells, and store at -80°C.
  • the bacteria were resuspended with 50mM sodium acetate buffer at pH 6, and 500 ⁇ L of whole-cell catalysis was performed, wherein PLP 0.1mM, lysine hydrochloride 1M, bacterial suspension OD was 1.5, 45°C, 500rpm for 1h, and 12000rpm for 2min centrifugation , take the supernatant and dilute to detect the content of lysine hydrochloride and pentamethylene diamine.
  • the engineering strain EDW obtained in Example 1 was cultured in 5 mL of LB medium supplemented with 100 mg/L ampicillin at 37° C. overnight to obtain seed liquid.
  • the seed solution was transferred into 50 mL of LB medium with 100 mg/L ampicillin at 1% volume, and cultured at 37 °C.
  • OD 600 was 0.6
  • IPTG IPTG with a final concentration of 0.1 mM was added for induction, and the temperature was 20 °C.
  • centrifuge 4000 rpm to collect bacterial cells and store at -80°C.
  • EDW is the highest when the pH of the reaction system reaches 6.5. At this time, the conversion rate can reach 99.47%, and lysine hydrochloride is basically completely converted.
  • AER reached the highest at pH 6 of the reaction system, and the conversion rate reached 92.26% at this time.
  • the pH increased to 7.5 the yield of pentamethylenediamine decreased to 86.92%.
  • the engineering strain EDW obtained in Example 1 was cultured in 5 mL of LB medium supplemented with 100 mg/L ampicillin at 37° C. overnight to obtain seed liquid.
  • the seed solution was transferred into 50 mL of LB medium with 100 mg/L ampicillin at 1% volume, and cultured at 37 °C. When the OD 600 was 0.6, IPTG with a final concentration of 0.1 mM was added for induction. Continue to culture for 20h, centrifuge at 4000rpm, collect the cells, and store at -80°C.
  • the bacteria were resuspended with pH 8 buffer, and 500 ⁇ L of whole cell catalysis was performed, wherein PLP was 0.1 mM, lysine hydrochloride was 1 M, and the OD of the bacterial suspension was 1.5.
  • Catalysis was carried out at 35-65°C and 500 rpm for 1 h, centrifuged at 12,000 rpm for 2 min, and the content of pentamethylene diamine was detected after the supernatant was diluted.
  • the whole-cell catalysis results of EDW and AER at 35-65 °C are shown in Table 2. It is not difficult to find that the production of pentamethylenediamine first increases and then decreases with the increase of temperature. is 95.95%.
  • the engineering strains EDW and AER obtained in Example 1 were cultured in 5 mL of LB medium supplemented with 100 mg/L ampicillin at 37° C. overnight to obtain seed liquid.
  • the seed solution was transferred into 50 mL of LB medium with 100 mg/L ampicillin at 1% volume, and cultured at 37 °C. When the OD 600 was 0.6, IPTG with a final concentration of 0.1 mM was added for induction. Continue to culture for 20h, centrifuge at 4000rpm, collect the cells, and store at -80°C.
  • the 20mL shake flask whole-cell catalysis was carried out with phosphate buffer of pH 8 and temperature of 50°C.
  • the OD of the concentrated bacteria in the system was about 10, and the substrate concentration was 2M.
  • Samples were taken at 0h, 1h, and 2h, respectively. The content of pentamethylenediamine was detected after the supernatant was diluted.
  • the engineering strains EDW and AER obtained in Example 1 were cultured in 5 mL of LB medium supplemented with 100 mg/L ampicillin at 37° C. overnight to obtain seed liquid.
  • the seed solution was transferred into 50 mL of LB medium with 100 mg/L ampicillin at 1% volume, and cultured at 37 °C.
  • OD 600 was 0.6
  • IPTG IPTG with a final concentration of 0.1 mM was added for induction, and the temperature was 20 °C.
  • centrifuge 4000 rpm to collect bacterial cells and store at -80°C.
  • the cells were disrupted with a sonicator, the power was 40-60%, centrifuged at 8000 rpm, the cell debris was precipitated, filtered with a 0.22 ⁇ m filter, and the protein was purified using a 5 ml Histrap purification column on an AKTA protein purifier, followed by 5 mL HiTrap Desalting desalting column was used to replace the preservation solution, and the concentration of purified lysine decarboxylase LdcEdw and LdcAer was determined by BCA protein quantification method.
  • the in vitro catalytic reaction system of lysine decarboxylase LdcEdw is 500 ⁇ L, the concentration of lysine hydrochloride is 1.5M, and the concentration of PLP is 0.1 mM.
  • pH 6.5 catalyzed at 500rpm for 1h, centrifuged at 12,000rpm for 2min, took the supernatant and diluted it to detect the content of pentamethylenediamine.
  • the reaction system was 500 ⁇ L, the concentration of lysine hydrochloride was 1.5 M, and the concentration of PLP was 0.1 mM.
  • the pure LdcAer enzyme was diluted 50 times and added to 190 ⁇ L.
  • the lysine decarboxylase LdcEdw provided by the present application can efficiently catalyze the synthesis of pentamethylenediamine.
  • the optimal catalytic temperature for whole-cell catalysis is 50 °C
  • the optimal pH of the catalytic system is 6.5
  • the catalytic strength can reach 204 g. /L/h, can achieve complete conversion of high concentration of lysine hydrochloride
  • the optimal catalytic temperature of LdcAer whole cell catalysis is 50 °C
  • the optimal pH of the catalytic system is 6, and the conversion rate of pentamethylene diamine can reach 97.2%.
  • a recombinant engineering bacterium is constructed with the amino acid sequences shown in SEQ ID NO.3 and 4 at the same time, which can also express pentamethylenediamine efficiently. Due to space limitations and for the sake of simplicity, only the experimental results are indicated here: SEQ ID NO.
  • the amino acid sequences shown in ID NO.3 and SEQ ID NO.4 were constructed by the methods of Examples 1 and 2.
  • the pH of the system was 5-10, and the temperature was The reaction is stable at 40-55°C, the optimum catalytic temperature is 50°C, the optimum pH of the catalytic system is 6 and 7.5 respectively, and the conversion rate of pentamethylene diamine can reach 72.5%.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biomedical Technology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

提供了一种用于合成戊二胺的赖氨酸脱羧酶及其应用，包括该赖氨酸脱羧酶基因和蛋白序列、构建的表达载体和基因工程菌株并在生物基戊二胺合成中的应用。通过构建表达载体和基因工程菌，诱导表达赖氨酸脱羧酶，全细胞催化合成戊二胺。所述赖氨酸脱羧酶可实现高浓度赖氨酸盐酸盐100％转化，戊二胺生产强度可达204g/L/h。

Description

一种用于合成戊二胺的赖氨酸脱羧酶及其应用 技术领域
本申请属于基因工程技术领域，具体涉及一种用于合成戊二胺的赖氨酸脱羧酶及其应用，尤其涉及一种稳定高效合成戊二胺的赖氨酸脱羧酶基因序列、蛋白序列及其构建的表达载体和重组工程菌。
背景技术
尼龙因具有优良的机械性能、耐热性、耐腐蚀性等性质而在纤维和工程塑料等多个领域被广泛应用，近年来中国尼龙产量、产能和需求量均呈增长趋势。其中尼龙66的产量和消费量巨大，但由于其核心单体己二胺的前驱体己二腈的合成技术一直被国外企业垄断，资源紧张，成本波动巨大，且主要来源于石油。
戊二胺为赖氨酸脱羧的产物，与己二胺互为同系物，由戊二胺和二元酸可以合成多种尼龙5X产品，如尼龙52、5T、54、56、510、516、518等，具有更佳的轻质减重、吸湿排汗、耐温性、耐磨性、染色性及本质阻燃等特性，具有广阔的发展前景。生物尼龙5X的合成不仅能够降低对石油资源的依存度，而且能够打破跨国企业对己二胺产品的输出和技术的垄断，在国防和航天等领域具有广阔的应用前景。
生物基尼龙5X的关键是其核心单体戊二胺的高效合成。高效稳定的赖氨酸脱羧酶是生物基戊二胺合成的核心。赖氨酸脱羧酶来源广泛，目前报道存在赖氨酸脱羧酶的微生物主要是大肠杆菌(E.coli)、蜂房哈夫尼菌(Hafnia alvei)、耐碱芽胞杆菌(Bacillus halodurans)、蜡样芽胞杆菌(Bacillus cereus)、尸杆菌(Bacterium cadaveris)、伯克霍尔德氏菌(Burkholderia vietnamensia)、青紫色素杆菌(Chromobacterium violaceum)、霍乱弧菌(Vibrio cholerae)、毛链霉菌(Streptomyces polosus)、反刍动物月形单胞菌(Selenomonas ruminantium)、鼠伤寒沙门氏菌(Salmonella typhimurium)等细菌，但目前仅对少数来源的赖氨酸脱羧酶进行深入研究，如来自大肠杆菌和蜂房哈夫尼菌的赖氨酸脱羧酶。
CN105316270B、CN105368766A和CN104498519A中公开了利用大肠杆菌的诱导型赖氨酸脱羧酶CadA构建不同的基因工程菌株，全细胞催化合成戊二胺。同时，天津科技大学使用温度调控型启动子pR-pL和信号肽pelBs对过表达载体进行改造，中国科学院微生物研究所将T7CadB整合至底盘细胞基因组中，提高了戊二胺产量。CN106148373A、EP3118312B1与US7189543对大肠杆菌的诱导型赖氨酸脱羧酶CadA进行序列改造，日本味之素公司通过对CadA的定向进化，筛选到热稳定性更高的突变体，三井化学公司也在其专利中公开了活性提高10-20％的突变体。
但这些突变酶系均仅局限于对大肠杆菌诱导型赖氨酸脱羧酶CadA下改造，来源非常单一，突变菌株在赖氨酸高浓度催化转化过程中活性和催化强度低，细胞循环利用率差，增加了操作时间、生产成本，降低了戊二胺得率而制约了工业化发展。
发明内容
鉴于现有技术中存在的问题，本申请的目的在于提供一种用于合成戊二胺的赖氨酸脱羧酶及其应用，包括赖氨酸脱羧酶的氨基酸序列、编码其的核苷酸序列、基因表达载体以及重组工程菌。
为达此目的，本申请采用以下技术方案：
第一方面，本申请提供一种用于合成戊二胺的赖氨酸脱羧酶，可催化L-赖氨酸生成戊二胺，所述赖氨酸脱羧酶具有(I)、(II)或(III)中的任意一个所示的氨基酸序列：
(I)如SEQ ID NO.1～4任一项所示的氨基酸序列；
(II)与SEQ ID NO.1～4中任一项所示的氨基酸序列具有≥75％同源性的氨基酸序列；
(II)如SEQ ID NO.1～4任一项所示的氨基酸序列经修饰、取代、缺失或添加至少一个氨基酸获得的氨基酸序列。
本申请提供了一种新型高效赖氨酸脱羧酶，所述赖氨酸脱羧酶可以催化L-赖氨酸生成戊二胺，该赖氨酸脱羧酶可以通过构建表达载体、基因工程菌后诱导表达得到，全细胞催化赖氨酸盐酸盐合成戊二胺。同时，本申请提供的赖氨酸脱羧酶在较高的温度以及pH时仍然稳定，且产量较高。
本申请中，将SEQ ID NO.1代表的赖氨酸脱羧酶记为LdcEdw，所述氨基酸序列与大肠杆菌CadA的相似度为86.7％；将SEQ ID NO.2代表的赖氨酸脱羧酶命名为LdcAer，其与大肠杆菌CadA相似度75.77％；将SEQ ID NO.3代表的赖氨酸脱羧酶命名为LdcSal，所述氨基酸序列与大肠杆菌CadA的相似度为92.3％；将SEQ ID NO.4代表的赖氨酸脱羧酶命名为LdcKle，所述氨基酸序列与大肠杆菌CadA的相似度为94.4％。而氨基酸序列对于酶的立体结构以及酶学性质有较大影响，有时少量氨基酸的不同也可能会导致两个酶之间有较大的性质差异，其造成的影响是不可预期的。因此，而正是这两者的差异性赋予了LdcEdw与大肠杆菌CadA不同的酶学性质，也正是这些差异使LdcEdw能够实现对高浓度赖氨酸盐酸盐的高效转化。
同时，除以上四条序列外，与SEQ ID NO.1～4中任一项所示的氨基酸序列具有≥75％(例如同源性大于75％、78％、80％、84％、85％、88％、90％、92％、94％、96％、98％或99％等)同源性的氨基酸序列也可实现赖氨酸盐酸盐的转化。
本申请所述的赖氨酸脱羧酶的来源于蜂房哈夫尼菌(Hafnia alvei)、耐碱芽胞杆菌(Bacillus halodurans)、蜡样芽胞杆菌(Bacillus cereus)、尸杆菌(Bacterium cadaveris)、伯克霍尔德氏菌(Burkholderia vietnamensia)、青紫色素杆菌(Chromobacterium violaceum)、迟缓爱德华氏菌(Edwardsiella tarda)、霍乱弧菌(Vibrio cholerae)、毛链霉菌(Streptomyces polosus)、反刍动物月形单胞菌(Selenomonas ruminantium)、鼠伤寒沙门氏菌(Salmonella typhimurium)、邦戈沙门氏菌(Salmonella bongori)、沙雷氏菌属(Serratia)、博代氏杆菌属(Bordetella)、霍乱弧菌(Vibrio cholerae)、气单胞菌属(Aeromonas)、克雷伯氏菌属(Klebsiella)等细菌的赖氨酸脱羧酶基因及其突变体。
优选地，所述赖氨酸脱羧酶来源于迟缓爱德华氏菌(Edwardsiella tarda)、克雷伯氏菌属(Klebsiella)、气单胞菌属(Aeromonas)或邦戈沙门氏菌(Salmonella bongori)。
第二方面，一种编码第一方面所述的赖氨酸脱羧酶的核苷酸，所述核苷酸具有(i)、(ii)或(iii)中的任意一个所示的核苷酸序列：
(i)编码第一方面所述的赖氨酸脱羧酶的核苷酸序列；
(ii)编码SEQ ID NO.1～4任一项所示的赖氨酸脱羧酶的核苷酸序列；
(iii)如SEQ ID NO.5～8任一项所示的核苷酸序列。
所述的LdcEdw其核酸序列为SEQ ID NO.5所示的序列。所述的核酸序列经密码子优化，GC含量43％，与大肠杆菌CadA核酸序列相似度74.5％。所述的LdcAer其核酸序列为SEQ ID NO.6所示的序列。所述的核酸序列经密码子优化，GC含量45％，与大肠杆菌CadA核酸序列相似度69.2％。所述的LdcSal其核酸序列为SEQ ID NO.7所示的序列。所述的核酸序列经密码子优化，GC含量43％，与大肠杆菌CadA核酸序列相似度78.1％。所述的LdcKle其核酸序列为SEQ ID NO.8所示的序列。所述的核酸序列经密码子优化，GC含量43％，与大肠杆菌CadA核酸序列相似度78.6％。
第三方面，本申请还提供了一种基因表达载体。所述基因表达载体包括：编码如第一方面所述的氨基酸序列的核苷酸序列或如第二方面所述的核苷酸。
优选地，所述基因表达载体为pET质粒，优选为pETDuet质粒。所述表达载体可以是pETDuet，也可以是大肠杆菌中表达目的基因的本领域常用的各种表达载体。优选地，所述基因表达载体还包括编码赖氨酸戊二胺反向转运蛋白的核苷酸序列。
第四方面，本申请还提供一种如第三方面所述的基因表达载体的构建方法，所述构建方法包括如下步骤：
将编码如第一方面所述的氨基酸序列的核苷酸序列或如第二方面所述的核苷酸***质粒的限制性酶切位点之间，得到所述基因表达载体。
所述构建方法还包括***赖氨酸戊二胺反向转运蛋白基因的操作。
优选地，所述赖氨酸戊二胺反向转运蛋白基因前还包括信号肽。
优选地，所述信号肽包括大肠杆菌周质空间分泌信号肽。
所述的表达载体中以pelB信号肽为例，但不仅限于该信号肽，可以是大肠杆菌常见的，如dsbA、hlyA、lamB、malE、ompA、ompF、ompT、phoA等。
示例性的，所述构建方法具体包括如下步骤：
将赖氨酸脱羧酶基因ldc和编码赖氨酸戊二胺反向转运蛋白基因cadB，分别***pETDuet质粒的NcoI/SacI、Bgl Ⅱ/Pac Ⅰ限制性酶切位点之间，构建质粒pETDuet-ldc-cadB，再在cadB序列前引入pelB，通过NdeI/Bgl Ⅱ限制性酶切位点连接，最终，构建好的表达载体为pETDuet-ldc-pelB-cadB。
第五方面，一种用于合成戊二胺的重组工程菌，包含如第三方面所述的基因表达载体和/或编码第一方面所述的赖氨酸脱羧酶的核苷酸。所述工程菌可以是E.coli BL21(DE3)。
第六方面，本申请还提供一种利用如第五方面所述的宿主细胞制备戊二胺的方法，所述方法包括如下步骤：
将所述重组工程菌培养、诱导后的得到的菌液离心并重悬，得到菌悬液，所述菌悬液与含有赖氨酸盐酸盐和磷酸吡哆醛(PLP)的缓冲液混合反应、离心后得到所述戊二胺。
作为本申请优选的技术方案，所述反应的温度为35～65℃，例如可以是35℃、40℃、45℃、50℃、55℃、60℃或65℃等，时间为0.5-24h，例如可以是0.5h、1h、1.5h、2h、2.5h、3h、5h、6h、8h、10h、12h、15h、16h、18h、20h或24h等，优选为1～4h。
优选地，所述反应时的振荡速率为400～800rpm，例如可以是400rpm、500rpm、550rpm、600rpm、650rpm、700rpm或800rpm等。
优选地，所述离心时的转速为8000～12000rpm，例如可以是8000rpm、8500rpm、9000rpm、10000rpm、10500rpm、11000rpm或12000rpm等，时间为1～3min，例如可以是1min、1.5min、2min、2.5min或3min等。
优选地，所述缓冲液中赖氨酸盐酸盐的摩尔浓度为0.1～3M，例如可以是0.1M、0.5M、0.8M、1M、1.2M、1.5M、1.8M、2M、2.5M或3M等。
优选地，所述缓冲液中PLP的摩尔浓度为0.1～0.5mM，例如可以是0.1mM、0.15mM、0.2mM、0.25mM、0.3mM、0.35mM、0.4mM、0.45mM或0.5mM等。
优选地，所述缓冲液包括醋酸钠缓冲液、磷酸缓冲液、Tris-HCl缓冲液或碳酸钠缓冲液中的任意一种，优选为磷酸缓冲液。
优选地，所述缓冲液的pH为5～11，例如可以是5、6、6.4、7、7.2、7.5、8、9、10或11等，优选为6～10；所述缓冲液与重悬时使用的溶液相同。
优选地，所述诱导后的菌液离心后，还包括在冻存操作，所述冻存为：在-80℃下冻存1h以上。
优选地，所述制备戊二胺的方法包括如下步骤：
(1)将重组工程菌培养、诱导后得到的菌液离心，冻存后重悬得到菌悬液；
(2)将所述菌悬液与含有赖氨酸盐酸盐和磷酸吡哆醛的缓冲液混合，所述缓冲液中赖氨酸盐酸盐的摩尔浓度为0.1～3M，磷酸吡哆醛的摩尔浓度为0.1～0.5mM，所述缓冲液为磷酸缓冲液，pH为6～10；
(3)在35～65℃下以400～800rpm振荡反应1～4h，再以8000～12000rpm离心1～3min，得到戊二胺。
本申请中，在合成戊二胺后，检测赖氨酸和戊二胺的方法为：
(1)在反应体系中加入600μL 50mM pH 9的硼酸缓冲液、200μL甲醇、60μL稀释样品、130μL ddH 2O和10μL 1M乙氧基亚甲基丙二酸二乙酯(DEEMM)，室温下放置反应10min，转移至60～80℃水浴1～2h，终止反应；
(2)使用反相高效液相色谱紫外(nm)检测器进行检测，流动相A为100％的乙腈；流动相B为25mM pH 4.8醋酸钠缓冲溶液，流速0.5mL/min；检测柱为C18；柱检测温度：35℃； 进样量：2～10μL；波长：284nm。
采用梯度洗脱：0min A:B为20:80；2min A:B为25:75；27min A:B为62.5:37.5；27.01min A:B为20:80；37min A:B为20:80；37.01min结束，所述的梯度洗脱并不仅限于上述梯度洗脱比例。
第六方面，本申请还提供一种利用如第一方面所述的赖氨酸脱羧酶、如第三方面基因表达载体或如第五方面所述的重组的宿主细胞在生物基戊二胺合成中的应用。
本申请所述的数值范围不仅包括上述列举的点值，还包括没有列举出的上述数值范围之间的任意的点值，限于篇幅及出于简明的考虑，本申请不再穷尽列举所述范围包括的具体点值。
与现有技术相比，本申请至少具有以下有益效果：
(1)本申请提供了一种能够高效合成戊二胺的新型赖氨酸脱羧酶，所述新型高效赖氨酸脱羧酶同时该赖氨酸脱羧酶可以通过构建表达载体、基因工程菌后诱导表达得到，全细胞催化赖氨酸盐酸盐合成戊二胺时，在pH为5～9、温度为40～60℃下保持稳定，且催化效率较高，催化强度可达136～204g/L/h；
(2)本申请提供的新型赖氨酸脱羧酶可实现高浓度赖氨酸盐酸盐近完全转化，在pH 6.5、温度为50℃时，戊二胺转化率最大，可达100％，其催化强度可达204g/L/h，其活性和催化强度均明显高于现有报道的大肠杆菌CadA及其突变体，有利于高效合成高浓度戊二胺，有极高的工业应用前景。
附图说明
图1为实施例1中构建的pETDuet-ldcEdw-pelB-cadB表达载体示意图。
图2为实施例5中LdcEdw全细胞催化随反应时间的变化曲线图。
图3为实施例5中LdcAer全细胞催化随反应时间的变化曲线图。
具体实施方式
下面结合附图并通过具体实施方式来进一步说明本申请的技术方案，但下述的实例仅仅是本申请的简易例子，并不代表或限制本申请的权利保护范围，本申请的保护范围以权利要求书为准。
本申请中，对表达载体的种类没有特殊要求，可以为能够在大肠杆菌中表达目的基因的本领域常用的各种表达载体，例如质粒等。表达载体的构建方法可以采用本领域常用的各种方法，如将目的基因经过酶切处理后连接至载体中。
以下实施例中，采用的HPLC检测器为：SPD-20A二极管阵列检测器；检测柱为：C18柱(Shim-pack GIST-HP-C18 column，2.1×100mm，3μm particle size)。
本申请中所用的赖氨酸盐酸盐和戊二胺的检测方法具体步骤如下：
在反应体系中加入600μL 50mM pH为9硼酸缓冲液、200μL甲醇、60μL反应液和130μL ddH 2O和10μL 1M乙氧基亚甲基丙二酸二乙酯(DEEMM)，室温下反应10min后，转 移至70℃，静置2h终止反应，并用高效液相色谱(HPLC)进行检测。
HPLC检测器：SPD-20A二极管阵列检测器；检测柱：C18柱；检测温度：35℃；进样量：5μL，波长：284nm。
其中流动相A为乙腈；流动相B为25mM pH 4.8醋酸钠缓冲溶液；流速0.5mL/min；时间程序(流动相B所占比例)：0min 80％；2min 75％；22min 51.7％；22.01min 80％；27min 80％。
实施例1
本实施例提供含有赖氨酸脱羧酶的基因表达载体及表达其的工程菌株。
将所述赖氨酸脱羧酶命名为LdcEdw，其氨基酸序列为SEQ ID NO.1，与大肠杆菌CadA相似度86.7％；经密码子优化之后，合成LdcEdw的核苷酸序列为SEQ ID NO.5，所述核苷酸序列的GC含量43％，与大肠杆菌CadA核酸序列相似度74.5％。
将所述赖氨酸脱羧酶命名为LdcAer，其氨基酸序列为SEQ ID NO.2，与大肠杆菌CadA相似度75.77％；经密码子优化之后，合成LdcAer的核苷酸序列为SEQ ID NO.6，所述核苷酸序列的GC含量45％，与大肠杆菌CadA核酸序列相似度69.2％。
将所述赖氨酸脱羧酶命名为LdcSal，其氨基酸序列为SEQ ID NO.3，与大肠杆菌CadA相似度92.3％；经密码子优化之后，合成LdcSal的核苷酸序列为SEQ ID NO.7，所述核苷酸序列的GC含量43％，与大肠杆菌CadA核酸序列相似度78.1％。
将所述赖氨酸脱羧酶命名为LdcKle，其氨基酸序列为SEQ ID NO.4，与大肠杆菌CadA相似度94.4％；经密码子优化之后，合成LdcKle的核苷酸序列为SEQ ID NO.8，所述核苷酸序列的GC含量43％，与大肠杆菌CadA核酸序列相似度78.6％。
同时，为了比较本申请提供的赖氨酸脱羧酶的功能，本申请还将大肠杆菌来源的EcCadA(GenBank：WP_001295383.1)作为比较；并将LdcEdw、LdcAer、LdcSal、LdcKle和CadA分别构建在pETDuet质粒的NcoI/SacI之间；同时将CadB(GenBank：WP_000092909.1)基因构建在pETDuet质粒的BglⅡ/PacⅠ之间；编码蛋白的核苷酸序列之前均***His标签；
再在cadB序列前引入信号肽pelB，构建pETDuet-ldcEdw-pelB-cadB(如图1所示)、pETDuet-ldcAer-pelB-cadB、pETDuet-ldcSal-pelB-cadB、pETDuet-ldcKle-pelB-cadB和pETDuet-EccadA-pelB-cadB质粒，并分别转入E.coli BL21(DE3)底盘细胞，构建全细胞催化合成戊二胺的基因工程菌株，命名为EDW、AER、SAL、KLE和WT，并于-80℃保存。
实施例2
本实施例为分别含有赖氨酸脱羧酶LdcEdw、LdcAer与EcCadA的基因表达载体的全细胞催化比较。
将实施例1中获取的工程菌株EDW、AER和WT在5mL加入100mg/L氨苄抗生素的LB培养基中，37℃，过夜培养，获得种子液。
然后，将种子液按1％体积的转接量分别转接入50mL加入100mg/L氨苄抗生素的LB培养基中，37℃培养，当OD 600为0.6时加入终浓度为0.1mM的IPTG(异丙基-β-D-硫代吡 喃半乳糖苷)进行诱导，20℃继续培养20h。4000rpm离心，收集菌体，-80℃保存。
用50mM pH 6的醋酸钠缓冲液将菌体重悬，进行500μL全细胞催化，其中PLP 0.1mM，赖氨酸盐酸盐1M，菌悬液OD为1.5，45℃，500rpm催化1h，12000rpm离心2min，取上清并稀释，检测赖氨酸盐酸盐和戊二胺的含量。
最终检测结果可知：EDW全细胞催化戊二胺的产量是109g/L，是WT产量的1.6倍；AER全细胞催化戊二胺的产量是89.4g/L是WT产量的1.3倍。
实施例3
本实施例中检测不同pH下赖氨酸脱羧酶LdcEdw和LdcAer的全细胞催化
将实施例1中获取的工程菌株EDW在5mL加入100mg/L氨苄抗生素的LB培养基中，37℃，过夜培养，获得种子液。
将种子液按1％体积的转接量转接入50mL加入100mg/L氨苄抗生素的LB培养基中，37℃培养，当OD 600为0.6时加入终浓度为0.1mM的IPTG进行诱导，20℃继续培养20h后，4000rpm离心，收集菌体，-80℃保存。
将菌体分别用50mM不同pH的缓冲液重悬后，进行500μL全细胞催化，其中PLP 0.1mM，赖氨酸盐酸盐1M，菌悬液OD为1.5。
45℃500rpm催化1h，12000rpm离心2min，取上清稀释后检测戊二胺的含量。EDW和AER在pH 4.5～7.5下的全细胞催化结果如下表1，其中，EDW在反应体系pH达到6.5时最高，此时转化率可达99.47％，赖氨酸盐酸盐基本完全被转化，之后体系pH再增高戊二胺产量开始下降；AER在反应体系pH 6时达到最高，此时转化率可达92.26％，当pH升高至7.5，戊二胺产量下降至86.92％。
表1
Figure PCTCN2021076305-appb-000001
实施例4
本实施例中检测不同温度对赖氨酸脱羧酶LdcEdw和LdcAer的全细胞催化影响。
将实施例1中获取的工程菌株EDW在5mL加入100mg/L氨苄抗生素的LB培养基中，37℃，过夜培养，获得种子液。
将种子液按1％体积的转接量转接入50mL加入有100mg/L氨苄抗生素LB培养基中，37℃培养，当OD 600为0.6时加入终浓度为0.1mM的IPTG进行诱导，20℃继续培养20h，4000rpm离心，收集菌体，-80℃保存。
用pH为8的缓冲液将菌体重悬，进行500μL全细胞催化，其中PLP 0.1mM，赖氨酸盐酸盐1M，菌悬液OD为1.5。
在35～65℃，500rpm条件下催化1h，12000rpm离心2min，取上清稀释后检测戊二胺的含量。EDW和AER在35～65℃下的全细胞催化结果如下表2，不难发现戊二胺产量随着温度的升高先增加后减小，在50℃时，EDW戊二胺转化率最大，为95.95％。
表2
Figure PCTCN2021076305-appb-000002
实施例5
本实施例中验证赖氨酸脱羧酶LdcEdw和LdcAer的摇瓶全细胞催化能力。
将实施例1中获取的工程菌株EDW和AER在5mL加入100mg/L氨苄抗生素的LB培养基中，37℃，过夜培养，获得种子液。
将种子液按1％体积的转接量转接入50mL加入有100mg/L氨苄抗生素LB培养基中，37℃培养，当OD 600为0.6时加入终浓度为0.1mM的IPTG进行诱导，20℃继续培养20h，4000rpm离心，收集菌体，-80℃保存。
用pH为8的磷酸缓冲液、温度为50℃的条件进行20mL摇瓶全细胞催化，体系中浓缩菌体的OD约为10，底物浓度为2M，在0h、1h、2h分别取样，取上清稀释后检测戊二胺的含量。
LdcEdw的检测结果如图2所示，其横坐标为时间(h)，纵坐标为含量(单位mM)，全细胞催化反应前30min就有明显气泡产生。由图可知，赖氨酸盐酸盐的量随时间增加逐渐减少，相对的，戊二胺的含量逐渐增加，反应2h后，赖氨酸盐酸盐已几近完全转化，戊二胺产量为202.3g/L。将体系中浓缩菌体的OD增加至15，底物浓度为2M，反应1h后，赖氨酸盐酸盐已几近完全转化，戊二胺产量为204g/L，戊二胺生产强度为204g/L/h。
LdcAer的检测结果如图3所示，其横坐标为时间(h)，纵坐标为含量(单位mM)，全细胞催化反应前30min就有明显气泡产生，反应4h，戊二胺产量为198.4g/L，催化速率最高可达136g/L/h。
实施例6
本实施例中验证赖氨酸脱羧酶LdcEdw和LdcAer在不同浓度下的体外催化能力。
将实施例1中获取的工程菌株EDW和AER在5mL加入100mg/L氨苄抗生素的LB培养基中，37℃，过夜培养，获得种子液。
将种子液按1％体积的转接量转接入50mL加入100mg/L氨苄抗生素的LB培养基中，37℃培养，当OD 600为0.6时加入终浓度为0.1mM的IPTG进行诱导，20℃继续培养20h后，4000rpm离心，收集菌体，-80℃保存。
用超声破碎仪将细胞破碎，功率40-60％，8000rpm离心，沉淀细胞碎片，用0.22μm的滤膜进行过滤，使用5ml的Histrap纯化柱在AKTA蛋白纯化仪上进行蛋白纯化，再用5mL HiTrap Desalting脱盐柱置换保藏液，使用BCA蛋白定量方法测定纯化得到的赖氨酸脱羧酶LdcEdw和LdcAer的浓度。
赖氨酸脱羧酶LdcEdw的体外催化反应体系为500μL，赖氨酸盐酸盐浓度为1.5M，PLP浓度为0.1mM，将LdcEdw纯酶分别稀释10倍和50倍后，加入190μL稀释的纯酶到反应体系。在50℃，pH 6.5，500rpm条件下催化1h，12000rpm离心2min，取上清稀释后检测戊二胺的含量。赖氨酸脱羧酶LdcEdw体外酶催化中，添加60μg纯酶催化反应1h，戊二胺转化率可达到100％，即60μg纯酶能够催化1.5M的赖氨酸盐酸盐，使其完全转换为戊二胺。
赖氨酸脱羧酶LdcAer体外酶催化中，反应体系为500μL，赖氨酸盐酸盐浓度为1.5M，PLP浓度为0.1mM，将LdcAer纯酶稀释50倍加入190uL。在50℃，pH 6，500rpm条件下催化1h，12000rpm离心2min，取上清并稀释，用实施方式中所述方法进行检测，添加60μg纯酶催化反应1h，戊二胺转化率可达到94.43％。
综上所述，本申请提供的赖氨酸脱羧酶LdcEdw能够高效催化合成戊二胺，其全细胞催化的最佳催化温度为50℃，催化体系的最佳pH为6.5，催化强度可达204g/L/h，能够实现高浓度的赖氨酸盐酸盐的完全转化；LdcAer全细胞催化的最佳催化温度为50℃，催化体系的最佳pH为6，其戊二胺转化率可达到97.2％。本申请中，同时以SEQ ID NO.3和4所示的氨基酸序列构建重组工程菌，其同样能够高效表达戊二胺，限于篇幅及出于简明的考虑，此处仅写明实验结果：SEQ ID NO.3和SEQ ID NO.4所示的氨基酸序列通过实施例1和2的方法构建菌株，在添加1M赖氨酸盐酸盐的全细胞催化中，体系pH为5～10、温度为40～55℃下反应稳定，且最佳催化温度为50℃，催化体系的最佳pH分别为6和7.5，戊二胺的转化率可达72.5％。
申请人声明，以上所述仅为本申请的具体实施方式，但本申请的保护范围并不局限于此，所属技术领域的技术人员应该明了，任何属于本技术领域的技术人员在本申请揭露的技术范围内，可轻易想到的变化或替换，均落在本申请的保护范围和公开范围之内。

Claims (11)

  1. 一种用于合成戊二胺的赖氨酸脱羧酶，其具有(I)、(II)或(III)中的任意一个所示的氨基酸序列：
    (I)如SEQ ID NO.1～4任一项所示的氨基酸序列；
    (II)与SEQ ID NO.1～4中任一项所示的氨基酸序列具有≥75％同源性的氨基酸序列；
    (III)如SEQ ID NO.1～4任一项所示的氨基酸序列经修饰、取代、缺失或添加至少一个氨基酸获得的氨基酸序列。
  2. 根据权利要求1所述的赖氨酸脱羧酶，其中，所述赖氨酸脱羧酶来源于迟缓爱德华氏菌、蜂房哈夫尼菌、耐碱芽胞杆菌、蜡样芽胞杆菌、尸杆菌、伯克霍尔德氏菌、青紫色素杆菌、霍乱弧菌、毛链霉菌、反刍动物月形单胞菌、鼠伤寒沙门氏菌、邦戈沙门氏菌、沙雷氏菌属、博代氏杆菌属、霍乱弧菌、气单胞菌属或克雷伯氏菌属中的任意一种。
  3. 根据权利要求2所述的赖氨酸脱羧酶，其中，所述赖氨酸脱羧酶来源于迟缓爱德华氏菌、克雷伯氏菌属、气单胞菌属或邦戈沙门氏菌。
  4. 一种编码权利要求1至3中任一项所述的赖氨酸脱羧酶的核苷酸，其具有(i)、(ii)或(iii)中的任意一个所示的核苷酸序列：
    (i)编码权利要求1至3中任一项所述的赖氨酸脱羧酶的核苷酸序列；
    (ii)编码SEQ ID NO.1～4任一项所示的赖氨酸脱羧酶的核苷酸序列；
    (iii)如SEQ ID NO.5～8任一项所示的核苷酸序列。
  5. 一种用于合成戊二胺的基因表达载体，其包括：编码如权利要求1至3中任一项所述的赖氨酸脱羧酶的核苷酸序列或如权利要求4所述的核苷酸；
    优选地，所述基因表达载体为pET质粒，优选为pETDuet质粒；
    优选地，所述基因表达载体还包括编码赖氨酸戊二胺反向转运蛋白的核苷酸序列。
  6. 一种如权利要求5所述的基因表达载体的构建方法，其中，所述构建方法包括如下步骤：
    将编码如权利要求1至3中任一项所述的赖氨酸脱羧酶的核苷酸序列或如权利要求4所述的核苷酸***质粒的限制性酶切位点之间，得到所述基因表达载体。
  7. 根据权利要求6所述的构建方法，其中，所述构建方法还包括***赖氨酸戊二胺反向转运蛋白基因的操作；
    优选地，所述赖氨酸戊二胺反向转运蛋白基因前还包括信号肽；
    优选地，所述信号肽包括大肠杆菌周质空间分泌信号肽；
    优选地，所述信号肽包括dsbA、hlyA、lamB、malE、ompA、ompF、ompT、phoA或pelB中的任意一种，优选为pelB。
  8. 一种用于合成戊二胺的重组工程菌，其中，包含如权利要求5所述的基因表达载体和/或编码权利要求1至3中任一项所述的赖氨酸脱羧酶的核苷酸。
  9. 一种利用如权利要求8所述的重组工程菌制备戊二胺的方法，其中，所述方法包括如下步骤：
    将所述重组工程菌培养、诱导后的得到的菌液离心并重悬，得到菌悬液，所述菌悬液与含有赖氨酸盐酸盐和磷酸吡哆醛的缓冲液混合反应、离心后得到所述戊二胺。
  10. 根据权利要求9所述的方法，其中，所述反应的温度为35～65℃，时间为0.5～24h；
    优选地，所述混合反应的时间为1～4h；
    优选地，所述反应时的振荡速率为400～800rpm；
    优选地，所述离心时的转速为8000～12000rpm，时间为1～3min；
    优选地，所述缓冲液中赖氨酸盐酸盐的摩尔浓度为0.1～3M；
    优选地，所述缓冲液中磷酸吡哆醛的摩尔浓度为0.1～0.5mM；
    优选地，所述缓冲液包括醋酸钠缓冲液、磷酸缓冲液、Tris-HCl缓冲液或碳酸钠缓冲液中的任意一种，优选为磷酸缓冲液；
    优选地，所述缓冲液的pH为5～11，优选为6～10；
    优选地，所述方法包括如下步骤：
    (1)将重组工程菌培养、诱导后得到的菌液离心，冻存后重悬得到菌悬液；
    (2)将所述菌悬液与含有赖氨酸盐酸盐和磷酸吡哆醛的缓冲液混合，所述缓冲液中赖氨酸盐酸盐的摩尔浓度为0.1～3M，磷酸吡哆醛的摩尔浓度为0.1～0.5mM，所述缓冲液为磷酸缓冲液，pH为6～10；
    (3)在35～65℃下以400～800rpm振荡反应1～4h，再以8000～12000rpm离心1～3min，得到戊二胺。
  11. 如权利要求1至3中任一项所述的赖氨酸脱羧酶、如权利要求6或7所述的基因表达载体或如权利要求8所述的重组工程菌在生物基合成戊二胺中的应用。
PCT/CN2021/076305 2020-07-02 2021-02-09 一种用于合成戊二胺的赖氨酸脱羧酶及其应用 WO2022001121A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010634482.1A CN113881657B (zh) 2020-07-02 2020-07-02 一种用于合成戊二胺的赖氨酸脱羧酶及其应用
CN202010634482.1 2020-07-02

Publications (1)

Publication Number Publication Date
WO2022001121A1 true WO2022001121A1 (zh) 2022-01-06

Family

ID=79012824

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/076305 WO2022001121A1 (zh) 2020-07-02 2021-02-09 一种用于合成戊二胺的赖氨酸脱羧酶及其应用

Country Status (2)

Country Link
CN (2) CN117737102A (zh)
WO (1) WO2022001121A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024000368A1 (zh) * 2022-06-30 2024-01-04 江南大学 一种重组大肠杆菌及其构建方法与合成1,5-戊二胺的方法

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112746067B (zh) * 2021-01-26 2023-10-31 洛阳华荣生物技术有限公司 用于制备d-鸟氨酸的赖氨酸脱羧酶突变体
CN114990045B (zh) * 2022-06-30 2023-08-22 江南大学 一种重组大肠杆菌及其构建方法与合成1,5-戊二胺的方法

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1175280A (zh) * 1994-12-09 1998-03-04 味之素株式会社 新的赖氨酸脱羧酶基因以及生产l－赖氨酸的方法
CN107177641A (zh) * 2016-11-03 2017-09-19 中国科学院天津工业生物技术研究所 新的赖氨酸脱羧酶及其应用
CN109402189A (zh) * 2018-12-06 2019-03-01 宁夏伊品生物科技股份有限公司 发酵生产戊二胺的方法及其提取方法
CN110291101A (zh) * 2016-12-30 2019-09-27 上海凯赛生物技术研发中心有限公司 修饰的赖氨酸脱羧酶
CN110291192A (zh) * 2016-12-30 2019-09-27 上海凯赛生物技术研发中心有限公司 在可滴定氨基酸具有修饰的赖氨酸脱羧酶
CN110546255A (zh) * 2017-05-16 2019-12-06 上海凯赛生物技术股份有限公司 对赖氨酸脱羧酶酶类的修饰
CN111117940A (zh) * 2019-12-04 2020-05-08 天津大学 一种高产戊二胺的大肠杆菌工程菌与方法

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104498519A (zh) * 2014-12-19 2015-04-08 南京工业大学 一种表达重组载体及其应用

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1175280A (zh) * 1994-12-09 1998-03-04 味之素株式会社 新的赖氨酸脱羧酶基因以及生产l－赖氨酸的方法
CN107177641A (zh) * 2016-11-03 2017-09-19 中国科学院天津工业生物技术研究所 新的赖氨酸脱羧酶及其应用
CN110291101A (zh) * 2016-12-30 2019-09-27 上海凯赛生物技术研发中心有限公司 修饰的赖氨酸脱羧酶
CN110291192A (zh) * 2016-12-30 2019-09-27 上海凯赛生物技术研发中心有限公司 在可滴定氨基酸具有修饰的赖氨酸脱羧酶
CN110546255A (zh) * 2017-05-16 2019-12-06 上海凯赛生物技术股份有限公司 对赖氨酸脱羧酶酶类的修饰
CN109402189A (zh) * 2018-12-06 2019-03-01 宁夏伊品生物科技股份有限公司 发酵生产戊二胺的方法及其提取方法
CN111117940A (zh) * 2019-12-04 2020-05-08 天津大学 一种高产戊二胺的大肠杆菌工程菌与方法

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DATABASE PROTEIN 11 February 2021 (2021-02-11), ANONYMOUS : "lysine decarboxylase CadA [Salmonella bongori]", XP055883719, retrieved from NCBI Database accession no. WP_001100654 *
DATABASE PROTEIN 11 February 2021 (2021-02-11), ANONYMOUS : "MULTISPECIES: lysine decarboxylase CadA [Klebsiella]", XP055883721, retrieved from NCBI Database accession no. WP_095123982 *
DATABASE PROTEIN 16 July 2020 (2020-07-16), ANONYMOUS : "MULTISPECIES: lysine decarboxylase CadA [Aeromonas]", XP055883718, retrieved from NCBI Database accession no. WP_069526264 *
DATABASE PROTEIN 29 April 2018 (2018-04-29), ANONYMOUS : "lysine decarboxylase CadA [Edwardsiella tarda]", XP055883717, retrieved from NCBI Database accession no. WP_005282982 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024000368A1 (zh) * 2022-06-30 2024-01-04 江南大学 一种重组大肠杆菌及其构建方法与合成1,5-戊二胺的方法

Also Published As

Publication number Publication date
CN113881657B (zh) 2024-02-02
CN113881657A (zh) 2022-01-04
CN117737102A (zh) 2024-03-22

Similar Documents

Publication Publication Date Title
WO2022001121A1 (zh) 一种用于合成戊二胺的赖氨酸脱羧酶及其应用
Schneider et al. Improving putrescine production by Corynebacterium glutamicum by fine-tuning ornithine transcarbamoylase activity using a plasmid addiction system
US11225675B2 (en) D-lactate dehydrogenase, engineered strain containing D-lactate dehydrogenase and construction method and use of engineered strain
KR101318422B1 (ko) D-사이코스 에피머화 효소, 및 이를 이용하는 사이코스 생산방법
JP5774493B2 (ja) ジオールの生産方法
WO2019157921A1 (zh) 腈水解酶突变体及其应用
WO2021147558A1 (zh) 一种腈水解酶突变体及其在制备抗癫痫药物中间体中的应用
US20140065697A1 (en) Cells and methods for producing isobutyric acid
WO2018121458A1 (zh) 编码丙谷二肽生物合成酶的基因及其应用
Liu et al. Rapid production of l‐DOPA by Vibrio natriegens, an emerging next‐generation whole‐cell catalysis chassis
CN104212850B (zh) 利用腈水解酶工程菌制备1‑氰基环己基乙酸的方法
US9512420B2 (en) Use of N-acetylneuraminic acid aldolase in catalytic synthesis of N-acetylneuraminic acid
JP2017512460A (ja) イソプレンモノマーの製造方法
Zhou et al. Enhanced production of N-acetyl-d-neuraminic acid by whole-cell bio-catalysis of Escherichia coli
US11408015B2 (en) Expression vector, recombinant microorganism and method for producing 1,5-diaminopentane
CN115125229B (zh) 一种用于合成戊二胺的赖氨酸脱羧酶突变体
CN112941003A (zh) 一种双酶偶联全细胞催化马来酸合成l-丙氨酸的方法
CN113881719A (zh) 一种全细胞催化合成1,5-戊二胺的方法
CN116064494B (zh) 一种谷氨酸脱羧酶突变体、基因及其应用
Yeo et al. Production, purification, and characterization of soluble NADH-flavin oxidoreductase (StyB) from Pseudomonas putida SN1
CN103088090A (zh) 一种n-乙酰葡萄糖胺异构酶在生产n-乙酰甘露糖胺中的应用
WO2023186037A1 (zh) 一种利用苏氨酸制备甘氨酸、乙酰辅酶a及乙酰辅酶a衍生物的方法
CN115505581B (zh) 一种芳胺-n乙酰转移酶的突变体及其应用
CN115247144B (zh) 生产l-苏式-3-羟基天冬氨酸的基因工程菌及其应用
WO2023056699A1 (zh) 一种生产l-丙氨酸的基因工程菌株及其构建方法和应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21832779

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21832779

Country of ref document: EP

Kind code of ref document: A1