WO2024000368A1 - 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 - Google Patents

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 Download PDF

Info

Publication number
WO2024000368A1
WO2024000368A1 PCT/CN2022/102717 CN2022102717W WO2024000368A1 WO 2024000368 A1 WO2024000368 A1 WO 2024000368A1 CN 2022102717 W CN2022102717 W CN 2022102717W WO 2024000368 A1 WO2024000368 A1 WO 2024000368A1
Authority
WO
WIPO (PCT)
Prior art keywords
lysine
cada
pentanediamine
escherichia coli
coli
Prior art date
Application number
PCT/CN2022/102717
Other languages
English (en)
French (fr)
Inventor
饶志明
乔郅钠
刘祖怡
徐美娟
杨套伟
张显
Original Assignee
江南大学
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 江南大学 filed Critical 江南大学
Priority to PCT/CN2022/102717 priority Critical patent/WO2024000368A1/zh
Publication of WO2024000368A1 publication Critical patent/WO2024000368A1/zh

Links

Images

Classifications

    • 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
    • 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
    • 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
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli

Definitions

  • the invention belongs to the field of genetic engineering technology, and in particular relates to a recombinant Escherichia coli and its construction method and a method for synthesizing 1,5-pentanediamine.
  • 1,5-Pentanediamine also known as 1,5-diaminopentane and cadaverine
  • 1,5-diaminopentane and cadaverine is a natural polyamine with various biological activities. It can be formed by lysine decarboxylase catalyzing the direct decarboxylation of L-lysine. Widely used in agriculture, medicine and industry.
  • the production methods of pentanediamine mainly include microbial fermentation or whole-cell bioconversion.
  • Microbial fermentation methods are usually modified from Corynebacterium glutamicum and Escherichia coli that have the ability to produce lysine.
  • the main problems are long fermentation cycles and low conversion rates.
  • the fermentation system is complex, has many impurities, and contains pentanediamine. Separation and purification are difficult, which increases production costs.
  • the difference between the whole-cell bioconversion method and the microbial fermentation method is that the bacterial strain is first enriched through fermentation culture, and then centrifuged to obtain a whole-cell whole-cell catalyst, which is used for biocatalytic substrate L-lysine or L-lysine hydrochloride. It is converted into pentanediamine, which has the advantage that the system has less impurities and is easy to purify.
  • the latest research generally uses overexpression of CadA in Escherichia coli to produce 1,5-pentanediamine.
  • the US7189543 patent of Japan's Ajinomoto Company protects the use of dicarboxylic acid to adjust the pH and the conversion of lysine to 1,5-pentanediamine through cell overexpression of the wild-type CadA enzyme of Escherichia coli to produce 1,5-pentanediamine, with a yield of 69g/L.
  • Shanghai Kaiser converts lysine by overexpressing the Escherichia coli wild-type CadA enzyme in Hafnia alveoli, thereby achieving the preparation of pentamethylene diamine and downstream polymers.
  • the EP3118312 patent of Japan's Ajinomoto Company discloses E. coli CadA mutation sites Val3, Ala590 and Glu690 with improved thermal stability.
  • the US2015132808 patent of Mitsui Chemicals of Japan protects multiple E. coli CadA mutants with increased activity. However, the activity improvement of these CadA mutants is less than 20%, and even the activity improvement of most CadA mutants is less than 10%. , therefore the application value of these CadA mutants in actual production is very limited.
  • Lysine decarboxylase is a catalyst that catalyzes the production of 1,5-pentanediamine from lysine. Improving the activity of lysine decarboxylase can reduce the amount of catalyst or shorten the reaction time, thereby reducing production costs.
  • the industrialization of diamines has an important impact.
  • lysine decarboxylase catalyzes lysine to generate 1,5-pentanediamine, an additional cofactor PLP is required. PLP is expensive, resulting in increased production costs. Therefore, there is an urgent need to construct a strain for efficient production of pentanediamine to achieve industrial production of 1,5-pentanediamine.
  • the object of the present invention is to provide a recombinant Escherichia coli and a construction method thereof and a method for synthesizing 1,5-pentanediamine.
  • the recombinant Escherichia coli of the present invention can efficiently produce 1,5-pentanediamine and simultaneously prepare When using 1,5-pentanediamine, there is no need to add additional cofactor PLP, no buffer, no need to use hydrochloric acid for neutralization, the conversion time is short, and the production cost is reduced.
  • the invention provides a recombinant Escherichia coli, which contains lysine decarboxylase mutant CadA P530L/M569V , lysine/cadaverine antiporter CadB and pyridoxal kinase in sequence.
  • the pyridoxal kinase is pyridoxal kinase I and pyridoxal kinase II.
  • the amino acid sequence of the lysine decarboxylase mutant CadA P530L/M569V is shown in SEQ ID No. 15; the nucleotide sequence encoding the lysine decarboxylase mutant CadA P530L/M569V is shown in SEQ ID No. Shown in No.16.
  • the amino acid sequence of the lysine/cadaverine antiporter CadB is shown in SEQ ID No. 19; the nucleotide sequence of the gene cadB encoding the lysine/cadaverine antiporter is as follows Shown as SEQ ID No.20.
  • the recombinant E. coli contains lysine decarboxylase mutant CadA P530L/M569V , lysine/cadaverine antiporter CadB, pyridoxal kinase I and pyridoxal kinase II in sequence.
  • the pyridoxal kinase I amino acid sequence is shown in SEQ ID No. 25; the nucleotide sequence encoding the pyridoxal kinase I gene pdxK is shown in SEQ ID No. 26.
  • the pyridoxal kinase II amino acid sequence is shown in SEQ ID No. 27; the nucleotide sequence encoding the pyridoxal kinase II gene pdxY is shown in SEQ ID No. 28.
  • the starting strain of the recombinant E. coli is E. coli BL21.
  • the present invention also provides an expression vector containing the amino acid sequences of the above-mentioned lysine decarboxylase mutant CadA P530L/M569V , lysine/cadaverine antiporter CadB and pyridoxal kinase.
  • the expression vector includes any one of pETDuet1, pACYCDuet1, and pET28a.
  • the invention also provides a method for constructing the above-mentioned recombinant Escherichia coli, which includes the following steps: combining the genes of lysine decarboxylase mutant CadA P530L/M569V , lysine/cadaverine antiporter CadB and pyridoxal kinase The sequences were sequentially introduced into E. coli.
  • the gene sequences of lysine decarboxylase mutant CadA P530L/M569V , lysine/cadaverine antiporter CadB, pyridoxal kinase I and pyridoxal kinase II are introduced into E. coli in sequence.
  • the invention also provides the use of the above-mentioned recombinant Escherichia coli or the recombinant Escherichia coli obtained by the above-mentioned construction method in the synthesis of 1,5-pentanediamine.
  • the invention also provides a method for synthesizing 1,5-pentanediamine. After fermenting and culturing the above-mentioned recombinant Escherichia coli or the recombinant Escherichia coli obtained by the above-mentioned construction method, it is inoculated into the transformation medium containing L-lysine hydrochloride. In the system, L-lysine hydrochloride is converted into 1,5-pentanediamine.
  • the transformation system includes components at the following concentrations: L-lysine hydrochloride 1.5-2.5M, Mn 2+ 45-55mM, vitamin B 60.2-0.3mM, and the pH is natural.
  • the conversion temperature is 38-42°C, and the conversion time is 2.5-3.5 hours.
  • the fermentation culture method includes:
  • the fermentation temperature is 32-40°C
  • the rotation speed is 550-650rpm
  • the ventilation volume is 3-5vvm
  • the feeding time is controlled at 10 to 13 hours.
  • IPTG inducer is added, and the fermentation temperature is set to 27 to 29°C.
  • the fermentation is completed.
  • the fermentation medium is a TY medium, which includes yeast extract 8g/L, tryptone 12g/L, K 3 PO 4 4.02g/L, NaCl 3g/L, and citric acid monohydrate. 2.1g/L, ferric ammonium citrate 0.3g/L, glycerin 10g/L, (NH 4 ) 2 SO 4 2.5g/L, MgSO 4 ⁇ 7H 2 O 0.5g/L, and adjust the pH to 7.2 with ammonia.
  • the feed medium includes 400g/L glycerol, 50g/L yeast powder, and 25g/L tryptone.
  • the present invention has the following beneficial effects:
  • the present invention provides a recombinant Escherichia coli, a construction method thereof, and a method for synthesizing 1,5-pentanediamine.
  • the recombinant Escherichia coli contains lysine that catalyzes L-lysine to generate 1,5-pentanediamine.
  • the method for synthesizing 1,5-pentanediamine provided by the invention uses recombinant Escherichia coli as a catalyst, does not require the addition of additional cofactor PLP, does not use buffers (no need to adjust pH), and does not require the use of hydrochloric acid. And, the synthetic conversion time of 1,5-pentanediamine is short and the production cost is low, which provides an efficient and economical method for the industrial production of 1,5-pentanediamine.
  • Figure 1 shows the SDS-PAGE results of wild-type lysine decarboxylase and its mutant enzyme
  • Figure 2 shows the HPLC spectrum of lysine standard
  • FIG. 3 shows the HPLC spectrum of 1,5-pentanediamine standard
  • Figure 4 is the HPLC spectrum of E.coli BL21/pETDuet1-cadA P530L/M569V strain synthesizing 1,5-pentanediamine;
  • Figure 5 is the HPLC spectrum of E.coli BL21/pETDuet1-cadA P530L/M569V -cadB strain synthesizing 1,5-pentanediamine;
  • Figure 6 is the HPLC chromatogram of 1,5-pentanediamine synthesized by E.coli BL21/pETDuet1-cadA P530L/M569V -cadB-pdxK and E.coli BL21/pETDuet1-cadA P530L/M569V -cadB-pdxY strains, where, A HPLC chromatogram of 1,5-pentanediamine synthesized by E.coli BL21/pETDuet1-cadA P530L/M569V -cadB-pdxK strain; B is synthesized by E.coli BL21/pETDuet1-cadA P530L/M569V -ca dB-pdxY strain 1 , HPLC spectrum of 5-pentanediamine;
  • Figure 7 shows the HPLC chromatogram of 1,5-pentanediamine synthesized by E.coliBL21/pETDuet1-cadA P530L/M569V -cadB-pdxK-pdxY strain;
  • Figure 8 shows the HPLC chromatogram of 1,5-pentanediamine synthesized by the recombinant E. coli E.coli BL21/pETDuet1-cadA P530L/M569V- cadB-pdx K-pdxY strain;
  • Figure 9 is a schematic diagram of the recombinant pETDuet1-cadA P530L/M569V -cadB-pdxK-pdxY plasmid.
  • the invention provides a recombinant Escherichia coli, which contains lysine decarboxylase mutant CadA P530L/M569V , lysine/cadaverine antiporter CadB and pyridoxal kinase in sequence.
  • the lysine decarboxylase mutant CadA P530L/M569V is derived from Serratia marcescens and is the optimal lysine decarboxylase of the lysine decarboxylase CadA derived from Serratia marcescens.
  • Mutant CadA P530L/M569V the lysine decarboxylase activity of CadA P530L/M569V is high, with a specific enzyme activity of 292.54U/mg, which is 1.63 times that of wild-type CadA.
  • the amino acid sequence of the lysine decarboxylase mutant CadA P530L/M569V is preferably shown in SEQ ID No. 15; the nucleotide encoding the lysine decarboxylase mutant CadA P530L/M569V The sequence is preferably as shown in SEQ ID No. 16.
  • the recombinant E. coli also contains the lysine/cadaverine antiporter CadB, which facilitates the entry/exit of substrates/products into the cell.
  • the amino acid sequence of the lysine/cadaverine antiporter CadB is preferably shown in SEQ ID No. 19; the core of the gene cadB encoding the lysine/cadaverine antiporter
  • the nucleotide sequence is preferably as shown in SEQ ID No. 20.
  • the recombinant E. coli also provides a self-supplying system for the cofactor PLP, that is, pyridoxal kinase.
  • the pyridoxal kinase is preferably pyridoxal kinase I and pyridoxal kinase II, and the pyridoxal kinase I amino acid sequence is preferably as shown in SEQ ID No. 25; the encoding pyridoxal kinase
  • the nucleotide sequence of the I gene pdxK is preferably as shown in SEQ ID No. 26; the pyridoxal kinase II amino acid sequence is preferably as shown in SEQ ID No.
  • the nucleotide sequence is preferably as shown in SEQ ID No. 28.
  • the PLP self-supply system constructed by the recombinant Escherichia coli expressing pyridoxal kinase I and II of the present invention enhances the level of intracellular cofactor PLP.
  • 1,5-pentanediamine there is no need to add expensive additional cofactor PLP. That is, 1,5-pentanediamine can be synthesized efficiently and production costs can be reduced.
  • the recombinant E. coli contains lysine decarboxylase mutant CadA P530L/M569V , lysine/cadaverine antiporter CadB, pyridoxal kinase I and pyridine in sequence. Doral kinase II.
  • the starting strain of the recombinant E. coli is preferably E. coli BL21.
  • the present invention also provides an expression vector containing the amino acid sequences of the above-mentioned lysine decarboxylase mutant CadA P530L/M569V , lysine/cadaverine antiporter CadB and pyridoxal kinase.
  • the expression vector preferably includes any one of pETDuet1, pACYCDuet1, and pET28a.
  • the invention also provides a method for constructing the above recombinant Escherichia coli, which includes the following steps: combining the genes of lysine decarboxylase mutant CadA P530L/M569V , lysine/cadaverine antiporter CadB and pyridoxal kinase The sequences were sequentially introduced into E. coli.
  • the gene sequences of lysine decarboxylase mutant CadA P530L/M569V , lysine/cadaverine antiporter CadB, pyridoxal kinase I and pyridoxal kinase II are sequentially Introduction into E. coli.
  • the Escherichia coli is preferably Escherichia coli E.coliBL21.
  • the nucleotide sequence encoding the lysine decarboxylase mutant CadA P530L/M569V is preferably shown in SEQ ID No. 16; the nucleotide sequence of the gene cadB encoding the lysine/cadaverine antiporter The sequence is preferably as shown in SEQ ID No.
  • nucleotide sequence encoding the pyridoxal kinase I gene pdxK is preferably as shown in SEQ ID No. 26; the nucleic acid sequence encoding the pyridoxal kinase II gene pdxY
  • the nucleotide sequence is preferably as shown in SEQ ID No. 28.
  • the invention also provides the use of the above-mentioned recombinant Escherichia coli or the recombinant Escherichia coli obtained by the above-mentioned construction method in the synthesis of 1,5-pentanediamine.
  • the invention also provides a method for synthesizing 1,5-pentanediamine. After fermenting and culturing the above-mentioned recombinant Escherichia coli or the recombinant Escherichia coli obtained by the above-mentioned construction method, it is inoculated into the transformation medium containing L-lysine hydrochloride. In the system, L-lysine hydrochloride is converted into 1,5-pentanediamine.
  • the transformation system preferably includes components at the following concentrations: L-lysine hydrochloride 1.5-2.5M, Mn 2+ 45-55mM, vitamin B 60.2-0.3mM, and the pH is natural.
  • the conversion temperature is preferably 38 to 42°C
  • the conversion time is preferably 2.5 to 3.5 hours.
  • the reaction system does not need to add a buffer, adjust the pH, and do not need to use hydrochloric acid for neutralization, and the conversion time is short.
  • the fermentation culture method includes: inoculating the seed liquid of recombinant Escherichia coli into a fermentation medium for fermentation, with a fermentation temperature of 32 to 40°C, a rotation speed of 550 to 650 rpm, and a ventilation rate of When the temperature is 3 to 5 vvm and the OD 600 is 7 to 9, start feeding the feeding medium at a constant speed.
  • the feeding time is controlled at 10 to 13 hours.
  • induction culture for 20 to 26 hours at 29°C the fermentation was completed.
  • the fermentation medium is a TY medium
  • the TY medium preferably includes yeast extract 8g/L, tryptone 12g/L, K 3 PO 4 4.02g/L, NaCl 3g/L, Citric acid monohydrate 2.1g/L, ferric ammonium citrate 0.3g/L, glycerol 10g/L, (NH 4 ) 2 SO 4 2.5g/L, MgSO 4 ⁇ 7H 2 O 0.5g/L, adjust pH with ammonia to 7.2.
  • the feed medium preferably includes 400g/L glycerol, 50g/L yeast powder, and 25g/L tryptone.
  • the present invention has no special limitations on the preparation methods of TY culture medium and feed culture medium, and methods known in the art can be used.
  • the synthesized 1,5-pentanediamine has a high content and a high molar conversion rate. There is no need to add a buffer, no need to adjust the pH, and no need to add an additional expensive cofactor PLP.
  • the synthesis method is efficient and economical, achieving Industrialized production of 1,5-pentanediamine.
  • LB liquid medium peptone 10g/L, yeast extract 5g/L, NaCl 10g/L.
  • (2) LB solid medium peptone 10g/L, yeast extract 5g/L, NaCl 10g/L, agar 15g/L.
  • Feeding medium glycerol 400g/L, yeast powder 50g/L, tryptone 25g/L.
  • Enzyme activation reaction system 500mM, pH 6.0 sodium acetate buffer (350 ⁇ L), 100mM substrate L-lysine (100 ⁇ L), 0.25mM cofactor PLP (50 ⁇ L) and pure enzyme (100 ⁇ L), react at 40°C for 1 hour. Boil at 100°C for 5 minutes, and detect the pentanediamine content by HPLC.
  • Enzyme activity definition The amount of enzyme required to generate 1 ⁇ mol of pentanediamine per minute is 1 U.
  • Sample derivatization treatment Centrifuge the sample at 10,000 rpm for 20 min, take 75 ⁇ L each of the supernatant and standard (lysine and pentanediamine) and add them to a solution containing diethyl ethoxymethylene malonate ( DEEMM), 70.5 ⁇ L of distilled water, 150 ⁇ L of 100% methanol, 450 ⁇ L of 50mM, pH 9.0 borate buffer, vortex to mix and incubate at 70°C for 2 hours to remove excess DEEMM and side effects. product.
  • DEEMM diethyl ethoxymethylene malonate
  • the derivatized sample was centrifuged at 10,000 rpm for 5 min, and the supernatant was filtered through a 0.22 ⁇ m membrane and used for HPLC detection.
  • HPLC detection conditions are: UV-VIS detector, chromatographic column (Platisil ODS 5 ⁇ m, 250 ⁇ 4.6mm), column temperature: 35°C, flow rate 1mL/min, detection wavelength: 284nm.
  • Mobile phase A 100% acetonitrile
  • mobile phase B 25mM
  • pH 4.8 sodium acetate aqueous solution gradient: 0 ⁇ 2min: 20 ⁇ 25%A
  • 2 ⁇ 20min 25 ⁇ 60%A
  • 20 ⁇ 25min 60 ⁇ 20%A.
  • HotSpotWizard 3.0 https://loschmidt.chemi.muni.cz/hot spotwizard
  • cadA-F and cadA-R as primers and the Serratia marcescens JNB5-1 genome as a template
  • the cadA gene fragment was amplified by PCR. Its size was 2139 bp.
  • the purified cadA gene fragment was mixed with BamHI and EcoRI double enzymes.
  • the cut pETDuet1 linearized plasmid was ligated by homologous recombination, and the ligation product was transformed into E.coli BL21 competent cells.
  • the recombinant plasmid was verified by double enzyme digestion. If the verification was successful, it was sent to Suzhou Jinweizhi Co., Ltd. for sequencing analysis. The sequencing results were analyzed by SnapGene 3.2.1 software.
  • the correctly sequenced strain was named E.coli BL21/pETDuet1-cadA.
  • the primers are as follows:
  • Serratia marcescens JNB5-1 genome was used as a template for PCR amplification to obtain cadA gene fragments containing N120A, S165R, P530L, M569V, M678A and T691P mutation points. The sizes were all 2139bp.
  • the purified cadA gene fragments containing N120A, S165R, The cadA gene fragments of P530L, M569V, M678A and T691P mutation points were connected by homologous recombination with the pETDuet1 linearized plasmid digested by BamHI and SalI, respectively, and the ligation products were transformed into E.coliBL21 competent cells. The recombinant plasmid was verified by double enzyme digestion.
  • BL21/pETDuet1-cadA S165R E.coli BL21/pETDuet1-cadA P530L
  • E.coli BL21/pETDuet1-cadA M569V E.coli BL21/pETDuet1-cadA M678A
  • E.coli BL21/pETDuet1-cadA T691P Among them, the primers are listed in Table 1 below (capital letters are mutation sites):
  • the constructed recombinant Escherichia coli E.coli BL21/pETDuet1-cadA and its mutant strains were streaked and activated on a solid LB plate containing 50 ⁇ g/mL kanamycin. After culturing for 12-24 hours at 37°C, single colonies were picked. , transferred to 10mL LB liquid medium supplemented with 50 ⁇ g/mL ampicillin, and cultured at 37°C, 180r/min for 12-24h; then inoculated with 1% (v/v) inoculation volume into 50mL LB liquid supplemented with 50 ⁇ g/mL ampicillin.
  • Lanes 1 to 8 were E.coli BL21/pETDuet1-cadA N120A , E.coli BL21/pETDuet1-cadA S165R , E.coli BL21/pETDuet1-cadA P530L , respectively.
  • E.coli BL21/pETDuet1-cadA M569V E.coli BL21/pETDuet1-cadA M678A , E.coli BL21/pETDuet1-cadA T691P , E.coli BL21/pETDuet1-cadA P530L/M569V and E.coli BL21/pETDuet1 cell disruption
  • the results of the supernatant showed that there was an obvious protein band at 75kDa, indicating that the wild-type lysine decarboxylase was successfully expressed, and that there was an obvious protein band at 75kDa, indicating that both lysine decarboxylase mutant enzymes were successfully expressed (see figure 1).
  • the lysine decarboxylase was purified to study the enzymatic properties.
  • the method of protein purification is to use nickel column affinity chromatography, and the specific process is carried out according to the instrument operating procedures provided by the company.
  • the pure enzyme directly purified by a protein purifier contains a large amount of imidazole, which will reduce the enzyme activity of lysine decarboxylase. Therefore, in order to remove the large amount of imidazole contained in the pure enzyme, use 0.05M, pH 7.0 Tris-HCl buffer The pure enzyme was dialyzed as dialysate, and the dialyzed pure enzyme was used for subsequent activity determination.
  • the enzyme activity of wild-type lysine decarboxylase and its mutants was determined: after reacting for 1 hour under the optimal temperature of 40°C and optimal pH of 6.0, immediately place the enzyme in a water bath at 100°C for 5 minutes to terminate the enzyme reaction, and detect the generated enzyme by HPLC. Pentylenediamine content. The enzyme activity was determined using the above-mentioned enzyme activity assay method for lysine decarboxylase.
  • the results of the enzyme activity measurement of wild-type lysine decarboxylase and its mutants showed that the specific enzyme activities of wild-type lysine decarboxylase CadA and mutant enzymes CadA N120A , CadA S165R , CadA P530L , CadA M569V , CadA M678A and CadA T691P were respectively were 179.01, 165.45, 172.23, 232.78, 254.32, 180.25 and 176.62U/mg. It can be seen that the specific enzyme activities of CadA P530L and CadA M569V were improved, which were 1.3 and 1.42 times that of wild-type CadA respectively.
  • the enzyme activity measurement results showed that the specific enzyme activities of mutant enzymes CadA P530L and CadA M569V were improved, which were 1.3 and 1.42 times that of wild-type CadA respectively. Therefore, these two mutation sites were combined to mutate in order to further improve the enzyme activity level of lysine decarboxylase.
  • cadA-F/M569V-R and M569V-F/cadA-R as primers and the recombinant plasmid containing the P530L mutation site as the template
  • PCR amplification was performed to obtain the cadA gene fragments containing the M569V and P530L double mutation sites respectively. Both are 2139bp.
  • the purified cadA gene fragment containing the M569V and P530L double mutation sites was ligated by homologous recombination with the pETDuet1 linearized plasmid digested by BamHI and EcoRI. The ligation product was transformed into E.coli BL21 competent cells.
  • the recombinant plasmid was verified by double enzyme digestion, and those successfully verified were sent to Suzhou Jinweizhi Co., Ltd. for sequencing analysis.
  • the sequencing results were analyzed by SnapGene 3.2.1 software.
  • the strains with correct sequencing were named E.coli BL21/pETDuet1-cadA P530L/M569V .
  • the amino acid sequence of the lysine decarboxylase mutant CadA P530L/M569V is shown in SEQ ID No. 15, and the nucleotide sequence encoding the lysine decarboxylase mutant CadA P530L/M569V is shown in SEQ ID No. Shown in No.16.
  • Streak activation after culturing at 37°C for 12 to 24 hours, pick a single colony, transfer it to 10 mL LB liquid culture medium supplemented with 50 ⁇ g/mL ampicillin, and culture at 37°C and 180 r/min for 12 to 24 hours; then use 1% (v /v) inoculum amount was inoculated into 50 mL LB liquid culture medium supplemented with 50 ⁇ g/mL ampicillin. After culturing for 2 hours at 37°C and 180r/min, IPTG was added to a final concentration of 0.5mmol/L, and continued at 28°C and 180r/min.
  • the bacterial solution after induction was centrifuged at 4°C to collect the cells.
  • Figure 2 is the HPLC spectrum of lysine standard. In this HPLC spectrum, the retention time of lysine is 11.725min.
  • Figure 3 is the HPLC spectrum of 1,5-pentanediamine standard. In this HPLC spectrum, The retention time of 1,5-pentanediamine is 24.111min.
  • the cadB gene fragment was obtained by PCR amplification. Its size was 1335 bp.
  • the purified cadB gene fragment was mixed with pETDuet1-cadA P530L/ which was double digested with BglII and XhoI.
  • M569V linearized plasmid was ligated by homologous recombination, and the ligation product was transformed into E.coli BL21 competent cells.
  • the recombinant plasmid was verified by double enzyme digestion. If the verification was successful, it was sent to Suzhou Jinweizhi Co., Ltd. for sequencing analysis. The sequencing results were analyzed by SnapGene 3.2.1 software.
  • the correctly sequenced strain was named E.coli BL21/pETDuet1-cadA P530L/M569V -cadB.
  • the primers are as follows:
  • the amino acid sequence of the lysine/cadaverine antiporter CadB is shown in SEQ ID No. 19, and the nucleotide sequence of the gene cadB encoding the lysine/cadaverine antiporter is shown in SEQ ID No. .20 shown.
  • the pdxK and pdxY gene fragments were obtained by PCR amplification using the Escherichia coli genome as the template. Their sizes were 852bp and 864bp respectively.
  • the purified pdxK and pdxY gene fragments were compared with those obtained by EcoRI. Homologous recombination was performed with the HindIII double-digested pETDuet1-cadA P530L/M569V -cadB linearized plasmid, and the ligation product was transformed into E.coli BL21 competent cells.
  • the amino acid sequence of pyridoxal kinase I is shown in SEQ ID No. 25, and the nucleotide sequence of the gene pdxK encoding pyridoxal kinase I is shown in SEQ ID No. 26; the pyridoxal kinase II The amino acid sequence of is shown in SEQ ID No. 27, and the nucleotide sequence of the gene pdxY encoding pyridoxal kinase II is shown in SEQ ID No. 28.
  • HPLC was used to detect pentamethylenediamine: the constructed recombinant E. coli E.coli BL21/pETDuet1-cadA P530L/M569V -cadB, E.coli BL21/pETDuet1-cadA P530L/M569V -cadB-pdxK and E.coli BL21/pETDuet1 -cadA P530L/M569V -cadB-pdxY was streaked and activated on a solid LB plate containing 50 ⁇ g/mL ampicillin.
  • pdxY-FF and pdxY-RR were used as primers and the E. coli genome was used as a template for PCR amplification to obtain pdxY gene fragments with NdeI and BglII restriction sites and homology arms added, respectively, and purified
  • the pdxY gene fragment with added NdeI and BglII restriction sites and homology arms was ligated by homologous recombination with the pETDuet1-cadA P530L/M569V -cadB-pdxK linearized plasmid double-digested by NdeI and BglII, and the ligation product was transformed into E .coliBL21 competent cells.
  • E. coli E.coliBL21/pETDuet1-cadA P530L/M569V- cadB-pdxK-pdxY was streaked and activated on a solid LB plate containing 50 ⁇ g/mL ampicillin. After culturing at 37°C for 12 to 24 hours, pick out For a single colony, transfer it to 10 mL LB liquid medium supplemented with 50 ⁇ g/mL ampicillin, and culture it at 37°C and 180 r/min for 12 to 24 hours; then inoculate it with 1% (v/v) inoculum into the medium supplemented with 50 ⁇ g/mL ampicillin.
  • Recombinant Escherichia coli E.coli BL21/pETDuet1-cadA P530L/M569V -cadB-pdxK-pdxY was streaked and activated on a solid LB plate containing 50 ⁇ g/mL ampicillin. After culturing for 12-24 hours at 37°C, single colonies were picked. Transfer 10 mL LB liquid medium supplemented with 50 ⁇ g/mL ampicillin, and culture at 37°C and 180 r/min for 12 to 24 hours; then inoculate 1% (v/v) inoculum into 200 mL LB liquid medium supplemented with 50 ⁇ g/mL ampicillin.
  • the culture medium culture at 37°C and 180r/min for 18h to obtain the seed liquid.
  • a 5L fermentation tank containing 2L fermentation medium (TY medium) was transferred to a 5L fermentation tank with an inoculation amount of 10%.
  • the fermentation conditions were as follows: the temperature was set to 37°C, the rotation speed was set to 600rpm, and the ventilation volume was 4vvm.
  • the OD 600 is 7 to 9
  • IPTG inducer final concentration 0.5 mM
  • the temperature of the fermentor was set to 28°C.
  • the fermentation was stopped, and the cells were collected by centrifugation at 4°C.

Landscapes

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

Abstract

本发明提供了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，属于基因工程技术领域，该重组大肠杆菌含有催化L-赖氨酸生成1,5-戊二胺的赖氨酸脱羧酶最优突变体CadA P530L/M569V、促进底物/产物进/出细胞的赖氨酸/尸胺反向转运蛋白CadB以及提供辅因子PLP自供应吡哆醛激酶I和II，将该重组大肠杆菌作为催化剂，得到的高产量的1,5-戊二胺，摩尔转化率高达98.67％。本发明提供的合成1,5-戊二胺的方法，该方法以重组大肠杆菌作为催化剂，不需要额外添加辅因子PLP，不使用缓冲液（无需调节pH），无需使用盐酸进行中和，合成转化1,5-戊二胺的时间短，生产成本低，为1,5-戊二胺的工业化生产提供了一种高效且经济的方法。

Description

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 技术领域
本发明属于基因工程技术领域，尤其涉及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。
背景技术
1,5-戊二胺，又名1,5-二氨基戊烷、尸胺，是具有多种生物活性的天然多胺，可通过赖氨酸脱羧酶催化L-赖氨酸直接脱羧形成，在农业，医药和工业中应用广泛。
目前，戊二胺的生产方法主要包括微生物发酵法或全细胞生物转化法。微生物发酵法通常由具有生产赖氨酸能力的谷氨酸棒杆菌和大肠杆菌改造而来，存在的主要问题是发酵周期长，转化率低等；此外，发酵体系复杂，杂质多，戊二胺分离纯化困难，进而增加了生产成本。而全细胞生物转化法，与微生物发酵法区别是先通过发酵培养富集菌株，再离心获得全细胞全细胞催化剂，用于生物催化底物L-赖氨酸或L-赖氨酸盐酸盐转化生成戊二胺，优点在于体系杂质少，易于纯化。
最新的研究普遍采用过表达大肠杆菌的CadA来生产1,5-戊二胺。日本味之素公司的US7189543专利中保护了以二羧酸调节pH并通过细胞过表达大肠杆菌的野生型CadA酶转化赖氨酸产生1,5-戊二胺，产量达69g/L。上海凯赛在其专利CN102851307A中通过在蜂房哈夫尼菌中过量表达大肠杆菌野生型CadA酶来转化赖氨酸，从而实现戊二胺和下游聚合物的制备。日本味之素公司的EP3118312专利中公开了热稳定性提高的大肠杆菌CadA突变位点Val3、Ala590和Glu690。日本三井化学公司的US2015132808专利保护了多个活性增加的大肠杆菌CadA突变体，然而，这些CadA突变体的活性提高程度均低于20％，甚至大多数CadA突变体的活性提高程度不到10％，因此这些CadA突变体在实际生产上的应用价值非常有限。
赖氨酸脱羧酶作为催化赖氨酸生产1,5-戊二胺的催化剂，提升赖氨酸脱羧酶的活性可以减少催化剂的用量或缩短反应时间，进而降低生产成本，对1,5-戊二胺的工业化有重要的影响。其次，赖氨酸脱羧酶在催化赖氨酸生成1,5-戊二胺时需要额外添加辅因子PLP，PLP价格昂贵，导致生 产成本升高。因此急需构建一株戊二胺高效生产菌株，实现1,5-戊二胺的工业化生产。
发明内容
有鉴于此，本发明的目的在于提供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，本发明的重组大肠杆菌能够高效生产1,5-戊二胺，同时制备1,5-戊二胺时，不需要额外添加辅因子PLP，不使用缓冲液，无需使用盐酸进行中和，转化时间短，降低生产成本。
为了实现上述发明目的，本发明提供了以下技术方案：
本发明提供了一种重组大肠杆菌，所述重组大肠杆菌依次含有赖氨酸脱羧酶突变体CadA P530L/M569V、赖氨酸/尸胺反向转运蛋白CadB和吡哆醛激酶。
优选地，所述吡哆醛激酶为吡哆醛激酶I和吡哆醛激酶II。
优选地，所述赖氨酸脱羧酶突变体CadA P530L/M569V的氨基酸序列如SEQ ID No.15所示；所述编码赖氨酸脱羧酶突变体CadA P530L/M569V的核苷酸序列如SEQ ID No.16所示。
优选地，所述赖氨酸/尸胺反向转运蛋白CadB的氨基酸序列如SEQ ID No.19所示；所述编码赖氨酸/尸胺反向转运蛋白的基因cadB的核苷酸序列如SEQ ID No.20所示。
优选地，所述重组大肠杆菌依次含有赖氨酸脱羧酶突变体CadA P530L/M569V、赖氨酸/尸胺反向转运蛋白CadB、吡哆醛激酶I和吡哆醛激酶II。
优选地，所述吡哆醛激酶I氨基酸序列如SEQ ID No.25所示；所述编码吡哆醛激酶I基因pdxK的核苷酸序列如SEQ ID No.26所示。
优选地，所述吡哆醛激酶II氨基酸序列如SEQ ID No.27所示；所述编码吡哆醛激酶II基因pdxY的核苷酸序列如SEQ ID No.28所示。
优选地，所述重组大肠杆菌的出发菌株为大肠杆菌E.coli BL21。
本发明还提供了一种含有上述赖氨酸脱羧酶突变体CadA P530L/M569V、赖氨酸/尸胺反向转运蛋白CadB和吡哆醛激酶的氨基酸序列的表达载体。
优选地，所述表达载体包括pETDuet1、pACYCDuet1、pET28a中的任意一种。
本发明还提供了一种上述重组大肠杆菌的构建方法，包括如下步骤：将赖氨酸脱羧酶突变体CadA P530L/M569V、赖氨酸/尸胺反向转运蛋白CadB和吡哆醛激酶的基因序列依次导入大肠杆菌。
优选地，将赖氨酸脱羧酶突变体CadA P530L/M569V、赖氨酸/尸胺反向转运蛋白CadB、吡哆醛激酶I和吡哆醛激酶II的基因序列依次导入大肠杆菌。
本发明还提供了一种上述重组大肠杆菌或上述构建方法得到的重组大肠杆菌在合成1,5-戊二胺中的应用。
本发明还提供了一种合成1,5-戊二胺的方法，将上述重组大肠杆菌或上述构建方法得到的重组大肠杆菌进行发酵培养后，接种至含有L-赖氨酸盐酸盐的转化体系中，以转化L-赖氨酸盐酸盐为1,5-戊二胺。
优选地，所述的转化体系包括如下浓度的成分：L-赖氨酸盐酸盐1.5～2.5M、Mn 2+45～55mM、维生素B60.2～0.3mM，pH自然。
优选地，所述转化的温度为38～42℃，所述转化的时间为2.5～3.5h。
优选地，所述发酵培养的方法包括：
将重组大肠杆菌的种子液接种在发酵培养基中发酵，发酵温度为32～40℃、转速为550～650rpm、通气量为3～5vvm，OD 600为7～9时，开始匀速流加补料培养基，补料时间控制在10～13h，发酵7～9h后，添加IPTG诱导剂，并将发酵温度设为27～29℃，诱导培养20～26h后，发酵结束。
优选地，所述发酵培养基为TY培养基，所述TY培养基包括酵母提取物8g/L、胰蛋白胨12g/L、K 3PO 44.02g/L、NaCl 3g/L、一水合柠檬酸2.1g/L、柠檬酸铁铵0.3g/L、甘油10g/L、(NH 4) 2SO 42.5g/L、MgSO 4·7H 2O 0.5g/L，用氨水调pH至7.2。
优选地，所述补料培养基包括甘油400g/L、酵母粉50g/L、胰蛋白胨25g/L。
相对于现有技术，本发明具有如下有益效果：
(1)本发明提供了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，该重组大肠杆菌含有催化L-赖氨酸生成1,5-戊二胺的赖氨酸脱羧酶最优突变体CadA P530L/M569V、促进底物/产物进/出细胞的赖氨酸/尸 胺反向转运蛋白CadB以及提供辅因子PLP自供应吡哆醛激酶I和II，将该重组大肠杆菌作为催化剂，得到的高产量的1,5-戊二胺，摩尔转化率高达98.67％。
(2)本发明提供的合成1,5-戊二胺的方法，该方法以重组大肠杆菌作为催化剂，不需要额外添加辅因子PLP，不使用缓冲液(无需调节pH)，无需使用盐酸进行中和，合成转化1,5-戊二胺的时间短，生产成本低，为1,5-戊二胺的工业化生产提供了一种高效且经济的方法。
说明书附图
图1为野生型赖氨酸脱羧酶及其突变酶的SDS-PAGE结果图；
图2为赖氨酸标准品HPLC图谱；
图3为1,5-戊二胺标准品HPLC图谱；
图4为E.coli BL21/pETDuet1-cadA P530L/M569V菌株合成1,5-戊二胺的HPLC图谱；
图5为E.coli BL21/pETDuet1-cadA P530L/M569V-cadB菌株合成1,5-戊二胺的HPLC图谱；
图6为E.coli BL21/pETDuet1-cadA P530L/M569V-cadB-pdxK和E.coli BL21/pETDuet1-cadA P530L/M569V-cadB-pdxY菌株合成1,5-戊二胺的HPLC图谱，其中，A为E.coli BL21/pETDuet1-cadA P530L/M569V-cadB-pdxK菌株合成1,5-戊二胺的HPLC图谱；B为E.coli BL21/pETDuet1-cadA P530L/M569V-ca dB-pdxY菌株合成1,5-戊二胺的HPLC图谱；
图7为E.coliBL21/pETDuet1-cadA P530L/M569V-cadB-pdxK-pdxY菌株合成1,5-戊二胺的HPLC图谱；
图8为重组大肠杆菌E.coli BL21/pETDuet1-cadA P530L/M569V-cadB-pdx K-pdxY菌株合成1,5-戊二胺的HPLC图谱；
图9为重组pETDuet1-cadA P530L/M569V-cadB-pdxK-pdxY质粒示意图。
具体实施方式
下面结合实施例和附图对本发明进一步说明。
本发明提供了一种重组大肠杆菌，所述重组大肠杆菌依次含有赖氨酸脱羧酶突变体CadA P530L/M569V、赖氨酸/尸胺反向转运蛋白CadB和吡哆醛激酶。
在本发明中，所述赖氨酸脱羧酶突变体CadA P530L/M569V来源于粘质沙雷氏菌，是粘质沙雷氏菌来源的赖氨酸脱羧酶CadA的最优赖氨酸脱羧酶突变体CadA P530L/M569V，该CadA P530L/M569V的赖氨酸脱羧酶的酶活高，比酶活为292.54U/mg，是野生型CadA的1.63倍。在本发明中，所述赖氨酸脱羧酶突变体CadA P530L/M569V的氨基酸序列优选地如SEQ ID No.15所示；所述编码赖氨酸脱羧酶突变体CadA P530L/M569V的核苷酸序列优选地如SEQ ID No.16所示。
在本发明中，该重组大肠杆菌还赖氨酸/尸胺反向转运蛋白CadB，促进了促进底物/产物进/出细胞。在本发明中，所述赖氨酸/尸胺反向转运蛋白CadB的氨基酸序列优选地如SEQ ID No.19所示；所述编码赖氨酸/尸胺反向转运蛋白的基因cadB的核苷酸序列优选地如SEQ ID No.20所示。
在本发明中，该重组大肠杆菌还提供辅因子PLP自供应系统，即吡哆醛激酶。所述吡哆醛激酶优选地为吡哆醛激酶I和吡哆醛激酶II，所述所述吡哆醛激酶I氨基酸序列优选地如SEQ ID No.25所示；所述编码吡哆醛激酶I基因pdxK的核苷酸序列优选地如SEQ ID No.26所示；所述吡哆醛激酶II氨基酸序列优选地如SEQ ID No.27所示；所述编码吡哆醛激酶II基因pdxY的核苷酸序列优选地如SEQ ID No.28所示。本发明的重组大肠杆菌表达吡哆醛激酶I和II构建的PLP自供应系统，增强了胞内辅因子PLP水平，在合成1,5-戊二胺时，无需添加昂贵的添加辅因子PLP，即能高效合成1,5-戊二胺，降低生产成本。
在本发明中，作为一优选的实施方式，所述重组大肠杆菌依次含有赖氨酸脱羧酶突变体CadA P530L/M569V、赖氨酸/尸胺反向转运蛋白CadB、吡哆醛激酶I和吡哆醛激酶II。
在本发明中，所述重组大肠杆菌的出发菌株优选地为大肠杆菌E.coli BL21。
本发明还提供了一种含有上述赖氨酸脱羧酶突变体CadA P530L/M569V、赖氨酸/尸胺反向转运蛋白CadB和吡哆醛激酶的氨基酸序列的表达载体。
在本发明中，所述表达载体优选地包括pETDuet1、pACYCDuet1、pET28a中的任意一种。
本发明还提供了一种上述重组大肠杆菌的构建方法，包括如下步骤： 将赖氨酸脱羧酶突变体CadA P530L/M569V、赖氨酸/尸胺反向转运蛋白CadB和吡哆醛激酶的基因序列依次导入大肠杆菌。
作为本发明的一优选地实施方式，将赖氨酸脱羧酶突变体CadA P530L/M569V、赖氨酸/尸胺反向转运蛋白CadB、吡哆醛激酶I和吡哆醛激酶II的基因序列依次导入大肠杆菌。所述大肠杆菌优选为大肠杆菌E.coliBL21。所述编码赖氨酸脱羧酶突变体CadA P530L/M569V的核苷酸序列优选地如SEQ ID No.16所示；所述编码赖氨酸/尸胺反向转运蛋白的基因cadB的核苷酸序列优选地如SEQ ID No.20所示；所述编码吡哆醛激酶I基因pdxK的核苷酸序列优选地如SEQ ID No.26所示；所述编码吡哆醛激酶II基因pdxY的核苷酸序列优选地如SEQ ID No.28所示。
本发明还提供了一种上述重组大肠杆菌或上述构建方法得到的重组大肠杆菌在合成1,5-戊二胺中的应用。
本发明还提供了一种合成1,5-戊二胺的方法，将上述重组大肠杆菌或上述构建方法得到的重组大肠杆菌进行发酵培养后，接种至含有L-赖氨酸盐酸盐的转化体系中，以转化L-赖氨酸盐酸盐为1,5-戊二胺。
在本发明中，所述的转化体系优选地包括如下浓度的成分：L-赖氨酸盐酸盐1.5～2.5M、Mn 2+45～55mM、维生素B60.2～0.3mM，pH自然。在本发明中，所述转化的温度优选为38～42℃，所述转化的时间优选为2.5～3.5h。在本发明中，所述的反应体系不需要添加缓冲液，无需调节pH，也就无需使用盐酸进行中和，转化时间短。
作为本发明的一种优选地实施方式，所述发酵培养的方法包括：将重组大肠杆菌的种子液接种在发酵培养基中发酵，发酵温度为32～40℃、转速为550～650rpm、通气量为3～5vvm，OD 600为7～9时，开始匀速流加补料培养基，补料时间控制在10～13h，发酵7～9h后，添加IPTG诱导剂，并将发酵温度设为27～29℃，诱导培养20～26h后，发酵结束。在本发明中，所述发酵培养基为TY培养基，所述TY培养基优选地包括酵母提取物8g/L、胰蛋白胨12g/L、K 3PO 4 4.02g/L、NaCl 3g/L、一水合柠檬酸2.1g/L、柠檬酸铁铵0.3g/L、甘油10g/L、(NH 4) 2SO 4 2.5g/L、MgSO 4·7H 2O 0.5g/L，用氨水调pH至7.2。在本发明中，所述补料培养基优选地包括甘油400g/L、酵母粉50g/L、胰蛋白胨25g/L。本发明对TY培养基、补 料培养基的制备方法没有特殊限定，采用本领域公知的方法即可。在本发明中，合成的1,5-戊二胺含量高，摩尔转化率高，不需要添加缓冲液，无需调节pH，不需要额外添加昂贵的辅因子PLP，该合成方法高效且经济，实现了1,5-戊二胺的工业化生产。
下面结合实施例对本发明提供的技术方案进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。
下述实施例中涉及的培养基如下：
(1)LB液体培养基：蛋白胨10g/L、酵母膏5g/L、NaCl 10g/L。
(2)LB固体培养基：蛋白胨10g/L、酵母膏5g/L、NaCl 10g/L、琼脂15g/L。
(3)TB培养基：
A：酵母粉24g、蛋白胨12g、甘油4g
B：KH 2PO 4 2.3g、K 2HPO 4 16.4g
将A溶于900mL超纯水，高压灭菌；将B溶于100mL超纯水，高压灭菌，将900mLA和100mLB混合以制备1L TB培养基。
(4)TY培养基：酵母提取物8g/L、胰蛋白胨12g/L、K 3PO 4 4.02g/L、NaCl 3g/L、一水合柠檬酸2.1g/L、柠檬酸铁铵0.3g/L、甘油10g/L、(NH 4) 2SO 4 2.5g/L、MgSO 4·7H 2O 0.5g/L，用氨水调pH至7.2。
(5)补料培养基：甘油400g/L、酵母粉50g/L、胰蛋白胨25g/L。
下述实施例中涉及的检测方法如下：
赖氨酸脱羧酶的酶活测定方法：
酶活反应体系：500mM、pH 6.0乙酸钠缓冲液(350μL)，100mM底物L-赖氨酸(100μL)，0.25mM辅因子PLP(50μL)和纯酶(100μL)，40℃下反应1h，100℃煮沸5min，HPLC检测戊二胺含量。
酶活定义：每分钟生成1μmol的戊二胺所需的酶量为1U。
赖氨酸和尸胺的HPLC检测方法：
(1)样品衍生化处理：将样品以10000rpm离心20min，分别取上清液和标准品(赖氨酸和戊二胺)各75μL加入到含有乙氧基亚甲基丙二酸二乙酯(DEEMM)4.5μL、蒸馏水70.5μL、100％甲醇150μL、50mM、pH 9.0的硼酸盐缓冲液450μL的反应体系中，涡旋震荡混匀后在70℃下 孵育2h，以去除多余的DEEMM和副产物。将衍生后的样品以10000rpm离心5min，上清液经0.22μm膜过滤后用于HPLC检测。
(2)HPLC检测条件为：UV-VIS检测器、色谱柱(Platisil ODS 5μm，250×4.6mm)、柱温：35℃、流速1mL/min、检测波长：284nm。流动相A：100％乙腈、流动相B：25mM、pH 4.8的乙酸钠水溶液，梯度为：0～2min：20～25％A、2～20min：25～60％A、20～25min：60～20％A。
实施例1
1.1粘质沙雷氏菌来源赖氨酸脱羧酶突变位点的选择
使用在线工具HotSpotWizard 3.0(https://loschmidt.chemi.muni.cz/hot spotwizard)生成可行的突变热点。其中，选取了6个突变位点N120A、S165R、P530L、M569V、M678A和T691P，可能会提高赖氨酸脱羧酶的酶活水平，有利于1,5-戊二胺的生物合成。
1.2重组大肠杆菌E.coli BL21/pETDuet1-cadA及其突变株的构建
(1)重组大肠杆菌E.coliBL21/pETDuet1-cadA的构建
以cadA-F和cadA-R为引物，以粘质沙雷氏菌JNB5-1基因组为模板PCR扩增获得cadA基因片段，其大小为2139bp，纯化后的cadA基因片段与经BamHI和EcoRI双酶切的pETDuet1线性化质粒进行同源重组连接，连接产物转化E.coli BL21感受态细胞。重组质粒进行双酶切验证，验证成功的送苏州金唯智有限公司进行测序分析，SnapGene 3.2.1软件分析测序结果，测序正确的菌株命名为E.coli BL21/pETDuet1-cadA。其中，引物如下：
cadA-F：
Figure PCTCN2022102717-appb-000001
Figure PCTCN2022102717-appb-000002
(下划线部分为BamHI，SEQ ID NO.1)
cadA-R：
Figure PCTCN2022102717-appb-000003
Figure PCTCN2022102717-appb-000004
(下划线部分为EcoRI，SEQ ID NO.2)
(2)突变菌株的构建
以cadA-F/N120A-R、N120A-F/cadA-R，cadA-F/S165R-R、S165R-F/cadA-R，cadA-F/P530L-R、P530L-F/cadA-R，cadA-F/M569V-R、 M569V-F/cadA-R，cadA-F/M678A-R、M678A-F/cadA-R和cadA-F/T691P-R、T691P-F/cadA-R为引物，以粘质沙雷氏菌JNB5-1基因组为模板PCR扩增分别获得含N120A、S165R、P530L、M569V、M678A和T691P突变点的cadA基因片段，其大小均为2139bp，纯化后的含N120A、S165R、P530L、M569V、M678A和T691P突变点的cadA基因片段分别与经BamHI和SalI双酶切的pETDuet1线性化质粒进行同源重组连接，连接产物转化E.coliBL21感受态细胞。重组质粒进行双酶切验证，验证成功的送苏州金唯智有限公司进行测序分析，SnapGene 3.2.1软件分析测序结果，测序正确的菌株分别命名为E.coli BL21/pETDuet1-cadA N120A、E.coli BL21/pETDuet1-cadA S165R、E.coli BL21/pETDuet1-cadA P530L、E.coli BL21/pETDuet1-cadA M569V、E.coli BL21/pETDuet1-cadA M678A和E.coli BL21/pETDuet1-cadA T691P。其中，引物如下表1所列(大写字母为突变位点)：
表1 各个引物的引物序列
引物名称 引物序列(5’-3’) 序列编号
N120A-F gccaagatcaaacagGCGaccgacgaatatatc SEQ ID NO.3
N120A-R gatatattcgtcggtCGCctgtttgatcttggc SEQ ID NO.4
S165R-F aaaagcccggtcggcAGActgttctacgatttc SEQ ID NO.5
S165R-R gaaatcgtagaacagTCTgccgaccgggctttt SEQ ID NO.6
P530L-F gtcgagaaaaccgggCTGtacaacctgttgttc SEQ ID NO.7
P530L-R gaacaacaggttgtaCAGcccggttttctcgac SEQ ID NO.8
M569V-F ctgcgggtgaaaaacGTActgccttcgctgtat SEQ ID NO.9
M569V-R atacagcgaaggcagTACgtttttcacccgcag SEQ ID NO.10
M678A-F ctggagttcctgcagGCActgtgcgaaatcggc SEQ ID NO.11
M678A-R gccgatttcgcacagTGCctgcaggaactccag SEQ ID NO.12
T691P-F tatccgggctttgaaCCGgacattcacggcgcc SEQ ID NO.13
T691P-R ggcgccgtgaatgtcCGGttcaaagcccggata SEQ ID NO.14
1.3野生型赖氨酸脱羧酶及突变酶的表达与酶学性质研究
将构建好的重组大肠杆菌E.coli BL21/pETDuet1-cadA及其突变株在含50μg/mL卡那霉素的固体LB平板上进行划线活化，37℃培养12-24h 后，挑取单菌落，转接添加50μg/mL氨苄青霉素的10mLLB液体培养基，于37℃、180r/min培养12-24h；之后以1％(v/v)接种量接种至添加50μg/mL氨苄青霉素的50mL LB液体培养基中，于37℃、180r/min培养2h后，添加IPTG至终浓度为0.5mmol/L，继续于28℃、180r/min诱导表达16h；最后，将诱导完成后的菌液在4℃下离心，收集菌体；将菌体用浓度为0.1mol/L、pH为7.4的PBS缓冲液洗涤两次后，重新悬浮于浓度为0.1mol/L、pH为7.4的PBS缓冲液中，得到浓缩菌液；将浓缩菌液利用超声破碎仪破碎，得到破碎液；将破碎液在4℃下离心20min，收集上清，此上清即为粗酶液。
对细胞破碎上清液进行SDS-PAGE分析，其中，泳道1～8分别为E.coli BL21/pETDuet1-cadA N120A、E.coli BL21/pETDuet1-cadA S165R、E.coli BL21/pETDuet1-cadA P530L、E.coli BL21/pETDuet1-cadA M569V、E.coli BL21/pETDuet1-cadA M678A、E.coli BL21/pETDuet1-cadA T691P、E.coli BL21/pETDuet1-cadA P530L/M569V和E.coli BL21/pETDuet1细胞破碎上清液结果显示，在75kDa有一条明显的蛋白带，表明野生型赖氨酸脱羧酶成功实现表达，在75kDa有一条明显的蛋白带，表明赖氨酸脱羧突变酶均成功实现表达(见图1)。
接下来，则进行赖氨酸脱羧酶的纯化以进行酶学性质研究。蛋白纯化的方法是采用镍柱亲和层析，具体过程根据公司提供的仪器操作步骤进行。经蛋白纯化仪直接纯化获得的纯酶中含有大量咪唑，会降低赖氨酸脱羧酶的酶活，因此，为去除纯酶中含有的大量咪唑，使用0.05M、pH 7.0的Tris-HCl缓冲液作为透析液对纯酶进行了透析处理，经透析处理的纯酶用于后续的活性测定。
研究了不同pH(4.0～10.0，间隔1.0)、不同温度(30、35、40、45、50和60℃)下野生型赖氨酸脱羧酶及其突变体的酶学性质差异，结果显示，在pH 6.0～9.0范围内，野生型赖氨酸脱羧酶及其突变体均具有活性，且在pH 6.0时转化率最佳。野生型赖氨酸脱羧酶及其突变体的最佳反应温度均为40℃。
最后，测定野生型赖氨酸脱羧酶及其突变体酶活：在最适温度40℃和最适pH 6.0条件下反应1h后，立即于100℃下水浴5min以终止酶反 应，HPLC检测生成的戊二胺含量。采用上述赖氨酸脱羧酶的酶活测定方法测定酶活。
野生型赖氨酸脱羧酶及其突变体酶活测定结果显示，野生型赖氨酸脱羧酶CadA及突变酶CadA N120A、CadA S165R、CadA P530L、CadA M569V、CadA M678A和CadA T691P的比酶活分别为179.01、165.45、172.23、232.78、254.32、180.25和176.62U/mg，可见CadA P530L、CadA M569V的比酶活得到提高，分别是野生型CadA的1.3和1.42倍。
1.4最优突变体E.coli BL21/pETDuet1-cadA P530L/M569V的构建、酶活测定以及全细胞转化合成戊二胺
酶活测定结果显示，突变酶CadA P530L、CadA M569V的比酶活得到提高，分别是野生型CadA的1.3和1.42倍。因此，将这两个突变位点进行组合突变，以期进一步提高赖氨酸脱羧酶的酶活水平。
以cadA-F/M569V-R和M569V-F/cadA-R为引物，以含P530L突变位点的重组质粒为模板PCR扩增分别获得含M569V和P530L双突变位点的cadA基因片段，其大小均为2139bp，纯化后的含M569V和P530L双突变位点的cadA基因片段分别与经BamHI和EcoRI双酶切的pETDuet1线性化质粒进行同源重组连接，连接产物转化E.coli BL21感受态细胞。重组质粒进行双酶切验证，验证成功的送苏州金唯智有限公司进行测序分析，SnapGene 3.2.1软件分析测序结果，测序正确的菌株分别命名为E.coli BL21/pETDuet1-cadA P530L/M569V。
经测序，所述赖氨酸脱羧酶突变体CadA P530L/M569V的氨基酸序列如SEQ ID No.15所示，所述编码赖氨酸脱羧酶突变体CadA P530L/M569V的核苷酸序列如SEQ ID No.16所示。
之后按1.3所述方法对赖氨酸脱羧酶进行诱导表达及酶活测定，结果显示，CadA P530L/M569V的比酶活为292.54U/mg，是野生型CadA的1.63倍。
采用HPLC检测戊二胺：将构建好的重组大肠杆菌E.coli BL21/pETDuet1-cadA或重组大肠杆菌E.coli BL21/pETDuet1-cadA P530L/M569V在含50μg/mL氨苄青霉素的固体LB平板上进行划线活化，37℃培养12～24h后，挑取单菌落，转接添加50μg/mL 氨苄青霉素的10mL LB液体培养基，于37℃、180r/min培养12～24h；之后以1％(v/v)接种量接种至添加50μg/mL氨苄青霉素的50mL LB液体培养基中，于37℃、180r/min培养2h后，添加IPTG至终浓度为0.5mmol/L，继续于28℃、180r/min诱导表达16h；最后，将诱导完成后的菌液在4℃下离心，收集菌体。在控制菌体浓度一样的前提下(OD 600＝4.0)，在500mM、pH 6.0乙酸钠缓冲液中加入0.5M底物L-赖氨酸盐酸盐、1％50mM Mn 2+、0.25mM PLP，40℃下反应20min，得到转化液。然后将转化液采用上述戊二胺HPLC检测方法检测戊二胺及其含量。
其中，图2为赖氨酸标准品HPLC图谱，在该HPLC图谱中，赖氨酸的保留时间为11.725min；图3为1,5-戊二胺标准品HPLC图谱，在该HPLC图谱中，1,5-戊二胺的保留时间为24.111min。
结果显示，E.coliBL21/pETDuet1-cadA菌株合成了3.15g/L的戊二胺，E.coliBL21/pETDuet1-cadA P530L/M569V菌株合成了3.76g/L的戊二胺(见图4)，由此可见，该菌株可以用于进一步改造来提高戊二胺的合成能力。
1.5赖氨酸/尸胺反向转运蛋白过表达促进戊二胺的生物合成
以cadB-F和cadB-R为引物，以大肠杆菌基因组为模板PCR扩增获得cadB基因片段，其大小为1335bp，纯化后的cadB基因片段与经BglII和XhoI双酶切的pETDuet1-cadA P530L/M569V线性化质粒进行同源重组连接，连接产物转化E.coli BL21感受态细胞。重组质粒进行双酶切验证，验证成功的送苏州金唯智有限公司进行测序分析，SnapGene 3.2.1软件分析测序结果，测序正确的菌株命名为E.coli BL21/pETDuet1-cadA P530L/M569V-cadB。其中，引物如下：
cadB-F：
Figure PCTCN2022102717-appb-000005
Figure PCTCN2022102717-appb-000006
(下划线部分为BglII，SEQ ID No.17)
cadB-R：
Figure PCTCN2022102717-appb-000007
(下划线部分为XhoI，SEQ ID No.18)
所述赖氨酸/尸胺反向转运蛋白CadB的氨基酸序列如SEQ ID No.19 所示，所述编码赖氨酸/尸胺反向转运蛋白的基因cadB的核苷酸序列如SEQ ID No.20所示。
采用HPLC检测戊二胺：将构建好的重组大肠杆菌E.coli BL21/pETDuet1-cadA P530L/M569V及E.coliBL21/pETDuet1-cadA P530L/M569V-c adB在含50μg/mL氨苄青霉素的固体LB平板上进行划线活化，37℃培养12～24h后，挑取单菌落，转接添加50μg/mL氨苄青霉素的10mL LB液体培养基，于37℃、180r/min培养12～24h；之后以1％(v/v)接种量接种至添加50μg/mL氨苄青霉素的50mL LB液体培养基中，于37℃、180r/min培养2h后，添加IPTG至终浓度为0.5mmol/L，继续于28℃、180r/min诱导表达16h；最后，将诱导完成后的菌液在4℃下离心，收集菌体。在控制菌体浓度一样的前提下(OD 600＝4.0)，在500mM、pH 6.0乙酸钠缓冲液中加入1M底物L-赖氨酸盐酸盐和1％50mM Mn 2+，40℃下反应20min，得到转化液1。在控制菌体浓度一样的前提下(OD 600＝4.0)，在500mM、pH 6.0乙酸钠缓冲液中加入1M底物L-赖氨酸盐酸盐、1％50mM Mn 2+、0.25mM PLP，40℃下反应20min，得到转化液2。将收集的转化液1和转化液2采用上述戊二胺HPLC检测方法检测戊二胺及其含量。
结果显示，在额外添加和不添加PLP辅因子条件下，E.coli BL21/pETDuet1-cadA P530L/M569V-cadB菌株可合成15.95g/L和5.57g/L的戊二胺，而E.coli BL21/pETDuet1-cadA P530L/M569V菌株仅合成了3.76g/L和1.47g/L的戊二胺(见图5)。由此可见，赖氨酸/尸胺反向转运蛋白的过表达有利于底物L-赖氨酸进入细胞和产物戊二胺运出细胞，提高了戊二胺的合成效率，其次，额外添加辅因子PLP提高了戊二胺的产量，可以看出，辅因子PLP供应不足将不利于戊二胺的合成。
1.6 PLP辅因子自循环系统构建
以pdxK-F/R和pdxY-F/R为引物，以大肠杆菌基因组为模板PCR扩增获得pdxK和pdxY基因片段，其大小分别为852bp和864bp，纯化后的pdxK和pdxY基因片段与经EcoRI和HindIII双酶切的pETDuet1-cadA P530L/M569V-cadB线性化质粒进行同源重组连接，连接产物转化E.coli BL21感受态细胞。转化子验证成功的送苏州金唯智有限公司 进行测序分析，SnapGene 3.2.1软件分析测序结果，测序正确的菌株分别命名为E.coli BL21/pETDuet1-cadA P530L/M569V-cadB-pdxK和E.coli BL21/pETDuet1-cadA P530L/M569V-cadB-pdxY。其中，引物如下：
pdxK-F：
Figure PCTCN2022102717-appb-000008
Figure PCTCN2022102717-appb-000009
(下划线部分为EcoRI，SEQ ID No.21)
pdxK-R：
Figure PCTCN2022102717-appb-000010
Figure PCTCN2022102717-appb-000011
(下划线部分为HindIII，SEQ ID No.22)
pdxY-F：
Figure PCTCN2022102717-appb-000012
Figure PCTCN2022102717-appb-000013
(下划线部分为EcoRI，SEQ ID No.23)
pdxY-R：
Figure PCTCN2022102717-appb-000014
Figure PCTCN2022102717-appb-000015
(下划线部分为HindIII，SEQ ID No.24)
所述吡哆醛激酶I的氨基酸序列如SEQ ID No.25所示，所述编码吡哆醛激酶I的基因pdxK核苷酸序列如SEQ ID No.26所示；所述吡哆醛激酶II的氨基酸序列如SEQ ID No.27所示，所述编码吡哆醛激酶II的基因pdxY的核苷酸序列如SEQ ID No.28所示。
采用HPLC检测戊二胺：将构建好的重组大肠杆菌E.coli BL21/pETDuet1-cadA P530L/M569V-cadB、E.coli BL21/pETDuet1-cadA P530L/M569V-cadB-pdxK和E.coli BL21/pETDuet1-cadA P530L/M569V-cadB-pdxY在含50μg/mL氨苄青霉素的固体LB平板上进行划线活化，37℃培养12～24h后，挑取单菌落，转接添加50μg/mL氨苄青霉素的10mL LB液体培养基，于37℃、180r/min培养12～24h；之后以1％(v/v)接种量接种至添加50μg/mL氨苄青霉素的50mLLB液体培养基中，于37℃、180r/min培养2h后，添加IPTG至终浓度为0.5mmol/L，继续于28℃、180r/min诱导表达16h；最后，将诱导完成后的菌液在4℃下离心，收集菌体。在控制菌体浓度一样的前提下(OD 600＝4.0)，在500mM、pH 6.0乙酸钠缓冲液中加入1M底物L- 赖氨酸盐酸盐，40℃下反应20min，得到转化液。将转化液采用上述戊二胺HPLC检测方法检测戊二胺及其含量。
结果显示，在不额外添加PLP条件下，E.coli BL21/pETDuet1-cadA P530L/M569V-cadB-pdxK和E.coli BL21/pETDuet1-cadA P530L/M569V-cadB-pdxY菌株分别合成了11.88g/L和14.43g/L的戊二胺，分别是E.coli BL21/pETDuet1-cadA P530L/M569V-cadB(5.57g/L)的2.13和2.59倍(见图6中的A和B)。因此，可以看出，吡哆醛激酶I和II编码基因pdxK和pdxY的过表达提高了辅因子PLP自供应水平，有利于戊二胺的生物合成。
为进一步提高辅因子PLP自供应能力，以pdxY-FF和pdxY-RR为引物，以大肠杆菌基因组为模板PCR扩增分别获得添加NdeI和BglII酶切位点和同源臂的pdxY基因片段，纯化后的添加NdeI和BglII酶切位点和同源臂的pdxY基因片段与经NdeI和BglII双酶切的pETDuet1-cadA P530L/M569V-cadB-pdxK线性化质粒进行同源重组连接，连接产物转化E.coliBL21感受态细胞。转化子验证成功的送苏州金唯智有限公司进行测序分析，SnapGene 3.2.1软件分析测序结果，测序正确的菌株分别命名为E.coli BL21/pETDuet1-cadA P530L/M569V-cadB-pdxK-pdxY。其中，引物如下：
pdxY-FF：
Figure PCTCN2022102717-appb-000016
Figure PCTCN2022102717-appb-000017
(下划线部分为NdeI，SEQ ID No.29)
pdxY-RR：
Figure PCTCN2022102717-appb-000018
Figure PCTCN2022102717-appb-000019
(下划线部分为BglII，SEQ ID No.30)
将构建好的重组大肠杆菌E.coliBL21/pETDuet1-cadA P530L/M569V-cadB-pdxK-pdxY在含50μg/mL氨苄青霉素的固体LB平板上进行划线活化，37℃培养12～24h后，挑取单菌落，转接添加50μg/mL氨苄青霉素的10mL LB液体培养基，于37℃、180r/min培养12～24h；之后以1％(v/v)接种量接种至添加50μg/mL氨苄青霉素的50mL LB液体培养基中，于37℃、180r/min培养2h后，添加IPTG至终浓度为0.5mmol/L，继续于 28℃、180r/min诱导表达16h；最后，将诱导完成后的菌液在4℃下离心，收集菌体。在控制菌体浓度一样的前提下(OD 600＝4.0)，在500mM、pH 6.0乙酸钠缓冲液中加入1M底物L-赖氨酸盐酸盐，40℃下反应20min，得到转化液。将转化液采用上述戊二胺HPLC检测方法检测戊二胺及其含量。
结果显示，在不额外添加PLP条件下，E.coli BL21/pETDuet1-cadA P530L/M569V-cadB-pdxK-pdxY菌株合成了18.52g/L的尸胺(见图7)，使尸胺产量得到了较高程度提高。
1.7全细胞催化生物合成1,5-戊二胺
重组大肠杆菌E.coli BL21/pETDuet1-cadA P530L/M569V-cadB-pdxK-pdxY在含50μg/mL氨苄青霉素的固体LB平板上进行划线活化，37℃培养12-24h后，挑取单菌落，转接添加50μg/mL氨苄青霉素的10mL LB液体培养基，于37℃、180r/min培养12～24h；之后以1％(v/v)接种量接种至添加50μg/mL氨苄青霉素的200mL LB液体培养基中，于37℃、180r/min培养18h，获得种子液。之后按10％接种量转接装有2L发酵培养基(TY培养基)的5L发酵罐，发酵条件：温度设为37℃、转速设为600rpm、通气量为4vvm。OD 600为7～9时，开始匀速流加补料培养基，补料时间控制在12h。发酵8h后，添加IPTG诱导剂(终浓度0.5mM)，并将发酵罐温度设为28℃，诱导培养24h后，停止发酵，4℃下离心收集菌体。
全细胞转化体系(1L)：离心收集的菌体用500mL水悬浮后(OD 600为50～70)，之后投加500mL含2M(365.3g/L)工业级L-赖氨酸盐酸盐、1％(质量浓度1g/100mL)50mM Mn 2+、0.25mM维生素B6的水溶液，40℃转化3h，得到转化液，采用上述戊二胺HPLC检测方法检测戊二胺及其含量。
由图8的结果表明，重组大肠杆菌E.coli BL21/pETDuet1-cadA P530L/M569V-cadB-pdxK-pdxY合成了201.65g/L的1,5-戊二胺，摩尔转化率高达98.67％，反应中不使用缓冲液，无需使用盐酸进行中和，转化时间短，为1,5-戊二胺的工业化生产提供了一种高效且经济的方法。
以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。对这些实施例的多种修改对本领域的专业技术人员来说是显而易见的，本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下在其它实施例中实现。因此，本发明将不会被限制于本文所示的这些实施例，而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。

Claims (10)

  1. 一种重组大肠杆菌，其特征在于，所述重组大肠杆菌依次含有赖氨酸脱羧酶突变体CadA P530L/M569V、赖氨酸/尸胺反向转运蛋白CadB和吡哆醛激酶。
  2. 根据权利要求1所述的重组大肠杆菌，其特征在于，所述吡哆醛激酶为吡哆醛激酶I和吡哆醛激酶II；
    优选的，所述重组大肠杆菌依次含有赖氨酸脱羧酶突变体CadA P530L/M569V、赖氨酸/尸胺反向转运蛋白CadB、吡哆醛激酶I和吡哆醛激酶II；
    优选的，所述吡哆醛激酶II氨基酸序列如SEQ ID No.27所示；所述编码吡哆醛激酶II基因pdxY的核苷酸序列如SEQ ID No.28所示；
    优选的，所述吡哆醛激酶I氨基酸序列如SEQ ID No.25所示；所述编码吡哆醛激酶I基因pdxK的核苷酸序列如SEQ ID No.26所示。
  3. 根据权利要求1所述的重组大肠杆菌，其特征在于，所述赖氨酸脱羧酶突变体CadA P530L/M569V的氨基酸序列如SEQ ID No.15所示；所述编码赖氨酸脱羧酶突变体CadA P530L/M569V的核苷酸序列如SEQ ID No.16所示；所述赖氨酸/尸胺反向转运蛋白CadB的氨基酸序列如SEQ ID No.19所示；所述编码赖氨酸/尸胺反向转运蛋白的基因cadB的核苷酸序列如SEQ ID No.20所示。
  4. 根据权利要求1～3任意一项所述的重组大肠杆菌，其特征在于，所述重组大肠杆菌的出发菌株为大肠杆菌E.coli BL21。
  5. 含有权利要求1所述赖氨酸脱羧酶突变体CadA P530L/M569V、赖氨酸/尸胺反向转运蛋白CadB和吡哆醛激酶的氨基酸序列的表达载体，其特征在于，所述表达载体包括pETDuet1、pACYCDuet1、pET28a中的任意一种。
  6. 一种权利要求1～4任意一项所述重组大肠杆菌的构建方法，其特征在于，将赖氨酸脱羧酶突变体CadA P530L/M569V、赖氨酸/尸胺反向转运蛋白CadB和吡哆醛激酶的基因序列依次导入大肠杆菌；
    优选的，将赖氨酸脱羧酶突变体CadA P530L/M569V、赖氨酸/尸胺反向转 运蛋白CadB、吡哆醛激酶I和吡哆醛激酶II的基因序列依次导入大肠杆菌。
  7. 权利要求1～4任意一项所述重组大肠杆菌或权利要求6所述构建方法得到的重组大肠杆菌在合成1,5-戊二胺中的应用。
  8. 一种合成1,5-戊二胺的方法，其特征在于，将权利要求1～4任意一项所述重组大肠杆菌或权利要求6所述构建方法得到的重组大肠杆菌进行发酵培养后，接种至含有L-赖氨酸盐酸盐的转化体系中，以转化L-赖氨酸盐酸盐为1,5-戊二胺；
    优选的，所述的转化体系包括如下浓度的成分：L-赖氨酸盐酸盐1.5～2.5M、Mn 2+45～55mM、维生素B6 0.2～0.3mM，pH自然。
    优选的，所述转化的温度为38～42℃，所述转化的时间为2.5～3.5h。
  9. 根据权利要求8所述的方法，其特征在于，所述发酵培养的方法包括：
    将重组大肠杆菌的种子液接种在发酵培养基中发酵，发酵温度为32～40℃、转速为550～650rpm、通气量为3～5vvm，OD 600为7～9时，开始匀速流加补料培养基，补料时间控制在10～13h，发酵7～9h后，添加IPTG诱导剂，并将发酵温度设为27～29℃，诱导培养20～26h后，发酵结束。
  10. 根据权利要求9所述的方法，其特征在于，所述发酵培养基为TY培养基，所述TY培养基包括酵母提取物8g/L、胰蛋白胨12g/L、K 3PO 44.02g/L、NaCl 3g/L、一水合柠檬酸2.1g/L、柠檬酸铁铵0.3g/L、甘油10g/L、(NH 4) 2SO 42.5g/L、MgSO 4·7H 2O 0.5g/L，用氨水调pH至7.2；所述补料培养基包括甘油400g/L、酵母粉50g/L、胰蛋白胨25g/L。
PCT/CN2022/102717 2022-06-30 2022-06-30 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 WO2024000368A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2022/102717 WO2024000368A1 (zh) 2022-06-30 2022-06-30 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

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2022/102717 WO2024000368A1 (zh) 2022-06-30 2022-06-30 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

Publications (1)

Publication Number Publication Date
WO2024000368A1 true WO2024000368A1 (zh) 2024-01-04

Family

ID=89383728

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/102717 WO2024000368A1 (zh) 2022-06-30 2022-06-30 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

Country Status (1)

Country Link
WO (1) WO2024000368A1 (zh)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105316270A (zh) * 2014-06-27 2016-02-10 中国科学院微生物研究所 一种催化生产1, 5-戊二胺的工程菌及其应用
CN106148373A (zh) * 2016-05-27 2016-11-23 南京工业大学 一种基因工程菌及其在生产1,5‑戊二胺中的用途
CN108138160A (zh) * 2015-08-06 2018-06-08 首尔大学校产学协力团 赖氨酸脱羧酶的突变型开发方法及其应用
CN113444713A (zh) * 2021-06-28 2021-09-28 南京工业大学 一种L-赖氨酸脱羧酶SpLDC及其在产1，5-戊二胺上的应用
CN113881719A (zh) * 2020-07-02 2022-01-04 中国科学院过程工程研究所 一种全细胞催化合成1,5-戊二胺的方法
WO2022001121A1 (zh) * 2020-07-02 2022-01-06 中国科学院过程工程研究所 一种用于合成戊二胺的赖氨酸脱羧酶及其应用

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105316270A (zh) * 2014-06-27 2016-02-10 中国科学院微生物研究所 一种催化生产1, 5-戊二胺的工程菌及其应用
CN108138160A (zh) * 2015-08-06 2018-06-08 首尔大学校产学协力团 赖氨酸脱羧酶的突变型开发方法及其应用
CN106148373A (zh) * 2016-05-27 2016-11-23 南京工业大学 一种基因工程菌及其在生产1,5‑戊二胺中的用途
CN113881719A (zh) * 2020-07-02 2022-01-04 中国科学院过程工程研究所 一种全细胞催化合成1,5-戊二胺的方法
WO2022001121A1 (zh) * 2020-07-02 2022-01-06 中国科学院过程工程研究所 一种用于合成戊二胺的赖氨酸脱羧酶及其应用
CN113444713A (zh) * 2021-06-28 2021-09-28 南京工业大学 一种L-赖氨酸脱羧酶SpLDC及其在产1，5-戊二胺上的应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
JIE DENG, GAO ZHEN; GAO HUA; WU QIAOFEN; JIANG CHENGJIAN: "Research Progress of the Biosynthesis of 1,5-Diaminopentane ", GUANGXI SCIENCES, vol. 24, no. 1, 16 January 2017 (2017-01-16), pages 40 - 47, XP093122091 *
MA, WEICHAO ET AL.: "Advances in Cadaverine Bacterial Production and Its Applications", ENGINEERING, vol. 3, no. 3, 15 June 2017 (2017-06-15), XP055603579, ISSN: 2095-8099, DOI: 10.1016/J.ENG.2017.03.012 *

Similar Documents

Publication Publication Date Title
CN108467860B (zh) 一种高产γ-氨基丁酸的方法
CN109777763B (zh) 一株用于l-茶氨酸生产的基因工程菌及其构建与应用
CN109609475A (zh) 草铵膦脱氢酶突变体及其合成l-草铵膦的应用
JP6431205B2 (ja) 新規リシンデカルボキシラーゼ、及びそれを利用してカダベリンを生産する方法
WO2022174597A1 (zh) 一种用于l-肌氨酸生产的基因工程菌及构建方法与应用
CN116286700A (zh) 一种亚胺还原酶突变体及用途
CN112831488B (zh) 一种谷氨酸脱羧酶及γ-氨基丁酸高产菌株
CN111748548B (zh) 一种精氨酸脱羧酶突变体及其在生产胍基丁胺中的应用
CN113025542B (zh) 产l-谷氨酰胺的重组大肠杆菌及其构建方法与应用
CN114525268B (zh) 一种pH耐受性提高的谷氨酸脱羧酶突变体及其在γ-氨基丁酸合成中的应用
CN113564090B (zh) 一种用于产四氢嘧啶重组菌的构建方法及其应用
WO2024140379A1 (zh) 酶、生产红景天苷的菌株及生产方法
CN112980906B (zh) 一种用于制备β-烟酰胺单核苷酸的酶组合物及其应用
CN113462669A (zh) 酮泛解酸羟甲基转移酶突变体、编码基因及其应用
WO2024000368A1 (zh) 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
JP2002330785A (ja) ヒダントイナーゼをコードするｄｎａ、ｎ−カルバミル−ｌ−アミノ酸ハイドロラーゼをコードするｄｎａ、組み換えｄｎａ、形質転換された細胞、タンパク質の製造方法および光学活性アミノ酸の製造方法
CN114317510B (zh) 一种马来酸顺反异构酶突变体
CN114990045B (zh) 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
CN116064445A (zh) 一种亮氨酸脱氢酶突变体及其在生产l-2-氨基丁酸中的应用
CN112481320A (zh) 一种催化效率高的制备(-)γ-内酰胺的方法
CN114107270B (zh) 一种L-天冬氨酸β-脱羧酶突变体
CN116064494B (zh) 一种谷氨酸脱羧酶突变体、基因及其应用
CN116042682B (zh) 一株产2’-岩藻糖基乳糖的工程菌及其构建方法和应用
CN114250237B (zh) 一种酪氨酸酚裂解酶突变体、工程菌及在催化合成左旋多巴中的应用
CN107201355B (zh) 一种高立体选择性苯丙氨酸脱氨酶突变体及其应用

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: 22948480

Country of ref document: EP

Kind code of ref document: A1