Carboxyspermidine decarboxylase mutant and application thereof

Abstract

The invention discloses carboxyl spermidine decarboxylase, the amino acid sequence of the carboxyl spermidine decarboxylase is as shown in SEQ ID NO. 1, and the nucleotide sequence of the carboxyl spermidine decarboxylase is as shown in SEQ ID NO. 2. The invention also discloses an application of the carboxyl spermidine decarboxylase in enzymatic synthesis of spermidine. The carboxyl spermidine decarboxylase provided by the invention has higher catalytic activity and protein expression quantity, and the concentration of a spermidine product can be remarkably improved and is far greater than the yield disclosed in the general prior art.

Description

Technical Field

[0001] The present invention belongs to the technical field of biocatalysis, and specifically relates to a carboxyspermidine decarboxylase mutant and its application. Background Art

[0002] Spermidine is widely distributed in organisms and is biosynthesized from putrescine (butanediamine) and carboxylated S-adenosylmethionine. Spermidine has the effects of delaying aging, preventing Alzheimer's disease and protecting nerve cells. Currently, the main preparation methods include chemical synthesis and extraction from wheat germ. In addition, there are also reports on preparation by fermentation and enzymatic methods.

[0003] In 2021, researchers from Seoul National University first reported the production of spermidine by fermentation. They obtained a genetically engineered strain, which could produce 224 mg / L of spermidine after 68 h of fermentation under the condition of co-fermentation of glucose (restricted) and xylose, with a conversion rate of 2.2 mg / g (Kim, Bosu, B. R. Cho, and J. S. Hahn. "Metabolic engineering of Saccharomyces cerevisiae for the production of 2-phenylethanol via Ehrlich pathway." Biotechnology and Bioengineering 111.1 (2014).). Jens Nielsen et al. from the Technical University of Denmark fermented under glucose-restricted conditions in Saccharomyces cerevisiae, and the product concentration could reach 2.3 g / L (Qin, J., et al. "Engineering yeast metabolism for the discovery and production of polyamines and polyamine analogues." Nature Catalysis.). In 2022, the team of Cai Yujie from Jiangnan University co-expressed homoserine dehydrogenase (scHSD, Saccharomyces cerevisiae), carboxyarginine amidinohydrolase (afCASDH, Rhizobium radiobacter), and carboxyspermidine decarboxylase (bcCASDC, Butyrivibrio fibrisolvens), constructed a three-enzyme coupled catalytic system, and in the presence of cofactor NADP+, it could produce 4.15 g / L of spermidine after 24 h of catalysis, which is the highest concentration reported so far (Xinxin Liang et al. “Highly efficient biosynthesis of spermidine from L homoserine and putrescine using an engineered Escherichia coli with NADPH self-sufficient system.” Applied Microbiology and Biotechnology (2022) 106:5479–5493).

[0004] However, although the yield of spermidine synthesized by the enzymatic method is the highest, the research on the rate-limiting enzyme in the enzymatic synthesis process has not been reported in this field. Based on this, the present invention studied the rate-limiting enzyme in the enzymatic synthesis of spermidine, and significantly improved the yield and recovery rate of spermidine. Summary of the Invention

[0005] The inventors screened a carboxylspermidine decarboxylase that can significantly increase the concentration of spermidine products, and through site-directed mutagenesis, improved the soluble protein expression level of the enzyme and the concentration of spermidine products.

[0006] Specifically, the present invention includes the following technical solutions:

[0007] In a first aspect, the present invention provides a carboxylspermidine decarboxylase, and the amino acid sequence of the carboxylspermidine decarboxylase is as shown in SEQ ID NO.1:

[0008] MGMTELATPYYLIDESRLKRNLEIIRQVRERSGAKSVLALKCFSTWCVFDLMRQYMDGTTSSSLYEARLGHEEFGGETHAYSVGFSRAEVLAVREFADKLIFNSVSQLRQFYPEVAGLNLGLRVNPGISYSRFDLADPARRYSRLGVRNKAEISEALPWIKGLMFHFNCENADIEHFRAALATIGREYGELLHRMEWVSLGGGLSFTQEGYPLETFCEALRTFGQSFDIQVYLEPGEAAVTQSTELVTTVLDVVHNEIDIAIVDASLEAHMLDHLIYRTNPRLASPPPGEHRMMIAGRTCLAGDVFGDFKLACPLQIGDEVRIADAGGYTMVQVNWFNGVSMPAIVVRRLDGQVECVRTFGYEDFKRSLS*

[0009] In a second aspect, the present invention provides a gene encoding the carboxylspermidine decarboxylase as described above.

[0010] In one or more embodiments, the nucleotide sequence of the carboxylspermidine decarboxylase is as shown in SEQ ID NO.2:

[0011]

[0012] In a third aspect, the present invention provides an expression vector comprising the gene as described above. Preferably, the vector is a PET series vector.

[0013] In a fourth aspect, the present invention provides a host cell comprising the carboxy-spermidine decarboxylase as described above. Preferably, the host cell is selected from Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae, Yarrowia lipolytica or Bacillus subtilis.

[0014] In a fifth aspect, the present invention provides the use of the carboxy-spermidine decarboxylase as described above or the host cell as described above in the enzymatic synthesis of spermidine.

[0015] In a sixth aspect, the present invention provides a method for preparing spermidine, the method comprising the step of using homoserine and putrescine as substrates and performing a one-pot synthesis of spermidine using homoserine dehydrogenase, carboxy-spermidine dehydrogenase and carboxy-spermidine decarboxylase, wherein the nucleotide sequence of the carboxy-spermidine decarboxylase is as shown in SEQ ID NO.2.

[0016] In the preparation method, homoserine dehydrogenase dehydrogenates homoserine to generate aspartic semialdehyde and NADH, carboxy-spermidine dehydrogenase uses NADH as a coenzyme to synthesize carboxy-spermidine from aspartic semialdehyde and putrescine, and realizes coenzyme regeneration to generate NAD. Finally, carboxy-spermidine decarboxylase decarboxylates carboxy-spermidine to generate spermidine.

[0017] In one or more embodiments, the concentrations of the substrates homoserine and putrescine are both 100 mM.

[0018] In one or more embodiments, expression strains of homoserine dehydrogenase, carboxy-spermidine dehydrogenase and carboxy-spermidine decarboxylase are added to the substrates homoserine and putrescine, and the wet cell weights of the three expression strains are in a ratio of 1:1:3 - 1:1:5.

[0019] In one or more embodiments, the construction method of the three expression strains is as follows: first, plasmids of homoserine dehydrogenase, carboxy-spermidine dehydrogenase and carboxy-spermidine decarboxylase are constructed respectively, and then transformed into the BL21(DE3) strain; the three constructed plasmids are pET28a-scHSD, pET28a-afCASDH and pET28a-bcCASDC, and the corresponding nucleotide sequences are SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5 respectively.

[0020] In the present invention, the sources of the homoserine dehydrogenase and carboxy-spermidine dehydrogenase are not limited. Preferably, the source of the homoserine dehydrogenase can be Thermococcus sp., Thermocrinis minervae, Saccharomyces cerevisiae, etc.; the source of the carboxy-spermidine dehydrogenase can be Agrobacterium fabrum, Vibrio alginolyticus, Hydrogenimonas thermophila, etc.

[0021] Preferably, the homoserine dehydrogenase and carboxy-spermidine dehydrogenase are derived from Saccharomyces cerevisiae and Agrobacterium fabrum, respectively.

[0022] In the present invention, a carboxy-spermidine decarboxylase derived from Thermochromatium tepidum was screened and site-directed mutagenesis was performed on it, which not only increased the protein expression level of the enzyme, reduced the amount of bacterial cells used, but also increased the yield of spermidine. Therefore, this enzyme is suitable for industrial production of spermidine. Detailed implementation manners

[0023] The in vitro multi-enzyme synthesis system mimics the in vivo metabolic pathway, combines a series of enzymes and coenzymes in vitro to construct a biochemical reaction pathway, and is a new type of biological manufacturing method for efficiently catalyzing the synthesis of products from substrates. By in vitro design and assembly regulation of enzyme components and multi-enzyme complexes, a brand-new artificial biosynthesis pathway is constructed to reshape the natural production line, which has significant advantages such as diverse pathways, high controllability, high efficiency, and high atom economy, and is of great significance for changing the manufacturing mode of chemicals.

[0024] The enzymatic preparation of spermidine can adopt the following method, specifically: using homoserine and putrescine as substrates, homoserine dehydrogenase dehydrogenates homoserine to generate aspartic semialdehyde and NADH, carboxy-spermidine dehydrogenase uses NADH as a coenzyme to synthesize carboxy-spermidine from aspartic semialdehyde and putrescine, and realizes coenzyme regeneration to generate NAD. Finally, carboxy-spermidine decarboxylase decarboxylates carboxy-spermidine to generate spermidine.

[0025] By analyzing the related enzymes in the above enzymatic synthesis of spermidine, the inventors of the present invention found that carboxy-spermidine decarboxylase is the rate-limiting enzyme for the synthesis of spermidine. The inventors screened a carboxy-spermidine decarboxylase that can significantly increase the concentration of spermidine product, and through site-directed mutagenesis, increased the soluble protein expression level of the enzyme and the yield of spermidine product, and reduced the amount of bacterial cells used.

[0026] The carboxylspermine decarboxylase of the present invention, abbreviated as CASDC, can decarboxylate carboxylspermine to generate spermidine. The enzyme screened in the present invention is derived from Thermochromatium tepidum, and its amino acid sequence is shown in SEQ ID NO:1.

[0027] In this article, the terms "carboxylspermine decarboxylase", "CASDC enzyme", and "CASDC" all refer to the carboxylspermine decarboxylase with the amino acid sequence of SEQ ID NO:1. For convenience, the enzyme is generally described as CASDC in this article.

[0028] In order to improve the protein expression level of the carboxylspermine decarboxylase, the present invention also optimized the codons of some arginine, leucine, and isoleucine of the enzyme, and found that after site-directed mutation at the 51st amino acid of the enzyme, the protein expression level of the enzyme can be increased and the usage amount of wet cells can be reduced. Specifically, the nucleotide sequence of the enzyme after mutation is shown in SEQ ID NO:2.

[0029] In the present invention, the gene, expression cassette, plasmid, and transformant can be obtained by genetic engineering construction methods well-known to those skilled in the art.

[0030] In the present invention, the transformant host can be any microorganism suitable for expressing carboxylspermine decarboxylase, including bacteria and fungi. Preferred microorganisms are Bacillus subtilis, Pichia pastoris, Saccharomyces cerevisiae, Yarrowia lipolytica, or Escherichia coli, preferably Escherichia coli, and more preferably Escherichia coli BL21(DE3).

[0031] When used as a biocatalyst, the carboxylspermine decarboxylase of the present invention, as well as additional homoserine dehydrogenase and carboxylspermine dehydrogenase, can be added to the reaction system either in the form of an enzyme or in the form of cells. The forms of the enzyme include free enzyme, immobilized enzyme, including purified enzyme, crude enzyme, fermentation broth, enzyme immobilized on a carrier, etc.; the forms of the cells include viable cells, dead cells, immobilized cells, etc.

[0032] When microorganisms such as Bacillus subtilis, Pichia pastoris, Saccharomyces cerevisiae, or Escherichia coli are no longer used for fermentation and proliferation but for enzymatic catalysis reactions, they are a natural immobilized enzyme in themselves, and can be directly used as an enzyme preparation for catalytic reactions without undergoing cell disruption treatment, or even extraction and purification treatment. Since the reaction substrates and reaction products are all small molecule compounds, they can easily pass through the biological barrier of the cells - the cell membrane. Therefore, there is no need to perform cell disruption treatment on the cells, which is economically beneficial.

[0033] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are only used to illustrate the present invention and not to limit the scope of the present invention.

[0034] Example

[0035] In this article, the addition amounts, contents and concentrations of various substances are involved. Among them, the percentage content, unless otherwise specified, all refers to the mass percentage content.

[0036] Materials and Methods

[0037] The molecular biology experiments in the examples include plasmid construction, restriction digestion, ligation, preparation of competent cells, transformation, medium preparation, etc. These are mainly carried out with reference to the third edition of "Molecular Cloning: A Laboratory Manual" (edited by J. Sambrook, D.W. Russell (USA), translated by Huang Peitang et al., Science Press, Beijing, 2002). Specific experimental conditions can be determined through simple experiments when necessary.

[0038] The PCR amplification experiment is carried out according to the reaction conditions provided by the plasmid or DNA template supplier or the kit instruction manual. Adjustments can be made through simple experiments when necessary.

[0039] LB liquid medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride.

[0040] LB solid medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 20 g / L agar powder.

[0041] In the following implementation cases, when using a medium containing kanamycin (kan), the final concentration of kanamycin in the medium is 50 μg / ml; when using IPTG for induction, the final concentration of IPTG in the medium is 0.2 mM.

[0042] In the following examples, the sample derivatization conditions in HPLC detection are as follows:

[0043] 1. Add 1 mL of the supernatant dilution of the fermentation broth to 100 μL of NaOH solution (2 mol / L) and 200 μL of saturated NaHCO3 solution (to make the pH of the reaction system about 11).

[0044] 2. Add 3 mL of Dns-cL solution (10 mg / mL dissolved in acetone, stored at -20 °C), mix, and perform derivatization in a water bath at 55 °C for 60 min.

[0045] 3. After the water bath, wait for the mixed solution to cool, add 200 μL of ammonia water, and keep it in the dark for half an hour to terminate the reaction.

[0046] 4. Add 2 mL of saturated NaCl solution, mix well, then add 5 mL of ether. Vortex and extract for 5 min at room temperature. After standing for 30 min to separate layers, transfer the upper organic phase (ether layer) to a 50 mL centrifuge tube and dry it in a 42 °C water bath in the fume hood.

[0047] 5. Finally, make up the volume to 1 mL with acetonitrile and send it for inspection (the derivative product needs to be stored at -20 °C).

[0048] In the following examples, the reference conditions for liquid chromatography in HPLC detection are as follows:

[0049] The chromatographic column is a C18 column (column length 250 mm, column inner diameter 4.6 mm, column packing particle size 5 μm). The ultraviolet detection wavelength is 254 nm, the injection volume is 20 μL, and the column temperature is 35 °C;

[0050] Mobile phase A is 90% acetonitrile / 10% (0.01 mol / L ammonium acetate solution containing 0.1% acetic acid), mobile phase B is 10% acetonitrile / 90% (0.01 mol / L ammonium acetate solution containing 0.1% acetic acid), and the flow rate is 0.8 mL / min;

[0051] The gradient elution program is shown in Table 1:

[0052] Table 1 Gradient elution program table

[0053]

[0054] In the following examples, the homoserine dehydrogenase (HSD) and carboxyspermidine dehydrogenase (CASDH) involved are derived from Saccharomyces cerevisiae and Agrobacterium fabrum, respectively.

[0055] It should be noted that for the sake of convenience in description, in the following examples, the strain number, plasmid number, enzyme number, and enzyme-encoding gene number can share the same number, which is easily understood by those skilled in the art, that is, the same number can refer to different biological forms in different environments.

[0056] Example 1: In vitro construction of the spermidine synthesis pathway

[0057] Based on the reference (Xinxin Liang et al. “Highly efficient biosynthesis of spermidine from L-homoserine and putrescine using an engineered Escherichia coli with NADPH self-sufficient system.” Applied Microbiology and Biotechnology (2022) 106:5479–5493), the inventors commissioned General Biology (Anhui) Co., Ltd. to synthesize plasmids pET28a-scHSD, pET28a-afCASDH, and pET28a-bcCASDC (NcoI / XhoI). The full sequences are shown in SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5. Then, the synthesized plasmids were respectively transformed into the BL21(DE3) strain.

[0058] Single colonies were separately selected from the transformation LB plates of the three enzymes and inoculated into 5 ml of LB medium, and cultured overnight at 37 °C; inoculated into a 1000 ml flask containing 100 ml of LB medium at a ratio of 1% v / v, and cultured at 37 °C and 220 rpm for 4 - 6 h. When the OD 600 value reached 0.6 - 0.8, 0.2 mM IPTG was added for induction; then the temperature was lowered to 25 °C and cultured for another 3 - 4 h, and the cells were obtained by centrifugation; the cells of the three enzymes were collected, purified and recovered, the protein concentration was measured, and the reaction was carried out according to the catalytic system shown in Table 2. Incubated at 30 °C and 220 rpm for 30 min, catalyzed at 220 rpm, sampled at 6 h and 12 h respectively, heated at 95 °C for 3 min to end the reaction, and the catalytic samples were derivatized and then sent for detection. The detection results are shown in Table 3.

[0059] Table 2 Catalytic system

[0060] 500ul System System 1 System 2 System 3 System 4 L-Homoserine 50mM 50mM 50mM 50mM Putrescine 50mM 50mM 50mM 50mM NaDP+ 0.5mM 0.5mM 0.5mM 0.5mM PLP 0.5mM 0.5mM 0.5mM 0.5mM scHSD 1mg / mL 2mg / mL 1mg / mL 1mg / mL afCASDH 1mg / mL 1mg / mL 2mg / mL 1mg / mL bcCASDC 1mg / mL 1mg / mL 1mg / mL 2mg / mL PBS(7.5) 50mM 50mM 50mM 50mM

[0061] Table 3 Detection results

[0062] Reaction System Spermidine g / L Yield % System 1 2.3 31.7％ System 2 3.5 48.2％ System 3 1.9 26.2％ System 4 5.1 70.2％

[0063] From the test results, the spermidine yield and substrate conversion rate in System 4 were the highest. Therefore, it was speculated that CASDC was the rate-limiting enzyme, and then gene mining was carried out on this enzyme.

[0064] Example 2: Gene mining and activity identification of the rate-limiting enzyme CASDC

[0065] Using the CASDC sequence from Butyrivibrio fibrisolvens as a template, perform a BLAST search in Uniprot to screen for sequences with over 60% homology. Then, focus on screening for CASDC enzymes from thermophilic bacteria and archaea, and perform gene synthesis. The screened and synthesized CASDC enzymes are shown in Table 4.

[0066] Table 4 Screened and Synthesized CASDC Enzymes

[0067] Number Uniprot No. Strain CASDC1 A0A174REK2 Bacteroides thetaiotaomicron CASDC2 D4S136 Butyrivibrio crossotus CASDC3 A0A497H0S4 Thermoplasmata archaeon CASDC4 A0A1B1YEA2 Thermoclostridium stercorarium CASDC5 A0A1I1ZUR0 Thermophagus xiamenensis CASDC6 A0A6I6DZ58 Thermochromatium tepidum

[0068] Purify the enzyme after gene synthesis and detect its protein expression and catalytic ability (react according to System 4 in Example 1). The results are shown in Table 5.

[0069] Table 5 Detection Results of the Catalytic Ability of CASDC Enzymes

[0070] Number Purified Protein Amount mg / ml Spermidine Yield g / L (24h) CASDC1 38 2.81 CASDC2 5 0.98 CASDC3 41 2.81 CASDC4 41 2.30 CASDC5 3 0.66 CASDC6 8 6.89

[0071] From the catalytic results, Thermochromatium tepidum has a relatively high catalytic activity, and its amino acid sequence is shown in SEQ ID NO:1. However, from the perspective of the purified protein amount, its protein concentration is relatively low.

[0072] Example 3: Optimization of the Catalytic System

[0073] For industrial production, whole-cell catalysis is used (on the basis of Example 1, configure wet cell bodies in different ratios for catalytic reactions, only adjust the CASDC cell body and addition ratio), and at the same time, the substrate concentration is optimized. Set two substrate concentrations of 50 mM and 100 mM, the NADP+ concentration is 0.5 mM, the PLP concentration is 0.5 mM, a 50 mM PBS buffer system (pH 7.5), react for 18 h, and take samples for HPLC detection. The results are shown in Table 6.

[0074] Table 6 Catalytic Results under Different Conditions

[0075]

[0076]

[0077] From the above data, it can be seen that under the condition that the concentrations of the substrates homoserine and putrescine are 100 mM, when the specific ratio is 1:1:5 (CASDC6), the product concentration of spermidine is the highest, which can reach 13 g / L. The spermidine yield reaches 89.5%.

[0078] Example 4: Optimization of the Amino Acid Codons of CASDC6

[0079] As can be seen from Examples 2 and 3, although CASDC6 can increase the product concentration of spermidine, its protein expression level is low and becomes the rate-limiting step. Therefore, we optimized some amino acid codons in CASDC6 in order to increase its protein expression level and further increase the spermidine product concentration.

[0080] In this example, we chose to mutate the codons of some arginine, leucine, and isoleucine into rare codons. Specifically, we chose to introduce mutations at positions 6L, 12L, 13I, 17R, 51L, 108L, and 122L (commissioned GenScript Biotech for site-directed mutagenesis).

[0081] According to the method described in Example 1, the plasmid containing the site-directed mutagenized CASDC6 sequence was transferred into BL21(DE3) strain, induced for expression with IPTG, the protein was purified after cell disruption, and then the protein was quantified. The results are shown in Table 7.

[0082] Table 7 Quantitative detection results of CASDC6 protein concentration after different point mutations

[0083] Strain Purified Protein Concentration mg / ml Purified Volume ml Total Protein Amount mg CASDC6 8.3 4.2 34.9 CASDC6-M6 10.7 4.5 48.2 CASDC6-M12 9.8 4.1 40.2 CASDC6-M13 13.5 4.6 62.1 CASDC6-M17 15.4 4.2 64.7 CASDC6-M51 21.9 4.5 98.6 CASDC6-M108 9.7 4.3 41.7 CASDC6-M122 8.5 4.8 40.8

[0084] It can be seen from the experimental results that mutating the 51st amino acid into a rare codon can effectively improve the soluble expression of the protein. Therefore, a higher spermidine product concentration can be obtained with a reduced amount of its use. The DNA sequence of this mutant is shown in SEQ ID NO.2.

[0085] Example 5: Fermentation verification of CASDC6 mutant 51L

[0086] The above-mentioned mutant CASDC6-M51 strain was used to prepare the enzyme protein. In a 50 mM PBS buffer system (pH 7.5) containing 100 mM L-homoserine, 100 mM putrescine, 0.5 mM NADP+, and 0.5 mM PLP, and the ratio of the three kinds of bacteria was 1:1:3 (HSD:CASDH:CASDC), reacted at 30 °C for 18 h, sampled for HPLC detection, the spermidine concentration could reach 13.5 g / L, and the yield could reach 92.9%.

[0087] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention should be covered within the protection scope of the present invention.

Claims

1. A carboxy-spermidine decarboxylase, characterized in that, The amino acid sequence of the carboxy-spermidine decarboxylase is shown in SEQ ID NO.

1.

2. A gene encoding the carboxy-spermidine decarboxylase according to claim 1.

3. The gene according to claim 2, wherein Its nucleotide sequence is shown in SEQ ID NO.

2.

4. An expression vector containing the gene according to claim 2 or 3, preferably, the vector is a PET series vector.

5. A host cell containing the carboxy-spermidine decarboxylase according to claim 1, preferably, the host cell is selected from Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae, Yarrowia lipolytica or Bacillus subtilis.

6. Use of the carboxy-spermidine decarboxylase according to claim 1 or the host cell according to claim 5 in the enzymatic synthesis of spermidine.

7. A method for preparing spermidine, characterized in that, The method includes the step of one-pot synthesizing spermidine using homoserine and putrescine as substrates, and using homoserine dehydrogenase, carboxy-spermidine dehydrogenase and carboxy-spermidine decarboxylase, and the nucleotide sequence of the carboxy-spermidine decarboxylase is shown in SEQ ID NO.

2.

8. The preparation method according to claim 7, characterized in that, The concentrations of the substrates homoserine and putrescine are both 100 mM.

9. The preparation method according to claim 7, characterized in that, The expression strains of homoserine dehydrogenase, carboxy-spermidine dehydrogenase and carboxy-spermidine decarboxylase are added to the substrates homoserine and putrescine, and the wet cell weights of the three expression strains are in a ratio of 1:1:3 - 1:1:

5.

10. The preparation method according to claim 9, wherein The construction method of the three expression strains is: first construct plasmids of homoserine dehydrogenase, carboxy-spermidine dehydrogenase and carboxy-spermidine decarboxylase respectively, and then transform them into the BL21(DE3) strain; the three constructed plasmids are pET28a-scHSD, pET28a-afCASDH and pET28a-bcCASDC, and the corresponding nucleotide sequences are SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5 respectively.

Images