Multi-enzyme complex of S-adenosylmethionine decarboxylase / spermine synthetase and application of multi-enzyme complex in spermine production

Abstract

The invention discloses a multi-enzyme complex of S-adenosylmethionine decarboxylase / spermine synthetase and an application of the multi-enzyme complex in production of spermine. Belongs to the technical field of enzyme engineering. The purpose of the present invention is to increase the yield of spermine. The invention provides a multi-enzyme complex of S-adenosylmethionine decarboxylase / spermine synthetase. The multi-enzyme complex is obtained by connecting the S-adenosylmethionine decarboxylase and the spermine synthetase through a flexible linker. And a theoretical basis is provided for preparation of spermine.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering technology, specifically relating to a multi-enzyme complex of S-adenosylmethionine decarboxylase / spermine synthase and its application in the production of spermine. Background Technology

[0002] Spermine is a tetraamine molecule that plays a variety of key biological roles in organisms, including regulating ion channels, resisting oxidative stress, maintaining polyamine homeostasis, and participating in the immune and reproductive development systems. In recent years, spermine has gradually attracted attention due to its potential applications in medicine, nutrition and health, agriculture, and chemical industries. However, while the chemical synthesis methods for spermine are mature, they are costly, have low yields, and cause environmental pollution. Although the latest patent (CN116162029B) increases the overall yield of spermine to 62%, it still requires high-pressure hydrogenation, precious metal catalysts, and multi-step purification, resulting in high energy consumption and significant waste. Furthermore, the content of spermine in organisms is generally extremely low, and natural extraction is insufficient to meet industrial-scale demands. Therefore, utilizing synthetic biology and metabolic engineering to construct microbial cell factories to achieve green, efficient, and sustainable production of spermine is a highly promising alternative.

[0003] Currently, there are no publicly reported reports of spermine biosynthesis reaching industrial-scale or high-titer levels. Spermine biosynthesis relies on two cascade pathways: putrescine → spermine → spermine, with both stages sharing and competitively utilizing the aminopropyl donor dcAdoMet (decarboxylated S-adenosylmethionine). This metabolic conflict leads to an imbalance in the synthetic pathway, with a "strong upstream, weak downstream" dynamic. Even if the synthesis flux of the upstream precursor (such as spermine) is high, the synthesis flux of the downstream spermine cannot be effectively increased. Achieving a rational distribution of the metabolic flux of the common dcAdoMet precursor in this continuous cascade reaction is key to improving spermine yield. Summary of the Invention

[0004] The purpose of this invention is to increase the yield of spermine.

[0005] This invention provides a multi-enzyme complex of S-adenosylmethionine decarboxylase and spermine synthase, wherein the multi-enzyme complex is obtained by linking S-adenosylmethionine decarboxylase and spermine synthase through a flexible linker.

[0006] To further specify, the flexible linker is GGGS.

[0007] Further specifying, the amino acid sequence of S-adenosylmethionine decarboxylase is shown in SEQ ID NO.6; the amino acid sequence of spermine synthase is shown in SEQ ID NO.8.

[0008] The present invention provides a nucleotide sequence encoding the above-mentioned S-adenosylmethionine decarboxylase / spermine synthase multienzyme complex.

[0009] The present invention provides a recombinant vector containing the above-described nucleotide sequence.

[0010] The present invention provides a recombinant microbial cell containing the above-mentioned nucleotide sequence.

[0011] The present invention provides the application of the above-mentioned S-adenosylmethionine decarboxylase / spermine synthase multienzyme complex, the above-mentioned nucleotide sequence, the above-mentioned recombinant vector, or the above-mentioned recombinant microbial cells in increasing the yield of spermine and spermidine.

[0012] This invention provides a method for increasing the yield of spermine and spermidine by overexpressing the coding gene of the above-mentioned multi-enzyme complex in yeast and fermenting at 30 °C for 120 days.

[0013] Further specifying the components of the fermentation medium, the following are provided: (NH4)2SO4, KH2PO4, MgSO4•7H2O, pH adjusted to 5.8, trace metals, vitamins, glucose and uracil.

[0014] This invention provides a method for preparing a multi-enzyme complex of S-adenosylmethionine decarboxylase / spermamine synthase. The specific steps of the preparation method are as follows: the TEF1p, SPE2-GGGS-SPE4 and PRM9t genes are sequentially linked to obtain an expression cassette, which is then transformed into yeast for expression to obtain the multi-enzyme complex of S-adenosylmethionine decarboxylase / spermamine synthase; the TEF1p gene is shown in SEQ ID NO.1, the SPE2-GGGS-SPE4 gene is shown in SEQ ID NO.11 and the PRM9t gene is shown in SEQ ID NO.3.

[0015] Beneficial Effects: This invention addresses the bottleneck of unbalanced precursor metabolic flux distribution in spermine synthesis by proposing an innovative enzyme fusion regulation strategy. By rationally fusing SPE2 (S-adenosylmethionine decarboxylase) and SPE4 (spermine synthase), a highly efficient "substrate channel" is formed within the cell, promoting the directed transfer and utilization of the precursor dSAM in the continuous reaction, reducing the diffusion loss of intermediate products and the occurrence of side reactions. This achieves the redistribution and enhancement of key metabolic fluxes, providing a new pathway for the efficient microbial synthesis of spermine. The principle is as follows: Figure 1 As shown. Attached Figure Description

[0016] Figure 1 This is a diagram illustrating the complete synthetic metabolic pathway of spermine in the engineered Saccharomyces cerevisiae in this embodiment of the invention.

[0017] Figure 2This is a schematic diagram of substrate channel construction in an embodiment of the present invention; a is the construction of the fusion protein; b is the construction of the protein scaffold.

[0018] Figure 3 This is a schematic diagram illustrating the spermine enhancement factor in an embodiment of the present invention.

[0019] Figure 4 This is a graph showing the colony PCR results.

[0020] Figure 5 This is a graph showing the colony PCR results. Detailed Implementation

[0021] Delft medium: (NH4)2SO4, 7.5 g; KH2PO4, 14.4 g; MgSO4•7H2O, 0.50 g; trace metals, 1 ml; vitamins, 1 ml; 20 g / L glucose; 40 mg / L uracil, and sterilized at 121 °C for 20 min after adjusting the pH to 5.8 with 6 M KOH.

[0022] The trace metal solution consisted of the following (per liter): Na₂EDTA• 5H₂O, 19.0 g; ZnSO₄• 7H₂O, 0.45 g; MnCl₂• 4H₂O, 1 g; CoCl₂• 6H₂O, 0.3 g; CuSO₄• 5H₂O, 0.3 g; Na₂MoO₄• 2H₂O, 0.4 g; CaCl₂• 2H₂O, 0.45 g; FeSO₄• 7H₂O, 0.3 g; H₃BO₃, 1 g; and KI, 0.10 g. The pH of the trace metal solution was adjusted to 4.0 using 2 M NaOH. The trace metal solution was filtered, sterilized, and stored at 4 °C.

[0023] The vitamin solution contains (per liter): d-biotin, 0.05 g; para-aminobenzoic acid, 0.2 g; niacin, 1 g; calcium pantothenate, 1 g; pyridoxine hydrochloride, 1 g; thiamine hydrochloride, 1 g; and inositol, 25 g. Adjust the pH of the vitamin solution to 6.5 with 2 M NaOH. Filter and sterilize the vitamin solution and store at 4 °C. Example 1. Construction of a multi-enzyme complex of S-adenosylmethionine decarboxylase / spermine synthase Step 1: The principle of enzyme fusion is to link DNA sequences encoding two or more different functional proteins together using linker peptides through genetic engineering technology, thereby expressing a single multifunctional recombinant protein (i.e., a multienzyme complex). Using amplification primers, the SPE2 (S-adenosylmethionine decarboxylase EC 4.1.1.50) and SPE4 (seminate synthase EC 2.5.1.22) genes, along with 500 bp homologous sequences upstream and downstream of the integration site on chromosome XII-5, were cloned from the genome of *Saccharomyces cerevisiae* (CEN.PK113-11C). The regulatory elements, promoters TEF1p and PGK1p, and terminators PRM9t and CYC1t, are also included. These regulatory elements drive transcription initiation (promoter) and termination (terminator) of the genes, respectively, and are key genetic components for achieving efficient and stable expression of the target genes SPE2 and SPE4 in yeast. To ensure stable inheritance of the expression elements, chromosome integration was performed using the CRISPR / Cas9 gene editing system. The principle is as follows: A specific sgRNA is designed to guide the Cas9 nuclease to generate a double-strand break at the pre-defined XII-5 site in the genome. The Saccharomyces cerevisiae cell utilizes its own high-fidelity homologous recombination repair mechanism to precisely integrate the exogenous expression cassette, with homologous arms (500 bp upstream and downstream in this example) at both ends to the break site, into the specific site. The nucleotide and protein sequences of the aforementioned genes, homologous sequences, and regulatory elements are shown in Table 1.

[0024] Table 1. Nucleotide and protein sequences

[0025] Step 2: Assemble the above genes and regulatory elements according to the preset combination, and amplify them using overlap extension PCR (a polymerase chain reaction technique that uses DNA fragments with complementary ends to achieve precise splicing by using these fragments as primers and templates). Expression cassette ①PGK1p-SPE21-CYC1t-TEF1p-SPE41-PRM9t (abbreviated as SPE2+SPE4): In this expression cassette, SPE21 is driven by the PGK1p promoter and controlled by the CYC1t terminator, while SPE41 is driven by the TEF1p promoter and controlled by the PRM9t terminator, allowing the two genes to be expressed independently. This serves as a control group to evaluate the effect of independent expression of the two genes on product synthesis. The experimental group evaluated the effect of two gene fusion expression on product synthesis. Considering the linkage direction of SPE2 and SPE4, two different multi-enzyme complexes were designed, resulting in expression cassette ② TEF1p-SPE22-GGGS-SPE42-PRM9t (abbreviated as SPE2-SPE4): This expression cassette utilizes the promoter TEF1p to drive the fusion expression of SPE22 and SPE42 through a flexible linker peptide (GGGS), with PRM9t as the terminator, aiming to achieve co-expression of the two enzymes on a single polypeptide chain; Expression cassette ③ TEF1p-SPE43-GGGS-SPE23-PRM9t (abbreviated as SPE4-SPE2): Similar in structure to expression cassette ②, but SPE43 is located at the N-terminus of the fusion protein, and SPE23 is located at the C-terminus, used to study the effect of enzyme sequence on catalytic efficiency. In the above expression cassettes, SPE2 and SPE4 are the same gene, but the primers are designed with different homologous arms, distinguished by different subscripts. The primer sequence information involved is shown in Table 2. See the two connection methods for fusion expression. Figure 2 'a' in 'a'.

[0026] Table 2. Primer sequences

[0027] Step 3: Gene cassettes ①, ②, and ③ obtained in Step 1 were transformed into CEN.PK113-11C-JQ43 (CEN.PK113-11C-JQ43, URA-deficient, containing a plasmid carrying an HIS selection tag, derived from Qin, J., et al., Engineering yeast metabolism for the discovery and production of polyamines and polyamine analogues. Nature Catalysis, 2021.4(6): p. 498-509.) using plasmids carrying sgRNA (PMEL10, carrying a URA selection tag, Robert Mans., et al., CRISPR / Cas9: a molecular Swiss army knife for simultaneous introduction of multiple genetic modifications in Saccharomyces cerevisiae. FEMS Yeast Research, 2015. 15, 2015, fov004.) with plasmids carrying sgRNA. Only strains successfully transformed with sgRNA could grow on SC-URA plates. The strain itself contains a plasmid carrying the HIS tag, so it was selectively cultured on SC-URA-HIS plates according to the lithium acetate transformation method, transforming ①, ②, and ③ respectively (transformation results are shown in...). Figure 4 Eight single colonies were randomly selected and colony PCR was performed using the primers in Table 2 for JP-F and JP-R to verify successful integration of the expression cassette on the chromosome. The correct bands should be 4899bp (SPE2+SPE4), 3883bp (SPE2-SPE4), and 3883bp (SPE4-SPE2) (see colony PCR results for details). Figure 4 Selected single clones were sequenced to be free of mutations. After chromosome integration, to reduce the growth stress on the strains, the introduced sgRNA plasmid (containing the URA selection tag) needed to be further removed. The correct single clones were inoculated into 1 ml of Delft medium containing uracil and cultured for 12 h. They were then plated on plates containing 5-FOA + uracil. Only cells that had lost the sgRNA plasmid, lacking the URA3 gene, could not metabolize 5-fluoroorotic acid (5-FOA) in the medium into toxic 5-fluorouracil, and could utilize exogenous uracil for growth, thus surviving on the plates. Finally, engineered bacteria SPM1, SPM2, and SPM3, expressing SPE2+SPE4, SPE2-SPE4, and SPE4-SPE2 respectively, were obtained.

[0028] Step 4: The SPM1, SPM2, and SPM3 engineered bacteria obtained in Step 3 were cultured in deep-well plates, and the spermine content was determined by high-performance liquid chromatography (HPLC). First, a single colony was inoculated into 2 ml of Delft medium and cultured at 30 ℃ and 250 rpm for 24 h. Then, the initial OD was determined. 600 =0.05% inoculation was used to ferment cells in deep-well plates with 2 ml of culture medium at 30 °C and 250 rpm for 120 h (5 days).

[0029] Prepare a fermentation sample by taking 0.05 ml of the culture medium after fermentation. Preheat a test tube containing 0.95 ml of Delft medium in a 100 °C metal bath for 10 min. Then, add 0.05 ml of the fermentation sample, vortex to mix, and continue heating in the 100 °C metal bath for 30 min. Place the test tube on ice for 5 min. Centrifuge at 8000 rpm for 3 min, and use the supernatant directly for derivatization. For derivatization, the procedure is adapted from [Qin, J., et al., Engineering yeast metabolism for the discovery and production of polyamines and polyamine analogues. [NatureCatalysis, 2021.4(6): p. 498-509.] 0.25 ml of saturated NaHCO3 solution and 0.5 ml of dansyl chloride solution (5 mg / ml dissolved in acetone) were added to 0.25 ml of the sample. The reaction mixture was then incubated in the dark at 40 °C for 1 h with occasional shaking. The reaction was terminated by adding 0.275 ml of methanol. Spermine standard (>97%) was dissolved in 0.1 M HCl and serially diluted with Delft medium to 0.5, 1, 10, and 15 mg / L for use. Similarly, 0.25 ml of the standard solution was used to derivatize the sample. Both the standard and the sample were filtered through 0.22 μm nylon filters after derivatization for HPLC detection. The chromatographic conditions and elution procedures are shown in Tables 3 and 4. Table 3. Chromatographic conditions

[0030] Table 4 Elution Procedure

[0031] The results are as follows Figure 3 The results showed that, compared with the control strain (SPM1) co-expressing SPE2 and SPE4, the SPE2-SPE4 fusion protein strain (SPM2) increased the yield by 1.52-fold; while the SPE4-SPE2 fusion protein strain (SPM3) did not show an increase in yield. This indicates that the fusion protein successfully constructed an efficient substrate channel, but its function depends on the specific fusion orientation of SPE2-SPE4.

[0032] Comparative Example 1. Construction of a multi-enzyme complex of S-adenosylmethionine decarboxylase / spermine synthase using a protein scaffold. Step 1: The protein scaffold consists of two domains, PDZ and SH3, with corresponding ligands PDZlig and SH3lig, respectively. These two ligands can spontaneously and specifically bind to the corresponding domains of the protein scaffold, thereby forming a stable complex structure. Based on these characteristics, we fused the target enzymes SPE2 and SPE4 with PDZlig and SH3lig, respectively, using flexible linker peptides (GGGS). After fusion, the ligands can guide the target enzymes to specifically assemble with the protein scaffold, thereby forming a multi-enzyme complex. Using amplification primers, the genes for SPE2 (S-adenosylmethionine decarboxylase EC 4.1.1.50) and SPE4 (semamine synthase EC 2.5.1.22) were cloned from the genome of *Saccharomyces cerevisiae* (CEN.PK113-11C), along with the regulatory elements TEF1p and PGK1p promoters, and PRM9t and CYC1t terminators. The nucleotide and protein sequences of the above-mentioned regulatory elements and genes are the same as those shown in Table 1 of Example 1. The nucleotide and protein sequences of the newly added protein scaffold are shown in Table 5. Table 5. Nucleotide and protein sequences

[0033] Step 2: To assess the potential impact of fusion direction on assembly performance, the above-mentioned genes and regulatory elements were assembled according to a preset combination. Overlap extension PCR amplification was performed (same as in Example 1), and four complete expression cassettes were designed and constructed: PGK1p-PDZlig-GGGS-SPE24-CYC1t-TEF1p-SPE44-GGGS-SH3lig-PRM9t (expression cassette ④), PGK1p-PDZlig-GGGS-SPE25-CYC1t-TEF1p-SH3lig-GGGS-SPE45-PRM9t (expression cassette ⑤), PGK1p-SPE26-GGGS-PDZlig-CYC1t-TEF1p-SPE46-GGGS-SH3lig-PRM9t (expression cassette ⑥), and PGK1p-SPE27-GGGS-PDZlig-CYC1t-TEF1p-SH3lig-GGGS-SPE47-PRM9t (expression cassette ⑦). SPE2 and SPE4 in the four expression cassettes are the same gene, but the primers are designed with different homologous arms and are distinguished by different subscripts. The primer sequences used are shown in Table 6. To avoid excessive modification of the genome and to facilitate parallel testing of multiple combinations of ligands and key enzymes, the p426PGD plasmid vector (URA-deficient type, derived from Qin, J., et al., Engineering yeast metabolism for the discovery and production of polyamines and polyamine analogues. Nature Catalysis, 2021.4(6): p. 498-509.) was selected to assemble the above expression cassettes. The p426PGD was amplified using primer pairs Up-F and Down-R to obtain a linear plasmid vector, which was used to provide URA screening tags. The plasmid p423PDZ-SH3 with protein scaffold was optimized with codons of Saccharomyces cerevisiae and then synthesized by Sangon Biotech (Shanghai) Co., Ltd. A schematic diagram of the assembled protein scaffold recruiting the target enzyme is shown in Table 6. Figure 2 b in the text.

[0034] Table 6 Primer sequences

[0035] Step 3: The gene cassette and linear plasmid vector obtained in Step 1 were used to construct plasmids using a seamless cloning kit. These plasmids were then transformed into *E. coli* DH5α for amplification and extraction, yielding plasmids p426GPD-SPE2-PDZlig+SPE4-SH3lig, p426GPD-SPE2-PDZlig+SH3lig-SPE4, p426GPD-PDZlig-SPE2+SPE4-SH3li, and p426GPD-PDZlig-SP. After transforming E2+SH3lig-SPE4 and p426GPD-SPE2+SPE4 into E. coli DH5α, eight single colonies were randomly selected and colony PCR was performed using the primers in Table 4 for JP-F2 and JP-R2 to verify the expression cassette and linear plasmid vector. The correct bands were 4282bp (protein scaffold ①), 4285bp (protein scaffold ②), 4285bp (protein scaffold ③), and 4288bp (protein scaffold ④) (see colony PCR results for details). Figure 5 After selecting a subset of correct single clones and verifying the absence of mutations through sequencing, the correct plasmid (URA deficient type) was transformed into *Saccharomyces cerevisiae* along with the synthetically produced plasmid p423PDZ-SH3 (HIS deficient type). Each plasmid carries a deficient selection tag; only strains successfully transformed with both plasmids simultaneously could grow on SC-URA-HIS plates. Selection culture was performed on SC-URA-HIS plates using the lithium acetate transformation method, corresponding to transformations ④, ⑤, ⑥, and ⑦ (transformation results are shown in...). Figure 5 The engineered bacteria SPM4, SPM5, SPM6, and SPM7 were obtained respectively. Step 4: The engineered bacteria obtained in step 3 were subjected to deep-well plate fermentation culture, using the same method as step 4 of Example 1. The results are as follows: Figure 3 The results showed that the spermine production of all protein scaffold combinations (SPM4 to SPM7) was lower than that of the control strain (SPM1) co-expressing SPE2 and SPE4, indicating that the protein scaffold system failed to effectively enhance spermine synthesis under the conditions of this study.

Claims

1. A multi-enzyme complex of S-adenosylmethionine decarboxylase / spermine synthase, characterized in that, The multi-enzyme complex is obtained by linking S-adenosylmethionine decarboxylase and spermine synthase through enzyme fusion.

2. The multi-enzyme complex of S-adenosylmethionine decarboxylase / spermine synthase according to claim 1, characterized in that, The enzyme fusion method involves adding a linking peptide between the two proteins.

3. The multi-enzyme complex of S-adenosylmethionine decarboxylase / spermine synthase according to claim 1, characterized in that, The enzyme fusion is performed by sequentially connecting TEF1p, SPE2, GGGS, SPE42, and PRM9t.

4. The multi-enzyme complex of S-adenosylmethionine decarboxylase / spermine synthase according to claim 1, characterized in that, The amino acid sequence of S-adenosylmethionine decarboxylase is shown in SEQ ID NO.6; the amino acid sequence of spermine synthase is shown in SEQ ID NO.

8.

5. The nucleotide sequence encoding the multienzyme complex of S-adenosylmethionine decarboxylase / spermine synthase as described in claim 1.

6. A recombinant vector containing the nucleotide sequence of claim 5.

7. A recombinant microbial cell containing the nucleotide sequence of claim 5.

8. The use of the S-adenosylmethionine decarboxylase / spermine synthase multienzyme complex according to any one of claims 1-4, the nucleotide sequence according to claim 5, the recombinant vector according to claim 6, or the recombinant microbial cell according to claim 7 in increasing the yield of spermine and spermidine.

9. A method for increasing the yield of spermine and spermidine, characterized in that, After overexpressing the encoding gene of the multi-enzyme complex according to any one of claims 1-4 in yeast, fermentation was carried out at 30 °C for 120 days.

10. A method for preparing a multi-enzyme complex of S-adenosylmethionine decarboxylase / spermine synthase, characterized in that, The specific steps of the preparation method are as follows: TEF1p, SPE2-GGGS-SPE4 and PRM9t genes are sequentially linked to obtain an expression cassette, which is then transformed into yeast for expression to obtain a multi-enzyme complex of S-adenosylmethionine decarboxylase / spermine synthase. The TEF1p gene is shown in SEQ ID NO.1, the SPE2-GGGS-SPE4 gene is shown in SEQ ID NO.11, and the PRM9t gene is shown in SEQ ID NO.3.

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