New method for synthesizing vidarabine by enzyme method

Abstract

The invention discloses a novel method for synthesizing vidarabine by an enzyme method. The method comprises the following steps: connecting three genes for coding ribokinase, phosphopentose mutase and purine nucleoside phosphorylase in escherichia coli to a pET28a plasmid, and then transferring into host escherichia coli in which adenine is knocked out and converted into hypoxanthine; the obtained recombinant strain can be used for efficiently synthesizing vidarabine by directly utilizing adenine, D-arabinose and ATP (adenosine triphosphate) as substrates. Compared with the existing enzymatic conversion method, the method disclosed by the invention has the advantage that the production cost is obviously reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of biosynthesis, and in particular relates to a new method for synthesizing vidarabine by recombinant Escherichia coli. Background Art

[0002] Adenine arabinoside (Ara-A) is a nucleoside compound with important clinical applications. Current industrial production of Ara-A primarily relies on traditional chemical synthesis processes, which not only involve the use of large amounts of organic solvents but also carry significant risks of ecological and environmental pollution. Developing green, sustainable biosynthetic industrial routes is of great practical significance. Currently, biosynthesis of Ara-A primarily relies on the enzyme catalysis of uridine phosphorylase and purine nucleoside phosphorylase, using adenine (A) and arabinuridine (Ara-U) as substrates. Although the enzyme catalysis technology for this cascade reaction is nearly mature, Ara-U is relatively expensive in the synthesis of Ara-A.

[0003] Based on this, the present invention has developed a new enzymatic production process for Ara-A, which directly uses inexpensive D-arabinose and adenine as substrates and synthesizes Ara-A by adding ATP. Compared with existing processes, this process can significantly reduce the production cost of Ara-A. Summary of the Invention

[0004] Escherichia coli naturally contains ribokinase (RBKS, encoded by the gene rbsk), pentose phosphate mutase (PPM, encoded by the gene deoB), and purine nucleoside phosphorylase (PNP, encoded by the gene pnp). Theoretically, these three enzymes can catalyze the Figure 1 The reaction uses D-arabinose and adenine as substrates and synthesizes Ara-A through an enzymatic reaction by adding ATP.

[0005] However, native E. coli expresses these enzymes at low levels, and native E. coli contains other pathways for adenine metabolism, preventing the aforementioned reactions. The present invention aims to efficiently express the genes encoding these enzymes in E. coli and to knock out the pathway that converts adenine to hypoxanthine in E. coli, thereby constructing recombinant bacteria capable of directly synthesizing Ara-A using D-arabinose and adenine as substrates, achieving efficient biosynthesis of Ara-A. Compared to existing enzymatic conversion processes, the present method significantly reduces production costs.

[0006] The genes encoding related enzymes are: rbsk (NCBI No. ECK3746, encoding ribokinase), deoB (NCBI No. ECK4375, encoding pentose phosphate mutase), and pnp (NCBI No. ECK4376, encoding purine nucleoside phosphorylase).

[0007] The highly expressed three genes in E. coli refers to inserting the three genes rbsk, deoB and pnp into the multiple cloning site of the plasmid vector pET28a and introducing them into the host E. coli in which the adenine to hypoxanthine conversion pathway has been knocked out for high expression.

[0008] The host Escherichia coli for knocking out the pathway for converting adenine to hypoxanthine refers to an Escherichia coli strain in which the adeD gene (NCBI No. ECK3656) of Escherichia coli BL21 (DE3) is knocked out. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 The biosynthesis reaction equation of Ara-A is DETAILED DESCRIPTION

[0010] The present invention will be further described in detail below with reference to specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0011] In this example, the LB medium formula is as follows: 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L sodium chloride. 1.5% agar powder is additionally added to the solid medium.

[0012] In the example, the formula of the composite culture medium is as follows: 3 g / L tryptone, 6 g / L yeast extract, 15 g / L crude glycerol, 2.3 g / L potassium dihydrogen phosphate, and 16.4 g / L potassium dihydrogen phosphate trihydrate.

[0013] Example 1: The recombinant Escherichia coli strain containing the overexpressed single gene cannot directly synthesize Ara-A using D-arabinose.

[0014] 1. Construction of single gene vector plasmid

[0015] Take the construction of the rbsk-containing vector plasmid pET-rbsk as an example. Using the E. coli BL21 (DE3) strain as a template, upstream primers (5'-pET28a homologous sequence required for seamless cloning + rbsk upstream gene sequence-3') and downstream primers (5'-pET28a homologous sequence required for seamless cloning + EcoRI and XhoⅠ restriction sites + rbsk downstream gene sequence-3') were used to amplify the gene fragment containing rbsk. The pET28a plasmid was linearized using the EcoRI and XhoⅠ restriction endonucleases, and then seamlessly cloned to construct the plasmid pET-rbsk, abbreviated as pET-R. The plasmids pET-deoB and pET-pnp, abbreviated as pET-D and pET-P, were constructed using the same method.

[0016] 2. Construction of single gene recombinant strains

[0017] The plasmids pET-R, pET-D and pET-P were respectively introduced into the host strain BL21(DE3) by chemical transformation to obtain the recombinant strains pET-R / BL21(DE3), pET-D / BL21(DE3) and pET-P / BL21(DE3).

[0018] The cell culture process is as follows: 1% of the strain was inoculated from the stored glycerol tube into a test tube containing LB liquid medium and activated at 37°C for 12 hours. Then, 1% of the activated bacterial solution was inoculated into a shake flask containing a complex liquid medium and cultured at 37°C with shaking until the OD 600 Between 0.6-1.0, add inducer IPTG with a final concentration of 0.05mmol / L and induce at 37℃ for 4.5h.

[0019] Whole-cell catalytic process. Specifically as follows: After the induction of the recombinant strain is completed, use a high-speed centrifuge to harvest the bacteria by centrifugation at 6500rpm for 10 minutes at 4°C. Weigh a certain proportion of the bacteria and add 20mmol / L Tris-HCl buffer, 2mmol / L D-arabinose, 2mmol / L adenine and 2mmol / L ATP. The reaction temperature is 37°C and the reaction time is 24h. After the reaction is completed, samples are taken for high-performance liquid chromatography detection, and the molar conversion rate of Ara-A is calculated. Molar conversion rate of Ara-A (%) = (number of moles of product Ara-A / initial number of moles of substrate adenine) × 100.

[0020] Table 1 shows the whole-cell catalytic results of three recombinant strains containing single gene overexpression: pET-R / BL21(DE3), pET-D / BL21(DE3), and pET-P / BL21(DE3). The results indicate that overexpressing only rbsk, deoB, or pnp alone does not result in Ara-A synthesis.

[0021] Table 1 Whole-cell catalytic results of single gene overexpression recombinant strains

[0022]

[0023] Example 2: The recombinant Escherichia coli strain containing the double gene overexpression was also unable to utilize D-arabinose to directly synthesize Ara-A.

[0024] For example, the construction of the dual-gene rbsk and deoB plasmid vector pET-rbsk-deoB was performed. Using the E. coli iBL21(DE3) strain as a template, upstream primers (5'-pET-R homologous sequence required for seamless cloning + deoB upstream gene sequence-3') and downstream primers (5'-pET-R homologous sequence required for seamless cloning + EcoRI and XhoI restriction sites + deoB downstream gene sequence-3') were used to amplify the deoB gene fragment. The pET-R plasmid was linearized using the EcoRI and XhoI restriction enzymes, and then seamlessly cloned to construct the plasmid pET-rbsk-deoB, abbreviated as pET-RD. The plasmids pET-rbsk-pnp and pET-deoB-pnp, abbreviated as pET-RP and pET-DP, were constructed using the same method. The plasmid was then introduced into the recombinant strains pET-RD / BL21(DE3), pET-RP / BL21(DE3) and pET-DP / BL21(DE3), and cell culture was performed according to the method described in Example 1 to prepare whole-cell catalysts. A certain proportion of the cells were weighed to test the whole-cell catalytic ability of the recombinant strains.

[0025] Table 2 shows the whole-cell catalytic results of three recombinant strains containing dual-gene overexpression: pET-RD / BL21(DE3), pET-RP / BL21(DE3), and pET-DP / BL21(DE3). The results indicate that overexpressing only two genes in this cascade reaction does not result in Ara-A synthesis.

[0026] Table 2 Whole-cell catalytic results of the double-gene overexpression recombinant strain

[0027]

[0028] Example 3: The recombinant Escherichia coli strain containing the three genes overexpressed can directly synthesize Ara-A using D-arabinose, but the efficiency is not high.

[0029] Construction of the three gene rbsk, deoB and pnp plasmid vector pET-rbsk-deoB-pnp. Using the E. col iBL21 (DE3) strain as a template, the pnp fragment was amplified using upstream primers (5'-pET-RD homologous sequence required for seamless cloning + pnp upstream gene sequence-3') and downstream primers (5'-homologous sequence required for seamless cloning pET-RD + EcoRⅠ and XhoⅠ restriction sites + pnp downstream gene sequence-3'). The linearized plasmid is pET-RD, and the target plasmid pET-rbsk-deoB-pnp, abbreviated as pET-RDP, is constructed by seamless cloning. Then, the recombinant strain pET-RDP / BL21 (DE3) was constructed, and cell culture was carried out to prepare the whole-cell catalyst according to the method described in Example 1. A certain proportion of the bacteria was weighed to test the whole-cell catalytic ability of the recombinant strain.

[0030] Table 3 shows the whole-cell catalytic results of the recombinant strain pET-RDP / BL21(DE3). Overexpression of the rbsk, deoB, and pnp genes demonstrated the ability to synthesize Ara-A. As the reaction progressed, the catalytic activity of this recombinant strain initially increased and then plateaued. After 12 hours, the conversion rate reached 50.8%, but further increases in reaction time did not improve the conversion rate.

[0031] The recombinant Escherichia coli constructed by overexpressing the three genes can directly synthesize Ara-A using D-arabinose and adenine, but the efficiency is not high.

[0032] Table 3 pET-RDP / BL21 (DE3) whole cell catalysis results

[0033]

[0034] Example 4: The recombinant strain with the synthetic pathway of the byproduct hypoxanthine knocked out can directly use D-arabinose to efficiently synthesize Ara-A

[0035] In Case 3, three genes involved in synthesizing Ara-A were overexpressed in the BL21(DE3) strain, demonstrating that the cascade reaction could catalyze the synthesis of Ara-A from D-arabinose, but the conversion rate was 50.8%. Extensive research in our laboratory revealed the presence of adenine deaminase (AAH, encoded by the adeD gene) in Escherichia coli, which metabolizes adenine to hypoxanthine, resulting in adenine not being fully utilized for Ara-A synthesis. Therefore, Red homologous recombination was used to knock out the adeD gene (NCBI No. ECK3656) in the BL21(DE3) strain, thereby eliminating the pathway for adenine conversion to hypoxanthine. The recombinant strain, pET-RDP / BL21(DE3)ΔadeD, was then introduced into the plasmid pET-RDP.

[0036] The construction of BL21(DE3)△adeD utilizes Red homologous recombination technology. Specifically, overlapping PCR was used to construct a targeting fragment containing upstream and downstream homologous arms of the adeD gene and KN resistance (derived from the pKD4 plasmid). The constructed targeting fragment was introduced into the BL21(DE3) genome to achieve exchange and knockout of the target gene. To improve the efficiency of homologous recombination, the pKD46 plasmid carrying the recombinase was first introduced into BL21(DE3) by electroporation. The targeting fragment was then electroporated on this basis. After successful introduction of the targeting fragment, electroporation was used again to introduce the thermosensitive plasmid pCP20, using FLP recombinase to remove KN resistance from the targeting fragment, thereby achieving a seamless knockout and obtaining the host Escherichia coli BL21(DE3)△adeD that knocks out the adenine to hypoxanthine pathway. Furthermore, the plasmid pET-RDP was introduced on this basis to obtain the recombinant strain pET-RDP / BL21(DE3)△adeD.

[0037] Cell culture was performed according to the method described in Example 1 to prepare whole-cell catalysts, and a certain proportion of cells were weighed to test the whole-cell catalytic ability of the recombinant strain pET-RDP / BL21(DE3)ΔadeD.

[0038] Table 4 shows the whole-cell catalytic results of the recombinant strain pET-RDP / BL21(DE3)ΔadeD. The results demonstrate that whole-cell catalysis using pET-RDP / BL21(DE3)ΔadeD efficiently synthesizes Ara-A from D-arabinose and adenine, achieving a 100% molar conversion within 12 hours.

[0039] Table 4 pET-RDP / BL21(DE3) ΔadeD whole cell catalysis results

[0040]

Claims

1. A novel method for enzymatically synthesizing vidarabine, characterized in that: The three genes encoding ribokinase, pentose phosphate mutase, and purine nucleoside phosphorylase in Escherichia coli were connected to the pET28a plasmid and then transferred into the host Escherichia coli in which the adenine to hypoxanthine conversion pathway was knocked out. The resulting recombinant strain was able to directly use adenine, D-arabinose, and ATP as substrates to efficiently synthesize adenosine.

2. The method according to claim 1, characterized in that The three genes are: rbsk (NCBI No. ECK3746, encoding ribokinase), deoB (NCBI No. ECK4375, encoding pentose phosphate mutase), and pnp (NCBI No. ECK4376, encoding purine nucleoside phosphorylase).

3. The method according to claim 1, characterized in that The host Escherichia coli in which the pathway of adenine conversion to hypoxanthine is knocked out refers to an Escherichia coli strain in which the gene adeD (NCBI No. ECK3656) in the genome of Escherichia coli BL21 (DE3) is knocked out.

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