Cytochrome c mutant and its application in synthesis of organic germanium compounds

Abstract

The application belongs to the technical field of enzyme catalysis, and relates to a cytochrome C mutant and application of the cytochrome C mutant in synthesis of organic germanium compounds. The amino acid sequence of the mutant is shown as SEQ ID NO. 4. The application further provides a gene encoding the cytochrome C mutant, a recombinant plasmid containing the gene, a recombinant microorganism expressing the cytochrome C mutant, and application of the mutant and the recombinant microorganism in synthesis of chiral organic germanium compounds. The application further provides a method for asymmetrically synthesizing chiral organic germanium compounds by using the engineered cytochrome C mutant, which can catalyze construction of a carbon-germanium (C-Ge) bond between an organic germanium alkane compound and a diazo compound. The application can be efficiently performed under the isothermal conditions of normal temperature and an aqueous buffer solution by using a very low load of a biological catalyst, and has wide substrate applicability.

Description

Technical Field

[0001] This invention belongs to the field of enzyme catalysis technology and relates to a cytochrome C mutant and its application in the synthesis of organogermanium compounds. Background Technology

[0002] Germanium, along with carbon, silicon, tin, and lead, belongs to the carbon group and possesses similar physicochemical properties. In 2016, Arnold's team pioneered the use of natural heme protein to catalyze the formation of C-Si bonds via carbene transfer, thereby introducing silicon into bio-organic molecules. Germanium shares the same valence state and bonding mode as silicon but exhibits stronger metallic properties. Previous studies have shown that replacing carbon or silicon with germanium can precisely modulate the biological activity and other pharmacological properties of the resulting molecules. Therefore, organogerium compounds are increasingly becoming highly promising therapeutic agents and synthetic tools in drug discovery and molecular synthesis.

[0003] Currently, the synthesis of chiral organogermanium compounds mainly relies on transition metal catalysis. This process not only often uses halogenated solvents but also requires low-temperature environments to ensure catalytic performance. Theoretically, enzymes, with their precisely arranged active sites, inherent conformational flexibility, and highly adaptable microenvironments, may be particularly well-suited to the unique physicochemical properties of germanium. Furthermore, biocatalysis offers advantages such as mild reaction conditions, high selectivity, and environmental friendliness.

[0004] Heme proteins can catalyze a variety of non-natural carbene transfer reactions in aqueous media, including the insertion of NH, SH, BH, and Si-H. Directed evolution can significantly enhance their reactivity and catalytic selectivity. However, no naturally occurring enzymes have yet been found capable of catalyzing the formation of bonds between carbon and any metal element, making metal elements a "forbidden zone" for the biosynthesis of organometallic compounds.

[0005] In summary, chiral organogermanium compounds are an emerging and promising molecular group in the field of drug development. However, there is currently a lack of methods for preparing single chiral organogermanium compounds using biocatalysis, which greatly restricts the research, development, and application of chiral organogermanium compounds. Summary of the Invention

[0006] The purpose of this invention is to solve the problems existing in the prior art and provide a method for the asymmetric synthesis of chiral organogermanium compounds using cytochrome C mutants. The method utilizes the mechanism of heme protein catalyzing carbene insertion reaction and uses cytochrome C mutants to highly selectively catalyze the insertion of carbene into germanium hydrogen bonds to form carbon-germanium bonds, thereby producing a single chiral organogermanium compound.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is: a cytochrome C mutant, the amino acid sequence of which is shown in SEQ ID NO.4.

[0008] Furthermore, the present invention also provides a gene encoding the cytochrome C mutant, the nucleoside sequence of which is shown in SEQ ID NO.3.

[0009] Furthermore, the present invention also provides a recombinant plasmid carrying the said gene.

[0010] Furthermore, the present invention also provides a recombinant microorganism comprising the recombinant plasmid, the recombinant microorganism expressing the cytochrome C mutant.

[0011] Preferably, the recombinant microorganism uses pBBR1 plasmid as an expression vector and fluorescent Pseudomonas ATCC 17400 as an expression host.

[0012] Furthermore, the present invention also provides the application of the cytochrome C mutant or the recombinant microorganism in the synthesis of chiral organogermanium compounds.

[0013] Furthermore, the present invention also provides a method for synthesizing chiral organogermanium compounds using the cytochrome C mutant. This method uses wet bacterial cells, cell lysates, or the cytochrome C mutant protein obtained through fermentation culture of recombinant microorganisms containing the cytochrome C mutant encoding gene as a catalyst, and uses the organogermanium alkane compound shown in Formula 1 and the carbene precursor compound shown in Formula 2 as substrates. The reaction is carried out at room temperature. After the reaction is completed, a reaction solution containing the chiral organogermanium compound is obtained. The reaction solution is then separated and purified to obtain the chiral organogermanium compound shown in Formula 3. The general reaction formula is shown below: ; In Equation 1, R 1 -R 5 Each is independently selected from H, F, Cl, Me, methoxy, methylthio, or phenyl; or, R 1 -R 5 In formula 2, two adjacent groups and the carbon atom attached to them together form a naphthalene ring system; R 6 Selected from methyl or ethyl; R in Formula 2 7 Selected from ethyl, tert-butyl, or benzyl; The final substrate concentration of organogermanium compounds in the reaction system was 15-20 mM, the final substrate concentration of carbene precursor compounds was 20-30 mM, and the catalyst concentration was 0.5 μM-1.4 μM.

[0014] Furthermore, the OD of the bacterial culture in the whole-cell reaction system 600 The value is 60; the pyrolysis solution in the pyrolysis reaction system is composed of OD 600 It was obtained by thermal lysis of whole-cell bacterial culture with a concentration of 60.

[0015] Furthermore, 15%-20% v / v ethanol is added to the reaction system as a solvent for the substrate.

[0016] Furthermore, the reaction temperature was 30℃, the reaction time was 4h, and the reaction speed was 250rpm.

[0017] Compared with existing technologies, the advantages of this invention are as follows: This invention provides a method for the asymmetric synthesis of chiral organogermanium compounds using engineered cytochrome C mutants, which can catalyze the formation of carbon-germanium (C–Ge) bonds between organogrananes and diazo compounds. This invention can be carried out efficiently under mild conditions such as room temperature and aqueous buffer solutions using extremely low-load biocatalysts, and has broad substrate applicability. The chiral organogermanium compounds prepared by this invention have a chemical purity of over 99%, with very few byproducts, significantly simplifying the product separation and purification process, and providing an efficient and precise new method for the green biosynthesis of chiral organogermanium compounds. Attached Figure Description

[0018] Figure 1 This is a schematic diagram comparing the catalytic efficiency and product ee value of each group after optimizing the reaction conditions in the embodiments of the present invention, including wild-type cytochrome C, the first-round mutant M100E, the final mutant RccGe, and the group with optimized reaction conditions. Figure 2 The normal phase chromatogram of ethyl (R)-2-(dimethylphenylgermanium)propionate in Example 2; Figure 3 The normal phase chromatogram of ethyl (R)-2-(dimethyl-p-tolylgermanium)propionate in Example 3; Figure 4 The normal phase chromatogram of ethyl (R)-2-((3-methoxyphenyl)dimethylgermanium)propionate in Example 4; Figure 5 The normal phase chromatogram of ethyl (R)-2-((4-methoxyphenyl)dimethylgermanium)propionate in Example 5; Figure 6 The normal phase chromatogram of ethyl (R)-2-((2-methoxyphenyl)dimethylgermanium)propionate in Example 6; Figure 7 The normal phase chromatogram of ethyl (R)-2-((4-fluorophenyl)dimethylgermanium)propionate in Example 7; Figure 8 The normal phase chromatogram of ethyl (R)-2-((4-chlorophenyl)dimethylgermanium)propionate in Example 8; Figure 9 The normal phase chromatogram of ethyl (R)-2-(dimethyl(4-methylthiophenyl)germanium)propionate in Example 9; Figure 10The normal phase chromatogram of ethyl (R)-2-(dimethyl(naphthyl-1-yl)germanium)propionate in Example 10; Figure 11 The normal phase chromatogram of ethyl (R)-2-([1,1'-biphenyl]-4-yldimethylgermanium)propionate in Example 11; Figure 12 The normal phase chromatogram of (R)-2-(dimethylphenylgermanium)propionate tert-butyl ester in Example 12; Figure 13 The normal phase chromatogram of ethyl (R)-2-(dimethylphenylgermanium)butyrate in Example 13; Figure 14 This is the normal phase chromatogram of (R)-2-(dimethylphenylgermanium)propionate benzyl ester in Example 14. Detailed Implementation

[0019] To facilitate understanding of the present invention, the specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings and specific examples. The following examples or drawings are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0020] Example 1: Saturation Mutation and Screening of Cytochrome c (1) Construction of wild-type cytochrome C expression plasmid The wild-type cytochrome C gene (SEQ ID NO.1) is derived from *Haliotis diffusa*. Rhodothermus marinus The genome was synthesized by BGI Genomics Co., Ltd. in Shenzhen, and the ligation plasmid was pET22b. To enable protein expression in *Pseudomonas fluorescens*, it was constructed onto the pBBR1 plasmid via PCR. Primers were designed (primer sequences are shown in Table 1) for PCR preparation of the gene fragment and vector fragment. The gene fragment and vector fragment were then ligated via homologous recombination to construct the expression vector. The recombinant product was chemically transformed into *E. coli* DH5α competent cells, plated on LB agar plates containing antibiotics, and incubated at 37°C for 12 h. Single colonies were then picked for further culture and plasmid extraction, followed by sequencing. Plasmids with correct sequencing results contain proteins from... Rhodothermus marinus The plasmid of the wild-type cytochrome c gene.

[0021] (3) Expression of cytochrome C protein The correctly constructed plasmid was then transformed into *Pseudomonas fluorescens* via electroporation. Pseudomonas fluorescensIn ATCC 17400 competent cells, the cells were spread onto LB agar plates containing antibiotics and incubated at 30°C for 24-30 hours. Single colonies were then picked from the agar plates and transferred to liquid culture medium, incubated at 30°C and 220 rpm for 18 hours in a shaker. The colonies were then transferred at a 1:100 ratio to shake flasks for further incubation until OD (Organization Rate) was reached. 600 The concentration of rhamnose was set at 0.6-0.8, and a final concentration of 15 mg / mL and 200 μM of 5-aminolevulinic acid (5-ALA) were added as precursors for heme synthesis. Expression was induced for 20 hours in a constant-temperature shaker at 18℃ and 180 rpm. After culture, the bacterial cells were collected by centrifugation at 8000 rpm for 8 min to obtain cells expressing cytochrome C protein.

[0022] (3) Construction and screening of cytochrome C mutant library Contains from Rhodothermus marinus Using the plasmid of the wild-type cytochrome c gene as a template, the gene was modified and screened by iterative saturation mutagenesis targeting the M100 and M103 amino acids around the active site of heme iron in the amino acid sequence shown in SEQ ID NO.2.

[0023] First-round mutation (targeting the M100 site): Using the wild-type plasmid as a template, three sets of primers were designed at the M100 site to introduce the NDT, VHG, or TGG codons. The three forward primers were mixed in a molar ratio of 12:9:1 (NDT:VHG:TGG), and the corresponding reverse primers were mixed in the same proportion. PCR amplification was performed using high-fidelity DNA polymerase 2×Phanta Flash Master Mix, with two PCR reactions to generate overlapping DNA fragments. One reaction used a mixed forward primer pool with an inner reverse primer, and the other used a mixed reverse primer pool with an inner forward primer. The PCR products were purified by gel extraction, assembled via homologous recombination, and transformed into *E. coli* DH5α competent cells. The cells were plated on LB agar plates containing antibiotics and incubated at 37°C for 12 hours. Thirty independent single colonies were selected from the agar plates for sequencing. Mutants not obtained through sequencing were subsequently obtained through site-directed mutagenesis. The correctly sequenced plasmid was then transformed into *Pseudomonas fluorescens*. Pseudomonas fluorescens In ATCC 17400, fermentation expression was performed, and the collected bacterial cells were used to catalyze the reactions of dimethylphenylgermanane and ethyl 2-diazopropionate and their similar compounds, and the catalytic efficiency and stereoselectivity of the mutant reactions were compared. The activity and selectivity of the mutant catalytic reaction were compared, and the optimal mutant M100E at the M100 site was finally obtained.

[0024] The second round of mutation (targeting the M103 site): Using the superior mutant plasmid obtained in the first round of screening as a template, primers containing saturated mutations at the M103 site were designed. The above-mentioned PCR amplification, gel recovery, transformation, and sequencing verification process was repeated to construct a saturated mutant library at the M103 site. The above-mentioned comparison of the catalytic activity and selectivity of the mutants was repeated again, and finally the optimal mutant M100E-M103S was obtained, which was named RccGe. Its nucleotide sequence is shown in SEQ ID NO.3, and its amino acid sequence is shown in SEQ ID NO.4.

[0025] Table 1 shows the primer sequences used in the examples. .

[0026] Example 2: Asymmetric synthesis of chiral organogermanium compounds using cytochrome C mutants The reaction formula for this embodiment is: .

[0027] Reaction system: 380 μL of OD 600 A 400 μL reaction system was constructed using 60% cytochrome C mutant (RccGe) cells, 10 μL of 800 mM dimethylphenylgermanane substrate, and 10 μL of 1.2 M ethyl 2-diazopropionate substrate.

[0028] The recombinant microorganisms were inoculated into 5 mL of LB medium containing kanamycin resistance and cultured at 30°C and 220 rpm for 12-16 h to obtain the seed culture. The seed culture was then inoculated into 500 mL of fermentation medium at a ratio of 1:100 and cultured at 30°C and 220 rpm until OD reached the target value. 600 To reach a concentration of 0.5-0.6, induce with L-rhamnose at a final concentration of 15 mg / mL and 5-aminolevulinic acid at a final concentration of 200 μM, and incubate at 18℃ for 18-20 h.

[0029] Take 380 μL and dilute with M9-N buffer to culture until OD. 600Cytochrome C mutant cells of 60 were transferred to 2 mL vials. The vials were then placed under a nitrogen evaporator for 5 min and sealed with a rubber sieve. Next, 10 μL of dimethylphenylgermanane substrate and ethyl 2-diazopropionate substrate were added using a microsyringe, with final concentrations of 20 mM and 30 mM respectively. The vials were then sealed with an aluminum sieve. The vials were incubated at 30 °C and 250 rpm for 4 h. The reaction was then terminated with n-hexane. After vortexing, the upper organic phase was collected, and the product and peak area were analyzed using normal-phase chromatography. Based on the peak area versus concentration standard curve of the product standards, the enzyme's catalytic efficiency (TTN, Total Turnover Number) and the product's enantiomeric excess (ee, %) were calculated.

[0030] The M9-N buffer solution has the following composition: 47.7 mM Na2HPO4, 22.0 mM KH2PO4, 8.6 mM NaCl, 2.0 mM MgSO4 and 0.1 mM CaCl2, pH 7.4.

[0031] In addition, this embodiment also includes experimental groups for wild-type cytochrome C, the first-round mutant M100E, and those with optimized reaction conditions (adding 20% ​​v / v ethanol to the reaction system of the final mutant RccGe of this invention). The results of each group are compared as follows: Figure 1 As shown, the catalytic efficiency of the whole-cell enzyme of the cytochrome C mutant (RccGe) provided by this invention is significantly higher than that of the wild type and the first-round mutant M100E. Furthermore, the addition of 20% v / v ethanol to the reaction system can further improve the catalytic efficiency TTN and product ee value of the whole-cell enzyme of the cytochrome C mutant (RccGe).

[0032] Examples 3-14 used the same reaction system but different substrates to asymmetrically synthesize chiral organogermanium compounds, and calculated the enzyme catalytic efficiency and product ee value, as detailed in Table 2. Normal phase chromatograms of the products from Examples 2-14 are shown in Table 2. Figure 2-14 As shown.

[0033] Table 2. Two substrate types and enzyme catalytic efficiency, product ee values ​​in Examples 2-14 .

Claims

1. A cytochrome C mutant, characterized in that: The amino acid sequence is shown in SEQ ID NO.

4.

2. The gene encoding the cytochrome C mutant of claim 1, characterized in that: The nucleoside sequence is shown in SEQ ID NO.

3.

3. A recombinant plasmid, characterized in that: Carrying the gene described in claim 2.

4. A recombinant microorganism, characterized in that: It includes the recombinant plasmid as described in claim 3.

5. The recombinant microorganism according to claim 4, characterized in that: The recombinant microorganism was expressed using the pBBR1 plasmid as an expression vector and fluorescein-producing Pseudomonas ATCC 17400 as an expression host.

6. The use of any one of the cytochrome C mutant of claim 1 or the recombinant microorganism of claim 4 or 5 in the synthesis of chiral organogermanium compounds, characterized in that: The structural formula of the chiral organogermanium compound is as follows: ； Among them, R 1 -R 5 Each is independently selected from H, F, Cl, Me, methoxy, methylthio, or phenyl; or, R 1 -R 5 The two adjacent groups and the carbon atom attached to them together form a naphthalene ring system; R 6 Selected from methyl or ethyl; R 7 Selected from ethyl, tert-butyl, or benzyl.

7. A method for synthesizing chiral organogermanium compounds, characterized in that: This method uses the cytochrome C mutant protein of claim 1, or the whole-cell culture or cell lysate obtained by fermentation culture of the recombinant microorganisms of claim 4 or 5, as a catalyst, and the organogermanium alkane compound shown in formula 1 and the carbene precursor compound shown in formula 2 as substrates. The reaction is carried out at room temperature. After the reaction is completed, a reaction solution containing a chiral organogermanium compound is obtained. The reaction solution is separated and purified to obtain the chiral organogermanium compound shown in formula 3. The general reaction formula is shown below: ； In Equation 1, R 1 -R 5 Each is independently selected from H, F, Cl, Me, methoxy, methylthio, or phenyl; or, R 1 -R 5 In formula 2, two adjacent groups and the carbon atom attached to them together form a naphthalene ring system; R 6 Selected from methyl or ethyl; R in Formula 2 7 Selected from ethyl, tert-butyl, or benzyl; The final substrate concentration of organogermanium compounds in the reaction system was 15-20 mM, the final substrate concentration of carbene precursor compounds was 20-30 mM, and the catalyst concentration was 0.5 μM-1.4 μM.

8. The method according to claim 7, characterized in that: OD of bacterial culture in whole-cell reaction system 600 The value is 60; the pyrolysis solution in the pyrolysis reaction system is composed of OD 600 It was obtained by thermal lysis of whole-cell bacterial culture with a concentration of 60.

9. The method according to claim 7, characterized in that: The reaction system is supplemented with 15-20% v / v ethanol as a solvent for the substrate.

10. The method according to claim 7, characterized in that: The reaction temperature was 30℃, the reaction time was 4 hours, and the reaction speed was 250 rpm.

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