A squalene-hopaene cyclase mutant, encoding gene, recombinant plasmid, genetically engineered bacteria and application thereof

Abstract

The present invention relates to the field of genetic engineering technology, and discloses a squalene-hopaene cyclase mutant, a coding gene, a recombinant plasmid, a genetic engineering bacterium and an application thereof. By performing the combined mutation on the amino acid sequence of the original squalene-hopaene cyclase shown in SEQ ID NO.2, the amino acid and its related nucleotide sequence undergo directional structural and functional changes, which greatly improves the catalytic activity of the squalene-hopaene cyclase mutant and is conducive to industrial production. Among them, the specific enzyme activity of the squalene-hopaene cyclase mutant reaches 45.2 U / g; when the substrate concentration is 10 g / L, the yield of catalytically generated ambrox reaches 90.5%.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to a squalene-hopaene cyclase mutant, a coding gene, a recombinant plasmid, a genetic engineering bacterium and applications thereof. Background Art

[0002] Ambrox ((-)-Ambrox) is the most important aroma component of ambergris. It has a soft, lasting and stable animal-type ambergris aroma and a mild woody aroma. It is a very good fixative and is widely used in the cosmetics industry. At the same time, ambrox is also a food flavoring that can be used in various edible flavor formulations. At present, the industrial synthesis of ambrox mainly adopts a route using sclareol as a raw material. Sclareol has a carbon atom skeleton similar to ambrox. First, it undergoes an oxidation reaction to generate sclareolide, and then undergoes a reduction and cyclization reaction to generate ambrox.

[0003] In the past, sclareol was oxidized with potassium permanganate to produce sclareolide, but now it is fermented by biological fermentation. Sclareol is extracted from the sclareol plant. The flowers and leaves of the plant are steam distilled to obtain sclareol oil, which is then extracted and separated with alkanes and methanol to obtain sclareol. Both sclareol and sclareolide are very expensive, and the supply fluctuates greatly.

[0004] At present, there is a shortage of sclareol and sclareolide, and the research on their substitutes is increasing. In the prior art, sclareol is prepared by biosynthesis using geranyl pyrophosphate as a raw material. Geranyl pyrophosphate is first converted into lysine diol pyrophosphate monoester under the action of lysine diol pyrophosphate synthase (LDPP synthase), and then sclareol is generated under the action of sclareol synthase. The obtained sclareol is then converted into ambroxol through the above route. There are also more and more studies on the semi-biosynthesis of ambroxol. Using chemically synthesized homofarnesol as a raw material, the ambroxol product is obtained by ring closure under the catalysis of cyclase. These new biotransformation technologies are expected to be industrialized in the future. However, the catalytic activity of squalene and hopene cyclase mutants under the prior art is low and still cannot meet actual needs. Summary of the invention

[0005] The purpose of the present application is to provide a squalene-hopaene cyclase mutant, encoding gene, recombinant plasmid, genetically engineered bacteria and applications thereof, aiming to solve the technical problem of low catalytic activity of existing squalene-hopaene cyclase mutants.

[0006] In order to achieve the above purpose, the technical solution of this application is:

[0007] The first aspect of the present application provides a squalene-hopaene cyclase mutant, the amino acid sequence of the mutant is shown in SEQ ID NO: 4. The squalene-hopaene cyclase mutant is based on the amino acid sequence of the original squalene-hopaene cyclase shown in SEQ ID NO: 2 as a template (the encoding nucleotide sequence is the nucleotide sequence shown in SEQ ID NO: 1), and the amino acid sequence is subjected to the following combined mutations:

[0008] i: mutate the threonine at position 38 to alanine;

[0009] ii: mutate the leucine at position 80 to threonine;

[0010] iii: mutating the threonine at position 374 to tyrosine;

[0011] iv: mutate the glycine at position 437 to alanine;

[0012] v: Mutation of alanine at position 438 to serine.

[0013] The second aspect of the present application provides a gene encoding a squalene-hopaene cyclase mutant provided in the first aspect of the present application.

[0014] In an optional implementation of the second aspect, the nucleotide sequence of the gene encoding the squalene-hopaene cyclase mutant is shown in SEQ ID NO:3.

[0015] The third aspect of the present application provides a recombinant plasmid comprising a gene encoding the squalene-hopaene cyclase mutant described in the second aspect.

[0016] In an optional implementation of the third aspect, the expression vector of the recombinant plasmid is pET28a(+).

[0017] The fourth aspect of the present application provides a genetically engineered bacterium comprising a gene encoding the squalene-hopaene cyclase mutant described in the second aspect.

[0018] In an optional implementation of the fourth aspect, the expression host of the genetically engineered bacteria is E. coli BL21(DE3).

[0019] The fifth aspect of the present application provides the use of the genetically engineered bacteria described in the fourth aspect in catalyzing the synthesis of Ambrox from homofarnesol.

[0020] In an optional implementation of the fifth aspect, the application method includes:

[0021] S1: suspending the genetically engineered bacteria in a citric acid-sodium citrate buffer system to obtain a reaction system;

[0022] S2: After adding homofasyl alcohol and SDS into the reaction system, the reaction was oscillated at a temperature of 50 °C and a rotation speed of 200 r / min, and the reaction products were extracted and analyzed.

[0023] Compared with the prior art, the embodiments of the present invention have the following technical effects:

[0024] The squalene-hopaene cyclase mutant provided in the first aspect of the present application is a squalene-hopaene cyclase mutant, which is a squalene-hopaene cyclase mutant. The squalene-hopaene cyclase mutant is a squalene-hopaene cyclase mutant. The squalene-hopaene cyclase mutant is a squalene-hopaene cyclase mutant. The squalene-hopaene cyclase mutant has a specific enzyme activity of 45.2 U / g. When the substrate concentration is 10 g / L, the yield of catalytically generated ambrox reaches 90.5%. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0026] Figure 1 It is a comparison chart of the enzyme activity and catalytic yield of the original enzyme and mutant of squalene-hopaene cyclase provided in the examples of the present application. DETAILED DESCRIPTION

[0027] Example 1 A squalene-hopaene cyclase mutant.

[0028] A squalene-hopaene cyclase mutant, wherein the squalene-hopaene cyclase mutant is based on the amino acid sequence of the original squalene-hopaene cyclase shown in SEQ ID NO: 2, and the following amino acids are mutated:

[0029] i: mutate the threonine at position 38 to alanine;

[0030] ii: mutate the leucine at position 80 to threonine;

[0031] iii: mutating the threonine at position 374 to tyrosine;

[0032] iv: mutate the glycine at position 437 to alanine;

[0033] v: Mutation of alanine at position 438 to serine.

[0034] The gene sequence of the original squalene-hopaene cyclase, SEQ ID NO: 1, is as follows:

[0035]

[0036] The amino acid sequence of the original squalene-hopene cyclase, SEQ ID NO: 2, is as follows:

[0037]

[0038] The letters in the above amino acid sequence are single-letter abbreviations of amino acids. The amino acid names represented by the abbreviations in the specific amino acid sequences are shown in Table 1:

[0039] Table 1 Names and abbreviations of amino acids

[0040]

[0041] Therefore, the amino acid sequence of the original squalene-hopaene cyclase shown in SEQ ID NO: 2 is used as a carrier in the present embodiment, and the above-mentioned combined mutation is performed on the specific sites in the amino acid sequence of SEQ ID NO: 2, and the amino acid sequence of the obtained squalene-hopaene cyclase mutant is shown in SEQ ID NO. 4. Among them, the amino acid sequence of the mutant in the embodiment of the present application is represented by replacing the corresponding amino acid sites in the amino acid sequence of the original squalene-hopaene cyclase with mutant amino acids, and the number of the mutant site corresponds to the site of the amino acid sequence of the original squalene-hopaene cyclase.

[0042] The amino acid sequence of the squalene-hopaene cyclase mutant in the present application is SEQ ID NO.4, as follows:

[0043]

[0044] Those skilled in the art should understand that the above-mentioned combined mutation modification methods are all implemented in accordance with relevant genetic engineering techniques known in the art. The embodiments of the present application do not particularly limit the specific gene mutation technology, and it is subject to the ability of those skilled in the art to obtain the target mutant through the above-mentioned combined mutation.

[0045] In addition, the examples of the present application carried out individual mutations on each of the above mutation sites to verify the technical effect of the combined mutation described above. The results showed that individual mutations of each site could not achieve the catalytic synthesis and splitting effect of the combined mutation.

[0046] Among them, the embodiment of the present application performs the above-mentioned combined mutation on the amino acid sequence of the original squalene-hopaene cyclase shown in SEQ ID NO.2, so that the amino acid and its related nucleotide sequence produce directional structural and functional changes, thereby greatly improving the catalytic activity of the squalene-hopaene cyclase mutant, which is conducive to industrial production.

[0047] Example 2 A gene encoding a mutant of squalene-hopaene cyclase.

[0048] The squalene-hopaene cyclase mutant in Example 1 is encoded to obtain a coding gene of the squalene-hopaene cyclase mutant. The nucleotide sequence of the coding gene of the squalene-hopaene cyclase mutant is shown in SEQ ID NO. 3. At the same time, for the convenience of recording, the coding gene of the squalene-hopaene cyclase mutant is represented as SHC12 in the present embodiment.

[0049] The coding gene SEQ ID NO.3 of the squalene-hopaene cyclase mutant in the present application is as follows:

[0050]

[0051] At the same time, the embodiment of the present application provides the nucleotide sequence SEQ ID NO.1 of the original squalene-hopaene cyclase, that is, the gene sequence SEQ ID NO: 1 of the original squalene-hopaene cyclase. Based on the specific nucleotide sequences of each amino acid, after the above-mentioned combined mutations are performed in the embodiment of the present application, the nucleotide sequence of the encoding gene of the squalene-hopaene cyclase mutant obtained is shown in SEQ ID NO. 3. Among them, the nucleotide sequence of the mutant in the embodiment of the present application is represented by replacing the corresponding nucleotide sites in the nucleotide sequence of the original squalene-hopaene cyclase with the nucleotides of the mutant amino acid, and the number of the mutation site corresponds to the nucleotide sequence site of the original squalene-hopaene cyclase.

[0052] Example 3 A recombinant plasmid carrying a gene encoding a mutant of squalene-hopaene cyclase.

[0053] The present application embodiment provides a recombinant plasmid carrying a gene encoding a squalene-hopaene cyclase mutant. The preparation method of the recombinant plasmid preferably comprises:

[0054] Restriction endonucleases Nco I and XOtI After double digestion of the coding gene of the squalene-hopaene cyclase mutant and the expression vector pET28a(+) of Escherichia coli for 4-8 hours, the digestion products are recovered and connected with T4 DNA ligase at a temperature of 16°C for 16 hours to obtain a recombinant plasmid containing the coding gene of the squalene-hopaene cyclase mutant; at the same time, for the convenience of recording, the recombinant plasmid is represented as pET28a-SHC12 in the present embodiment.

[0055] It should be noted that the examples of this application use T4 DNA ligase and restriction endonuclease Nco I and XOt The specific source of I is not particularly limited. For example, it can be obtained from Fermetens by commercial means, or synthesized by genetic engineering methods known in the relevant field.

[0056] Example 4 A genetically engineered bacterium

[0057] A genetically engineered bacterium, using Escherichia coli as a host, expresses the recombinant plasmid described in Example 3.

[0058] Specific methods include:

[0059] S41: Transform the recombinant plasmid pET28a-SHC12 into E. coli BL21(DE3) recipient bacteria were plated on LB agar plates containing kanamycin (mass concentration of 50 mg / L) and cultured at 37 °C for 12 h to allow single colonies to grow on the plates. Single colonies were randomly picked and cloned, then inoculated into LB liquid culture medium for 12 h and plasmids were extracted for sequencing. Genetically engineered bacteria were screened out based on the sequencing results and were represented as positive clones. E. coli BL21(DE3) / pET28a-SHC12.

[0060] S42: The genetically engineered bacteria were inoculated into 4 mL of LB medium containing kanamycin at a mass concentration of 50 mg / L, and initially cultured at a temperature of 37°C and a rotation speed of 200 r / min for 12 h; the genetically engineered bacteria of the initial culture were inoculated into a new 30 mL of LB medium containing kanamycin at a mass concentration of 50 mg / L at an inoculum amount of 1 vt.%, and cultured at a temperature of 37°C and a rotation speed of 200 r / min to an optical density (OD600) of 0.6-0.8; then isopropyl-β-D-thiogalactopyranoside (IPTG) inducer was added to a final concentration of 0.1 mM, and induced culture was performed at a temperature of 25°C and a rotation speed of 200 r / min for 16 h; finally, the culture was centrifuged at 5000×g, 5 The cells were collected by centrifugation at 1000 × g for 1 min, resuspended and washed with Tris-SO4 buffer (pH 7.0), and centrifuged at 13,000 × g for 1 min to collect the wet cells, which were then stored at -20 °C for future use.

[0061] It should be noted that the composition of the LB agar plate includes: 10.0 g / L tryptone, 5.0 g / L yeast powder, and 10.0 g / L NaCl; the composition of the LB solid culture medium includes: 10.0 g / L tryptone, 5.0 g / L yeast powder, 10.0 g / L NaCl, and 15.0 g / L agar. The LB agar plate and the LB solid culture medium are both autoclaved at 121°C for 20 min.

[0062] The present embodiment utilizes a coding gene to encode the squalene-hopaene cyclase mutant described above, and expresses a recombinant plasmid carrying the coding gene to obtain the genetically engineered bacteria.

[0063] In a specific embodiment, the expression host of the genetically engineered bacteria is preferably E. coli BL21 (DE3).

[0064] Example 5 Use of a genetically engineered bacterium in catalyzing the synthesis of Ambrox from homofarnesol.

[0065] The specific method of using the genetically engineered bacteria expressed by the mutant of squalene-hopaene cyclase to catalyze the synthesis of ambroxan by homofarnesol includes:

[0066] Weigh 0.5g of genetically engineered bacteria and suspend them in 10mL of citric acid-sodium citrate buffer system (pH=6.0, 50mM), add 10g / L homofascinol and 0.05% (w / v) SDS, and shake for 24h at 50℃ and 200r / min. During the shaking reaction, take samples of 0.2mL at regular intervals, add 0.8ml of n-hexane:isopropanol (v:v=2:1) ​​to the samples for extraction, and analyze and detect with GC-FID.

[0067] To further illustrate the technical effect of the genetically engineered bacteria containing the squalene-hopaene cyclase mutant, the present application example provides an application test of the genetically engineered bacteria containing the original squalene-hopaene cyclase in catalyzing the synthesis of Ambrox from homofarnesol. The test method is the same as the specific operation and parameter setting described in Example 5, except that the genetically engineered bacteria containing the squalene-hopaene cyclase mutant is replaced by the genetically engineered bacteria containing the original squalene-hopaene cyclase.

[0068] Figure 1 This is a comparison chart of the enzyme activity and catalytic yield of the original enzyme and mutant of squalene-hopaene cyclase provided in the embodiment of the present application. It can be seen from the figure that the genetically engineered bacteria containing the original squalene-hopaene cyclase can also catalyze the synthesis of ambrox from homofarnesol. Among them, the specific enzyme activity of the original squalene-hopaene cyclase is 14.1 U / g, and the yield of ambrox catalyzed by it is 30.1%. After the genetically engineered bacteria is used to catalyze the synthesis of ambrox from homofarnesol, the specific enzyme activity reaches 45.2U / g, and the yield of ambrox generated by catalyzing 10 g / L of homofarnesol is 90.5%. In summary, the specific activity catalyzed by the genetically engineered bacteria containing the squalene-hopaene cyclase mutant is 3.2 times that of the original squalene-hopaene cyclase, based on the excellent performance of the squalene-hopaene cyclase mutant contained in the genetically engineered bacteria with high specific activity.

[0069] In the examples of the present application, the content of the product was analyzed by gas chromatography, and the experimental conditions specifically included:

[0070] A Shimadzu GC-14A system was used, and the column type was Zebron ZB-5 column (30 m × 0.32 mm × 0.25 mm); the carrier gas was hydrogen, and the detector temperature was 250 °C. The column temperature was increased from 100 °C to 200 °C at a rate of 15 °C / min, and from 200 °C to 240 °C at a rate of 120 °C / min, and the column temperature was maintained at 240 °C for 4 min.

[0071] The various embodiments in this specification are described in a progressive manner, and the same or similar parts between the various embodiments can be referenced to each other, and each embodiment focuses on the differences from other embodiments.

[0072] The above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit the present application. Although the present application has been described in detail with reference to the aforementioned embodiments, a person of ordinary skill in the art should understand that the technical solutions recorded in the aforementioned embodiments may still be modified, or some or all of the technical features therein may be replaced by equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the present application.

[0073] The above is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any technician familiar with the profession may use the above technical content as inspiration to change or modify it into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiment based on the technical essence of the present invention without departing from the technical solution of the present invention still belongs to the protection scope of the claims of the present invention.

Claims

1. A squalene-hopaene cyclase mutant, characterized in that: The amino acid sequence of the mutant is shown in SEQ ID NO:

4.

2. A coding gene, characterized in that The gene encodes the squalene-hopaene cyclase mutant according to claim 1.

3. A recombinant plasmid, characterized in that: The plasmid carries the gene according to claim 2.

4. The recombinant plasmid according to claim 3, characterized in that The expression vector of the recombinant plasmid is pET28a(+).

5. A genetically engineered bacterium, characterized in that: The genetically engineered bacteria comprises the gene encoding the squalene-hopaene cyclase mutant according to claim 2.

6. The genetically engineered bacterium according to claim 5, characterized in that: The expression host of the genetically engineered bacteria is E. coli BL21 (DE3).

7. Use of the genetically engineered bacteria according to claim 5 or 6 in catalyzing the synthesis of Ambrox from homofarnesol.

8. The use according to claim 7, comprising the following steps: S1: suspending the genetically engineered bacteria in a citric acid-sodium citrate buffer system to obtain a reaction system; S2: After adding homofasyl alcohol and SDS into the reaction system, the reaction was oscillated at a temperature of 50 °C and a rotation speed of 200 r / min, and the reaction products were extracted and analyzed.

Images