A high-activity gernien a synthase mutant, a high-throughput screening method for gernien a synthase mutation and application thereof

Abstract

The application discloses a kind of high-activity germacradiene A synthase mutant and germacradiene A synthase mutation high-throughput screening method and application, through eGFP fluorescence report system, first utilize FPP response type promoter, the activity of sesquiterpene synthase is associated with fluorescence level, establishes the enzyme mutation screening platform combined with random mutagenesis, can be applied to the screening of a variety of clear synthesis mechanism sesquiterpene natural product synthase.It is precise, large, specific to screen the screening method of the application, can directional screening high-yield AvGAS, overcome the current lack of sesquiterpene synthase screening technical means, screening efficiency is low, and cannot directional screening high-yield germacradiene A synthase bottleneck.

Description

Technical Field

[0001] This invention belongs to the field of microbial and bioengineering technology, specifically relating to a high-throughput screening method for highly active gemmaene A synthase mutants and sesquiterpene synthase mutants. Background Technology

[0002] β-elemene is a sesquiterpene compound naturally derived from the mature tubers of the representative plant, Curcuma longa. It is one of my country's domestically developed antitumor drugs with independent intellectual property rights. Due to its strong antitumor activity, broad spectrum of action, mild toxicity, and low likelihood of inducing drug resistance, it is widely used in the clinical treatment of various malignant tumors. Currently, β-elemene is mainly produced through the separation and extraction of plant volatile oils. However, the low content of β-elemene, the difficulty in separation and purification, the low yield, and the high cost severely hinder the large-scale production and application of β-elemene.

[0003] In recent years, with the development of synthetic biology, research on constructing cell factories using microorganisms for the biosynthesis of β-elemene has become a hot topic. Through the MVA pathway and the construction and expression of the key gene germarne A synthase in the host, the precursor of β-elemene, germarne A, is biosynthesized, and β-elemene can be converted in one step via Cope molecule rearrangement under heating conditions. Currently, researchers have initially achieved and continuously improved the heterologous biosynthesis yield of β-elemene using metabolic engineering, combinatorial biology, and genetic engineering. Among these methods, the key rate-limiting enzyme, germarne A synthase (GAS), has become a crucial link in the efficient production of β-elemene, and modification of GAS is expected to overcome the bottleneck limiting the increase in β-elemene yield.

[0004] Enzyme modification methods include rational design or random mutagenesis. For the large number of mutant libraries obtained, appropriate screening methods are needed to obtain enzymes with beneficial mutations. Currently, screening methods for terpene synthases are very limited. Conventional methods involve direct or indirect identification and screening of product concentration, including: gas chromatography or liquid chromatography detection, screening based on the accumulation of downstream tetraterpenoid compounds by visual color, and screening based on the corresponding absorption wavelengths of some terpene derivatives or complexes. However, for high-throughput screening of sesquiterpene synthases, these methods are unsuitable due to poor specificity, high time and cost, and the complex intracellular metabolic environment. Therefore, designing a reasonable and effective screening method is crucial.

[0005] Metabolic biosensors are recognition elements that are sensitive to and responsive to specific substances. When combined with output elements (such as fluorescent proteins), they can convert the concentration of corresponding metabolites into specific output signals. They are characterized by high sensitivity, strong specificity, and fast analysis speed, and can be used directly as ligands for target proteins or to achieve a response based on metabolite concentration. Using them in random mutagenesis and efficient screening strategies can overcome many limitations. By determining the cellular-level metabolite concentration and the catalytic efficiency of enzymes in metabolic pathways, screening throughput can be significantly improved, and screening time and costs can be effectively reduced.

[0006] Therefore, selecting a suitable metabolite biosensor and designing and constructing a corresponding high-throughput screening tool for screening sesquiterpene synthase gemmaene A synthase is very effective. Summary of the Invention

[0007] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0008] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0009] Therefore, the purpose of this invention is to overcome the shortcomings of the prior art and provide a highly active gemmaene A synthase (GAS) mutant.

[0010] To solve the above-mentioned technical problems, the present invention provides the following technical solution: including,

[0011] The mutant AvGAS-Y20H, with the sequence shown in SEQ ID NO.1, was obtained by mutating the tyrosine residue at position 20 to histidine.

[0012] The wild-type gemmaene A synthase is derived from Anabaena variabilis.

[0013] As a preferred embodiment of the highly active gemmaene A synthase mutant of the present invention, the mutant further includes:

[0014] The mutant AvGAS-Y20H-A277P, with the sequence shown in SEQ ID NO.3, was obtained by mutating alanine at position 277 to proline.

[0015] As a preferred embodiment of the highly active gemmaene A synthase mutant of the present invention, wherein: the nucleotide sequence encoding the wild-type gemmaene A synthase is shown in SEQ ID NO.2, and the nucleotide sequence encoding the mutant AvGAS-Y20H is shown in SEQ ID NO.4.

[0016] As a preferred embodiment of the highly active gemmaene A synthase mutant of the present invention, wherein the nucleotide sequence encoding the mutant AvGAS-Y20H-A277P is shown in SEQ ID NO.6.

[0017] Another object of the present invention is to provide an application of a highly active gemmaene A synthase mutant in increasing β-elemene yield.

[0018] As a preferred embodiment of the application of the highly active gemmaene A synthase mutant of the present invention in improving the yield of β-elemene, wherein the β-elemene yield of the strain containing the mutant AvGAS-Y20H is increased by more than 20% compared with the original strain.

[0019] As a preferred embodiment of the application of the highly active gemmaene A synthase mutant of the present invention in improving the yield of β-elemene, wherein the β-elemene yield of the strain containing the mutant AvGAS-Y20H-A277P is increased by more than 70% compared with the original strain.

[0020] Another objective of this invention is to provide a high-throughput screening method for gemmaene A synthase mutations.

[0021] To solve the above-mentioned technical problems, the present invention provides the following technical solution: including,

[0022] A recombinant plasmid pETDuet-T7-AvGAS-gadE-eGFP with the nucleotide sequence shown in SEQ ID NO.7 was constructed as a fluorescent reporter plasmid.

[0023] Using the recombinant plasmid pETDuet-T7-AvGAS-gadE-eGFP as a template, primers eAvGAS-F and eAvGAS-R were designed, with sequences shown in SEQ ID NO.8 and SEQ ID NO.9, respectively. The AvGAS mutant fragment was obtained by error-prone PCR amplification.

[0024] Using the recombinant plasmid pETDuet-T7-AvGAS-gadE-eGFP as a template, primers Temp-F and Temp-R were designed, with sequences shown in SEQ ID NO.10 and SEQ ID NO.11, respectively. A linear vector was obtained by reverse amplification.

[0025] Homologous recombination of the mutant fragment and the linear vector yielded the mutant library plasmid pETDuet-T7-eAvGAS-gadE-eGFP;

[0026] The mutant library plasmid pETDuet-T7-eAvGAS-gadE-eGFP and the pMVA plasmid carrying the MVA pathway enzyme gene were transformed into Escherichia coli BL21(DE3) competent cells to obtain transformants.

[0027] The nucleotide sequence of the pMVA plasmid is shown in SEQ ID NO.12;

[0028] Transformants were cultured at constant temperature followed by IPTG induction culture, and fluorescence was measured using a microplate reader. Blank background fluorescence was subtracted, and OD was calculated. 600 Normalization processing and screening of positive samples that exceed the fluorescence screening threshold enable high-throughput screening of gemmaene A synthase mutations.

[0029] As a preferred embodiment of the application of the highly active gemmaene A synthase mutant of the present invention in improving β-elemene yield, wherein: the method for constructing the recombinant plasmid pETDuet-T7-AvGAS-gadE-eGFP with the nucleotide sequence shown in SEQ ID NO.7 includes,

[0030] Using pETDuet-1 as a vector and controlled by the T7 promoter, primers AvGAS-F, AvGAS-R, MCS1-F, and MCS1-R were designed with sequences shown in SEQ ID NO.13 to SEQ ID NO.16, respectively, to clone the gene fragment of wild-type gemmaene A synthase AvGAS into the downstream coding region.

[0031] Using the same plasmid pETDuet-1 as a vector, the promoter P gadE To control the development of the gene, primers MCS2-F, MCS2-R, eGFP-F, and eGFP-R were designed, with sequences shown in SEQ ID NO.17 to SEQ ID NO.20, respectively. The eGFP gene was cloned into the downstream coding region, and the recombinant plasmid pETDuet-T7-AvGAS-gadE-eGFP was constructed as a fluorescent reporter plasmid.

[0032] The nucleotide sequence of pETDuet-1 is shown in SEQ ID NO.21, the nucleotide sequence of the gene fragment of wild-type gemmaene A synthase AvGAS is shown in SEQ ID NO.2, and the promoter P... gadE The nucleotide sequence of the eGFP gene is shown in SEQ ID NO.22, and the nucleotide sequence of the eGFP gene is shown in SEQ ID NO.23.

[0033] Another objective of this invention is to provide a high-throughput screening method for gemmaene A synthase mutations for the specific screening of strains with high β-elemene production.

[0034] Beneficial effects of this invention:

[0035] This invention, through the eGFP fluorescent reporter system, for the first time utilizes an FPP-responsive promoter to correlate the activity of sesquiterpene synthases with fluorescence levels, establishing an enzyme mutation screening platform combined with random mutagenesis. This platform can be applied to the screening of various sesquiterpene natural product synthases with well-defined synthesis mechanisms. Furthermore, the screening method of this invention is highly accurate, has high throughput, strong specificity, and can be used for targeted screening of high-yielding AvGAS, overcoming the current bottlenecks of limited sesquiterpene synthase screening techniques, low screening efficiency, and the inability to target high-yielding gemmaene A synthases. Attached Figure Description

[0036] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein:

[0037] Figure 1 This is a schematic diagram of the biosynthetic pathway of β-elemene.

[0038] Figure 2 This is a diagram illustrating the screening mechanism of FPP-responsive promoter fluorescent reporter plasmids.

[0039] Figure 3 Test the response range of the fluorescent reporter plasmid;

[0040] Figure 4 This was to verify the positive correlation between fluorescence and yield in some strains.

[0041] Figure 5 It refers to the beneficial mutation sites and yields obtained through screening. Detailed Implementation

[0042] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0043] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0044] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0045] Experimental methods not specified in detail in this invention are conventional methods, referring to the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations.

[0046] Unless otherwise specified, all materials used in this invention are commercially available in the field.

[0047] Example 1 establishes a positive correlation between the catalytic activity of AvGAS and the fluorescence intensity.

[0048] Reference Figure 1 The target protein is AvGAS, a key enzyme in the synthesis of β-elemene. This enzyme can catalyze and consume the cyclization of the sesquiterpene precursor skeleton FPP to form the β-elemene precursor gemmaene A.

[0049] like Figure 2 As shown, the promoter (P) for the FPP response was selected. gadE or P rstA Using sequences shown in SEQ ID NO.22 and SEQ ID NO.24 as the input element and eGFP as the output element, a fluorescent reporter plasmid was constructed. The fluorescence intensity changes of production strains containing different active AvGAS under the control of different FPP-responsive promoters were tested to identify and select P... gadE A positive correlation between controlled fluorescence expression levels and yield.

[0050] The results are as follows Figure 3 As shown, the two selected FPP response types P gadE / P rstA They were combined with two known AvGAS with different activities, respectively. Figure 3 As shown in Figure A, the production strains expressing two enzymes, P-GAS / S-GAS, yielded 463.31 and 6.46 mg / L, respectively. Figure 3 As shown in B, Pg represents the enzymes P-GAS and P gadE The combination, Sg represents the enzymes S-GAS and P gadE The combination, Pr, represents the enzymes P-GAS and P... rstA The combination, Sr represents the enzymes S-GAS and P rstACombinations. Three samples were selected as parallel controls for each group, and cultured and induced in 96-well plates with periodic fluorescence detection. Over time, combinations of enzymes with different activities and promoters showed significant fluorescence differences. Compared to low-activity S-GAS, the production strain containing high-activity P-GAS showed significantly higher fluorescence activity in P-GAS-producing strains. gadE Higher fluorescence levels were observed under controlled conditions, while at P rstA Lower fluorescence levels were observed under controlled conditions.

[0051] Subsequently, P, which has a larger fluorescence response range, was selected. gadE As a subsequent filtering tool.

[0052] Example 2

[0053] To further verify P gadE To assess the effectiveness of the fluorescent reporter plasmid system in practical operation, this embodiment selected several strain samples with different fluorescence levels from the mutant library obtained by error-prone PCR, carried out shake-flask amplification culture, and added isopropyl myristate to extract the organic phase of the fermentation product, and detected its yield at 72 h.

[0054] like Figure 4 As shown, the β-elemene production of the selected strains exhibited a certain positive correlation with fluorescence. These results indicate that P... gadE Fluorescent reporter plasmid systems have strong screening specificity.

[0055] Example 3

[0056] This embodiment provides a high-throughput screening method for gemmaene A synthase mutations, specifically:

[0057] 1) Construct a promoter P containing an FPP response gadE Controlled eGFP fluorescent reporter plasmid:

[0058] Using pETDuet-1 as a vector and controlled by the T7 promoter, primers AvGAS-F, AvGAS-R, MCS1-F, and MCS1-R were designed with sequences as shown in SEQ ID NO.13 to SEQ ID NO.16, respectively. The gene of AvGAS with the sequence shown in SEQ ID NO.2 was cloned into the downstream coding region.

[0059] Using the same plasmid pETDuet-1 as a vector, the promoter P gadETo control the flow, primers MCS2-F, MCS2-R, eGFP-F, and eGFP-R were designed, with sequences shown in SEQ ID NO.17 to SEQ ID NO.20, respectively. The eGFP gene with the sequence shown in SEQ ID NO.23 was cloned into the downstream coding region, and the recombinant plasmid pETDuet-T7-AvGAS-gadE-eGFP was constructed as a fluorescent reporter plasmid.

[0060] 2) Generating mutant libraries via error-prone PCR:

[0061] Using plasmid pETDuet-T7-AvGAS-gadE-eGFP as a template, primers eAvGAS-F and eAvGAS-R were designed, with sequences shown in SEQ ID NO.8 and SEQ ID NO.9, respectively. The AvGAS mutant fragment was obtained by error-prone PCR amplification.

[0062] Using plasmid pETDuet-T7-AvGAS-gadE-eGFP as a template, primers Temp-F and Temp-R were designed, with sequences shown in SEQ ID NO.10 and SEQ ID NO.11, respectively. The linear vector was obtained by reverse amplification.

[0063] The mutant fragment and the linear vector were then recombined via homologous recombination to obtain the mutant library plasmid pETDuet-T7-eAvGAS-gadE-eGFP.

[0064] 3) Fluorescence screening and identification of strains containing the AvGAS mutant library:

[0065] Subsequently, the mutant library plasmid pETDuet-T7-eAvGAS-gadE-eGFP and the pMVA plasmid with the nucleotide sequence shown in SEQ ID NO. 12 were transformed into E. coli BL21(DE3) competent cells. The pMVA plasmid carries the MVA pathway enzyme gene, which is used to supply the sesquiterpene precursor FPP. Transformants were picked and added to 250 μl of LB medium in 96-well plates and cultured at 37°C and 300 rpm for 6 h in a microplate shaker. 0.5 mM IPTG was added for induction, and the cells were cultured at 25°C and 300 rpm for another 17 h. Fluorescence was measured using a microplate reader (excitation wavelength 484 nm, emission wavelength 510 nm), and the background fluorescence was subtracted and OD was calculated. 600Normalization was performed to screen out positive samples that exceeded the fluorescence screening threshold. The selected positive sample strains were passaged, and the plasmid pMVA (containing chloramphenicol Cm marker) was lost to facilitate sequencing identification of the plasmid pETDuet-T7-eAvGAS-gadE-eGFP (containing ampicillin Amp marker). After sequencing, sequence alignment was performed to confirm the mutated amino acid sites, and the yield was further verified in shake flasks.

[0066] Example 4

[0067] This embodiment is used to identify highly active AvGAS beneficial mutation sites obtained through the FPP-responsive promoter screening platform constructed in Example 3. Specifically:

[0068] After induction and fluorescence screening in 96-well plates, we collected the strains in the wells that exceeded the set fluorescence threshold. We then inoculated them into LB medium containing only the monoclonal antibody Amp and passaged them to lose the Cm-labeled plasmid pMVA, which facilitated subsequent plasmid purification and sequencing.

[0069] The results are as follows Figure 5 As shown, in the second round of random mutation screening, the mutation site Y20H was identified, in which the tyrosine Tyr at position 20 of AvGAS was mutated to histidine His. Y20H increased the yield of β-elemene by 20.98%, reaching 560.51 mg / L.

[0070] Further mutation screening was conducted based on Y20H, and the mutation site A277P was identified. The alanine at position 277 of AvGAS was mutated from Ala to Proline (Pro). A277P further increased the yield by 41.84% compared to Y20H, reaching 795.03 mg / L. Finally, the β-elemene yield of the strain containing the double mutant AvGAS was increased by about 72% compared to the original strain.

[0071] In summary, this invention, through the eGFP fluorescent reporter system, for the first time utilizes an FPP-responsive promoter to correlate the activity of sesquiterpene synthases with fluorescence levels, establishing an enzyme mutation screening platform combined with random mutagenesis. This platform can be applied to the screening of various sesquiterpene natural product synthases with well-defined synthesis mechanisms. Furthermore, the screening method of this invention boasts high precision, high throughput, strong specificity, and the ability to target high-yielding AvGAS, overcoming the current bottlenecks of limited sesquiterpene synthase screening techniques, low screening efficiency, and the inability to target high-yielding gemmaene A synthases.

[0072] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A highly active gemmaene A synthase mutant, characterized in that: The mutant AvGAS-Y20H, with the amino acid sequence shown in SEQ ID NO.1, was obtained by mutating the tyrosine residue at position 20 to histidine. The alanine at position 277 of the mutant AvGAS-Y20H, with the amino acid sequence shown in SEQ ID NO.3, was mutated to proline to obtain the mutant AvGAS-Y20H-A277P, with the sequence shown in SEQ ID NO.

5. The wild-type gemmaene A synthase is derived from Anabaena variegata (… Anabaena variabilis ).

2. The application of the highly active gemmaene A synthase mutant as described in claim 1 in increasing β-elemene yield.

3. The application of the highly active gemmaene A synthase mutant as described in claim 1 in increasing β-elemene yield, characterized in that: The β-elemene yield of strains containing the mutant AvGAS-Y20H was increased by more than 20% compared to the original strain; or, The β-elemene production of the strain containing the mutant AvGAS-Y20H-A277P was increased by more than 70% compared with the original strain.

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