A method for activating the expression of silent secondary metabolite synthesis gene clusters in actinomycetes

Abstract

The present invention discloses a method for activating the expression of a silent secondary metabolite synthesis gene cluster (BGC) of actinomycetes, and relates to the technical field of microbial secondary metabolites. The technical key points are as follows: the method uses microbial quorum sensing signal molecules to stimulate the silent gene cluster of actinomycetes, so that the silent BGCs of actinomycetes are activated; the quorum sensing signal molecules used include but are not limited to Farnesol, Tyrosol, 3‑oxo‑C12‑HSL and Tryptophol. The method of the present invention is a novel method for activating the expression of silent gene clusters of actinomycetes, that is, a strategy for rapidly and efficiently activating silent BGCs in different actinomycetes. The method of the present invention will provide an effective way to activate the treasure house of silent BGCs existing in large quantities in nature and to deeply explore new actinomycete natural products, thereby contributing more and better new lead compounds to the research and development of multiple fields such as medicine and agriculture.

Description

Technical Field

[0001] The invention relates to the technical field of microbial secondary metabolites, and in particular to a method for activating the expression of a silent secondary metabolite synthesis gene cluster of actinomycetes. Background Art

[0002] Actinomycetes, as an important part of microorganisms, have complex morphological differentiation mechanisms and metabolic regulation networks. They can produce a large number of active substances with novel structures. They are an important source of natural medicinal active lead compounds and are widely used in basic theory and applied research. Taking Streptomyces, the main group of actinomycetes, as an example, a large number of whole genome sequencing analyses have shown that Streptomyces contains rich secondary metabolite synthesis gene clusters. Currently, about 6,000 antibiotics and pharmacologically active substances such as anti-tumor, anti-parasitic and immunosuppressive agents have been discovered. The genome sequencing results show that there are 20 to 40 biosynthetic gene clusters (BGCs) of secondary metabolites in the genome of each Streptomyces strain, which far exceeds the number of secondary metabolites actually isolated from Streptomyces. For example, the only secondary metabolites found in Streptomyces coelicolor A3(2) are actinomycin (ACT), calcium-dependent antibiotic (CDA), undecyl prodigiosin (RED) and methymycin (Mmy), while according to bioinformatics analysis, S. coelicolor A3(2) can produce more than 20 secondary metabolites. A considerable part of the reason is that most of the synthetic gene clusters are in a "silent" or low expression state due to the limitation of culture conditions, so that the corresponding secondary metabolites cannot be obtained. Therefore, how to effectively activate the silent secondary metabolite synthetic gene clusters that exist in large quantities in actinomycetes and discover new secondary metabolites is a research hotspot and difficulty in the field of microbial natural products.

[0003] In order to activate the expression of silent gene clusters in actinomycetes and obtain more new active secondary metabolites, finding and developing new technologies and methods to activate silent gene clusters has become a hot frontier in this field. At present, there are two common activation strategies for silent BGCs: one is to remove the inhibitory factors of silent BGCs in natural host bacteria, mainly including changing culture conditions, co-cultivation, editing regulatory systems, introducing strong promoters, etc.; the other is to avoid the complex regulatory system in the original bacteria, directly clone the operon encoding silent BGCs into a heterologous host for expression or reconstruct the control elements of silent BGCs and then transfer them to the heterologous host. Although these common strategies have shown certain advantages, they also show certain limitations: the traditional improvement of culture conditions and co-cultivation lack a high-throughput screening system, and the workload is large; the strategy of activating silent BGCs by genetic manipulation is not suitable for strains that are difficult to culture or difficult to genetically manipulate; although the activation strategy based on heterologous expression avoids the limiting factors of the natural host, the simple heterologous host environment usually cannot provide the regulatory network and metabolic requirements for the biosynthesis of secondary metabolites.

[0004] Studies have found that some chemicals can inhibit the activity of specific enzymes in Streptomyces, increase its growth pressure, and thus produce metabolic changes. For example, Craney et al. found that some small molecules can change the secondary metabolites of Streptomyces and increase the production of antibiotics by inhibiting fatty acid synthase. γ-Butyrolactones can increase the production of antifungal antibiotic Validamycin in S. hygroscopicus by 30%. Quorum sensing (QS) is an information exchange mechanism between microbial cells. Recent studies have found that QS not only plays a role in the communication of microbial cells of the same species, but also plays a role in the cross-species and even cross-kingdom recognition and communication of microbial cells. Therefore, QS signal molecules may be used as signal molecules for recognition between mutually antagonistic microbial cells, activating microbial antagonism-related genes for bacterial competition. Based on this hypothesis, we speculate that exogenous addition of QS signal molecules can be used to activate the expression of silent BGCs genes in actinomycetes, which is of great significance for exploring a large number of existing BGCs resources and promoting the discovery of new structural natural products.

[0005] The increasing incidence, mortality and drug resistance of pathogenic bacteria and fungal infections have made their prevention and treatment a global problem. Humans are about to face the point where there are no drugs available. It is urgent to explore new antimicrobial drugs and develop new treatment strategies. Quorum sensing signal molecules as drug targets have become the key to solving pathogenic infections and are gradually becoming a hot topic in the field of microbial quorum sensing. However, there are no reports on methods for using quorum sensing signal molecules to activate the silenced BGCs of actinomycetes. The model fungus Candida albicans and the bacterium Pseudomonas aeruginosa are common human conditional pathogens and have been one of the research hotspots in recent years. Their signal molecules such as farnesol, tyrosol and 3-oxo-C12-HSL have been widely studied for a long time and can be used as suitable representative signal molecules for testing the activation of BGCs expression of actinomycetes by quorum sensing signal molecules.

[0006] To this end, the present invention aims to provide a new method for activating the expression of silent secondary metabolite synthesis gene clusters in actinomycetes to solve the above-mentioned problems in the prior art. Summary of the invention

[0007] The purpose of the present invention is to solve the above problems and provide a method for activating the expression of silent secondary metabolite synthesis gene clusters in actinomycetes.

[0008] In order to achieve the above object, the technical solution of the present invention is as follows:

[0009] The present invention provides a method for activating the expression of silent secondary metabolite synthesis gene clusters in actinomycetes, the method comprising: using representative microbial quorum sensing signal molecules to stimulate silent actinomycete gene clusters, so that silent actinomycete BGCs are activated (the activation of gene expression is detected by qRT-PCR); wherein the quorum sensing signal molecules used in the experiment include but are not limited to Farnesol, Tyrosol, 3-oxo-C12-HSL and Tryptophol.

[0010] Furthermore, the above-mentioned quorum sensing signal molecules were used to stimulate the representative actinomycete Streptomyces coelicolor A3 (2).

[0011] The present invention also provides an application of the above-mentioned method for activating the expression of silent secondary metabolite synthesis gene clusters in actinomycetes, and the method is applied to activating silent BGCs in actinomycetes and to the synthesis of potential new antibiotics.

[0012] Furthermore, it was applied to the study of the growth status of Streptomyces coelicolor A3(2).

[0013] The present invention also provides an application of the above-mentioned method for activating the expression of a silent actinomycete secondary metabolite synthesis gene cluster, and the method is used to obtain novel actinomycete secondary metabolites and their encoded synthetic genes and proteins.

[0014] Furthermore, the nucleotide sequence encoding the synthetic protein is shown in SEQ ID NO:2.

[0015] SEQ ID NO:2:

[0016] 1.SCO3232 encodes a synthetic protein CDA peptide synthetase III, with a total length of 7254 bp. The specific nucleotide sequence is as follows:

[0017]

[0018] 2.SCO5088 encodes the synthetic protein actinorhodin polyketide beta-ketoacylsynthase subunit beta, with a total length of 1224 bp. The specific nucleotide sequence is as follows:

[0019]

[0020] 3.SCO5892 encodes a synthetic protein polyketide synthase RedL, with a total length of 6894 bp. The specific nucleotide sequence is as follows:

[0021]

[0022] 4.SCO6274 encodes a synthetic protein type I polyketide synthase, with a total length of 10731 bp. The specific nucleotide sequence is as follows:

[0023]

[0024] 5.SCO7221 encodes a synthetic protein polyketide synthase, with a total length of 1170 bp. The specific nucleotide sequence is as follows:

[0025]

[0026] 6.SCO7682 encodes a synthetic protein non-ribosomal peptide synthase, with a total length of 6690 bp. The specific nucleotide sequence is as follows:

[0027]

[0028] 7.SCO7683 encodes a synthetic protein non-ribosomal peptide synthase, with a total length of 5529 bp. The specific nucleotide sequence is as follows:

[0029]

[0030] In the scheme of the present invention, the present invention discovered that bacterial and fungal signal molecules from different sources can activate the up-regulation of expression of key secondary metabolites of Streptomyces coelicolor A3(2).

[0031] Compared with the prior art, this solution has the following beneficial effects:

[0032] The method of the present invention develops a method for activating the expression of silent gene clusters in actinomycetes by using common representative quorum sensing signal molecules Farnesol, Tyrosol, 3-oxo-C12-HSL and Tryptophol to activate silent BGCs in Streptomyces coelicolor A3 (2) (a model actinomycete research model), that is, a strategy for rapidly and efficiently activating silent BGCs in different actinomycetes. The method of the present invention will provide an effective way to fully explore new actinomycete natural products and contribute more and better new lead compounds to clinical drug research and development. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Schematic diagram of the prediction results of secondary metabolite synthesis gene clusters in the genome information of Streptomyces coelicolor A3(2) in the embodiment of the present invention;

[0034] Figure 2 is the growth curve of Streptomyces coelicolor A3(2) under normal conditions in the embodiment of the present invention;

[0035] Figure 3 It is a schematic diagram of the qRT-PCR results of activating the expression of the silent gene cluster of Streptomyces coelicolor A3(2) by using the Candida albicans signal molecules Farnesol and Tyrosol in the embodiment of the present invention;

[0036] Figure 4 Schematic diagram of qRT-PCR results of activating the expression of the silent gene cluster of Streptomyces coelicolor A3(2) by the copper-bearing Pseudomonas signal molecule 3-oxo-C12-HSL in the embodiment of the present invention;

[0037] Figure 5 Schematic diagram of qRT-PCR results of activating the expression of the silenced gene cluster of Streptomyces coelicolor A3(2) by the Saccharomyces cerevisiae signal molecule Tryptophol in an embodiment of the present invention. DETAILED DESCRIPTION

[0038] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solution of the present invention will be further described in detail below in conjunction with the embodiments of the present invention and the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0039] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present invention can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0040] Example 1: Prediction of secondary metabolites from the genome information of Streptomyces coelicolor A3(2)

[0041] The genome information of Streptomyces coelicolor A3(2) GCA_000203835.1_ASM20383v1 was predicted and analyzed using the antiSMASH online prediction website. At least 18 secondary metabolites were found, of which at least 12 may belong to PKS or NRPS antibiotics.

[0042] Example 2: Streptomyces coelicolor A3(2) growth assay

[0043] Streptomyces coelicolor A3(2) cultured to the stable phase was transferred to the fermentation medium at a ratio of 1:100, and samples were taken at fixed points. The growth curve of Streptomyces was measured and plotted by weighing the dry weight of Streptomyces cells. Streptomyces began to enter the logarithmic growth phase after 48 hours, reached the end of the logarithmic phase at around 96 hours, and entered the stable phase after 120 hours.

[0044] Example 3: Quantitative analysis of functional genes in the secondary metabolism gene cluster in Streptomyces coelicolor A3(2)

[0045] Streptomyces coelicolor A3(2) was cultured in a fermentation medium at 30°C to the mid-to-late logarithmic phase. Bacteria- and fungi-related signal molecules (100 μM Farnesol, 100 μM Tyrosol, 100 μM 3-oxo-C12-HSL, 100 μM Tryptophol) were added, respectively, and cultured for 2.5 h. The cells were then collected by centrifugation.

[0046] (1) Streptomyces coelicolor A3 (2) Total RNA was extracted using an RNA extraction kit (Tiangen Biotechnology, China) as follows:

[0047] ① Thoroughly resuspend the cells in 100 μL dd H2O containing 10 mg / mL lysozyme and incubate at room temperature for 15 min;

[0048] ② Add 350 μL of lysis buffer RL (β-mercaptoethanol has been added before use) and vortex to mix;

[0049] ③ Add 250 μL of anhydrous ethanol. Transfer the obtained solution and precipitate into the adsorption column CR3, centrifuge at 12000 rpm for 1 min, discard the waste liquid, and put the adsorption column back into the collection tube;

[0050] ④ Add 350 μL of deproteinized solution RW1 to the adsorption column CR3, centrifuge at 12000 rpm for 1 min, discard the waste liquid, and put the adsorption column back into the collection tube;

[0051] ⑤ Preparation of DNase I working solution: Take 10 μL of DNase I storage solution and put it into a new RNase-Free centrifuge tube, add 70 μL of RDD buffer and mix gently;

[0052] ⑥ Add 80 μL of DNase I working solution to the center of adsorption column CR3 and leave at room temperature for 15 minutes;

[0053] ⑦ Add 350 μL of rinse solution RW1 to the adsorption column CR3, centrifuge at 12000 rpm for 1 min, discard the waste liquid, and put the adsorption column back into the collection tube;

[0054] ⑧ Add 500 μL of deproteinized solution RW (ethanol has been added before use) to the adsorption column CR3, place at room temperature for 2 min, centrifuge at 12000 rpm for 1 min, discard the waste liquid, put the adsorption column back into the collection tube, and repeat once;

[0055] ⑨ Centrifuge at 12000 rpm for 2 min, discard the waste liquid, and place the adsorption column CR3 at room temperature for 10 min;

[0056] ⑩ Transfer the adsorption column CR3 into a new RNase-Free centrifuge tube, add 50 μL RNase-Free dd H2O to the middle part of the adsorption membrane, leave it at room temperature for 2 minutes, centrifuge it at 12000 rpm for 2 minutes, and use agarose gel electrophoresis to detect the integrity and purity of the RNA sample, and use an enzyme-labeled instrument to determine the concentration.

[0057] (2) RNA reverse transcription and quantitative analysis

[0058] First, the reverse transcription kit of TransGen was used. cDNA was generated according to the reverse transcription system in Table 1 and the reverse transcription program in Table 2. Then, the qRT-PCR reaction was performed using a LightCycler 96 thermal cycler (Roche, USA) according to the quantitative system in Table 3, the quantitative related primers in Table 4, and the quantitative program in Table 5. The hrdB gene of Streptomyces coelicolor A3 (2) was used as an internal reference.

[0059] Table 1 RNA reverse transcription system

[0060]

[0061]

[0062] Table 2 RNA reverse transcription procedure

[0063]

[0064] Table 3 Quantitative system

[0065]

[0066] Table 4 Quantitative related primers

[0067]

[0068]

[0069] Table 5 Quantification procedure

[0070]

[0071] Through the above embodiments of the present invention, the present invention has developed a method for activating the expression of silent gene clusters in actinomycetes by using a novel signal molecule by studying the effects of quorum sensing signal molecules Farnesol, Tyrosol, 3-oxo-C12-HSL and Tryptophol on the activation of silent BGCs in Streptomyces coelicolor A3 (2) (a model actinomycete research model). The method is a strategy for rapidly and efficiently activating silent BGCs in different actinomycetes, and will provide an effective way to activate a large number of silent BGCs and to further explore new actinomycete natural products, thereby contributing more and better new lead compounds to the research and development in multiple fields such as medicine and agriculture.

[0072] The above specific embodiments are merely explanations of the present invention and are not limitations of the present invention. After reading this specification, those skilled in the art may make modifications to the embodiments without any creative contribution as needed. However, such modifications are protected by the patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A method for activating the expression of a silent secondary metabolite synthesis gene cluster in actinomycetes, characterized by: The method comprises: using representative microbial quorum sensing signal molecules to stimulate the actinomycete silent gene cluster, so that the actinomycete silent BGCs are activated; wherein the microbial quorum sensing signal molecules are 100 μM Farnesol, 100 μM Tyrosol, 100 μM 3-oxo-C12-HSL, and 100 μM Tryptophol; Representative microbial quorum sensing signal molecules were used to stimulate the test actinomycete strain Streptomyces coelicolor A3 (2); The nucleotide sequence encoding the actinomycete silencing gene cluster is shown in SEQ ID NO:2.

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