Methods to enhance secondary metabolite secretion based on quorum sensing signaling molecules

Abstract

This invention relates to the field of gene technology, specifically disclosing a method for enhancing the secretion of secondary metabolites based on the regulation of quorum sensing signal molecules. This method activates the expression of bacterial secondary metabolite synthesis gene clusters through bacterial-fungal interaction, comprising the following steps: (a) co-culturing bacteria and fungi in contact or non-contact mode; (b) during co-culturing, the fungi secrete the quorum sensing signal molecule farnesol; (c) the bacteria sense farnesol through a two-component system, activating the expression of secondary metabolite synthesis genes. This invention effectively overcomes the gene silencing problem caused by metabolic load or lack of regulation in natural strains by activating the originally trace expression of secondary metabolite synthesis gene clusters in bacteria using the fungal-derived quorum sensing signal molecule farnesol.

Description

Technical Field

[0001] This invention belongs to the field of gene technology, specifically relating to a method for enhancing the secretion of secondary metabolites based on quorum sensing signaling molecular regulation. Background Technology

[0002] Secondary metabolites are a class of non-essential substances synthesized by microorganisms during specific growth stages. They belong to various chemical categories, such as nonribosomal peptides, polyketides, terpenes, bacteriocins, lasso peptides, and lanothiopeptides, and possess a variety of biological functions, such as lowering cholesterol, anti-tumor activity, and antibiotic activity. Although they do not directly participate in the basic metabolism of microorganisms, they play an irreplaceable role in fields such as healthcare, industrial production, agricultural applications, and ecological regulation. Therefore, secondary metabolites, especially bacterial secondary metabolites, have long been used as a major source for searching for novel antibiotics in drug repositories.

[0003] Despite the wide range of applications for secondary metabolites, their industrial development still faces multiple challenges. The yield of secondary metabolites in natural bacterial strains is typically low, and their synthetic pathways are governed by complex regulatory networks. The synthesis of most secondary metabolites often requires the synergistic action of large enzyme complexes such as polyketide synthases and non-ribosomal peptide synthases, and these gene clusters are easily silenced in host cells due to metabolic overload or the absence of regulatory factors. Furthermore, the synthesis of secondary metabolites is regulated by multiple genes, including transcription factors and signal transduction pathways. The expression of these genes is influenced by various factors such as environmental factors and nutritional conditions, and the regulatory networks are intricate. The complexity of their gene regulatory mechanisms limits a deep understanding and precise control of their synthetic processes. Regulating gene expression patterns through signal communication during interspecies interactions is a novel approach to activating bacterial secondary metabolite synthesis gene clusters that are originally expressed in trace amounts.

[0004] Quorum sensing is a unique form of communication among microbial cells. By secreting, sensing, and responding to specific signaling molecules, microorganisms can monitor population density and coordinate gene expression to regulate various physiological behaviors based on changes in cell number. This mechanism allows microorganisms to simultaneously regulate multiple physiological processes such as bioluminescence, virulence factor production, biofilm formation, and antibiotic synthesis in response to changes in cell number in the environment, thus adapting to environmental changes and enhancing their survival competitiveness. Several quorum sensing signaling molecules have been identified, such as AHL, AI-2, and oligopeptides. Microorganisms can also utilize quorum sensing systems to regulate the synthesis of antimicrobial substances. When cell density reaches a certain threshold, the quorum sensing system is activated, initiating the expression of genes related to antimicrobial substance synthesis, leading to the synthesis and secretion of antimicrobial substances to inhibit the growth of other surrounding microorganisms and enhance their own competitiveness in the ecological environment.

[0005] Despite significant progress in quorum sensing research, numerous challenges and limitations remain. Future research should further strengthen the discovery and identification of novel signaling molecules and delve deeper into the mechanisms by which quorum sensing regulatory networks and their interactions with other metabolic regulatory networks. This will be crucial for revealing the ecological functions and regulatory mechanisms of microbial communities, but current research is still incomplete. Summary of the Invention

[0006] The purpose of this invention is to provide a method for enhancing the secretion of secondary metabolites based on quorum sensing signaling molecules, so as to solve the problems mentioned in the background art.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] A method for enhancing secondary metabolite secretion based on quorum sensing signaling molecules activates the expression of bacterial secondary metabolite synthesis gene clusters through bacterial-fungal interactions, including the following steps:

[0009] (a) Co-culturing bacteria and fungi in contact or non-contact manner;

[0010] (b) During co-culture, the fungus secretes farnesol, a quorum sensing signaling molecule;

[0011] (c) Bacteria sense farnesol through a two-component system, activating the expression of genes that synthesize secondary metabolites.

[0012] Preferably, the bacteria are enzyme-producing bacilli, and the fungus is Candida, preferably Candida krusei.

[0013] Preferably, the non-contact co-culture uses a dialysis bag to separate bacteria and fungi, allowing signaling molecules to diffuse freely while preventing direct cell contact.

[0014] Preferably, the secondary metabolite is a heat-stable antifungal factor.

[0015] Preferably, the two-component system is a two-component system encoded by the LerscA / LerscB genes.

[0016] Preferably, the method includes overexpressing the two-component system by cloning the LerscA / LerscB genes and their promoters into the expression vector pBBR1 MCS-5 and transforming them into bacteria to enhance the ability to sense farnesol.

[0017] Preferably, the concentration of farnesol added is 10–100 μM.

[0018] Preferably, the fungus is a fungal extract, and its preparation method includes:

[0019] (i) After culturing the fungus, centrifuge to collect the supernatant;

[0020] (ii) Extract the secretions from the supernatant with ethyl acetate;

[0021] (iii) After drying, dissolve in DMSO to a final concentration of 10–100 μg / mL.

[0022] Preferably, the fungus includes Fusarium oxysporum, Aspergillus niger, Penicillium, Rhizopus, or Aspergillus fumigatus.

[0023] Preferably, the method is carried out in a fermenter, and the control conditions are: temperature 28°C, stirring speed 200–300 rpm, and aeration rate 1 vvm.

[0024] Compared with the prior art, the beneficial effects of the present invention are:

[0025] (1) This invention activates the gene clusters of secondary metabolite synthesis that were originally expressed in trace amounts in bacteria by farnesol, a fungal quorum sensing signal molecule. This effectively overcomes the problem of gene silencing caused by metabolic load or lack of regulation in natural strains. This cross-border signal recognition mechanism can precisely regulate the two-component system LerscA / LerscB and significantly enhance the activity of secondary metabolic pathways.

[0026] (2) This method is compatible with both contact and non-contact co-culture modes. In particular, the non-contact design can avoid the risk of strain contamination and achieve the directional transmission of signal molecules. Fermentation tank verification has confirmed that it maintains stability in large-scale production without the need to modify strains or adjust core process parameters. At the same time, various fungal secretions can induce the synthesis of target products, which significantly expands the application scope of this method in the development of microbial active substances such as antibiotics and antitumor drugs. Attached Figure Description

[0027] Figure 1 Schematic diagrams of bacterial-fungal contact co-culture and non-contact co-culture in Embodiment 1 of the present invention; A is a schematic diagram of contact co-culture; B is a schematic diagram of non-contact co-culture;

[0028] Figure 2 Schematic diagrams of HSAF qPCR and HPLC results from bacterial-fungal contact co-culture and non-contact co-culture in Example 1 of this invention; A shows the qPCR results of the co-cultured HSAF gene; B shows the HPLC detection of the co-cultured HSAF gene.

[0029] Figure 3 A schematic diagram illustrating the effect of different concentrations of fungal extracts on enzyme-producing lysozyme HSAF in Example 2 of this invention;

[0030] Figure 4 Schematic diagram of HPLC detection of fungal extracts in Example 3 of this invention;

[0031] Figure 5 A schematic diagram of the two-component system for sensing farnesol in Example 3 of this invention; A is the upregulated two-component in the transcriptome; B is the qPCR verification of the two-component system; C & D are the HSAF gene expression levels and HPLC detection of wild-type and two-component knockout strains after exogenous addition of farnesol;

[0032] Figure 6 Schematic diagram of the verification of the overexpression strain and the results of HSAF gene expression level and biosynthesis after overexpression in Example 4 of the present invention; A is the overexpression verification; B & C are the HSAF gene expression level and HPLC detection of the overexpression strain;

[0033] Figure 7 A schematic diagram illustrating the effect of other fungal secretions on HSAF biosynthesis in Example 5 of this invention;

[0034] Figure 8 A schematic diagram of HSAF yield verification under fermenter conditions in Embodiment 6 of the present invention. Detailed Implementation

[0035] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] Example 1: Activation of HSAF Synthesis by Bacterial-Fungal Contact / Non-Contact Co-culture

[0037] Inoculum preparation:

[0038] Bacteria: Lysobacter enzymogenes were cultured in 40% TSB medium at 28°C for 12 hours, and the OD was adjusted. 600 It is 1.0;

[0039] Fungi: Candida krusei was despored from SDA plates with sterile water and the hemocytometer was adjusted to 1×10^6 CFU / mL;

[0040] Bacteria and fungi were inoculated at a 1:1 ratio and at a 1% inoculum rate into 500 mL of 10% TSB medium for both contact and non-contact co-culture. The cultures were incubated at 28°C and 200 rpm, and OD values ​​were measured at 12 h, 24 h, 36 h, and 48 h. 600 RNA was extracted from 1 mL of bacterial cells, and 10 mL of bacterial cells were extracted. The yield of HSAF was determined by HPLC.

[0041] Contact co-culture involves directly inoculating the two microorganisms into the culture medium, while non-contact co-culture involves adding a pre-autoclaved (121℃, 20 min) dialysis bag to an Erlenmeyer flask, and then adding the corresponding concentration of Candida krusei to the dialysis bag. A detailed diagram is shown below. Figure 1 As shown;

[0042] The results showed that both contact co-culture and non-contact co-culture increased the expression level and biosynthesis of the HSAF gene. Figure 2 );

[0043] The dialysis bags MD34 (8000-14000D) were purchased from Beijing Solarbio Technology Co., Ltd., with the product number YA1072. They can trap macromolecules and bacteria, providing a non-contact function.

[0044] Example 2: Dose-effect of fungal extract in promoting HSAF synthesis.

[0045] Candida krusei was cultured in PDB medium at 28°C and 200 rpm for 48 h. After centrifugation, the supernatant was collected, and an equal volume of ethyl acetate was added to extract the fungal secretions. The extracts were then dried in a fume hood and dissolved in DMSO to prepare stock solutions with concentrations of 32 mg / mL, 16 mg / mL, and 8 mg / mL. Activated enzyme-producing lysinogens were inoculated into test tubes at a 1% inoculum concentration, and 10 μL of each of the different stock solutions was added to achieve final concentrations of 64 μg / mL, 32 μg / mL, and 16 μg / mL, respectively. The control group was treated with 10 μL of DMSO.

[0046] After 12 hours of incubation, bacteria were collected and their OD values ​​were measured. 600 1 mL of bacterial cells were centrifuged to extract RNA, and the remaining bacterial cells were centrifuged and then used to extract HSAF.

[0047] The results are as follows Figure 3 As shown, the synthesis and yield of HSAF both increase in a dose-dependent manner with increasing concentration of fungal secretions.

[0048] Example 3: Verification of the farnesol regulatory mechanism

[0049] The fungal extract and pure farnesol were analyzed by HPLC, and the results are as follows: Figure 4 As shown, the substances extracted in this study were found to contain farnesol;

[0050] The two-component system that may sense farnesol was identified by transcriptomics and real-time quantitative PCR and named LerscA / LerscB. The two-component system was knocked out by secondary homologous recombination to construct the gene knockout strain LeΔrscA / LeΔrscB, and the amount and yield of HSAF were measured under the condition of exogenous farnesol.

[0051] The results are as follows Figure 5 As shown, after the addition of farnesol, LeΔrscA / LeΔrscB no longer upregulated the expression and biosynthesis of enzyme-producing lysozyme HSAF;

[0052] As can be seen from the above, the two-component system LerscA / LerscB is the key two-component system for sensing farnesol.

[0053] Example 4: Overexpression enhancement effect in a two-component system

[0054] The LerscA / LerscB gene (including the promoter) was cloned into the pBBR1 MCS-5 vector, transformed into s17-1 competent cells, and conjugated with enzyme-producing lysobacteria. PCR was used to verify whether the introduction was successful.

[0055] The successfully overexpressing strains and wild-type strains were activated, and then farnesol (final concentration 50 μM) and fungal extract (final concentration 32 μg / mL) were added and cultured, respectively. The synthesis and expression levels of HSAF were measured.

[0056] The results are as follows Figure 6 As shown, compared with wild-type strains, the overexpression strains significantly upregulated the gene expression and biosynthesis of HSAF after the exogenous addition of farnesol or fungal extracts.

[0057] Example 5: Verification of the universality of other fungal extracts

[0058] According to the method in Example 2, the activated Fusarium oxysporum, Aspergillus niger, Penicillium, Rhizopus, and Aspergillus fumigatus were resuspended in physiological saline, filtered through autoclaved gauze, and the spore count was adjusted to 10^6 CFU / mL. They were then inoculated into PDB medium at a 1% inoculum rate and cultured at 28°C.

[0059] After culturing for 48 hours, the supernatant was collected by centrifugation, and extracted with an equal proportion of ethyl acetate. After drying, the supernatant was dissolved in DMSO to prepare a stock solution of 32 mg / mL. The stock solution was then added to the culture medium of enzyme-producing lysinogens at a final concentration of 64 μg / mL. After culturing for 48 hours, HSAF extraction was performed.

[0060] The results showed that, regardless of the type of fungal extract, it could increase the biosynthesis of HSAF to a certain extent. Figure 7 );

[0061] Example 6: Scale-up verification in a fermenter;

[0062] First, the wild-type strain was fermented. The fermentation conditions in the 10L fermenter were 28℃, 300rpm, and 1vvm aeration. The fermentation conditions were set as follows: wild-type, wild-type with fungal extract, wild-type with farnesol, overexpression strain with fungal extract, and overexpression strain with farnesol.

[0063] Compared with traditional fermentation conditions, the addition of farnesol and fungal extracts effectively increases the yield of HSAF. Figure 8 (This is consistent with laboratory conditions.)

[0064] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for enhancing the secretion of secondary metabolites based on quorum sensing signaling molecules, characterized in that, Activating the expression of bacterial secondary metabolite synthesis gene clusters through bacterial-fungal interactions includes the following steps: (a) Co-culturing bacteria and fungi in contact or non-contact manner; (b) During co-culture, the fungus secretes farnesol, a quorum sensing signaling molecule; (c) Bacteria sense farnesol through a two-component system, activating the expression of genes that synthesize secondary metabolites; The bacteria are enzyme-producing bacilli, and the fungus is Candida krusei. The two-component system is a two-component system encoded by the LerscA / LerscB genes, through which the bacteria sense farnesol and initiate intracellular signal transduction; This includes overexpressing the two-component system in the bacteria: cloning the bacteria's own LerscA / LerscB genes and their promoters into the expression vector pBBR1MCS-5, and transforming them into bacteria to enhance their ability to sense farnesol; The secondary metabolite is a heat-stable antifungal factor.

2. The method for enhancing the secretion of secondary metabolites based on quorum sensing signaling molecules according to claim 1, characterized in that, The non-contact co-culture method uses dialysis bags to separate bacteria and fungi, allowing signaling molecules to diffuse freely while preventing direct cell contact.

3. The method for enhancing the secretion of secondary metabolites based on quorum sensing signaling molecules according to claim 1, characterized in that, The method is carried out in a fermenter under the following conditions: temperature 28°C, stirring speed 200–300 rpm, and aeration rate 1 vvm.

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