A method for dynamically regulating the content of olfactory substances in the secondary metabolism of streptomyces

Abstract

The application discloses a method for dynamically regulating the content of smell and taste substances in the secondary metabolism process of streptomyces, and relates to the technical field of molecular biology. The method for dynamically regulating the content of smell and taste substances in the secondary metabolism process of streptomyces disclosed by the application determines a first promoter and an sgRNA sequence capable of targeting a first target gene affecting the synthesis of a target compound according to the types of microorganisms and the target compound; a first recombinant plasmid is constructed by using a molecular cloning method; meanwhile, a second promoter for starting the synthesis of the target compound and a gene sequence capable of encoding a Cas protein losing cutting activity are determined, and the two are connected to obtain a regulation unit; the linearized enzyme cutting product after double enzyme cutting of the first recombinant plasmid is connected with the regulation unit to obtain a time-sequential dynamic inhibition recombinant plasmid, and after the recombinant plasmid is introduced into streptomyces, a streptomyces strain with reduced content of smell and taste (geosmin) substances in the secondary metabolism process is obtained.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology, and more specifically, relates to a method for dynamically regulating the content of odor substances in the secondary metabolism process of Streptomyces. Background Art

[0002] Traditional static regulation strategies, including gene knockout and overexpression, often lead to an imbalance in metabolic flux between cell growth and product synthesis, affecting the efficiency of product synthesis. The dynamic regulation system is an emerging metabolic flux regulation method in the field of metabolic engineering. Its main feature, which distinguishes it from static regulation, is that during the fermentation process, the engineered strain will make corresponding enzyme activity adjustments based on fermentation time, physiological state, intracellular metabolite concentration, and changes in the extracellular environment, thereby affecting the metabolic flux distribution and improving product production capacity. The dynamic regulation system has the advantages of not requiring human regulation during the fermentation process and not requiring the addition of exogenous inducers, and has significant advantages in the production of high-value-added compounds. At present, dynamic regulation strategies mainly include the following three types: dynamic regulation systems based on exogenously added inducers, pathway-dependent dynamic regulation systems, and pathway-independent dynamic regulation systems.

[0003] Geosmin is a compound with a strong earthy odor produced during metabolism by many microorganisms, including most Streptomyces species and several cyanobacteria, slime molds, and fungi. It is associated with musty or off-flavored odors in drinking water, wine, fish, and other foods. Traditional methods to reduce geosmin production include physical adsorption, chemical oxidation, biodegradation, and altering culture conditions. Unfortunately, none of these methods can completely remove geosmin, and the processes are cumbersome and often result in the production of byproducts.

[0004] With the development of biotechnology, the key metabolic pathways and regulatory genes of geosmin have been largely clarified, and genetic engineering has gradually become the mainstream of bacterial strain modification technology. Modulating the synthesis of key enzymes in microorganisms through molecular biology is the key to reducing the production of secondary metabolic odorants.

[0005] Therefore, at this stage, there is an urgent need for a method to dynamically regulate target compounds such as geosmin by reducing the diversion of cellular metabolism to the synthesis of target compounds without affecting microbial growth and primary metabolism. Summary of the Invention

[0006] In response to the aforementioned problems in the prior art, the present invention aims to provide a promoter-controlled dynamic regulation system. Another technical problem addressed by the present invention is the application of this promoter-controlled dynamic regulation system to intervene in the metabolic network of Streptomyces at appropriate times, reshaping the metabolic flux distribution between product synthesis and cell growth, and reducing the yield of target compounds in Streptomyces secondary metabolites.

[0007] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0008] A method for dynamically regulating the content of odorous substances in the secondary metabolism process of Streptomyces, comprising:

[0009] 1) Determining a first target gene that affects the synthesis of the target compound based on the type of microorganism and target compound, and determining a first promoter and an sgRNA sequence that can target the first target gene based on the first target gene;

[0010] 2) Using molecular cloning, the sgRNA sequence targeting the first target gene is cloned into the starting plasmid to construct a first recombinant plasmid;

[0011] 3) Simultaneously determining a second promoter that initiates synthesis of the target compound and a gene sequence that encodes a Cas protein that loses cleavage activity;

[0012] 4) Connecting the second promoter to a gene sequence encoding a Cas protein that has lost its cleavage activity to obtain a regulatory unit;

[0013] 5) Connecting the linearized enzyme digestion product after double enzyme digestion of the first recombinant plasmid to the regulatory unit to obtain a temporal dynamic inhibition recombinant plasmid;

[0014] 6) The temporal dynamic inhibition recombinant plasmid is introduced into Streptomyces and positive transformants are screened to obtain a Streptomyces strain with reduced odorous substance content during secondary metabolism.

[0015] The nucleotide sequence of the first promoter kasOp* is shown in SEQ ID NO.1; the nucleotide sequence of the sgRNA sequence targeting the first target gene is shown in SEQ ID NO.2; the nucleotide sequence of the second promoter bolA is shown in SEQ ID NO.3; the nucleotide sequence of the gene encoding the Cas protein dCas9 that has lost its cleavage activity is shown in SEQ ID NO.4; the nucleotide sequence of the first target gene DXS is shown in SEQ ID NO.5.

[0016] The odorous substances include geosmin.

[0017] A temporal dynamic inhibition recombinant plasmid pSET-bolA-dCas9-sgRNA is used to dynamically regulate the content of olfactory substances in the secondary metabolism of Streptomyces.

[0018] A method for constructing a recombinant plasmid pSET-bolA-dCas9-sgRNA for dynamically regulating the content of olfactory substances in the secondary metabolism of Streptomyces, comprising the following steps:

[0019] 1) Determining a first target gene that affects the synthesis of the target compound based on the type of microorganism and target compound, and determining a first promoter and an sgRNA sequence that can target the first target gene based on the first target gene;

[0020] 2) Clone the sgRNA sequence targeting the first target gene into the starting plasmid to construct a first recombinant plasmid;

[0021] 3) Simultaneously determining a second promoter that initiates synthesis of the target compound and a gene sequence encoding a Cas protein that loses cleavage activity;

[0022] 4) Connecting the second promoter to a gene sequence encoding a Cas protein that has lost its cleavage activity to obtain a regulatory unit;

[0023] 5) The linearized enzyme digestion product after double enzyme digestion of the first recombinant plasmid is connected to the regulatory unit to obtain the temporal dynamic inhibition recombinant plasmid pSET-bolA-dCas9-sgRNA.

[0024] The nucleotide sequence of the first promoter kasOp* is shown in SEQ ID NO.1; the starting plasmid is the integrative plasmid pSET152; the nucleotide sequence of the sgRNA sequence targeting the first target gene is shown in SEQ ID NO.2; the nucleotide sequence of the second promoter bolA is shown in SEQ ID NO.3; the nucleotide sequence of the gene encoding the Cas protein dCas9 that has lost its cleavage activity is shown in SEQ ID NO.4; the nucleotide sequence of the first target gene DXS is shown in SEQ ID NO.5.

[0025] Recombinant engineering strain containing the temporal dynamic inhibition recombination plasmid pSET-bolA-dCas9-sgRNA.

[0026] Application of recombinant engineered strains in fermentation-based pharmaceuticals.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] 1) The present invention determines the first target gene that affects the synthesis of the target compound according to the type of microorganism and the target compound, and determines the first promoter and the sgRNA sequence that can target the first target gene according to the first target gene; uses molecular cloning to clone the positioning unit into the starting plasmid to construct a first recombinant plasmid; at the same time, determines the second target gene that initiates the synthesis of the target compound, and determines the second promoter that initiates the synthesis of the target compound and the gene sequence that can encode the Cas protein that has lost the cleavage activity according to the second target gene; the second promoter is connected to the gene sequence that can encode the Cas protein that has lost the cleavage activity to obtain a regulatory unit; the linearized enzyme digestion product after double enzyme digestion of the first recombinant plasmid is connected to the regulatory unit to obtain a temporal dynamic inhibition recombinant plasmid.

[0029] 2) The present invention utilizes a promoter with a temporal expression pattern to drive CRISPRi, thereby inhibiting the expression of the first target gene that affects the synthesis of the target compound. This does not require the addition of exogenous inducers or the development of biosensors that can perceive intermediate metabolites. Without affecting microbial growth and primary metabolism, this method diverts less cellular metabolism toward the synthesis of the target compound, thereby achieving dynamic regulation of the target compound. Furthermore, the method provided by the present invention is universal and is expected to become a universal plasmid for reducing the production of volatile odorous substances in industrial applications of Streptomyces.

[0030] 3) Based on the dynamic temporal characteristics of the expression of key genes in the secondary metabolic gene cluster of Streptomyces itself, the present invention uses a promoter with temporal expression patterns to drive the CRISPRi system to dynamically intervene in key nodes of primary metabolism, so that cell growth is not inhibited in the early stage, and in the later stage, genes related to the secondary metabolic biosynthesis pathway are underexpressed, thereby reducing the production of secondary metabolites of Streptomyces; and the method provided by the present invention is universal and is expected to become a universal strategy for the transformation of low-pollution genetically engineered strains in industrial fermentation of Streptomyces. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the metabolic flow chart of the positive transformants of this application;

[0032] Figure 2 Schematic diagram of the temporal dynamic inhibition plasmid pSET-bolA-dCas9-sgRNA constructed in the present invention;

[0033] Figure 3 This is a graph showing the statistical analysis results of the relative content of geosmin in the wild strain (WT) and mutant strain (TB) of Streptomyces avermitilis at different times (2nd, 4th, 6th and 10th days) detected by the present invention (ns indicates p>0.05, *** indicates p<0.001, **** indicates p<0.0001). DETAILED DESCRIPTION

[0034] In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. Unless otherwise specified in the following embodiments, the technical means used are conventional means well known to those skilled in the art.

[0035] Example 1

[0036] The recombinant plasmid constructed in this application refers to the product obtained by connecting the target gene with the starting plasmid.

[0037] 1. The starting plasmid can be either an integrative plasmid or an episomal plasmid, but episomal plasmids face the problem of plasmid instability during cell division. Therefore, the starting plasmid of this application is the integrative plasmid pSET152, which is well adapted to Streptomyces.

[0038] 2. The target gene includes a localization unit, a regulatory unit, a conjugation and transfer unit, and a screening unit, wherein:

[0039] Determine a first target gene that affects the synthesis of the target compound based on the types of the microorganism and the target compound, and determine a first promoter and an sgRNA sequence that can target the first target gene based on the first target gene;

[0040] 1) The localization unit includes a first promoter and an sgRNA sequence targeting a first target gene located downstream of the first promoter:

[0041] ① The first promoter is used to initiate transcription of the downstream sequence, and a suitable promoter can be selected according to conventional techniques in the art.

[0042] ② Downstream of the first promoter is the CRISPR array, which includes the sgRNA sequence. The sgRNA sequence is used to anchor the first target gene, and the RNA it transcribes precisely pairs with the first target gene.

[0043] 2) The regulatory unit includes a second promoter and a gene sequence downstream of the second promoter that encodes a Cas protein that loses cleavage activity:

[0044] ① The second promoter has a temporal expression pattern (low expression or no expression in the early stage, and high expression in the later stage of the cell endogenous promoter), and the second promoter is used to initiate the expression of the second target gene in the microorganism, and the second target gene can express an enzyme that synthesizes the target compound.

[0045] ② Based on the fact that the Cas protein used in the present invention is dCas9, dCas9 has lost the activity of the cutting functional domain of the Cas9 protein, but retains its ability to bind to DNA. It can utilize the targeting ability of the sgRNA sequence in the CRISPR array to recognize and bind to specific DNA chains, and utilize the "roadblock effect" of the dCas9 protein to target and inhibit the transcription of the first target gene.

[0046] Example 2

[0047] The method for constructing the temporal dynamic inhibition recombinant plasmid pSET-bolA-dCas9-sgRNA in this application is as follows:

[0048] 1. Determine the positioning unit

[0049] The olfactory substances in Streptomyces are mainly terpenoids, which are synthesized through the MEP (methylerythritol 4-phosphate) pathway in Streptomyces. Therefore, the first target gene DXS was determined to be the first rate-limiting enzyme gene of the MEP pathway, and its nucleotide sequence is shown in SEQ ID NO.5.

[0050] 1) Determine the first promoter

[0051] Based on the first target gene DXS, the first promoter of the present application was determined to be kasOp*, whose nucleotide sequence is shown in SEQ ID NO. 1. The constitutive promoter kasOp* has strong transcriptional activity from the beginning of strain growth and stably transcribes the gene it regulates.

[0052] 2) sgRNA sequence targeting the first target gene

[0053] According to the first target gene DXS, CHOPCHOP (https: / / chopchop.cbu.uib.no / ) was used to design an sgRNA sequence capable of targeting the first target gene DXS, and its nucleotide sequence is shown in SEQ ID NO.2.

[0054] The sgRNA sequence targeting the first target gene DXS is the targeting unit.

[0055] 2. Determine the control unit

[0056] 1) Determine the second promoter

[0057] The second promoter of the present application was determined to be bolA, and its nucleotide sequence is shown in SEQ ID NO. 3. The transcriptional activity of the dynamic promoter bolA was greatly increased after entering the stable phase, promoting the transcription and synthesis of its controlled protein.

[0058] 2) Encoding Cas proteins that lose cleavage activity

[0059] The Cas protein that has lost its cleavage activity is encoded in this application as dCas9, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.4.

[0060] The dynamic promoter bolA was connected to the dCas9 fragment to obtain a regulatory unit. The regulatory unit fragment was 4150 bp in length and consisted of the dynamic promoter bolA fragment (43 bp in length) and the dCas9 fragment (4107 bp in length).

[0061] 2. Construction of temporal dynamic inhibition plasmid pSET-bolA-dCas9-sgRNA

[0062] Figure 2 Schematic diagram of the temporal dynamic inhibition plasmid pSET-bolA-dCas9-sgRNA constructed in the present invention.

[0063] ①Construct the first recombinant plasmid pSET-sgRNA

[0064] The targeting unit (nucleotide sequence shown in SEQ ID NO. 2) was cloned into the pSET152 plasmid using conventional molecular cloning methods. The recombinase used was from the ClonExpress Ultra One-Step Cloning Kit, and the recombination system was configured according to the manufacturer's instructions. Recombination conditions: 37°C for 15 minutes. This yielded the first recombinant plasmid, pSET-sgRNA.

[0065] ②Construction of temporal dynamic inhibition recombination plasmid pSET-bolA-dCas9-sgRNA

[0066] The first recombinant plasmid pSET-sgRNA was double-digested with restriction endonucleases to obtain a linearized enzyme digestion product;

[0067] The linearized enzyme digestion product was connected to the regulatory unit to obtain the temporal dynamic inhibition recombinant plasmid pSET-bolA-dCas9-sgRNA ( Figure 2 ).

[0068] Example 3

[0069] The method for reducing the yield of target compounds in Streptomyces secondary metabolites, the specific steps are as follows:

[0070] 1. Introduce the temporal dynamic inhibition recombinant plasmid pSET-bolA-dCas9-sgRNA into Streptomyces (Streptomyces avermitilis)

[0071] E. coli ET12567 / pUZ8002 was streaked and activated on LB plates containing the corresponding antibiotics; one E. coli colony was picked and cultured overnight at 37°C in fresh LB medium containing antibiotics (chloramphenicol at a final concentration of 25 μg / mL, kanamycin at a final concentration of 25 μg / mL, and apramycin at a final concentration of 50 μg / mL);

[0072] Scrape the Streptomyces spores (2cm 2 ), add appropriate amount of sterile water and glass beads, shake and culture at 37℃ for 30min to fully break up the spores, use a 5mL syringe, remove the piston, and put 2mL of absorbent cotton inside to filter the spore suspension vertically without pressure. After filtration, use a hemocytometer to count, add one volume of 2×YT medium, heat shock at 50℃ for 10min, pre-germination at 37℃, and check under a microscope every 30min. Stop the culture after observing that most spores have grown germ tubes. The culture time in this article is 3h. At the same time, transfer the overnight cultured E. coli to fresh LB medium with the above three antibiotics according to the inoculum volume of 2% and culture to OD = 0.4-0.6. At this time, the concentration of E. coli is about 10 8 cfu / mL, the target range was reached after about 3 h of culture in this paper;

[0073] At the same time, collect 10 mL of Streptomyces spores and E. coli culture, centrifuge at 9000 rpm for 5 minutes, remove the supernatant, collect the cells, and rinse with equal volumes of sterile water and fresh LB suspension, respectively. Centrifuge again at 9000 rpm for 5 minutes, remove the supernatant, and resuspend the spores and cells in 1 / 10 volume of sterile water and fresh LB, respectively.

[0074] Dilute Streptomyces spores moderately (about 1:100), select an appropriate dilution, mix equal volumes of E. coli and Streptomyces, and spread on MS medium containing 10mM MgCl2. Incubate at 28°C until mist appears on the surface of the medium, which takes about 18h to 22h.

[0075] The culture was removed from the 28°C incubator and covered with 1 mL of sterile water containing 40 μg of apramycin and 90 μg of nalidixic acid; varying numbers of resistant colonies grew in about 5 days, and conjugates were obtained.

[0076] 2. Screening of positive transformants

[0077] The obtained conjugates were transferred to MS solid medium containing apramycin and cultured for three generations to obtain positive transformants.

[0078] 3. Culture of positive transformants

[0079] The positive transformants were picked up and placed in ISP-2 medium and cultured in a shaking incubator at 28°C and 180 rpm.

[0080] 4. Detect the relative yield of target compound (geosin) in Streptomyces secondary metabolites

[0081] HS-SPME-GC-MS was used to detect the relative content of geosmin in the wild strain (WT) and mutant strain (TB) of Streptomyces avermitilis at different times (2nd, 4th, 6th and 10th days).

[0082] The results are as follows Figure 3 As shown, the statistical analysis results showed that the geosmin content of the wild strain (WT) and the mutant strain (TB) decreased significantly on the 4th and 6th days, while the geosmin content of the wild strain (WT) and the mutant strain (TB) did not change significantly on the 2nd and 10th days.

[0083] The above description is only illustrative of the present invention and not restrictive. Those skilled in the art will understand that many modifications, changes or equivalents may be made without departing from the spirit and scope defined by the appended claims, but all of them will fall within the scope of protection of the present invention.

Claims

1. A method for dynamically regulating the content of odorous substances in the secondary metabolism of Streptomyces, characterized in that: include: 1) According to the type of microorganism and target compound, determine the first target gene DXS that affects the synthesis of the target compound, and determine the first promoter based on the first target gene DXS and an sgRNA sequence capable of targeting a first target gene; The first promoter The nucleotide sequence of is shown in SEQ ID NO.1; the nucleotide sequence of the sgRNA sequence targeting the first target gene is shown in SEQ ID NO.2; the nucleotide sequence of the first target gene DXS is shown in SEQ ID NO.5; 2) Clone the sgRNA sequence targeting the first target gene into the starting plasmid to construct the first recombinant plasmid; The starting plasmid is the integrative plasmid pSET152; 3) Simultaneously determine the second promoter, bolA, that initiates the synthesis of the target compound and the gene sequence that encodes the Cas protein dCas9 that loses its cleavage activity; The nucleotide sequence of the second promoter bolA is shown in SEQ ID NO.3; the nucleotide sequence of the gene encoding the Cas protein dCas9 that loses cleavage activity is shown in SEQ ID NO.4; 4) Connecting the second promoter bolA to the gene sequence encoding the Cas protein dCas9 that has lost its cleavage activity to obtain a regulatory unit; 5) The linearized enzyme digestion product after double enzyme digestion of the first recombinant plasmid is connected to the regulatory unit to obtain the temporal dynamic inhibition recombinant plasmid pSET-bolA-dCas9-sgRNA; 6) Introducing the sequential dynamic inhibition recombinant plasmid pSET-bolA-dCas9-sgRNA into Streptomyces avermitilis and screening for positive transformants, thereby obtaining a Streptomyces avermitilis strain with reduced odorant content during secondary metabolism; The odorous substances include geosmin.

2. A method for constructing a recombinant plasmid pSET-bolA-dCas9-sgRNA for dynamically regulating the content of olfactory substances in the secondary metabolism of Streptomyces, characterized in that: The specific steps include: 1) According to the type of microorganism and target compound, determine the first target gene DXS that affects the synthesis of the target compound, and determine the first promoter based on the first target gene DXS and an sgRNA sequence capable of targeting a first target gene; The first promoter The nucleotide sequence of is shown in SEQ ID NO.1; the nucleotide sequence of the sgRNA sequence targeting the first target gene is shown in SEQ ID NO.2; the nucleotide sequence of the first target gene DXS is shown in SEQ ID NO.5; 2) Clone the sgRNA sequence targeting the first target gene into the starting plasmid to construct the first recombinant plasmid; The starting plasmid is the integrative plasmid pSET152; 3) Simultaneously determine the second promoter, bolA, that initiates the synthesis of the target compound and the gene sequence that encodes the Cas protein dCas9 that loses its cleavage activity; The nucleotide sequence of the second promoter bolA is shown in SEQ ID NO.3; the nucleotide sequence of the gene encoding the Cas protein dCas9 that loses cleavage activity is shown in SEQ ID NO.4; 4) Connecting the second promoter bolA to the gene sequence encoding the Cas protein dCas9 that has lost its cleavage activity to obtain a regulatory unit; 5) The linearized enzyme digestion product after double enzyme digestion of the first recombinant plasmid is connected to the regulatory unit to obtain the temporal dynamic inhibition recombinant plasmid pSET-bolA-dCas9-sgRNA.

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