Recombinant streptomyces capable of producing terpenes at high yield and application of recombinant streptomyces

Abstract

The invention discloses recombinant streptomyces capable of producing terpenes at high yield and application of the recombinant streptomyces, and belongs to the technical field of microorganisms. According to the invention, a complete MVA approach is heterologously introduced into streptomyces for the first time, MVK / DPMD / PMK / IDI / HMG-CoA / HMGR / AACS key enzyme genes are amplified through PCR, an expression vector containing a kasOp promoter is constructed, and a recombinant strain is obtained through conjugational transfer. By taking lycopene as a visual marker, fermentation verification shows that the terpene yield of the recombinant strain is increased by about 50% compared with that of a control group. According to the invention, promoter activation and metabolic pathway optimization are combined, the limitation of single regulation is broken, and an efficient chassis is provided for synthesis of terpenoid derivatives.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a recombinant Streptomyces that produces high levels of terpenes and its applications. Background Technology

[0002] Terpenes are a general term for compounds containing isoprene units. They are widely found in nature; to date, more than 120,000 terpenes have been discovered in animals, plants, and microorganisms. These compounds possess numerous physiological activities and are widely used in the food, cosmetics, and pharmaceutical industries. Terpenes are mainly produced through methods such as natural product extraction, chemical synthesis, and microbial fermentation. Natural product extraction primarily involves extracting, purifying, and separating the corresponding terpenes from specific plants. However, this production method is affected by many uncontrollable factors such as climate, variety, geographical location, and maturity, exhibiting significant seasonality and unstable content. Furthermore, large-scale cultivation and breeding are costly, and the content is usually relatively low. In addition, purifying and separating specific terpenes from extracts containing multiple components is technically very difficult. These factors combined contribute to the high price of many compounds with good physiological activities. Chemical synthesis methods are limited by the numerous steps involved, the high difficulty of synthesis, and the potential for pollution, thus restricting the quality, safety, and application scope of the products. Microbial fermentation primarily utilizes the biological metabolism of microorganisms to convert basic metabolites such as glucose, starch, and soybean meal into corresponding terpenoid compounds. This method is unaffected by factors such as season, region, and climate. Raw materials are readily available, the production cycle is short, the process is simple, the cost is low, product quality is controllable, the product is easy to purify, and it has high safety. Furthermore, it causes less environmental pollution, solving the problem of occupying large amounts of arable land for planting and addressing the environmental drawbacks of chemical synthesis. Most importantly, terpenoid compounds produced by fermentation are natural products, and their activity is consistent with that of active ingredients extracted from natural plants, making it considered the most promising method for terpenoid compound production.

[0003] CN119120337A (2024.12.13) discloses a method to achieve high production of terpenoids by modifying the chassis of Streptomyces. The core idea is to use homologous recombination technology to enhance the expression of key rate-limiting enzymes in the methyl erythrose phosphate (MEP) pathway, such as DXS, DXR, and GGPS, at specific sites in the genome, thereby significantly improving the synthesis efficiency of sesquiterpenes and diterpenoid precursors, and ultimately increasing the overall yield of terpenoids. However, excessive enhancement of a single module (MEP pathway) may disrupt the inherent metabolic balance of cells, leading to the accumulation of toxic intermediates and inhibiting growth.

[0004] CN105176899A (December 23, 2015) proposes a strategy for constructing high-yielding recombinant strains, focusing on effectively enhancing the expression of target genes by introducing insulator sequences and artificially synthesized strong promoters into the genome. This method is also applied to the Streptomyces chassis, particularly targeting and regulating key genes such as farnesyl diphosphate synthase (FPS) to promote terpene synthesis. The patent's examples primarily focus on lycopene production, achieving a significant increase in target product yield through optimized metabolic pathways. However, the sustained high-energy expression of constitutive strong promoters may cause a significant metabolic burden, and long-term culture can easily lead to strain degeneration or decreased productivity.

[0005] In contrast, CN119875866A (April 25, 2025) focuses on recombinant yeast, primarily for the efficient fermentation production of the sesquiterpene compound β-elemene. Its technical approach involves heterologous expression of multiple key enzyme genes in yeast, such as ERG20 (farnesyl diphosphate synthase), LsLTC2 (sesquiterpene synthase), and RrFPS (bifunctional enzyme), while downregulating the expression of squalene synthase (ERG9) to reduce competition for metabolic flux to the sterol pathway. This metabolic flux optimization design significantly improved the synthesis level of β-elemene, demonstrating the potential for industrial applications. However, the catalytic efficiency, cofactor requirements, and subcellular localization of heterologous enzymes (such as RrFPS and LsLTC2) may not be compatible with the yeast's internal environment, potentially becoming a new bottleneck.

[0006] Existing technologies have two main drawbacks: First, the selection of metabolic pathways is limited. Most related studies focus on the modification of the MEP pathway, but have not given sufficient attention to the MVA pathway carried by Streptomyces itself and its application potential, which may lead to missing opportunities to further increase terpene production; Second, the utilization of Streptomyces chassis resources is insufficient.

[0007] The MVA pathway, or mevalonate pathway, is a core metabolic pathway for the synthesis of isoprene compounds in organisms.

[0008] Given that Streptomyces naturally contains a rich variety of terpenoid resources, developing a method to increase the yield of terpenoid compounds in Streptomyces using the MVA pathway would not only help increase the target yield, but also promote the discovery and application of new terpenoid synthases and post-modification enzymes. Summary of the Invention

[0009] To address the shortcomings of existing technologies, this invention provides a recombinant Streptomyces strain that produces high levels of terpenoids and its applications. The aim is to utilize the MVA pathway derived from Streptomyces to increase the yield of terpenoid compounds in the Streptomyces chassis, thus solving the problems of limited pathway selection and yield enhancement methods in existing technologies.

[0010] On the one hand, the present invention provides a recombinant strain that produces high levels of terpenoids by heterologously introducing the complete MVA pathway into the starting strain to obtain the recombinant strain.

[0011] Specifically, the introduction of the complete MVA pathway refers to the introduction of key genes in the MVA pathway. These key genes include MVK, DPMD, PMK, IDI, HMG-CoA, HMGR, and AACS. Preferably, the key genes are derived from Streptomyces durocortorensis, and their nucleotide sequences are shown in SEQ ID No: 1 to 7 or their degenerate sequences, respectively.

[0012] Specifically, the originating bacteria include wild-type or genetically engineered prokaryotes, preferably including Streptomyces, Escherichia coli and Actinomycetes, more preferably, the Streptomyces includes wild-type Streptomyces or Streptomyces obtained through modification, mutation, mutagenesis or gene recombination, preferably, the Streptomyces is a sky blue Streptomyces.

[0013] On one hand, the present invention provides an expression vector for constructing the recombinant bacteria, the vector comprising a promoter and key genes of the MVA pathway, the key genes of the MVA pathway including MVK, DPMD, PMK, IDI, HMG-CoA, HMGR, and AACS, preferably, the promoter being kasOp. The promoter, kasOp The nucleotide sequence of the promoter is shown in SEQ ID No:8.

[0014] On one hand, the present invention provides a method for constructing the recombinant bacteria, including the step of transferring the expression vector to Streptomyces cerevisiae M1154 via Escherichia coli conjugation and screening.

[0015] Specifically, the conjugation transfer was performed using MS solid medium containing MgCl2, and the screening was performed using double resistance screening with naridinone acid and hygromycin.

[0016] On the one hand, the present invention provides the application of the recombinant bacteria or the recombinant bacteria obtained by the construction method in the production of terpenoid compounds.

[0017] Specifically, the terpenoid compounds include lycopene.

[0018] On the other hand, the present invention provides a method for producing terpenoid compounds, comprising the following steps: fermenting the recombinant bacteria or the recombinant bacteria obtained by the construction method, centrifuging the fermentation product, ultrasonically crushing it, and organic extraction.

[0019] Specifically, the fermentation culture is carried out in SM medium, with fermentation conditions of 25-33℃ and 150-300 rpm for 4-10 days. Preferably, the fermentation conditions are 30℃ and 200-250 rpm for 5-8 days.

[0020] This invention is the first to heterologously introduce a complete MVA pathway into *Streptomyces cerevisiae* M1154, significantly increasing terpene yield. Furthermore, lycopene is used as a real-time visual marker, enabling intuitive monitoring of the yield increase. Simultaneously, the abundant P450 post-modification capability of *Streptomyces cerevisiae* M1154 creates favorable conditions for the synthesis of diverse terpene derivatives. This invention not only utilizes the promoter (kasOp...) This method activates the silent lycopene gene cluster and combines it with the optimization of the MVA pathway, thus breaking the limitations of a single regulatory strategy. The fermentation method is not limited by season or region, the raw materials are inexpensive, the process is simple, the product is easy to purify and has high safety, making it suitable for industrial applications. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the structure of the pIJ10257-MVA plasmid.

[0022] Figure 2 This is a schematic diagram of the structure of the pSOK-crt plasmid.

[0023] Figure 3 This study analyzed the lycopene production of Streptomyces M1154, which integrates the MVA pathway, after fermentation in SM liquid medium for 6 days. Detailed Implementation

[0024] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result of the description. However, these embodiments are merely illustrative and do not constitute any limitation on the scope of protection defined by the claims of the present invention.

[0025] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0026] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0027] In an embodiment of the present invention, the fermentation treatment is achieved by the following method: the microorganism is subjected to basic fermentation treatment in SM medium, wherein the fermentation treatment is carried out in SM medium containing: anhydrous glucose 60 g / L, ammonium sulfate 2 g / L, manganese sulfate heptahydrate 0.1 g / L, dipotassium hydrogen phosphate 0.5 g / L, sodium chloride 2 g / L, ferrous sulfate heptahydrate 0.05 g / L, zinc sulfate heptahydrate 0.05 g / L, manganese sulfate tetrahydrate 0.05 g / L, calcium carbonate 5 g / L, yeast extract 2 g / L, pH = 7.0.

[0028] The MS solid culture medium consisted of 20 g / L soybean meal, 20 g / L mannitol, 20 g / L agar powder, and pH = 6.8.

[0029] In an embodiment of the present invention, the extraction process includes: centrifuging, ultrasonically disrupting and organically extracting the fermentation product.

[0030] Example 1: MVA-based chassis construction

[0031] Using the mevalonate pathway of Streptomyces as a reference, the MVA pathway synthases in Streptomyces durocortorensis were identified. Through HMMER analysis and comparison, the original MVA pathway synthases MVK / DPMD / PMK / IDI / HMG-CoA / HMGR / AACS were screened from the Streptomyces durocortorensis genome sequencing file. The nucleotide sequences of the genes encoding these synthases are shown in SEQ ID No:1-7.

[0032] Expression vectors were constructed, and the genes were obtained by PCR amplification. The primers used are shown in Table 1.

[0033] Table 1. PCR primer sequence information

[0034]

[0035] Plasmid construction process: PCR amplification of the target fragment, restriction enzyme digestion of the vector to be inserted, and seamless cloning. The specific construction method is as follows:

[0036] Construction of pIJ10257-MVA plasmid: Using the genome of *Streptomyces durocortorensis* as a template, MVK / DPMD / PMK (nucleotide sequences as shown in SEQ ID Nos: 1-3) was amplified using primers MVA1-F / MVA1-R (primer sequences are shown in Table 1), and IDI / HMG-CoA / HMGR / AACS (nucleotide sequences as shown in SEQ ID Nos: 4-7) was amplified using primers MVA2-F / MVA2-R. The amplification conditions were: 98℃ for 1 min pre-denaturation, followed by 25 PCR cycles of 98℃ for 10 s; 60℃ for 5 s; 68℃ for 10 s; and a final extension at 68℃ for 1 min. The plasmid containing kasOp... pIJ10257-kasOp type promoter Linearization was achieved by double digestion with KpnI / NdeI, followed by digestion at 37°C for 1 h. After product purification via gel extraction, DNA concentration was determined. The two amplified fragments were then compared with the digested pIJ10257-kasOp. The linear plasmid fragments were assembled using seamless cloning technology at 50°C for 1 hour. The resulting plasmid was then transformed into *E. coli* DH5α, and plasmid extraction yielded plasmid pIJ10257-MVA containing the MVA1-MVA2 fragments. A schematic diagram of this plasmid is shown below. Figure 1 As shown, this plasmid controls the expression of the kasOp gene. Promoter (nucleotide sequence as shown in SEQ ID No:8).

[0037] Example 2: Transformation of recombinant plasmids

[0038] The constructed and verified recombinant plasmid was transformed into E. coli ET12567 competent cells. E. coli ET12567 cells containing the recombinant plasmid were then inoculated into 5 mL of LB broth containing 0.1% hygromycin and cultured at 37°C and 250 rpm in a shaker until OD500. 600 = 0.8. Transfer 1 mL of the cultured bacterial solution to a 1.5 mL EP tube and centrifuge at 6000 rpm for 2 min at room temperature to collect the bacterial cells, discarding the supernatant. Resuspend the bacterial cells in 1 mL LB liquid medium and centrifuge again. Repeat the operation twice to remove antibiotics from the surface of E. coli. Finally, resuspend the cells in 100 μL LB liquid medium for subsequent operations.

[0039] 50 μL of *Streptomyces cerevisiae* M1154 spores were collected in a 1.5 mL EP tube and centrifuged at 6000 rpm for 2 min at room temperature. The supernatant was discarded. The spores were resuspended in 1 mL of LB liquid medium and centrifuged again. This process was repeated twice to remove antibiotics from the surface of the *Streptomyces*. Finally, 100 μL of 2x YT liquid medium was added for resuspending, and the mixture was heat-shocked at 50 °C for 10 min. The heat-shocked *Streptomyces* spores were allowed to cool to room temperature before being mixed thoroughly with *E. coli* cultured in 100 μL of LB liquid medium. The mixture was then spread evenly on MS solid medium containing 20 mM MgCl2. After the medium dried, the mixture was incubated at 30 °C for 18 h.

[0040] After 18 hours, 30 μL of naphthiocarboxylic acid and 15 μL of hygromycin were added to 1 mL of sterile water, mixed thoroughly, and then evenly spread onto a plate. After drying, the plate was placed in a 30℃ incubator for 4-6 days.

[0041] Four days later, use a toothpick to pick up single colonies of Streptomyces on the plate and transfer them to MS solid medium containing 0.1% trimethoprim and 0.05% hygromycin (6-8 colonies of each type were picked up), and incubate at 30°C for 3 days.

[0042] Three days later, colonies from the resistant solid medium were picked and transferred to tryptone soybean broth liquid medium containing 0.1% naridinolone acid and 0.05% hygromycin, and cultured for 2 days in a constant temperature shaking incubator at 30°C.

[0043] Take 500 μL of Streptomyces cultured in the test tube, centrifuge, and use 20 μL of sample to prepare reagent (PrepMan). TM The plasmid was resuspended in Ultra, boiled at 100℃ for 10 min, and centrifuged at 12000 rpm for 15 min after releasing the genome. Subsequently, it was used as a template for bacterial culture PCR verification, and positive single-clone colonies were screened for fermentation experiments. The strains with positive verification results are those from which the constructed plasmid was successfully integrated into Streptomyces, thus obtaining recombinant strains, namely, the recombinant strain M1154-M containing the MVA pathway.

[0044] SM medium: anhydrous glucose 60 g / L, ammonium sulfate 2 g / L, magnesium sulfate heptahydrate 0.1 g / L, dipotassium hydrogen sulfate 0.5 g / L, sodium chloride 2 g / L, ferrous sulfate 0.05 g / L, zinc sulfate 0.05 g / L, manganese sulfate tetrahydrate 0.05 g / L, calcium carbonate 5 g / L, yeast extract 2 g / L, pH = 7.0.

[0045] MS medium: 20 g / L soybean meal, 20 g / L mannitol, 20 g / L agar powder, pH = 7.2.

[0046] The pSOK-crt plasmid containing the lycopene genes crtE, crtI, and crtB (nucleotide sequences shown in SEQ ID Nos: 9-11) and encoding proteins with NCBI accession numbers WP_387676372.1, WP_358994520.1, and WP_354894067.1 (its plasmid map is shown in...) was used. Figure 2 (As shown) The conjugates were transferred to M1154-M and M1154 without the MVA pathway and spread evenly on MS solid medium containing 20 mM MgCl2. After the medium dried, they were incubated at 30°C for 18-20 h. After 18-20 h, 30 μL of naphthiocarboxylic acid and 30 μL of apramycin were added to 1 mL of sterile water, mixed evenly, and spread evenly on the plates. After drying, they were incubated at 30°C for another 4-6 days. After red conjugates appeared on the plates, they were transferred to MS solid medium containing 0.1% trimethoprim and 0.1% apramycin (6-8 of each type of monoclonal antibody were picked) and incubated at 30°C for 3 days.

[0047] Example 3: Fermentation of recombinant strains

[0048] The *Streptomyces coelicolor* strain M1154-M (Juan Pablo Gomez‐Escribano; Mervyn J. Bibb, Engineering Streptomyces coelicolor for heterologous expression of secondary metabolite gene cluster, Microbial Biotechnology (2011), 4(2), 207–215) was verified to have produced abundant spores on MS solid medium containing its resistance. The lycopene production level of the *Streptomyces coelicolor* transformants was then tested. The experiment was conducted in triplicate, using M1154-crt without the MVA pathway as a negative control for M1154-M crt containing the MVA pathway. The transformed red colonies containing the crt pathway were picked and cultured in tryptone soybean broth containing 0.1% nalidixic acid and 0.1% apramycin in a 30°C shaking incubator for 2–3 days. The cultured bacterial suspension was transferred to 24-well plates (3% transfer volume, SM liquid medium) and incubated again at 30°C and 250 rpm on a shaker for 6 days to complete fermentation. The fermentation broth was then centrifuged at 4000 rpm at room temperature for 10 min, the supernatant was discarded, and a red precipitate was obtained. The precipitate was freeze-dried until completely dry, then sonicated with 1 mL of acetone for 30 min. Subsequent cycles of methanol:dichloromethane (1:1) were repeated until the precipitate was colorless. The extracts were combined, dried using a nitrogen evaporator, and then dissolved in 1 mL of dichloromethane. The yield was determined by HPLC.

[0049] HPLC detection of fermentation products: Shimadzu HPLC was used, with lycopene detection wavelength of 472 nm. The sample was eluted using isocratic elution, using a C18 column. The eluent was acetonitrile:methanol:isopropanol (70%:25%:5%), the elution time was 11 min, the injection volume was 10 μL, and the flow rate was 1 mL / min. Lycopene standards were used for the determination of the standard curve.

[0050] The above fermentation results are as follows Figure 3 As shown, Streptomyces M1154-M, which integrates the MVA pathway ( Figure 3 The lycopene yield of the strain M1154-M (as shown in the figure) after 6 days of fermentation in SM liquid medium was 129 mg / L, compared with 84 mg / L for the control strain that did not integrate the MVA pathway. Figure 3 The yield of terpene metabolites was increased by nearly 50% (as shown in M1154-crt), verifying that the introduction of the MVA pathway can significantly increase the yield of terpene metabolites.

[0051] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A recombinant bacterium that produces high levels of terpenoids, characterized in that, Recombinant strains were obtained by heterologously introducing the complete MVA pathway into the starting strain.

2. The recombinant bacteria according to claim 1, characterized in that, The introduction of the complete MVA pathway refers to the introduction of key genes for the MVA pathway. These key genes include MVK, DPMD, PMK, IDI, HMG-CoA, HMGR, and AACS genes. Preferably, the key genes are derived from Streptomyces durocortorensis, and their nucleotide sequences are shown in SEQ ID No: 1-7 or their degenerate sequences, respectively.

3. The recombinant bacteria according to claim 2, characterized in that, The starting bacteria include wild-type or genetically engineered prokaryotes, preferably including Streptomyces, Escherichia coli and Actinomycetes, more preferably, the Streptomyces includes wild-type Streptomyces or Streptomyces obtained through modification, mutation, mutagenesis or gene recombination, preferably, the Streptomyces is a sky blue Streptomyces.

4. An expression vector for constructing the recombinant bacteria of claim 1, characterized in that, The vector includes a promoter and key genes of the MVA pathway, wherein the key genes of the MVA pathway include MVK, DPMD, PMK, IDI, HMG-CoA, HMGR, and AACS genes. Preferably, the promoter is kasOp. The promoter, kasOp The nucleotide sequence of the promoter is shown in SEQ ID No:

8.

5. A method for constructing the recombinant bacteria according to claim 1, characterized in that, The method includes the step of transferring the expression vector of claim 4 to Streptomyces coli M1154 via Escherichia coli conjugation.

6. The construction method according to claim 5, characterized in that, The conjugation transfer was performed using MS solid medium containing MgCl2, and screening was conducted using double resistance screening with naridinone acid and hygromycin.

7. The use of the recombinant bacteria according to any one of claims 1-3 or the recombinant bacteria obtained by the construction method according to any one of claims 5-6 in the production of terpenoid compounds.

8. The application according to claim 7, characterized in that, The terpenoids include lycopene.

9. A method for producing terpenoid compounds, characterized in that, The process includes the following steps: fermenting and culturing the recombinant bacteria as described in any one of claims 1-3 or the recombinant bacteria obtained by the construction method as described in any one of claims 5-6, and centrifuging, ultrasonically disrupting, and organically extracting the fermentation product.

10. The production method according to claim 9, characterized in that, The fermentation culture is carried out in SM medium, with fermentation conditions of 25-33℃ and 150-300rpm for 4-10 days. Preferably, the fermentation conditions are 30℃ and 200-250rpm for 5-8 days.

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