Method for improving fermentation level of daunorubicin by modifying streptomyces albus ZD11-M4 strain

Abstract

The invention discloses a method for improving the fermentation level of daunorubicin by modifying a streptomyces albus ZD11-M4 strain. According to the invention, a transport protein gene drrAB2 and a DNA damage repair protein gene drrC2 from a streptomyces coeruleorubidus CICC 11043 strain are overexpressed in streptomyces albus ZD11-M4, so that the excretion efficiency and DNA damage repair capability of daunorubicin are remarkably enhanced, and the fermentation level of daunorubicin is improved: the yield of daunorubicin of an engineering strain overexpressed with drrAB2 is improved by at least 41%; and the daunorubicin yield of the engineering strain overexpressing drrC2 is increased by at least 64%. The invention provides an efficient and stable daunorubicin production strategy and has a wide industrial application prospect.

Description

Technical Field

[0001] The invention relates to the field of microbial fermentation and genetic engineering, and to the screening and overexpression of transport proteins and DNA damage repair proteins and their application in the efficient production of daunorubicin, and in particular to a method for improving the fermentation level of daunorubicin by transforming the Streptomyces albus ZD11-M4 strain. Background Art

[0002] Anthracycline antibiotics are an important class of anti-tumor drugs that can exert anti-tumor effects by embedding into DNA double strands, inhibiting topoisomerase II, and damaging cellular DNA and chromatin. Among this class of drugs, daunorubicin, doxorubicin, epirubicin, idarubicin, and valrubicin have been approved by the FDA for clinical use. As the earliest approved anthracycline antibiotic, daunorubicin is mainly produced by fermentation of Streptomyces peucetius and Streptomyces coeruleorubidus. In industrial production, the other four listed anthracycline antibiotics can be semi-chemically synthesized using daunorubicin as a precursor. Therefore, researching and promoting the efficient synthesis of daunorubicin has important industrial significance.

[0003] Streptomyces albus ZD11-M4 is a genetically engineered strain capable of heterologously synthesizing daunorubicin (patent number ZL202010905767.4). Our team cloned the daunorubicin biosynthetic gene cluster from Streptomyces coelicolor CICC 11043, and genetically engineered the gene cluster by replacing the promoter of pathway-specific regulatory factor genes, deleting synthetic branches, and blocking competitive synthetic pathways. The gene cluster was then integrated into the genome of Streptomyces albus ZD11-ΔA1 strain, and an engineered Streptomyces strain ZD11-M4 capable of heterologously synthesizing daunorubicin was constructed. The shake flask fermentation level of daunorubicin reached 22.62 mg / g, realizing the heterologous synthesis of daunorubicin for the first time.

[0004] As a drug targeting DNA, daunorubicin can not only inhibit tumor cells, but also has strong toxicity to its producing bacteria, mainly manifested in damage to its own DNA. In the middle and late stages of fermentation, daunorubicin-producing bacteria often experience a large number of mycelium deaths, resulting in the fermentation level of daunorubicin being much lower than that of antibiotics such as erythromycin, tylosin, and salinomycin. For the white Streptomyces ZD11-M4 strain that heterologously synthesizes daunorubicin, due to the natural lack of sufficient daunorubicin resistance elements, its resistance to daunorubicin is worse than that of the original daunorubicin producing bacteria S. peucetius and S. coeruleorubidus, which greatly limits the increase in the heterologous synthesis yield of daunorubicin. Therefore, it is crucial to discover daunorubicin resistance elements and transform and improve the tolerance of the engineered strain to daunorubicin for the efficient production of daunorubicin. Summary of the invention

[0005] The purpose of the present invention is to provide a method for improving the fermentation level of daunorubicin by genetically engineering the Streptomyces albus ZD11-M4 strain for heterologously synthesizing daunorubicin, targeting the important factor limiting the yield of daunorubicin, namely, the low efficiency of daunorubicin excretion by the Streptomyces albus ZD11-M4 engineered strain during the fermentation production of daunorubicin, and the resulting cell DNA damage.

[0006] To achieve the above object, the technical solution of the present invention is to provide a method for improving the fermentation level of daunorubicin by transforming the Streptomyces albus ZD11-M4 strain, and the improvement of the fermentation level of daunorubicin is achieved by any of the following methods:

[0007] (1) Overexpressing the ATP-binding cassette (ABC) transporter encoding gene drrAB2 in Streptomyces albus ZD11-M4 strain by genetic engineering to obtain an engineered strain with enhanced daunorubicin efflux capacity;

[0008] (2) Through genetic engineering methods, the DNA damage repair protein encoding gene drrC2 was overexpressed in the Streptomyces albus ZD11-M4 strain to obtain an engineered strain with enhanced DNA damage repair ability, ultimately achieving an improvement in the fermentation level of daunorubicin.

[0009] Furthermore, the amino acid sequence of the transport protein DrrAB2 is shown in SEQ ID NO.1, which is derived from Streptomyces coeruleorubidus CICC 11043, and its whole genome sequence has been uploaded to NCBI, with the genome number ASM3321621v1.

[0010] Furthermore, in (1), the genetic engineering method is specifically as follows: using the genomic DNA of Streptomyces coelicolor CICC 11043 as a template, amplifying the drrAB2 gene and its terminator fragment by PCR; connecting drrAB2 into the downstream of the kasOp* promoter of pSOK804P by seamless cloning to obtain the plasmid pdrrAB2; and introducing the plasmid pdrrAB2 into Streptomyces albus ZD11-M4 by conjugation transfer to obtain the engineered strain ZD11-M5 overexpressing drrAB2.

[0011] Furthermore, the amino acid sequence of the DNA damage repair protein DrrC2 is shown in SEQ ID NO.2, which is derived from Streptomyces coelicolor CICC 11043, and its whole genome sequence has been uploaded to NCBI, with the genome number ASM3321621v1.

[0012] Furthermore, in (2), the genetic engineering method is specifically as follows: using the genomic DNA of Streptomyces coelicolor CICC 11043 as a template, amplifying the drrC2 gene and its terminator fragment by PCR; connecting drrC2 into the downstream of the ermE*p promoter of pSOK804E by seamless cloning to obtain the plasmid pdrrC2; and introducing the plasmid pdrrC2 into Streptomyces albus ZD11-M4 by conjugation transfer to obtain the engineered strain ZD11-M6 overexpressing drrC2.

[0013] The beneficial effects of the present invention are as follows:

[0014] The present invention utilizes an innovative genetic engineering method to significantly enhance the efflux efficiency and DNA damage repair ability of daunorubicin, thereby improving the fermentation level of daunorubicin: the daunorubicin yield of the engineered strain overexpressing drrAB2 increased by 41%; the yield of the engineered strain overexpressing drrC2 increased by 64%. The DrrAB2 protein reduces the accumulation of daunorubicin in cells by enhancing the efflux ability of daunorubicin, thereby reducing cytotoxicity and increasing yield; the DrrC2 protein improves the stability and tolerance of the strain by repairing the DNA damage caused by daunorubicin. The present invention not only effectively solves the key problems in the prior art, but also provides a more efficient and stable method for producing daunorubicin. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings:

[0016] Figure 1 Schematic diagram of the construction of plasmid for overexpression of drrAB2;

[0017] Figure 2This is a comparison chart of the fermentation levels of daunorubicin between the engineered strain ZD11-M5 overexpressing drrAB2 and the starting strain ZD11-M4;

[0018] Figure 3 Schematic diagram of the construction of plasmid for overexpression of drrC2;

[0019] Figure 4 This is a comparison of the daunorubicin fermentation levels of the engineered strain ZD11-M6 overexpressing drrC2 and the starting strain ZD11-M4. DETAILED DESCRIPTION

[0020] As a DNA-targeted antibiotic, daunorubicin is autotoxic to its producing bacteria. If the daunorubicin synthesized by the producing bacteria cannot be quickly transported out of the cell, it will accumulate in the cell, resulting in the inhibition of the growth of the producing bacteria or even death. This endogenous toxicity reduces the vitality and growth rate of daunorubicin-producing bacteria, thereby significantly affecting the production of daunorubicin. Therefore, improving the efflux efficiency of daunorubicin is a key strategy to enhance cell growth and increase production.

[0021] In addition, daunorubicin can cause severe DNA damage by embedding into the DNA double helix structure and interfering with DNA replication. This damage activates the cell's stress response, leading to an excessive burden on the DNA repair system, which in turn consumes a large amount of cellular resources and reduces the energy and metabolite supply for daunorubicin synthesis. Continuous DNA damage not only causes mutations and apoptosis, but may also lead to a decrease in the genetic stability of the strain during the fermentation process, further limiting the efficient production of daunorubicin. Therefore, enhancing the DNA damage repair ability of daunorubicin-producing bacteria can help solve the above problems, thereby significantly improving the fermentation level of daunorubicin.

[0022] In view of this, the present invention improves the fermentation level of daunorubicin by screening and overexpressing two key genes, namely the transporter gene drrAB2 and the DNA damage repair protein gene drrC2, from the daunorubicin CICC 11043 strain. Specifically: First, by genetic engineering means, the drrAB2 and drrC2 genes are cloned from the daunorubicin CICC 11043, and integrated into a suitable expression vector, and transferred into the white Streptomyces ZD11-M4 strain for overexpression. This genetic engineering modification significantly improves the fermentation level of daunorubicin. Specific experimental results show that in the engineered strain overexpressing drrAB2, the shake flask fermentation level of daunorubicin is increased by 41%; in the engineered strain overexpressing drrC2, the shake flask fermentation level of daunorubicin is increased by 64%.

[0023] The present invention is further described below by way of examples. This example is implemented on the premise of the technical solution of the present invention, and detailed implementation methods and processes are given, but the protection scope of the present invention is not limited to the following examples. The experimental methods in the following examples without specifying specific conditions are based on conventional conditions or the conditions recommended by the manufacturer.

[0024] Embodiment 1

[0025] This example is a specific process for obtaining an engineered strain that overexpresses the daunorubicin transporter gene drrAB2, and its fermentation level evaluation. The specific steps are as follows:

[0026] Step 1: Construction of overexpression plasmid pdrrAB2

[0027] (1.1) Using the genomic DNA of Streptomyces coelicolor CICC 11043 (GeneBank assembly accession: ASM3321621v1) as a template, primers AB2-F / R with 20 bp homology arms introduced at both ends were used to amplify the drrAB2 gene and its terminator fragment (a total of 1883 bp) by high-fidelity PCR;

[0028] (1.2) Linearize the pSOK804 derivative plasmid pSOK804P (Ostash B, et al.. 2009. Plasmid 61: 171–175.) containing VWB integrase, spectinomycin resistance, kasOp* promoter and conjugative transfer element with restriction endonucleases AseI and EcoRV;

[0029] (1.3) By seamless cloning, drrAB2 was linked to the downstream of the kasOp* promoter of pSOK804P to obtain plasmid pdrrAB2. Figure 1 shown.

[0030] Step 2: Introduce the pdrrAB2 plasmid constructed in the first step into Streptomyces albus ZD11-M4 by conjugation transfer

[0031] (2.1) pdrrAB2 was transformed into the Escherichia coli ET12567 (pUZ8002) host by chemical transformation. A single clone was picked and inoculated into LB liquid culture medium containing 25 μg / mL chloramphenicol, 50 μg / mL kanamycin and 50 μg / mL abramycin sulfate. After overnight culture at 37°C and 220 rpm, it was transferred to fresh LB liquid culture medium containing the corresponding antibiotics at a ratio of 1:100, and then cultured at 37°C and 220 rpm until OD600≈0.6. Take the above 1.5 mL of culture solution into a sterilized Ep tube, centrifuge at 8000 rpm for 1 min, discard the supernatant, add 1 mL of antibiotic-free LB liquid culture medium to wash once, centrifuge again, repeat the wash twice, and resuspend the bacteria in 500 μL of antibiotic-free LB liquid culture medium for use;

[0032] (2.2) Take the frozen glycerol suspension of Streptomyces albus ZD11-M4 spores in an Ep tube, melt it on ice, wash it twice with TES buffer, and then resuspend the spores with 500μL TES buffer. After heat shock at 50℃ for 10min, rinse the outer surface of the Ep tube with tap water and cool it to room temperature. Add 500μL 2×YT and shake culture at 37℃ and 220rpm for germination for 2 hours. After centrifugation, remove the supernatant from the germinated spore suspension, mix it with the above 500μL Escherichia coli suspension, and spread it all on the ISP4 plate culture medium containing 10mM MgCl2. After inverted culture at 30℃ for 16-20h, spread the surface of the plate with a final concentration of 25μg / mL of nicotinic acid and a final concentration of 50μg / mL of abramycin sulfate, and continue to culture for 4-6 days until the conjugants grow;

[0033] (2.3) The conjugate was transferred to ISP4 solid culture medium containing 50 μg / mL abramycin sulfate, and after inverted culture at 30°C for 4-5 days, the mycelium was picked up and the conjugate was verified by PCR using verification primers AB2-det-F and AB2-det-R to obtain the engineered strain ZD11-M5 overexpressing drrAB2.

[0034] The primer sequences used in the above steps 1 and 2 are shown in the following table:

[0035]

[0036] Step 3: Fermentation of ZD11-M5 engineered strain

[0037] (3.1) The activated mycelium of the ZD11-M5 strain and the starting strain ZD11-M4 was inoculated into the primary seed culture medium and cultured at 30°C and 220 rpm for 30-40 hours;

[0038] (3.2) Transfer to the secondary seed culture medium at a 5% inoculum level and culture at 30°C and 220 rpm for 24-36 h;

[0039] (3.3) 10% of the inoculum was transferred to the fermentation medium. After fermentation at 30°C and 220 rpm for 5 days, the fermentation liquid was taken for extraction and yield determination.

[0040] The primary seed culture medium in step 3 above includes 3 w / v% tryptone soy broth (TSB).

[0041] The secondary seed culture medium described in the above step 3 includes 1 w / v% yeast extract, 4 w / v% glucose, 3 w / v% soybean powder, and 0.2 w / v% calcium carbonate.

[0042] The fermentation medium in the above step 3 includes 4w / v% glucose, 0.4w / v% yeast extract, 1w / v% malt extract, 5w / v% dextrin, and 5v / v% soybean oil.

[0043] Step 4: Detection of daunorubicin fermentation level of ZD11-M5 engineered strain

[0044] After fermentation, 1 mL of the fermentation broth was mixed with 9 mL of anhydrous methanol, allowed to stand for 12 h, centrifuged at 12,000 rpm for 10 min, and 200 μL of the supernatant was taken for HPLC detection.

[0045] The HPLC detection conditions are:

[0046] The HPLC detection used Shimadzu LC-20A instrument, the HPLC column selected was ZORBAX XDB-C18, the mobile phase A was 0.01 M ammonium acetate aqueous solution (pH about 4.2), the mobile phase B was pure acetonitrile, the detection wavelength was 254 nm, the column temperature was 37°C, and the elution conditions were: 0-15 min, B%=20%-50%; 15-18 min, B%=50%-90%; 28-22 min, B%=90%; 22-25 min, B%=90%-20%; 25-33 min, B%=20%.

[0047] The results are as follows Figure 2 As shown, after 5 days of fermentation in a 50 mL shake flask, the daunorubicin fermentation level of the engineered strain overexpressing drrAB2 reached 605 mg / L, which was 41% higher than that of the starting strain ZD11-M4.

[0048] Embodiment 2

[0049] This example is a specific process for obtaining an engineered strain that overexpresses the DNA damage repair protein gene drrC2, and its fermentation level evaluation. The specific operation steps are as follows:

[0050] Step 1: Construction of overexpression plasmid pdrrC2

[0051] (1.1) Using the genomic DNA of Streptomyces coelicolor CICC 11043 as a template and primers C2-F / R with 20 bp homology arms introduced at both ends, the drrC2 gene and its terminator fragment (a total of 2660 bp) were amplified by high-fidelity PCR;

[0052] (1.2) Linearize the pSOK804E derived plasmid containing VWB integrase, spectinomycin resistance, ermE*p promoter and conjugative transfer element (Ostash B, et al., 2009. Plasmid 61: 171–175) with restriction endonucleases NdeI and HindIII;

[0053] (1.3) Connect drrC2 to the downstream of the ermE*p promoter of pSOK804E by seamless cloning to obtain plasmid pdrrC2, as shown in Figure 3 shown.

[0054] Step 2: Introduce the pdrrC2 plasmid constructed in the first step into Streptomyces albus ZD11-M4 by conjugation transfer

[0055] The conjugative transfer method was consistent with that described in step 2 of Example 1, and the primers used to verify the conjugate were C2-det-F and C2-det-R. The conjugate was verified by PCR to obtain the engineered strain ZD11-M6 overexpressing drrC2.

[0056] The primer sequences used in the above steps 1 and 2 are shown in the following table:

[0057]

[0058] Step 3: Fermentation of ZD11-M6 engineered strain

[0059] (3.1) The activated mycelium of the ZD11-M6 strain and the starting strain ZD11-M4 was inoculated into the primary seed culture medium and cultured at 30°C and 220 rpm for 30-40 hours;

[0060] (3.2) Transfer to the secondary seed culture medium at a 5% inoculum level and culture at 30°C and 220 rpm for 24-36 h;

[0061] (3.3) 10% of the inoculum was transferred to the fermentation medium, and after fermentation at 30°C and 220 rpm for 7 days, the fermentation liquid was extracted and the yield was determined. The method for determining the fermentation level of daunorubicin was consistent with step 4 in Example 1.

[0062] The results are as follows Figure 4 As shown, after 7 days of fermentation in a 50 mL shake flask, the daunorubicin fermentation level of the engineered strain overexpressing drrC2 reached 730 mg / L, which was 64% higher than that of the starting strain ZD11-M4.

[0063] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A method for improving the fermentation level of daunorubicin by transforming the Streptomyces albus ZD11-M4 strain, characterized in that: The improvement of daunorubicin fermentation level can be achieved by any of the following methods: (1) Overexpressing the ATP-binding cassette transporter encoding gene drrAB2 in the Streptomyces albus ZD11-M4 strain by genetic engineering methods to obtain an engineered strain with enhanced daunorubicin efflux capacity; (2) The DNA damage repair protein encoding gene drrC2 was overexpressed in the Streptomyces albus ZD11-M4 strain through genetic engineering methods to obtain an engineered strain with enhanced DNA damage repair ability.

2. The method for improving the fermentation level of daunorubicin by transforming the albus Streptomyces ZD11-M4 strain according to claim 1, characterized in that: The amino acid sequence of the transporter protein DrrAB2 is shown in SEQ ID NO.1, and is derived from Streptomyces coelicolor CICC 11043.

3. The method for improving the fermentation level of daunorubicin by transforming the albus Streptomyces ZD11-M4 strain according to claim 1, characterized in that: In the above (1), the genetic engineering method is specifically as follows: using the genomic DNA of Streptomyces coelicolor CICC 11043 as a template, amplifying the drrAB2 gene and its terminator fragment by PCR; connecting drrAB2 into the downstream of the kasOp* promoter of pSOK804P by a seamless cloning method to obtain the plasmid pdrrAB2; and introducing the plasmid pdrrAB2 into Streptomyces albus ZD11-M4 by a conjugation transfer method to obtain the engineered strain ZD11-M5 overexpressing drrAB2.

4. The method for improving the fermentation level of daunorubicin by transforming the albus Streptomyces ZD11-M4 strain according to claim 1, characterized in that: The amino acid sequence of the DNA damage repair protein DrrC2 is shown in SEQ ID NO.2, and is derived from Streptomyces coelicolor CICC 11043.

5. The method for improving the fermentation level of daunorubicin by transforming the albus Streptomyces ZD11-M4 strain according to claim 1, characterized in that: In (2), the genetic engineering method is specifically as follows: using the genomic DNA of Streptomyces coelicolor CICC 11043 as a template, amplifying the drrC2 gene and its terminator fragment by PCR; connecting drrC2 into the downstream of the ermE*p promoter of pSOK804E by seamless cloning to obtain the plasmid pdrrC2; and introducing the plasmid pdrrC2 into Streptomyces albus ZD11-M4 by conjugation transfer to obtain the engineered strain ZD11-M6 overexpressing drrC2.

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