CN110066744B - Genome rearrangement high-yield bacterium and application thereof in production of natamycin - Google Patents
Genome rearrangement high-yield bacterium and application thereof in production of natamycin Download PDFInfo
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Abstract
The invention discloses a genome rearrangement high-yielding strain and application thereof in producing a tiansimycinStreptomyces sp.CB03234-GS26 has been preserved in China Center for Type Culture Collection (CCTCC) on 16.7.2018, with the preservation number of CCTCC M2018485. The invention is based on the existing high-yield strainStreptomyces sp.CB03234-S (preservation number is CCTCC M2017538) and gentamicin resistant ribosome engineering mutant strainStreptomyces sp.The genome of CB03234-G is rearranged to obtain a stable high-yield strain CB03234-GS26 of a novel alkene diyne natural product, namely, tianicin-A (TNM-A), wherein the fermentation yield of the TNM-A exceeds 38 mg/L.
Description
Technical Field
The invention relates to a genome rearrangement high-yield bacterium and application thereof in production of natamycin, belonging to the technical field of biological medicines.
Background
Natural enediynes have unique conjugated alkyne-alkene-alkyne molecular structure and super-strong biological activity, and are the most promising antitumor antibiotics, and can be divided into nine-membered cyclic enediynes including Neocarzinostatin (NCS), lidamycin (C-1027), kedarcidin, maduropepti and the like, and ten-membered cyclic enediynes including Calicheamicin (CAL), esperamicin, dynemicin (DYN) and Uncialamycin (UCM) according to the core structure. The biological activity of the enediyne antitumor antibiotics mainly depends on a DNA damage mechanism induced by the enediyne antitumor antibiotics, namely, temporary benzene ring diradicals are formed through electronic rearrangement, free radicals taking deoxyribonucleic acid carbon chains as centers are formed after nucleophilic attack is carried out on DNA minor grooves, and single-chain or double-chain DNA is broken under the action of molecular oxygen. As a class of molecules discovered to date with the strongest cytotoxicity, the enediyne natural product can be used as a warhead molecule of an antitumor antibody coupling drug (ADC), and has extremely high patent drug prospect. Of the 13 enediyne molecules currently discovered, NCS and CAL have been developed as clinical drugs, of which smacs (polystyrene-maleic acid conjugated NCS) developed in japan is mainly used for treating liver cancer, while ADC drugs, mylotarg (CD 33 monoclonal antibody conjugated CAL) and bespossa (CD 22 monoclonal antibody conjugated CAL), recently developed by the american pfeizu company, are used for treating acute myelogenous leukemia and adult relapsed or refractory B-cell precursor acute lymphoblastic leukemia, respectively.
The tiansimycin-A (tiancicin-A, abbreviated as TNM-A) is a novel ten-membered cycloenediyne antitumor antibiotic which is separated and found from a fermentation product of Streptomyces sp.CB03234 (CB 03234 for short) in 2016 by a genome mining technology, and has a structure shown as follows:
the right-hand cross in the formula has no carbon atoms.
The Tiancimycin-A has similar structure with UCM and DYN, has ultrahigh activity on various malignant tumor cells, nearly thousands of times more than the prior clinical first-line chemotherapy drug mitomycin, shows more rapid and complete tumor cell killing power, and is an ideal warhead drug molecule of an anti-tumor ADC drug, and the achievement is published in M Bio of International microorganism Authority journal (2016 (6): pii: e 02104-16). The yield of TNM-A in an original strain (Streptomyces sp.CB03234) is extremely low, only about 0.3mg/L, and the yields of other enediyne natural products reaching the industrial preparation level at present are all more than 20mg/L, so that the existing TNM-A source can not meet the application requirements of clinical research and industrial production. Meanwhile, TNM-A cannot be obtained by a traditional chemical synthesis method due to the complex and unique molecular structure of TNM-A, and is the most feasible means for preparing TNM-A at present through microbial fermentation.
Genome rearrangement mainly utilizes protoplast fusion to carry out fragment recombination and exchange in the whole genome range on microorganisms, and beneficial mutations are continuously accumulated through multiple rounds of recursive screening of fusions, so that forward evolution of target strains is realized, and the method has a plurality of obvious advantages compared with the traditional breeding method. Mutation is carried out on an original TNM-A producing strain Streptomyces sp.CB03234 based on ribosome engineering, mutant strains with different antibiotic resistances are respectively obtained, and the yield of TNM-A is obviously improved compared with that of the original strain. On the basis, the ribosome engineering mutant strain is used as a parent, genome rearrangement based on protoplast fusion is used, and a high-yield strain with further improved TNM-A yield is obtained by screening, so that an important foundation is laid for realizing large-scale production and preparation of the strain, promoting clinical early-stage researches such as TNM-A subsequent anticancer activity analysis, action mechanism and the like, and developing related anti-tumor ADC new drugs.
Disclosure of Invention
The invention solves the technical problem of improving the yield of TNM-A so as to meet the application requirement of industrial production.
The invention obtains a high-yield strain CB03234-G of TNM-A by carrying out mutagenesis on an original production strain Streptomyces sp.CB03234 of a novel enediyne natural product (TNM-A) with extremely strong activity based on gentamicin resistance, and finally obtains the high-yield strain CB03234-GS26 of TNM-A by screening based on a genome rearrangement based on protoplast fusion of CB03234-G and CB03234-S based on a high-yield strain CB03234-S of existing TNM-A (the fermentation yield reaches about 7mg/L, the strain is preserved in China center for type culture collection in 2017, 9 and 25 months and the preservation number is CCTCC M2017538).
The technical scheme of the invention is to provide a genome rearrangement high-yield mutant strain, which also belongs to Streptomyces, wherein the Streptomyces is Streptomyces sp.CB03234-GS26, is preserved in China Center for Type Culture Collection (CCTCC) at 7-16 months in 2018, and has the preservation number of CCTCC M2018485.
The preparation method of the strain CB03234-GS26 comprises the following steps:
1) Gentamicin resistance ribosome engineering mutagenesis of streptomycete CB 03234;
2) High-throughput screening of streptomycete CB03234 mutant strain bioactivity and obtaining of TNM-A high-yield strain CB 03234-G;
3) The strain CB03234-S and the strain CB03234-G are subjected to genome rearrangement to obtain the TNM-A high-yield strain CB03234-GS26.
The streptomycete CB03234-GS26 can be applied to the preparation of the natamycin-A and the derivatives thereof, and the structural formula of the natamycin-A is as follows:
the streptomyces is utilized to ferment and prepare the tiancins-A, and the culture medium used in the fermentation process comprises the following components: 40g/L soluble starch, 20g/L cottonseed meal and 0.1g/L CuSO 4 ·5H 2 O,0.005g/L NaI and 2g/L CaCO 3 The pH of the medium was 7.0, and 10g of HP20 macroporous adsorbent resin was added per liter of the medium.
According to the invention, stable TNM-A high-yield strain CB03234-G is obtained through ribosome engineering mutagenesis and high-throughput bioactivity screening, the fermentation yield of TNM-A reaches about 3.7mg/L, the strain CB03234-S and the strain CB03234-G are subjected to genome rearrangement screening to obtain the TNM-A high-yield strain CB03234-GS26, and the yield reaches about 38 mg/L.
Preservation information
The strain name is as follows: streptomycete CB03234-GS26; latin Wen Shuming: streptomyces sp.; the preservation number is: CCTCC No.: m2018485; the preservation date is as follows: 7, 7 and 16 in 2018; the preservation unit: china center for type culture Collection; the address of the depository: wuhan, wuhan university, china.
Detailed Description
The present invention will be further explained and illustrated with reference to the following examples, wherein the percentages of the eluent and the mobile phase are by volume, and the other percentages are by mass, unless otherwise specified.
Example 1: culturing and fermenting streptomycete CB03234 and detecting biological activity of TNM-A
Streptomyces CB03234 is inoculated to a Gauss No. 1 (G1) solid culture medium (the G1 solid culture medium is 10G/L of soluble starch and 0.5G/L of MgSO 4 ·7H 2 O、0.5g/L K 2 HPO 4 、1g/L NaCl、1g/L KNO 3 、0.01g/L FeSO 4 ·7H 2 O, 20g/L agar, pH = 7.0), culturing at 30 ℃ for about 8-15 days, collecting spores with a sterile 20% glycerol solution to obtain a spore suspension, and refrigerating at-80 ℃ for later use. To obtain the desired product TNM-A, 50. Mu.L of CB03234 spore suspension was inoculated into 50mL Tryptone Soya Broth (TSB) seed medium (17 g/L tryptone, 3g/L phytone, 2.5g/L K) 2 HPO 4 5g/L NaCl, 2.5g/L glucose, pH = 7.3), after 48 hours of incubation at 30 ℃ and 200rpm, 5mL of seeds were transferred to a medium containing 50mL of production medium (the production medium is: 10g/L soluble starch, 5g/L cottonseed meal and 2g/L CaCO 3 、0.05g/L CuSO 4 0.005g/L NaI, and 10g of HP20 macroporous adsorbent resin was added per liter of the medium) was cultured at 30 ℃ and 200rpm in a 250mL Erlenmeyer flask for 7 days. Centrifuging the obtained fermentation liquor, taking supernatant, performing paper sheet method test (20 mu L of fermentation supernatant/sheet) on a standard LB medium plate by taking Micrococcus luteus (Micrococcus luteus) ATCC10240 as a biological activity indicator, and preliminarily estimating the content of TNM-A in the fermentation liquor by measuring the size of a bacteriostatic zone.
Example 2: gentamicin resistant ribosome engineering mutagenesis of streptomycete CB03234
Inoculating streptomyces CB03234 to a G1 solid culture medium inclined plane, culturing for about 8-15 days at a constant temperature of 30 ℃, collecting spores by using a sterile 20% glycerol solution, filtering the obtained spore mixed solution by using a sterile sand core funnel after shaking and scattering, and counting the spores by plate sparse coating to finally prepare a spore suspension with uniform concentration. Meanwhile, preparing a gentamicin (Gen) aqueous solution with the concentration of 10mg/mL, filtering and sterilizing, and preparing G1 solid culture medium plates with different concentrations of Gen on the basis; after diluting the spore suspension of CB03234 by 50-100 times, 100 mu L of the spore suspension is taken to be evenly mixed with 5mL of G1 soft agar culture medium (other components of the culture medium are the same as G1, and the agar is 10G/L) containing Gen with different concentrations, the mixture is spread on a G1 solid culture medium plate (3 plates are cultured in parallel at each Gen concentration) containing Gen with corresponding concentration, the survival condition of colonies is observed after the culture is carried out for 4-5 days at 30 ℃, and finally the Minimum Inhibitory Concentration (MIC) of the Gen is determined to be 15mg/L. On the basis of the above studies, 4 different Gen concentrations (15 mg/L, 20mg/L, 40mg/L, 60mg/L, 80 mg/L) were selected for ribosome engineering mutagenesis of Streptomyces CB03234 (Table 2). 10G 1 plates were inoculated in parallel for each concentration according to the above inoculation procedure, and after incubation at 30 ℃ for 7-8 days, single colonies were picked.
Example 3: high-throughput screening of biological activity of streptomycete CB03234 mutant strain
Single colonies of the selected CB03234 and related mutant strains thereof are respectively inoculated into two 96-well plates G1 solid growth culture media in parallel. Culturing an inoculated 96-well plate at the constant temperature of 30 ℃ for 8-15 days, selecting an agar block corresponding to a single colony, placing the agar block on a perforated LB plate, taking Micrococcus luteus (Micrococcus luteus) ATCC10240 as a biological activity test indicator bacterium, uniformly mixing 1mL of ATCCC 10240 bacterium liquid and 4mL of LB soft agar culture medium, pouring the mixture on the LB plate with the single colony agar block to be screened, culturing the mixture at the constant temperature of 37 ℃ overnight after the mixture is solidified, taking an original strain as a reference, rapidly screening potential TNM-A high-yield strains by measuring the size of an inhibition zone corresponding to the single colony agar block, and finally screening 34 strains from 59 mutant strains for further fermentation verification (Table 2).
TABLE 2 results of ribosome engineering mutagenesis and bioactive high-throughput screening of CB03234 Strain
| Gen screening concentration (mg/L) | 15 | 20 | 40 | 60 | 80 |
| Number of growing Single colony | 10 | 20 | 4 | 18 | 7 |
| The antibacterial zone is larger than the original strain number | 3 | 9 | 0 | 17 | 5 |
Example 4: determination of TNM-A high-producing strain CB03234-G
The mutant single colony with the inhibition zone larger than that of the original strain CB03234 is transferred to 50mL Tryptone Soybean Broth (TSB) seed culture medium with corresponding concentration Gen (the TSB seed culture medium is 17g/L tryptone, 3g/L plant peptone and 2.5g/L K) 2 HPO 4 5G/L NaCl, 2.5G/L glucose, pH = 7.3), at 30 ℃ and 200rpm for 48 hours, sampling for cryopreservation and transferring to G1 solid medium slant; (remainder)The part is transferred to a medium containing 50mL of production medium (10 g/L soluble starch, 5g/L cottonseed meal and 2g/L CaCO) according to the inoculation amount of 10 percent 3 、0.1g/L CuSO 4 0.005g/L NaI, and 0.5g (1% by mass/volume, all the resin used in the examples are mass (g) to volume (mL)) of HP20 macroporous adsorbent resin was added to the flask, and the mixture was incubated at 30 ℃ and 200rpm for 7 to 10 days. After fermentation, macroporous resin is collected, methanol is soaked, the eluent is combined and concentrated to 2mL after ultrasonic elution, the TNM-A yield of mutant strains is calculated through HPLC analysis, and finally G-60-10 (CB 03234-G for short) mutant strains are screened from 34 potential high-yield strains, wherein the TNM-A yield of the mutant strains reaches about 3.7mg/L (Table 3).
TABLE 3 fermentation yields of a portion of the potential TNM-A highly productive strains
| Bacterial strains | Yield (mg/L) | Bacterial strains | Yield (mg/L) |
| G-60-3 | 1.4±0.3 | G-80-6 | 2.8±0.3 |
| G-60-7 | 2.6±0.5 | G-80-7 | 3.1±0.4 |
| G-60-10 | 3.7±0.2 | G-80-14 | 2.2±0.3 |
| G-60-14 | 1.9±0.3 | CB03234 | 0.8±0.1 |
Example 5: genomic rearrangements based on CB03234-S and CB03234-G
The TNM-A high-yield strains CB03234-S and CB03234-G are used as parents to respectively prepare corresponding high-quality protoplasts, and the main process is as follows: (1) Inoculating 0.2mL of the filtered spore suspension into 50mL of seed culture medium (added with 3g of fine glass beads and 0.1% of glycine), and culturing for 36-40 hours under the condition of a constant temperature shaker at 30 ℃ and 200 rpm; (2) After collecting mycelia by centrifugation, 15mL of P-buffer (103 g/L sucrose, 0.25g/L K as the buffer solution of P-buffer) was added 2 SO 4 、2g/L MgCl 2 ·6H 2 O，3.7g/L CaCl 2 ·2H 2 O，0.05g/L KH 2 PO 4 ) Repeatedly sucking and beating with a gun head to loosen the mycelia, and repeatedly cleaning for 3 times; (3) Suspending mycelia with 5mL of P-buffer per 0.5g mycelia, adding 100 μ L of helicase and 50 μ L of lysozyme (both 10mg/mL, prepared from P-buffer), slightly mixing, and performing enzymolysis for 3 hr at 32 deg.C under constant temperature shaking table at 100 rpm; (4) Adding 10mL of P-buffer again, sucking and beating slowly, filtering mycelium by using a sterile filter funnel (made of four layers of lens wiping paper), collecting protoplast by low-speed centrifugation (1000 rpm), cleaning by using 10mL of P-buffer, and repeating for 2 times; (5) Finally, the protoplasts were resuspended in 5mL of P-buffer and dispensed into 500. Mu.L/tube (10. Mu.L/tube) 7 -10 8 /mL). Mixing the prepared protoplast suspensions of the two parents in equal volume (500 mu L each), centrifuging at low speed, removing the supernatant, adding 5mL of 50-percent PEG-1000 fusion buffer solution, uniformly suspending, and standing in a constant-temperature water bath at 37 ℃ for 10min (gently mixing once every 3 min) for fusion; followed immediately by 5mL of P-buffer rinse,the fused protoplasts were resuspended in 5mL of P-buffer, diluted appropriately and spread on regenerated plates containing 50mg/L streptomycin and 50mg/L gentamicin (103 g/L sucrose; 10g/L glucose; 0.1g/L acid casein hydrolysate; 5g/L yeast powder; 0.25 g/LKH) 2 PO 4 ；10.12g/L MgCl 2 ·6H 2 O;5.73g/L TES;20g/L agar. Packaging and sterilizing in 100mL volume, and sequentially adding the following components: 1mL 5g/L KH 2 PO 4 ；0.8mL 367.5g/L CaCl 2 ·2H 2 O;0.7ml 1M NaOH), cultured in a 30 ℃ incubator for 7-10 days, and single colonies are selected for subsequent screening.
Example 6: screening and verification of TNM-A high-yield strain CB03234-GS26
The mutant single colony with the inhibition zone larger than that of the original strain CB03234 is transferred to 50mL Tryptone Soy Broth (TSB) seed culture medium containing streptomycin and gentamicin with corresponding concentrations (the TSB seed culture medium is 17g/L tryptone, 3g/L plant peptone and 2.5g/L K) 2 HPO 4 5g/L NaCl, 2.5g/L glucose, pH = 7.3), at 30 ℃ and 200rpm for 36-48 hours. Transferred to a medium containing 50mL of production medium (10 g/L soluble starch, 5g/L cottonseed meal, 2g/L CaCO) at an inoculum size of 10% 3 、0.1g/L CuSO 4 0.005g/L NaI, and 0.5g (1% by mass-to-volume, all examples using the resin in a mass (g) to volume (mL)) of HP20 macroporous adsorbent resin was added to the flask, and the flask was incubated at 30 ℃ and 200rpm for 7 to 10 days. After fermentation, macroporous resin is collected, methanol is soaked, the eluent is combined and concentrated to 2mL after ultrasonic elution, the TNM-A yield of mutant strains is calculated through HPLC analysis, and finally CB03234-GS26 is screened from 26 fusion mutant strains, wherein the TNM-A yield of the mutant strains reaches about 16mg/L (shown in table 4), and is improved by nearly 2.5 times compared with the yield of the original TNM-A high-yield strain CB 03234-S.
TABLE 4 TNM-A fermentation yields of partial GS fusion mutants
Subsequent stability tests show that the TNM-A yield of CB03234-GS26 in four consecutive generations under the condition of the original production medium is about 16mg/L (Table 5), and the genetic stability is good.
TABLE 5 genetic stability validation of CB03234-GS26
| Generation of spores | First generation | Second generation | Third generation | Fourth generation |
| Yield (mg/L) | 16.1±0.3 | 16.0±0.2 | 16.2±1.8 | 16.7±2.3 |
In addition, CB03234-GS26 is cultured in an optimized culture medium (the optimized culture medium is 40g/L of soluble starch, 20g/L of cottonseed meal and 0.1g/L of CuSO) 4 ·5H 2 O,0.005g/L NaI and 2g/L CaCO 3 (pH 7.0)) was further increased to 38.7. + -. 0.2mg/L, which is about 48-fold higher than the yield of 0.8mg/L of original bacterium CB 03234.
Example 7: analysis of genetic variation of CB03234-GS26
The total DNA of CB03234-GS26 was extracted for genome re-sequencing and the sequencing results were compared to the reference genome sequence of CB03234 to look for gene differences.
TABLE 6 analysis of genetic variations of CB03234-GS26
The results showed that 230 single nucleotide polymorphism mutations (SNPs) were detected in CB03234-GS26, wherein 96 non-synonymous mutations resulted in amino acid sequence changes of proteins encoded by 75 genes (Table 6); deletion of 3 gene coding regions and 2 open reading frame-shift mutations were detected, resulting in amino acid sequence changes in the 4 gene-encoded proteins (Table 6), and these mutations were uniformly distributed throughout the genome of CB03234-GS26. In addition, the genome structure of CB03234-GS26 is obviously changed, and a plurality of DNA fragments are changed by deletion, inversion, ectopy and the like. The results show that the genome rearrangement high-producing strain CB03234-GS26 has obvious genetic difference with the original strain CB 03234.
Claims (3)
1. A strain of high-producing strain with rearranged genome is characterized in that the high-producing strain with rearranged genome is streptomyces (StreptomycesStreptomyces sp.) CB03234-GS26, which is preserved in China Center for Type Culture Collection (CCTCC) at 7 and 16 months in 2018 with the preservation number of CCTCC M2018485.
2. The use of the recombinant highly productive genome strain of claim 1 in the preparation of natamycin-a, the structural formula of said natamycin-a is:
3. the use of claim 2, wherein the natamycin-A is prepared by fermentation of the recombinant highly productive genome strain of claim 1, the culture medium used in the fermentation process comprises the following components: 40g/L soluble starch, 20g/L cottonseed meal and 0.1g/L CuSO 4 ·5H 2 O，0.005g/L NaI and 2g/L CaCO 3 The pH of the medium was 7.0, and 10g of HP20 macroporous adsorbent resin was added per liter of the medium.
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