WO2022262384A1 - 一种提高刺糖多孢菌多杀菌素产量的方法 - Google Patents

一种提高刺糖多孢菌多杀菌素产量的方法 Download PDF

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WO2022262384A1
WO2022262384A1 PCT/CN2022/085682 CN2022085682W WO2022262384A1 WO 2022262384 A1 WO2022262384 A1 WO 2022262384A1 CN 2022085682 W CN2022085682 W CN 2022085682W WO 2022262384 A1 WO2022262384 A1 WO 2022262384A1
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gene
saccharopolyspora spinosa
spnp
spinosa
spnf
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刘天罡
刘然
邓子新
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武汉大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C12P19/60—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin
    • C12P19/62—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin the hetero ring having eight or more ring members and only oxygen as ring hetero atoms, e.g. erythromycin, spiramycin, nystatin

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  • the invention belongs to the field of genetic engineering, and in particular relates to a method for increasing the yield of spinosad of Saccharopolyspora spinosa.
  • Spinosyns are secondary metabolites obtained after aerobic fermentation of Saccharopolyspora spinosa, and are a new type of green broad-spectrum biopesticides, belonging to the Ester antibiotics.
  • the unique insecticidal mechanism of spinosyn shows the safety of biological pesticides and the rapidity of chemical pesticides, and due to the easy degradation of spinosyns, it is harmless to the environment, crops and mammals, and has no carcinogenicity, teratogenicity, or mutagenicity or neurotoxicity, so it has won the "Presidential Green Chemicals Challenge Award" in the United States three times.
  • spinosyns are still produced by aerobic fermentation by S. spinosa.
  • the purpose of the present invention is to solve the problem of low yield of spinosads produced by fermentation of Saccharopolyspora spinosa, and provide a method for increasing the yield of spinosads of Saccharopolyspora spinosa.
  • a method for improving spinosyn production of S. spinosa is to overexpress spnF gene and/or spnP gene in S. spinosa.
  • the spnF gene catalyzes the intermolecular cross-linking reaction of spinosad macrolides.
  • the spnP gene catalyzes the loading of forosamine in spinosad.
  • a Saccharopolyspora spinosa genetic engineering bacterium with high spinosyn production is the Saccharopolyspora spinosa overexpressing spnF gene and/or spnP gene.
  • the method for increasing the spinosyn production of S. spinosa is to overexpress spnF gene and/or spnP gene in S. spinosa WHU1107 strain.
  • the S. spinosyn genetically engineered strain with high spinosyn production is obtained by overexpressing spnF gene and/or spnP gene in S. spinosyn WHU1107 strain.
  • Saccharopolyspora spinosa strain WHU1107 was deposited in the China Center for Type Culture Collection (CCTCC for short, address: China. Wuhan. Wuhan University) on March 31, 2021.
  • the classification name is Saccharopolyspora spinosa WHU1107, and the preservation number is CCTCC NO : M 2021307.
  • the yield of spinosyn produced by fermentation of the S. spinosa WHU1107 strain is much higher than that of the existing reported strains. Overexpressing the spnF gene and/or spnP gene in the WHU1107 strain can further increase the spinosad yield.
  • a method for producing spinosyns comprising the following steps: inoculating the above-mentioned Saccharopolyspora spinosyn genetic engineering bacteria with high yield of spinosyns into a fermentation medium, and obtaining a fermented product containing spinosyns through fermentation.
  • the formula of the fermentation medium is as follows: 8g of glucose, 2g of cottonseed meal, 1g of protein powder, 0.5g of yeast powder, 0.4g of trisodium citrate, 0.2 g of dipotassium hydrogen phosphate, 0.3 g of calcium carbonate, 0.2 g of ammonium sulfate, 5 g of rapeseed oil, pH 7.0.
  • the fermentation conditions are: 250rpm, 28°C, and 60% humidity.
  • the present invention finds that the overexpression of spnF gene and/or spnP gene can significantly improve the spinosyn production of Saccharopolyspora spinosa.
  • the invention provides a new transformation method and new materials for the industrial production of spinosyns.
  • Fig. 1 is a graph showing the results of LC-MS detection of components in the fermentation liquid of Saccharopolyspora spinosa WHU1107 strain.
  • Fig. 2 is a graph showing the results of HPLC determination of the spinosyn content in the fermentation broth of Saccharopolyspora spinosa WHU1107 strain.
  • Fig. 3 is a construction diagram of pIB139-spnF plasmid.
  • A schematic diagram of pIB139-spnF plasmid;
  • B pIB139-spnF plasmid digestion verification analysis diagram.
  • Fig. 4 is a construction diagram of pSET152-spnP plasmid.
  • A schematic diagram of pSET152-spnP plasmid;
  • B pSET152-spnP plasmid digestion verification analysis diagram.
  • Fig. 5 is a schematic diagram of pIB139-spnF-spnP plasmid.
  • Fig. 6 is a graph showing the results of spinosad contents in different S. spinosa fermentation broths.
  • Saccharopolyspora spinosa strain WHU1107 used in the following examples has been preserved in the China Center for Type Culture Collection (CCTCC for short, address: China. Wuhan. Wuhan University) on March 31, 2021, and the classification is named Saccharopolyspora spinosa WHU1107, the deposit number is CCTCC NO: M 2021307.
  • the spinosyn output of the strain in a culture bottle filled with 25mL of fermentation medium is as high as 4g/L or more after fermentation and culture.
  • the separation method is: fill the soil with sterile water to 0.1g/mL, suspend the soil with sterile water and add 10 glasses Beads were then serially diluted 10-fold, and 10 pieces of separation medium were coated for each concentration.
  • the basic formula of the separation medium is: add 5g of glucose, 3g of yeast extract, and 10g of enzymatic casein (N-Z amine type A) to 1L of distilled water, mix and dissolve fully and adjust the pH to 7.0 with NaOH; 200mL aliquots, add 4g of agar to each bottle; sterilize at 115°C for 30min.
  • nalidixic acid to a final concentration of 25 ⁇ g/mL to inhibit the growth of Gram-negative bacteria
  • nystatin to a final concentration of 50 ⁇ g/mL to inhibit fungal growth.
  • Streptomyces samples with spore-producing colonies were selected for species preservation, and a total of 78 strains were collected.
  • Seed medium Add 1g of glucose, 1g of yeast extract, 0.2g of N-Z amine type A, 2.5g of cottonseed meal, 2g of cornstarch, 0.2g of magnesium sulfate heptahydrate, 0.1g of sulfate per 100mL of distilled water Ammonium, mix well and dissolve, then adjust the pH to 7.0 with NaOH; distribute 25mL seed medium in each bottle for seed culture; sterilize at 121°C for 30min.
  • Fermentation medium Add 8g of glucose, 2g of cottonseed cake powder, 1g of protein powder (Dolphin brand), 0.5g of yeast powder, 0.4g of trisodium citrate, 0.2g of dipotassium hydrogen phosphate, 0.3g of calcium carbonate, 0.2g of ammonium sulfate, 5g of rapeseed oil; after mixing and dissolving, adjust the pH to 7.0 with NaOH; sterilize at 121°C for 30min; divide 25mL of fermentation medium into each bottle of fermentation culture; Note: Calcium carbonate Dispensed into each bottle.
  • the second-level seeds When the second-level seeds are cultivated for 60 hours, perform a microscopic examination of the second-level seeds and observe whether the seeds are infected with bacteria.
  • the amount was transferred to a culture bottle equipped with a fermentation medium, and each bottle of bacteria was done in three parallels; the fermentation culture conditions were: 250rpm, 28°C, and 60% humidity for 12 days.
  • strain WHU1107 was identified.
  • the 16s rRNA sequence of the strain WHU1107 is shown in SEQ ID NO.1, which has the highest sequence similarity with GENBANK ACCESSION NR_024839.1, with a similarity of 99%. According to the above results, strain WHU1107 belongs to Saccharopolyspora spinosa.
  • the shake flask yield of the high-yielding strain of Mutocidal obtained through mutagenesis and other methods reported in previous literature or patents is about 1-2 g/L, which is far lower than the yield of 4 g/L of the WHU1107 strain of the present invention.
  • Saccharopolyspora spinosa genetically engineered bacterium of embodiment 2 overexpressing spnF gene and/or spnP gene
  • the attB-attP site is used for integration
  • the promoter ermEp is selected to overexpress spnF
  • the original promoter PspnP is selected to overexpress spnP.
  • the target band spnF perform Gibson assembly with the target band and the pIB139 vector digested with NdeI/EcoRV to obtain a vector for overexpressing spnF with a strong ermEp promoter, transform the assembled product into Escherichia coli DH10B competent, and randomly pick large intestine After the bacillus was cultured as a single clone, the plasmid was extracted and identified by
  • the clone fragment spnF primer (upstream primer: 5'-gtgccggttggtaggatccacatatggtgttgccaggtggcgcaccaac-3', downstream primer: 5'-cgtcgcctgctcagccgaccggcttccgcgcgcgtc-3') was used to amplify the 864bp
  • the clone fragment Pspnp-spnP primer upstream primer: 5'-ggtcggctgagcaggcgacgatcagtcttc-3', downstream primer: 5'-aacagctatgacatgattacgaattctcacggatggccatcagactg-3'
  • the assembled product is transformed into E. coli DH10B competent. After the single clone of E. coli is randomly selected and the plasmid is extracted, the correct one of the plasmids is identified by enzyme digestion After the sequencing was correct, the plasmid pIB139-spnF-spnP was successfully constructed ( FIG. 5 ).
  • the plasmids constructed above and pSET152 were respectively transformed into competent E.cooli ET12567/pUZ8002, and single clones were picked and expanded in LB medium to be used as donor bacteria for conjugative transfer, and the genus of the second parent was carried out with S. spinosa WHU1107.
  • the conjugants were picked out in ABB13 (5g/L soluble starch, 5g/L soybean peptone, 2.1g/L MOPS, 3g/L CaCO 3 , 0.01g/L thiamine hydrochloride, 0.046g/L FeSO 4 7H 2 O, 20g/L agar) plate transfer expansion culture, then pick some colonies of each zygote to TSB-M liquid medium (30g/L soybean trypsin broth, 50g/L mannitol) for activation 3 Day, extract genomic DNA as a template, use upstream primer: 5'-cagcggtggagtgcaatgtcgt-3', downstream primer: 5'-cagaggcgggatgcgaagaatg-3', the strain that can amplify the abramycin resistance gene with a size of 750bp is correct
  • the strains overexpressing spnF or spnP or spnF and spnP were named WHU1107/
  • the screened strains overexpressing spnF and spnP were fermented and tested according to the method in Example 1.
  • the results (Fig. 6) showed that the spnF gene spinosyn was overexpressed with the strong promoter ermEp compared with the control strain WHU1107/pSET1524 105 mg/L spinosyn production in a culture flask containing 25 mL of fermentation medium.
  • the yield reached 4410mg/L, and the yield increased by 7.4%; the spnP gene overexpressed with spnP original promoter produced spinosad and the yield reached 4499mg/L, and the yield increased by 9.6%.
  • the yield of spinosyn by overexpressing spnF and spnP genes at the same time can reach 4725mg/L, and the yield is increased by 15.1%.
  • Both SpnF and spnP belong to the modified genes after polyketide skeleton synthesis in spinosyn biosynthesis.
  • the spinosyn biosynthesis genes in addition to spnF and spnP, there are 7 genes that also belong to the modifier genes after polyketide skeleton synthesis , including spnJ, spnM, spnG, spnL, spnI, spnK and spnH, among which in the literature (GaoYi Tan, et al., Heterologous Biosynthesis of Spinosad: An Omics-Guided Large Polyketide Synthase Gene Cluster Reconstitution in Streptomyces[J].Acs Synth Biol, 2017.) confirmed that overexpression of spnI in Streptomyces albicans heterologously expressing spinosyn can increase the production of spinosyn.

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Abstract

本发明公开了一种提高刺糖多孢菌多杀菌素产量的方法，属于基因工程领域。本发明方法为在刺糖多孢菌中过表达spnF基因和/或spnP基因，通过在刺糖多孢菌中过表达spnF基因和/或spnP基因可获得高产多杀菌素的刺糖多孢菌基因工程菌。本发明发现过表达spnF基因和/或spnP基因可使刺糖多孢菌的多杀菌素产量得到提升。

Description

一种提高刺糖多孢菌多杀菌素产量的方法 技术领域
本发明属于基因工程领域，具体涉及一种提高刺糖多孢菌多杀菌素产量的方法。
背景技术
多杀菌素类化合物(spinosyns)是由刺糖多孢菌(Saccharopolyspora spinosa)进行有氧发酵后获得的一种次级代谢产物，是一种新型的绿色广谱生物杀虫剂，属于大环内酯类抗生素。多杀菌素独特的杀虫机理表现出生物农药的安全性和化学农药的快速性，并且由于多杀菌素的易降解性，对环境、农作物和哺乳动物无害，没有致癌、致畸、致突变或神经毒性，因此曾三次获得美国“总统绿色化学品挑战奖”。目前，多杀菌素仍由刺糖多孢菌通过有氧发酵进行生产。尽管有多个专利报道通过改良培养基或发酵罐的控制方法来提升多杀菌素的产量，但由于菌种本身发酵产量不佳，因此通过发酵优化也很难实现规模化生产，因此我国至今尚未实现该品种的产业化。
发明内容
本发明的目的在于解决现有刺糖多孢菌发酵生产多杀菌素产量低的问题，提供一种提高刺糖多孢菌多杀菌素产量的方法。
本发明的目的通过下述技术方案实现：
一种提高刺糖多孢菌多杀菌素产量的方法，为在刺糖多孢菌中过表达spnF基因和/或spnP基因。
所述的spnF基因催化多杀菌素大环内酯的分子间交联反应。
Figure PCTCN2022085682-appb-000001
所述的spnP基因催化多杀菌素中福乐糖胺的加载。
Figure PCTCN2022085682-appb-000002
一种高产多杀菌素的刺糖多孢菌基因工程菌，为过表达spnF基因和/或spnP基因的刺糖多孢菌。
在一些实施方案中，所述的提高刺糖多孢菌多杀菌素产量的方法，为在刺糖多孢菌WHU1107菌株中过表达spnF基因和/或spnP基因。所述的高产多杀菌素的刺糖多孢菌基因工程菌，通过在刺糖多孢菌WHU1107菌株中过表达spnF基因和/或spnP基因获得。
上述刺糖多孢菌WHU1107菌株已于2021年3月31日保藏于中国典型培养物保藏中心(简称CCTCC，地址：中国.武汉.武汉大学)，分类命名为Saccharopolyspora spinosa WHU1107，保藏编号为CCTCC NO：M 2021307。所述的刺糖多孢菌WHU1107菌株发酵生产多杀菌素的产量远高于现有已报道菌株，在WHU1107菌株中过表达spnF基因和/或spnP基因，可进一步提高多杀菌素的产量。
上述高产多杀菌素的刺糖多孢菌基因工程菌在生产多杀菌素中的应用。
一种生产多杀菌素的方法，包括以下步骤：将上述高产多杀菌素的刺糖多孢菌基因工程菌接种到发酵培养基中，通过发酵得到含多杀菌素的发酵物。
在一些实施方案中，所述的发酵培养基的配方为：每100mL水中加入8g的葡萄糖、2g的棉籽饼粉、1g的蛋白粉、0.5g的酵母粉、0.4g的柠檬酸三钠、0.2g的磷酸氢二钾、0.3g的碳酸钙、0.2g的硫酸铵、5g的菜籽油，pH 7.0。
在一些实施方案中，所述的发酵的条件为：250rpm、28℃、湿度60％。
本发明的优点和有益效果：本发明发现过表达spnF基因和/或spnP基因可使刺糖多孢菌的多杀菌素产量得到显著提升。本发明为多杀菌素的产业化生产提供了新的改造方法和新的材料。
附图说明
图1是LC-MS检测刺糖多孢菌WHU1107菌株发酵液成分的结果图。
图2是HPLC测定刺糖多孢菌WHU1107菌株发酵液中多杀菌素含量的结果图。
图3是pIB139-spnF质粒的构建图。A：pIB139-spnF质粒示意图；B：pIB139-spnF质粒酶切验证分析图。
图4是pSET152-spnP质粒的构建图。A：pSET152-spnP质粒示意图；B：pSET152-spnP质粒酶切验证分析图。
图5是pIB139-spnF-spnP质粒示意图。
图6是不同刺糖多孢菌发酵液中多杀菌素含量的结果图。
具体实施方式
以下实施例用于进一步说明本发明，但不应理解为对本发明的限制。若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段。
下述实施例中所使用的刺糖多孢菌WHU1107菌株已于2021年3月31日保藏于中国典型培养物保藏中心(简称CCTCC，地址：中国.武汉.武汉大学)，分类命名为Saccharopolyspora spinosa WHU1107，保藏编号为CCTCC NO：M 2021307。该菌株在装有25mL发酵培养基的培养瓶中，经发酵培养后多杀菌素的摇瓶产量高达4g/L以上。
实施例1刺糖多孢菌WHU1107菌株的筛选
1、多杀菌素菌株分离
2017年7月从多个地区采集土壤样品，将其进行放线菌分离，分离方法为：将土壤用无菌水分装成0.1g/mL，用无菌水将土壤进行悬浮后加入10颗玻璃珠，然后将其按照递减10倍进行逐级稀释，每个浓度涂布10块分离培养基。其中分离培养基的基础配方为：在1L的蒸馏水中加入5g的葡萄糖、3g的酵母提取物、10g的酶解酪蛋白(N-Z amine type A)，混合充分溶解后用NaOH调节pH为7.0；按照200mL分装，每瓶加入琼脂4g；115℃灭菌30min。灭菌完成后冷却至65℃左右，加入萘啶酮酸至终浓度25μg/mL以抑制革兰氏阴性菌生长、制霉菌素至终浓度50μg/mL以抑制真菌生长。最终能长出的菌落中，挑选菌落形态为有孢子产生的链霉菌样本进行保种，共收集78株菌。
每5株菌进行混合发酵，发酵方法如下：
(1)相关培养基
种子培养基：每100mL蒸馏水中加入1g的葡萄糖、1g的酵母提取物、0.2g的N-Z amine typeA、2.5g的棉籽饼粉、2g的玉米淀粉、0.2g的七水硫酸镁、0.1g的硫酸铵，混合充分溶解后用NaOH调节pH为7.0；种子培养每瓶分装25mL种子培养基；121℃灭菌30min。
发酵培养基：每100mL蒸馏水中加入8g的葡萄糖、2g的棉籽饼粉、1g的蛋白粉(海豚牌)、0.5g的酵母粉、0.4g的柠檬酸三钠、0.2g的磷酸氢二钾、0.3g的碳酸钙、0.2g的硫酸铵、5g的菜籽油；混合充分溶解后用NaOH调节pH为7.0；121℃灭菌30min；发酵培养每瓶分装25mL发酵培养基；注：碳酸钙分装到每瓶。
(2)发酵方法
将每个分离菌株挑取单菌落划菌于另一新平板生长，待生长到适合状态，划取1cm*1cm左右大小的菌块于种子培养基中，5株分离菌株接种于一个培养瓶(共16瓶)，250rpm、28℃、60％的湿度下培养一级种子96h后镜检观察一级种子是否染菌、以及种子的生长状态，未染菌且生长状态良好则按1％的转接量转接二级种子；二级种子培养条件同一级种子，培养到60h的时候进行二级种子镜检并观察种子是否染菌，若正常则进行发酵的转接，按5％的转接量转接到装有发酵培养基的培养瓶中，每瓶菌做三个平行；发酵培养条件为：250rpm、28℃、湿度60％的条件下培养12天。
参考文献(GaoYi Tan,et al.,Heterologous Biosynthesis of Spinosad:An Omics-Guided Large Polyketide Synthase Gene Cluster Reconstitution in Streptomyces[J].Acs Synth Biol,2017.)中的检测方法，分析发酵液中是否含有多杀菌素。发现在第1瓶和第7瓶中有多杀菌素产生，将第1瓶和第7瓶的共10株菌按照上述方法进行逐一发酵，最终发现3株可以产生多杀菌素的菌株，其产量分别为51.2mg/L、253mg/L和336mg/L，分别命名为WHU1100、WHU1101和WHU1102。
2、菌株诱变
将WHU1102的菌株斜面用生理盐水制备成孢子悬液，脱脂棉过滤后调整孢子浓度为10 -6～10 -7个/mL，取单孢子悬液10mL于无菌的直径9cm平皿内，在距15W紫外灯20cm处照射30s，然后按照递减10倍进行逐级稀释涂板。
将每20株菌分为一组，按照上述方法进行发酵筛选，共筛选约3000株菌，将其中产量最高的1组，共20株选为初步筛选菌株。将这20株菌株进行混合再按照上述方法进行第二轮诱变，同样筛选约3000株，选多杀菌素产量最高的一组20株为待筛选菌株(分别命名为WHU1103-WHU1122)。
将这20株菌株按照上述方法进行逐一发酵，其中最高产量的菌株为WHU1107，参考文献(GaoYi Tan,et al.,Heterologous Biosynthesis of Spinosad:An Omics-Guided Large Polyketide Synthase Gene Cluster Reconstitution in Streptomyces[J].Acs Synth Biol,2017.)中的检测方法，利用LC-MS检测发酵物的成分，结果(图1)显示，在发酵液中能检测到多杀菌素A和多杀菌素D；随后用HPLC(图2)分析，多杀菌素A和多杀菌素D的产量总和约为4.1g/L，其中A组分约占95％，D组分约占5％。
3、菌株WHU1107的鉴定
对菌株WHU1107进行鉴定，菌株WHU1107的16s rRNA序列如SEQ ID NO.1所示，与 GENBANK ACCESSION NR_024839.1的序列相似性最高，相似性为99％。根据以上结果，菌株WHU1107属于刺糖多孢菌(Saccharopolyspora spinosa)。
4、菌株WHU1107的保藏
菌株WHU1107，属于刺糖多孢菌(Saccharopolyspora spinosa)，已于2021年3月31日保藏于中国典型培养物保藏中心(简称CCTCC，地址：中国.武汉.武汉大学)，保藏编号为CCTCC NO：M 2021307。
5、菌株WHU1107和其他刺糖多孢菌菌株产量比较
前人文献或专利报道的经过诱变等方法获得的多杀高产菌株摇瓶产量约为1-2g/L，远低于本发明WHU1107菌株4g/L的产量。按照上述发酵方法检测市场上可购买到的刺糖多孢菌野生型菌株NRRL18395产多杀菌素的情况，其产量为78.7mg/L，其中A组分约占84％，D组分约在16％，也远低于WHU1107菌株4g/L的产量。这些结果说明WHU1107的产量高达4g/L是其自身高产多杀菌素的表现，其可作为多杀菌素高产的工业化菌株进行开发。
实施例2过表达spnF基因和/或spnP基因的刺糖多孢菌基因工程菌的构建
(1)质粒pIB139-spnF、pSET152-spnP、pIB139-spnF-spnP的构建
本实施例用attB-attP位点进行整合，选用启动子ermEp过表达spnF和选用原始启动子PspnP过表达spnP。
1)质粒pIB139-spnF构建
以提取的刺糖多孢菌WHU1107的基因组DNA作为PCR模板，用克隆片段spnF引物(上游引物：5’-gtgccggttggtaggatccacatatggtgttgccaggtggcgcaccaac-3’，下游引物：5’-tatgacatgattacgaattcgatatctcagccgaccggcttccgcgccgtc-3’)扩增大小为880bp的目的条带spnF，将该目的条带与用NdeI/EcoRV酶切后的pIB139载体进行Gibson组装期望得到用ermEp强启动子过表达spnF的载体，将组装产物转化大肠杆菌DH10B感受态，随机挑大肠杆菌单克隆培养后提取质粒后，用NcoI和EcoRI双酶切鉴定(图3B)，得到大小为4462bp，2256bp2个目的片段，挑选出酶切鉴定正确的质粒。随后用一代测序方法进行验证，验证成功的质粒即为pIB139-spnF(图3A)。
2)质粒pSET152-spnP构建
以提取的刺糖多孢菌WHU1107的基因组DNA作为PCR模板，用克隆片段Pspnp-spnP引物(上游引物：5’-gactctagagcaggcgacgatcagtcttcgcgc-3’，下游引物：5’-cgcgcggccgctcacggatggccatcagactgcccag-3’)扩增大小为1719bp的目的片段Pspnp-spnP，将该目的条带与pSET152载体同时用XbaI和NotI酶切回收后再用T4连接酶进行酶连期望得 到用spnP原始启动子PspnP过表达spnP的载体，用EcoRV酶切鉴定(图4B)得到大小为6012bp、1396bp 2个目的片段即成功构建质粒pSET152-spnP。随后用一代测序方法进行验证，验证成功的质粒即为pSET152-spnP(图4A)。
3)质粒pIB139-spnF-spnP构建
以提取的刺糖多孢菌WHU1107的基因组DNA作为PCR模板，用克隆片段spnF引物(上游引物：5’-gtgccggttggtaggatccacatatggtgttgccaggtggcgcaccaac-3’，下游引物：5’-cgtcgcctgctcagccgaccggcttccgcgccgtc-3’)扩增大小为864bp的目的条带spnF，用克隆片段Pspnp-spnP引物(上游引物：5’-ggtcggctgagcaggcgacgatcagtcttc-3’，下游引物：5’-aacagctatgacatgattacgaattctcacggatggccatcagactg-3’)扩增大小为1735bp的目的条带spnP，将该目的条带spnF和Pspnp-spnP与用NdeI/EcoRI酶切后的pIB139载体进行Gibson组装，组装产物转化大肠杆菌DH10B感受态，随机挑大肠杆菌单克隆培养后提取质粒后，酶切鉴定正确的其中一个质粒测序正确后即成功构建质粒pIB139-spnF-spnP(图5)。
(2)过表达spnF、spnP菌株的筛选
将上述构建的质粒和pSET152分别转化至感受态E.cooli ET12567/pUZ8002中，挑取单克隆用LB培养基扩大培养作为接合转移的供体菌，与刺糖多孢菌WHU1107进行二亲本的属间接合转移，挑取出接合子在ABB13(5g/L可溶性淀粉、5g/L大豆蛋白胨、2.1g/L MOPS、3g/L CaCO 3、0.01g/L盐酸硫胺素、0.046g/L FeSO 4·7H 2O、20g/L琼脂)平板转移扩大培养后，再挑取各个接合子的部分菌落至TSB-M液体培养基(30g/L大豆胰蛋白酶肉汤、50g/L甘露醇)活化3天，提取基因组DNA作模板，使用上游引物：5’-cagcggtggagtgcaatgtcgt-3’、下游引物：5’-cagaggcgggatgcgaagaatg-3’能扩增大小为750bp的阿伯拉霉素抗性基因的菌株即为正确的过表达spnF或spnP或spnF和spnP的菌株，分别命名为WHU1107/pIB139-spnF、WHU1107/pSET152-spnP、WHU1107/pIB139-spnF-spnP，以WHU1107/pSET152菌株作为对照组。
(3)过表达spnF、spnP菌株的发酵
将筛选的过表达spnF、spnP的菌株按照实施例1中的方式进行发酵培养并检测。结果(图6)表明，在装有25mL发酵培养基的培养瓶中进行发酵培养，与对照菌株WHU1107/pSET1524105mg/L的多杀菌素产量相比，用强启动子ermEp过表达spnF基因多杀菌素产量达到4410mg/L，产量提高7.4％；用spnP原始启动子过表达spnP基因多杀菌素产量达到4499mg/L，产量提高9.6％。同时过表达spnF和spnP基因多杀菌素产量达到4725mg/L，产量提高15.1％。
SpnF和spnP均属于多杀菌素生物合成中合成聚酮骨架后的修饰基因，在多杀菌素生物 合成基因中，除了spnF和spnP以外，还有7个基因也属于合成聚酮骨架后的修饰基因，包括spnJ、spnM、spnG、spnL、spnI、spnK和spnH，其中在文献(GaoYi Tan,et al.,Heterologous Biosynthesis of Spinosad:An Omics-Guided Large Polyketide Synthase Gene Cluster Reconstitution in Streptomyces[J].Acs Synth Biol,2017.)中证实在异源表达多杀菌素的白色链霉菌中过表达spnI能提升多杀菌素的产量。因此我们尝试在WHU1107中按照上述方法，以pIB139为载体分别构建利用ermEp过表达spnI、spnH和spnJ的质粒，并按照上述的接合转移方法将其转入WHU1107中，经检测，在过表达spnI的菌株中多杀菌素产量为2317mg/L，在过表达spnJ的菌株中多杀菌素产量为2259mg/L，在过表达spnH的菌株中多杀菌素产量为2301mg/L，三种菌株产量均比原始菌株产量更低。这些结果表明，在现有技术的基础上无法合理预料多杀菌素生物合成相关基因的过表达与提升刺糖多孢菌多杀菌素产量之间存在联系。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。

Claims (8)

  1. 一种提高刺糖多孢菌多杀菌素产量的方法，其特征在于：为在刺糖多孢菌中过表达spnF基因和/或spnP基因。
  2. 根据权利要求1所述的提高刺糖多孢菌多杀菌素产量的方法，其特征在于：所述的刺糖多孢菌为保藏编号为CCTCC NO：M 2021307的刺糖多孢菌(Saccharopolyspora spinosa)WHU1107。
  3. 一种高产多杀菌素的刺糖多孢菌基因工程菌，其特征在于：为过表达spnF基因和/或spnP基因的刺糖多孢菌。
  4. 根据权利要求3所述的高产多杀菌素的刺糖多孢菌基因工程菌，其特征在于：通过在保藏编号为CCTCC NO：M 2021307的刺糖多孢菌WHU1107中过表达spnF基因和/或spnP基因获得。
  5. 权利要求3或4所述的刺糖多孢菌基因工程菌在生产多杀菌素中的应用。
  6. 一种生产多杀菌素的方法，其特征在于：包括以下步骤：将权利要求3或4所述的刺糖多孢菌基因工程菌接种到发酵培养基中，通过发酵得到含多杀菌素的发酵物。
  7. 根据权利要求6所述的生产多杀菌素的方法，其特征在于：所述的发酵培养基的配方为：每100mL水中加入8g的葡萄糖、2g的棉籽饼粉、1g的蛋白粉、0.5g的酵母粉、0.4g的柠檬酸三钠、0.2g的磷酸氢二钾、0.3g的碳酸钙、0.2g的硫酸铵、5g的菜籽油，pH 7.0。
  8. 根据权利要求6所述的生产多杀菌素的方法，其特征在于：所述的发酵的条件为：250rpm、28℃、湿度60％。
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