农杆菌介导转化及原生质体融合法选育红曲菌Monacolin K高产株
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摘要
红曲菌发酵产物中含有一种胆固醇合成抑制剂-Monacolin K,它与土曲霉中发现的lovastatin系同一物质,都具有抑制生物体内胆固醇合成途径中关键酶HMG-CoA还原酶活性的作用,从而调节生物体内异常血脂,由于历史上红曲产品具有可直接食用的特性,因此含Monacolin K的红曲产品更为消费者接受和喜爱。三十年多来,已经发现紫色红曲菌(Monascus purpureus)、发白红曲菌(M. albidus)、丛毛红曲菌(M. pilosus)等可产生Monacolin K,但不同菌种产Monacolin K的性能不同。在长期的研究或生产过程中菌种由于不断的移接传代,而导致生产性能的退化,且与土曲霉发酵产Lovstatin的产率相比,红曲菌Monacolin K产率较低,因此需要持续不断地进行菌种选育获得高产Monacolin K的红曲菌株。为了解红曲菌基因组中Monacolin K合成相关基因,为红曲菌遗传改造奠定基础,并找出更为简便的高产Monacolin K红曲菌种的筛选方法,将分子克隆与原生质体融合育种技术相结合也许是一条可行的途径。
本课题的目的是建立根癌农杆菌介导转化红曲菌的方法,将携带遗传标记的T-DNA插入红曲菌基因组中,改变红曲菌遗传信息的排列,影响其生长和代谢,筛选高产Monacolin K突变转化子;从高产Monacolin K红曲菌突变株基因组中,根据已知的T-DNA两端保守序列,扩增出T-DNA插入位点侧翼序列,从而寻找影响Monacolin K合成的带T-DNA标签的突变基因,为研究红曲菌Monacolin K合成功能基因组及代谢调控提供基础信息;通过原生质体融合的方式选育红曲菌Monacolin K高产株并研究适合于遗传改造变异株高产Monacolin K的发酵工艺,为红曲菌工业化生产Monacolin K打下基础。
主要研究结果如下:
(1)构建了含有gpdA启动子、潮霉素抗性基因hph和trpC终止子的双元载体pCAMBIA 3300-gpdA-hph-trpC并转入根癌农杆菌GV3101;利用根癌农杆菌介导转化技术成功将潮霉素抗性基因转入发白红曲菌(M. albidus)9901,实验优化了抗生素浓度,发白红曲菌孢子浓度,根癌农杆菌细胞密度,共培养温度和时间,以及乙酰丁香酮浓度等转化条件,最终转化效率可达520个转化子/10~6个红曲孢子。对转化子进行潮霉素抗性基因PCR鉴定,结果进一步证实外源T-DNA已整合至转化子的染色体基因组中。在诸多转化子中,通过发酵实验筛选得到了一株比出发菌株发白红曲菌9901高产Monacolin K的T-DNA插入突变转化子H1。采用反向PCR方法对发白红曲菌转化子H1基因组T-DNA插入位点侧翼序列进行克隆,获得了一段0.88 kb的DNA片段,暂命名为mkWL,对DNA片段mkWL进行测序,通过与NCBI公布的丛毛红曲菌Monacolin K合成基因簇比对分析,发现与其中mkH基因(1.46 kb)部分基因序列同源性为97%。由此推断基因mkWL包含发白红曲菌Monacolin K合成关联基因。
(2)对烟色红曲菌(M. fumeus) 9908和发白红曲菌转化子H1原生质体制备与再生的条件进行研究,考察了菌龄、渗透压稳定剂、裂解酶组合、酶解时间和再生培养基等条件对这两株红曲菌原生质体制备和再生的影响。将携带潮霉素抗性的转化子H1的原生质体灭活后,与烟色红曲菌9908原生质体融合,以潮霉素抗性作为筛选标记,得到了多株融合子,并对融合子固态发酵产Monacolin K能力进行考察,筛选得到了固态发酵高产Monacolin K的红曲菌融合子HH1,此菌株Monacolin K的产量较亲本烟色红曲菌9908和转化子H1分别提高了404%和27%。
(3)对影响融合子HH1固态发酵产Monacolin K的因素进行了单因素考察,根据优化得到的发酵条件,融合子HH1以小米为原料固态发酵20 d后,Monacolin K的产量可达17.50 mg/g。采用Box-Behnken响应面分析法,进一步研究了拌料水加量、拌料水pH和接种量这三个因素对Monacolin K产量的影响,并建立了模型,模型预测的理论值与实验所得的实际值无显著性差异,说明此模型能够较好的模拟发酵实验。
本研究克隆得到了发白红曲菌Monacolin K合成相关基因的DNA片段mkWL,并采用根癌农杆菌介导转化和原生质体融合技术筛选到红曲菌Monacolin K高产株,为发白红曲菌基因遗传改造及Monacolin K工业化生产奠定良好基础。
One of cholesterol synthesis inhibitor, Monacolin K, was found in Monascus fermented products. It is the same substance as lovastatin in Aspergillus terreus. It inhibits the activity of hydroxymethyglutaryl coenzyme A reductase, which catalyzes the rate-limiting step in cholesterol biosynthesis and regulate abnormal blood liqid in organism. Over the last 30 years, many Monascus spp such as M.purpureus, M.albidus and M.pilosus were found to produce Monacolin K. Although the ability of synthesizing Monacolin K by Monascus spp degenerated with the generation passing on, and yield of Monacolin K from Monascus spp is less than A. terreus, but with its edibility, Monacolin K produced from Monascus spp seems more acceptable than from A. terreus. Constant selection and breeding of Monascus spp are necessary. In order to investigate the related gene in Monacolin K synthesis pathway and find out a simple and effective method to screen strains of Monascus spp with high Monacolin K production, the approach of combinated techniques with gene cloning and protoplast fusion may be a feasible choice.
The aim of this work was to develop the Agrobacterium tumefaciens-mediated transformation (ATMT) method in Monascus spp, and transfer the T-DNA with genetic marker into Monascus spp, expect the insertion of T-DNA would change the host DNA sequence arrangement and affect the growth and metabolism of Monascus transformants, and then the Monascus transformants with high Monanolin K could be selected and analysed. The flanking sequence of T-DNA from these transformants will be amplified to find out the related Monacolin K biosynthetic gene. Monascus fusants with enhanced Monacolin K production could be screened via proroplast fusion between parental strains with gene hph and the ability of Monacolin K production respectively, the fermentation conditions by Monascus fusants during solid-state fermentation will be optimized, and provide finally some data for industrialization of Monacolin K production.
The related results are as following:
(1) The binary vector pCAMBIA 3300-gpdA-hph-trpC was constructed and transformed into Agrobacterium tumefaciens GV3101. The exogenous gene gpdA-hph-trpC was transformed into M. albidus by ATMT. Various influencing factors on transformation efficiency were optimized including the concentration of antibiotics, concentration of M. albidus spores, cell density of A. tumefaciens, co-cultivation time, temperature and the concentration of acetosyringone. The highest transformation frequency was about 5.2×104 . Some of the transformants were selected for PCR analysis, and the results further confirmed that the transformants tested were arbitrarily integrated with exogenous T-DNA in genome. Among many transformants, one transformant H1 with high Monacolin K production was selected, from which the flanking sequence mkWL (0.88 kb) of T-DNA by inverse PCR was obtained. The result of homology between the gene mkWL and Monacolin K biosynthetic gene cluster from M. albidus in the Genbank was analyzed, which revealed that there was identity of 97% at DNA sequence homology. The gene mkWL was deduced to be the related gene in Monacolin K biosynthetic pathway of M. albidus.
(2) Main factors such as mycelium age, osmotic stabilizer, different enzymatic combinations, duration of enzyme treatment and regeneration medium for efficient protoplast formation and regeneration from transformant H1 and M. fumeus 9908 were investigated. Monascus fusants HH1 with high Monacolin K production were selected via protoplast fusion between tansformant H1 and M. fumeus 9908, and the concentration of Monacolin K in fermented millet by fusant HH1 increased by 404% and 27% compared with that produced by parental Monascus spp.
(3) The fermentation conditions for promotion Monacolin K production by solid-state fermentation were optimized, and the Monacolin K yield by fusant HH1 reached to 17.50 mg/g after fermentation for 20 d. The response surface methodology of Box-Behnken was applied to further optimize the fermentation conditions including water volume, pH of water and inoculant volume. A model was constructed, and further experiments were carried out to verify the accuracy and reliability of the model, the results showed that the predicted value was agreed well to the experimental value.
The gene mkWL related to Monacolin K biosynthesis in M. albidus was amplified, and Monascus fusants with high Monacolin K production were bred via Agrobacterium -mediated transformation and protoplast fusion, these works laid a good foundation for genetic modification and industrialization of Monacolin K fermentation of M. albidus.
本课题的目的是建立根癌农杆菌介导转化红曲菌的方法,将携带遗传标记的T-DNA插入红曲菌基因组中,改变红曲菌遗传信息的排列,影响其生长和代谢,筛选高产Monacolin K突变转化子;从高产Monacolin K红曲菌突变株基因组中,根据已知的T-DNA两端保守序列,扩增出T-DNA插入位点侧翼序列,从而寻找影响Monacolin K合成的带T-DNA标签的突变基因,为研究红曲菌Monacolin K合成功能基因组及代谢调控提供基础信息;通过原生质体融合的方式选育红曲菌Monacolin K高产株并研究适合于遗传改造变异株高产Monacolin K的发酵工艺,为红曲菌工业化生产Monacolin K打下基础。
主要研究结果如下:
(1)构建了含有gpdA启动子、潮霉素抗性基因hph和trpC终止子的双元载体pCAMBIA 3300-gpdA-hph-trpC并转入根癌农杆菌GV3101;利用根癌农杆菌介导转化技术成功将潮霉素抗性基因转入发白红曲菌(M. albidus)9901,实验优化了抗生素浓度,发白红曲菌孢子浓度,根癌农杆菌细胞密度,共培养温度和时间,以及乙酰丁香酮浓度等转化条件,最终转化效率可达520个转化子/10~6个红曲孢子。对转化子进行潮霉素抗性基因PCR鉴定,结果进一步证实外源T-DNA已整合至转化子的染色体基因组中。在诸多转化子中,通过发酵实验筛选得到了一株比出发菌株发白红曲菌9901高产Monacolin K的T-DNA插入突变转化子H1。采用反向PCR方法对发白红曲菌转化子H1基因组T-DNA插入位点侧翼序列进行克隆,获得了一段0.88 kb的DNA片段,暂命名为mkWL,对DNA片段mkWL进行测序,通过与NCBI公布的丛毛红曲菌Monacolin K合成基因簇比对分析,发现与其中mkH基因(1.46 kb)部分基因序列同源性为97%。由此推断基因mkWL包含发白红曲菌Monacolin K合成关联基因。
(2)对烟色红曲菌(M. fumeus) 9908和发白红曲菌转化子H1原生质体制备与再生的条件进行研究,考察了菌龄、渗透压稳定剂、裂解酶组合、酶解时间和再生培养基等条件对这两株红曲菌原生质体制备和再生的影响。将携带潮霉素抗性的转化子H1的原生质体灭活后,与烟色红曲菌9908原生质体融合,以潮霉素抗性作为筛选标记,得到了多株融合子,并对融合子固态发酵产Monacolin K能力进行考察,筛选得到了固态发酵高产Monacolin K的红曲菌融合子HH1,此菌株Monacolin K的产量较亲本烟色红曲菌9908和转化子H1分别提高了404%和27%。
(3)对影响融合子HH1固态发酵产Monacolin K的因素进行了单因素考察,根据优化得到的发酵条件,融合子HH1以小米为原料固态发酵20 d后,Monacolin K的产量可达17.50 mg/g。采用Box-Behnken响应面分析法,进一步研究了拌料水加量、拌料水pH和接种量这三个因素对Monacolin K产量的影响,并建立了模型,模型预测的理论值与实验所得的实际值无显著性差异,说明此模型能够较好的模拟发酵实验。
本研究克隆得到了发白红曲菌Monacolin K合成相关基因的DNA片段mkWL,并采用根癌农杆菌介导转化和原生质体融合技术筛选到红曲菌Monacolin K高产株,为发白红曲菌基因遗传改造及Monacolin K工业化生产奠定良好基础。
One of cholesterol synthesis inhibitor, Monacolin K, was found in Monascus fermented products. It is the same substance as lovastatin in Aspergillus terreus. It inhibits the activity of hydroxymethyglutaryl coenzyme A reductase, which catalyzes the rate-limiting step in cholesterol biosynthesis and regulate abnormal blood liqid in organism. Over the last 30 years, many Monascus spp such as M.purpureus, M.albidus and M.pilosus were found to produce Monacolin K. Although the ability of synthesizing Monacolin K by Monascus spp degenerated with the generation passing on, and yield of Monacolin K from Monascus spp is less than A. terreus, but with its edibility, Monacolin K produced from Monascus spp seems more acceptable than from A. terreus. Constant selection and breeding of Monascus spp are necessary. In order to investigate the related gene in Monacolin K synthesis pathway and find out a simple and effective method to screen strains of Monascus spp with high Monacolin K production, the approach of combinated techniques with gene cloning and protoplast fusion may be a feasible choice.
The aim of this work was to develop the Agrobacterium tumefaciens-mediated transformation (ATMT) method in Monascus spp, and transfer the T-DNA with genetic marker into Monascus spp, expect the insertion of T-DNA would change the host DNA sequence arrangement and affect the growth and metabolism of Monascus transformants, and then the Monascus transformants with high Monanolin K could be selected and analysed. The flanking sequence of T-DNA from these transformants will be amplified to find out the related Monacolin K biosynthetic gene. Monascus fusants with enhanced Monacolin K production could be screened via proroplast fusion between parental strains with gene hph and the ability of Monacolin K production respectively, the fermentation conditions by Monascus fusants during solid-state fermentation will be optimized, and provide finally some data for industrialization of Monacolin K production.
The related results are as following:
(1) The binary vector pCAMBIA 3300-gpdA-hph-trpC was constructed and transformed into Agrobacterium tumefaciens GV3101. The exogenous gene gpdA-hph-trpC was transformed into M. albidus by ATMT. Various influencing factors on transformation efficiency were optimized including the concentration of antibiotics, concentration of M. albidus spores, cell density of A. tumefaciens, co-cultivation time, temperature and the concentration of acetosyringone. The highest transformation frequency was about 5.2×104 . Some of the transformants were selected for PCR analysis, and the results further confirmed that the transformants tested were arbitrarily integrated with exogenous T-DNA in genome. Among many transformants, one transformant H1 with high Monacolin K production was selected, from which the flanking sequence mkWL (0.88 kb) of T-DNA by inverse PCR was obtained. The result of homology between the gene mkWL and Monacolin K biosynthetic gene cluster from M. albidus in the Genbank was analyzed, which revealed that there was identity of 97% at DNA sequence homology. The gene mkWL was deduced to be the related gene in Monacolin K biosynthetic pathway of M. albidus.
(2) Main factors such as mycelium age, osmotic stabilizer, different enzymatic combinations, duration of enzyme treatment and regeneration medium for efficient protoplast formation and regeneration from transformant H1 and M. fumeus 9908 were investigated. Monascus fusants HH1 with high Monacolin K production were selected via protoplast fusion between tansformant H1 and M. fumeus 9908, and the concentration of Monacolin K in fermented millet by fusant HH1 increased by 404% and 27% compared with that produced by parental Monascus spp.
(3) The fermentation conditions for promotion Monacolin K production by solid-state fermentation were optimized, and the Monacolin K yield by fusant HH1 reached to 17.50 mg/g after fermentation for 20 d. The response surface methodology of Box-Behnken was applied to further optimize the fermentation conditions including water volume, pH of water and inoculant volume. A model was constructed, and further experiments were carried out to verify the accuracy and reliability of the model, the results showed that the predicted value was agreed well to the experimental value.
The gene mkWL related to Monacolin K biosynthesis in M. albidus was amplified, and Monascus fusants with high Monacolin K production were bred via Agrobacterium -mediated transformation and protoplast fusion, these works laid a good foundation for genetic modification and industrialization of Monacolin K fermentation of M. albidus.
引文
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