温度和蓝光对红曲霉莫纳可林K代谢调控的影响

发布时间：2018-01-18 08:53

  本文关键词：温度和蓝光对红曲霉莫纳可林K代谢调控的影响　出处：《天津科技大学》2015年硕士论文　论文类型：学位论文

  更多相关文章： 红曲霉 温度 蓝光 莫纳可林K 代谢调控 桔霉素

【摘要】：红曲霉次生代谢产物中的莫纳可林K(Monacolin K)是有效的胆固醇合成抑制剂,而其产率普遍不高和红曲霉代谢产物中桔霉素的存在,限制了红曲产品的开发与应用。红曲霉的次生代谢产物受温度、光照、pH、溶氧量等环境因子的影响。本文主要研究了温度和蓝光对红曲霉发酵过程中莫纳可林K产量的影响,并采用RT-qPCR技术对莫纳可林K相关合成基因的表达情况进行分析,探讨莫纳可林K代谢调控的机理；对功能红曲菌株的产桔霉素特性进行安全性评价,为构建高产莫纳可林K不产桔霉素的基因工程菌提供依据。采用固态发酵方式,研究不同温度对高产莫纳可林K的红曲霉MC2菌株发酵过程中莫纳可林K产量的影响。结果显示,在30-C条件下培养到第25 d时莫纳可林K产量达到21.2 μg/g干重；在20-C条件下培养到第28 d时莫纳可林K产量达到2917.7μg/g干重,两者相差137倍。采用液态静置发酵方式,研究蓝光对莫纳可林K产量的影响,结果发现,蓝光和黑暗条件均在第28 d产量达到最高,分别为519.5 μg/g干重和1213.4 μg/g干重。蓝光照射培养条件下,莫纳可林K的最高产量仅为黑暗培养条件下的43%。设计引物,克隆红曲霉MC2基因组DNA中与莫纳可林K合成相关的10个基因的部分片段,将PCR产物测序结果与NCBI上报道的桔霉素合成相关基因序列进行BLAST比对,结果显示,同源性均在99%以上。采用RT-qPCR方法,比较30℃与20℃条件下,以糯糙米为基质,在发酵过程中红曲霉MC2菌株的莫纳可林K合成相关基因表达量的差异,结果表明：低温条件提高了mokA、mokC、mokD、mokE、mokF、mokG和MpLaeA的表达量,降低了mokB、mokH和mokI基因的表达量。采用RT-qPCR方法,比较蓝光和黑暗培养条件下,莫纳可林K合成相关基因表达量的差异,发现蓝光照射培养条件下,除mokB外,所有与红曲霉MC2菌株莫纳可林K合成相关基因的表达量均低于黑暗培养条件,从而导致蓝光下莫纳可林K产量降低,其中mokA、mokC、mokD、mokE、mokG是莫纳可林K合成过程中九酮化合物合成途径中的相关基因,温度和蓝光都是通过影响莫纳可林K九酮化合物的合成来影响莫纳可林K的最终产量。分析了红曲霉MC2、C100和M9菌株在不同培养基中的产桔霉素情况,发现红曲霉MC2是一株不产桔霉素的菌株,该菌株缺少桔霉素合成的关键基因ctnA、pksCT和orf3。对红曲霉C100和M9两株正常产桔霉素菌株进行培养,发现在添加一定量NH4NO3的大米粉培养基中不产桔霉素,利用RT-PCR方法分析不产桔霉素时相关基因的表达情况,发现桔霉素合成基因簇中相关基因均有表达。
[Abstract]:Monacolin (Monacolin K), a secondary metabolite of Monascus, is an effective inhibitor of cholesterol synthesis, but its yield is generally low and citrinin is present in the metabolites of Monascus. The development and application of Monascus products were limited. The secondary metabolites of Monascus were subjected to temperature, light and pH. The effects of temperature and blue light on the yield of Monacolin K during Monascus fermentation were studied. RT-qPCR technique was used to analyze the expression of monacolin K related synthetic gene, and the mechanism of the regulation of monacolin K metabolism was discussed. The safety evaluation of citrinin-producing strain was carried out to provide the basis for the construction of genetically engineered bacteria with high yield of Monacolin K and no citrinin production. Solid state fermentation was adopted. The effects of different temperatures on the yield of Monacolin K during the fermentation of Monascus MC2 strain with high yield of Monacolin K were studied. The yield of Monacolin K reached 21.2 渭 g / g dry weight at the 25th day under 30-C condition. The yield of Monacolin K reached 2917.7 渭 g / g dry weight at the 28th day under 20-C condition, and the difference was 137 times. The effect of blue light on the yield of Monacolin K was studied. The results showed that both blue light and dark conditions reached the highest yield on the 28th day. Dry weight of 519.5 渭 g / g and 1213.4 渭 g / g of dry weight respectively. Under the condition of blue light irradiation, the maximum yield of Monacolin K was only 43 under dark culture. The partial fragments of 10 genes related to the synthesis of Monacolin K were cloned from the genomic DNA of Monascus MC2. The sequencing results of PCR products were compared with the related gene sequences of citrinin synthesis reported on NCBI. The results showed that the homology was above 99%. RT-qPCR method was used. The relative gene expression of Monacolin K biosynthesis of Monascus MC2 strain was compared at 30 鈩，

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