【作者】 陈涛；

【导师】 赵学明；

【作者基本信息】 天津大学 ， 生物化工， 2004， 博士

【摘要】 本文通过构建不同的整合型和游离型核黄素质粒得到一系列产核黄素B.subtilis 基因工程菌,研究了核黄素操纵子的增加方式和剂量对枯草芽孢杆菌核黄素合成的影响,成功地通过增加核黄素操纵子的剂量而稳定地提高工程菌的核黄素合成能力,并通过两轮基因组重排改进了基因工程菌的生产性状。最后本文建立了 B.subtilis 的生化反应网络,对第二轮基因组重排前后的三株B.subtilis 产核黄素工程菌在间歇培养过程中的代谢通量分布作了比较分析。得到的主要结果如下: 将整合型核黄素质粒分别以两种不同的整合方式在宿主菌染色体的 amyE、thrC 和 rib 位点的进行整合,在整合方式的比较中发现双交换方式整合比单交换方式整合更能有效地提高核黄素的产量。在整合位点的比较中发现:对于B.subtilis RH13,以单交换方式在 amyE 位点整合对提高核黄素产量的效果要好于其它两个位点;对于 B.subtilis 24R7,以单交换方式在上述三个位点进行整合的效果基本相同。 基于基因扩增的原理,将解调节的核黄素操纵子在宿主菌染色体上进行不同程度的扩增,结果表明:对于 B.subtilis RH13,通过扩增可以使核黄素产量由0.43g/L 增加到约 3.0g/L,而当核黄素操纵子剂量超过 6-7 个时,核黄素产量不能进一步得到提高。对于 B.subtilis 24R7,其染色体上的同向串联重复结构的稳定性较差,通过 pRB63 的扩增仅使核黄素的产量增加约 70%。过多的核黄素合成基因剂量对过量合成核黄素的菌株具有显著的生长抑制作用,甚至具有致死性。对于 B.subtilis 24R7,转化入高稳定性的游离型核黄素质粒 pMX45 使核黄素的产量从 1.1g/L 提高到约 3g/L。游离型核黄素质粒 pRB49 稳定性较差,转化入受体菌株后并不能有效地提高菌株的核黄素产量。 本文基于基因组重排的原理来改进产核黄素工程菌的生产性状,通过两轮基因组重排产生的 B.subtilis RH33/pMX45 在以蔗糖为碳源的间歇发酵条件下可产核黄素 4.2g/L,比核黄素工业生产菌 B.subtilis 24/pMX45 的核黄素产量提高了约 40%,并能更快地同化利用葡萄糖来合成核黄素,在以葡萄糖为碳源的间歇发酵条件下可产核黄素 4.4g/L,约为 B.subtilis 24/pMX45 核黄素产量的 2 倍。 本文建立了 B.subtilis 产核黄素基因工程菌的生化反应网络及计量学模型,模型共包括 34 个方程和 39 个变量。通过对第二轮基因组重排前后的三株产核黄素 B.subtilis 在间歇发酵过程中的代谢通量分析表明:PP 途径的通量对核黄素合成的影响不大,只有增加从 5-磷酸核酮糖到核黄素的一系列反应的代谢通量才能有效地提高合成核黄素的通量。第二轮基因组重排后产生的 B.subtilisRH33/pMX45 中核黄素合成的主要通量限制因素存在于从 5-磷酸核酮糖到 GTP的一系列反应中。更多还原

【Abstract】 A series of genetic engineering Bacillus subtilis for riboflavin production wasobtained by transforming integration or autonomous rib-plasmids. The effect ofriboflavin operon dosage and its integration modes on riboflavin production wereresearched in this article. By increasing riboflavin operon dosage, riboflavinproduction of engineering strain was improved stably. The traits of riboflavin-producing B.subtilis were improved by two rounds of genome shuffling. Abiochemical reaction network was assembled with metabolites, then the analysis andcomparison of metabolic fluxes in batch cultures of three different engineering strainsabout the second round genome shuffling were conducted. The main results presentedin this work are as follows: Integration rib-plasmids were inserted in host chromosome at amyE、thrC and ribloci by two different modes, and the result indicated that integration of rib-plasmid bya double-cross event cause higher riboflavin yield than a single-cross event. As forhost B.subtilis RH13, integration of rib-plasmid at amyE locus cause more riboflavinyield than the other two loci, and as for another host B.subtilis 24R7, integration ofrib-plasmid at three loci almost cause equal increase of riboflavin production. Based on theory of gene amplification, deregulated riboflavin operon wasamplified to different copies in host chromosome. It shows that with amplification ofthe riboflavin operon in B.subtilis RH13, riboflavin production is raised from 0.43g/Lto 3.0g/L. When riboflavin operon dosage reached 6~7, amplifying more riboflavinoperons is no use for further improvement of yields of riboflavin. With amplificationof the riboflavin operon in B.subtilis 24R7, riboflavin production only had an increaseof 70% because of the instability of the reiterated sequences in the chromosome of24R7. Excessive riboflavin operon dosage in riboflavin-overproducing strains exertremarkable negative effect on cell growth, even is fatal to the host cells. Withautonomous rib-plasmid pMX45 being transformed, riboflavin production inB.subtilis 24R7 was raised from 1.1g/L to about 3g/L. The instability of anotherautonomous rib-plasmid pRB49 made it very difficult to enhance riboflavinproduction efficiently. Based on the theory of genome shuffling, a recombinant engineering strainB.subtilis RH33/pMX45 with improved phenotype was obtained by two rounds of II<WP=4>ABSTRACTgenome shuffling. In batch culture with sucrose as the carbon source, it could produceabout 4.2g/L riboflavin, which is 43% more than that of B.subtilis 24/pMX45, a strainfor commercial production of riboflavin. In addition, B.subtilis RH33/pMX45 couldassimilate glucose quickly for riboflavin biosynthesis, in batch culture with glucose asthe carbon source, it could produce about 4.4g/L riboflavin, which is about 100%more than that of B.subtilis 24/pMX45. A biochemical reaction network of engineering Bacillus subtilis was assembledwith metabolites, and a stoichiometric model including 34 equations and 39 variableswas used to analyze metabolic fluxes in batch cultures of three different engineeringstrains. The result of metabolic fluxes analysis indicated that flux in PP pathway hadno significant effect on riboflavin production, and the fluxes in the reactions fromRibulose-5-P to riboflavin must be enhanced for further increase of riboflavinproduction. The primary limiting factor for improvement of riboflavin production inB.subtilis RH33/pMX45 lies in the reactions between 5-P-ribulose and GTP.更多还原