【作者】 李小兵；

【导师】 元英进；

【作者基本信息】 天津大学 ， 生物化工， 2006， 博士

【摘要】 为了提高利迪链菌素的发酵水平,对利迪链霉菌(Streptomyces lydicus)进行了选育改良,通过LC/MS分析,研究了高产突变株过度表达的原因及利迪链菌素的生物合成途径,并在30 L发酵罐上进行了放大培养,取得了如下结果:基于生物合成前体的代谢抑制/阻遏作用,筛选前体的抗性/忍耐突变株,其原理在于只有生物合成途径发生了改变、消除了代谢抑制/阻遏作用的突变株才能生长,因此具有有效性状的突变株而被筛选到;筛选的正向突变率达83.5%,其中丙酸盐抗性突变株P28的产量提高了267%。通过LC/ESI–MS检测,采用全扫描、选择离子扫描、ESI–MS、MS/MS和二极管矩阵(PDA)等检测模式,对比分析了突变株P28及其出发菌株的代谢产物。结果显示,两个分支途径的阻断及一个中间产物积累的显著下降导致了突变株P28的过度表达,且这一中间产物(m/z 363.1 [M– H]ˉ)是承接利迪链菌素的聚酮部分与Tetramic acid部分生物合成的关键途径中间产物。应用LC/MS/MS技术研究了利迪链菌素的生物合成途径。首先基于同位素标记的研究,提出多条生物合成的可能路线;S. lydicus代谢产物的ESI–MS谱中以选择离子扫描模式进行分子量搜索,确定了唯一的生物合成路线,并对其中化合物的结构进行特征确认。由已知结构的终产物利迪链菌素及各化合物的MS/MS或MS~3碎片离子信息、亚结构断点位置,鉴别了17个途径中间产物的结构,阐明了利迪链菌素聚酮部分及Tetramic acid部分的生物合成途径。糖代谢是利迪链菌素发酵过程调控的关键,葡萄糖对S. lydicus代谢的pH、菌浓及利迪链菌素合成的影响非常敏感,针对这些代谢特点,以菌浓为基准在30 L发酵罐上进行了放大培养,发酵过程探索采用了葡萄糖、氨水和pH的串级调控策略,调控与菌体的代谢没有滞后性,各参数控制非常稳定,可使发酵液的糖浓度、pH、菌浓及补氨水量达到最佳的控制,利迪链菌素的平均发酵水平提高了52.7%。更多还原

【Abstract】 The comparative LC/ESI–MS analysis was successfully used for the characterization of reasons resulting in the yield increase in the selected mutant after strain improvement of Streptomyces lydicus AS 4.2501, and the biosynthetic pathway of streptolydigin was investigated by the LC/MS/MS analysis of culture extracts of this parent strain and its mutants. The results of this study were as follows:Isolated agar media plus propionate, glucose and valine with MIC were used to screen the precursor-tolerant/resistant mutants, and the positive frequency of 83.5% was obtained. Especially, a propionate-resistant mutant P28 showed an increase of 267% in streptolydigin titer over the starting strain S. lydicus AS 4.2501. In order to characterize the metabolic variations in intermediates, metabolites and biosynthetic pathways, culture extracts of the mutant P28 and its parent strain were parallel analyzed by LC/ESI–MS following the strain improvement of S. lydicus AS 4.2501. The results showed that two branch pathways blocked in the mutant and the accumulation of an intermediate decreased dramatically in the mutant cultures resulted in the streptolydigin overproduction in the mutant P28. Moreover, the compound at m/z 363.1 [M– H]ˉwas a key and significant intermediate being both the final product of polyketide biosynthesis and starting compound of tetramic acid moiety biosynthesis.In order to investigate the biosynthetic pathway of streptolydigin, the culture extracts using four strains S. lydicus AS 4.2501, P28, L8 and DSM 40461 were used for the LC/MS/MS analysis. Three possible pathways for the streptolol moiety and four for the tetramic acid moiety were proposed on the basis of the previous studies. Whether the hypothetical compounds existed in the culture extracts or not were decided by the combination of the extracted ion chromatogram (EIC) and ESI–MS scans on the corresponding molecular masses, thus, the unexpected pathways were obviously excluded and the exact pathway could be rapidly distinguished. The subsequent MS/MS or MS3 experiments provided direct evidences for the structural identification of the proposed pathway intermediates, via their substructural breakpoints as well as fragment ions along with structural origins更多还原