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目的探讨红色糖多孢菌KR6基因突变体生物合成3-脱氧-3羰基-赤酮酸内酯B(DOEB)产量显著降低的原因。方法以KR6基因突变体M菌株为研究对象,采用相对实时荧光定量RT-PCR技术、抗原-抗体检测技术和高分辨LC-MS分析技术等,分别从基因的转录水平、翻译水平以及聚酮化合物水平等层面研究了DOEB的生物合成量。结果红色糖多孢菌M菌株与出发菌株A226相比,两者聚酮合酶(PKS)基因簇在转录水平和翻译水平上的表达差异均非造成DOEB产量降低的限速因子;而DOEB的前体转化能力却大幅度降低,因此,DOEB合酶的活力降低是造成生物合成DOEB产量降低的关键因素。结论 DOEB合酶的活力是生物合成DOEB的限速因子,本研究为进一步探讨利用基因工程途径提高新酮内酯类化合物产量奠定了基础。
Objective To investigate the reasons for the significant decrease of the yield of 3-deoxy-3-oxo-3-oxolactone B (DOEB) biosynthesis in KRZ mutant of Saccharomyces. Methods Mutant M strain of KR6 gene was used as the research object. Relative real-time fluorescent quantitative RT-PCR, antigen-antibody detection and high-resolution LC-MS analysis were used to analyze the gene transcription level, translation level and polyketide Level and other aspects of the biosynthesis of DOEB. Results Compared with the original strain A226, the transcriptional levels and translational levels of the polyketide synthase (PKS) gene cluster of the two strains did not cause the rate-limiting factor for the decrease of DOEB production. However, the DOEB The conversion ability of the precursor is greatly reduced. Therefore, the decrease of the DOEB synthase activity is the key factor for the decrease of the yield of the biosynthetic DOEB. Conclusions DOEB synthase activity is the rate limiting factor for biosynthesis of DOEB. This study will lay the foundation for further exploration of improving the yield of neotoketolactone by genetic engineering.