构建高产紫杉醇基因工程菌株及其代谢途径与调控的研究

Funded by the national Natural Science Foundation of China (30570025) which has been finished and the subsequent national Natural Science Foundation of China (30970090), we have constructed SSH cDNA library, high proportion of full-length cDNA library and their high efficiency of genetic transformation of Taxol-producing fungi during Taxol synthesis phase substracting nonsynthethsis phase, the mutons have been screened with changed Taxol yields. The TAIL-PCR method was used to screen genes of five key enzymes controlling endophytic fungi Taxol-biosynthesis. We have also constructed the expression vectors for Taxol-producing fungi HDFS4-26 (the Taxol yields were 516.37μg/L. the related patent has been authorized). This item will introduce the screened gene complexes of key enzymes controlling endophytic fungi Taxol-biosynthesis into HDFS4-26 Taxol-producing fungi through overexpression of genes, then construct gene-engineering fungi with high Taxol yield. The metabolic pathway of microbes-based taxol synthesis will be studied by combining the classical methods for metabolic pathway study such as precursor feeding, resting cell, isotopic tracer, blocked mutant with mRNA differential display and protein spectrum expression differential analysis. The item may provide theoretic reference for elucidating the Taxol biosynthesis mechanisms from endophytic fungi.

在已完成的国家自然科学基金（30570025）和后续国家自然科学基金（30970090）资助下，成功构建了紫杉醇产生菌紫杉醇合成期消减非合成期的SSH cDNA文库和高比例全长cDNA文库及其高效的遗传转化体系，在突变文库中筛选了紫杉醇产量发生变化的突变子，用TAIL-PCR方法筛选到了5个内生真菌紫杉醇生物合成的关键酶基因。本研究将构建的紫杉醇产生菌HDFS4-26（紫杉醇产量为516.37μg/L，专利已授权）表达载体，采用基因过量表达手段将筛选到的内生真菌紫杉醇合成过程中的关键酶基因复合体导入HDFS4-26中，构建高产紫杉醇基因工程菌株，通过前体饲喂法、静息细胞法、同位素示踪法、阻断突变株法等经典的代谢途径研究手段与mRNA差异显示技术和蛋白谱表达差异分析技术相结合，探索微生物合成紫杉醇的代谢途径，为解析内生真菌紫杉醇的生物合成机制提供理论依据。

本研究在以前工作基础上，构建了紫杉醇产生菌HDFS4-26表达载体pBI121-43，并将pBI121-43转入到菌株HDFS4-26原生质体中，对pBI121-43稳定性和表达活性进行了检测。将pBI121-43与筛选到的内生真菌紫杉醇合成相关基因双酶切后分别连接，并将所获得的重组质粒利用根癌农杆菌介导遗传转化法分别转入菌株HDFS4-26，获得了4株产紫杉醇基因工程菌株，分别为含紫杉烷-13α-羟化酶基因的工程菌株，含GGPP合成酶基因的工程菌株，含紫杉二烯5α-醇-乙酰基转运酶基因的工程菌株和含紫杉二烯-5α-羟化酶基因的工程菌株。. 另外，本研究分别通过单因子试验、Plackett-Burman试验设计、Box-Benhnken中心组合试验设计研究了菌株HDFS4-26紫杉醇侧链的生物合成途径。结果表明，确定的丝氨酸、水杨酸、乙酸铵最优化浓度分别为41.95mg/L、15.57mg/L、15.28mg/L，试验因子对响应值的影响的出回归方程为Ytaxol= 389.38 + 4.88*X1 + 1.84*X2 + 3.79*X3 - 4.65*X1*X1 + 17.20*X1*X2+ 10.41*X1*X3 - 21.15*X2*X2 + 25.86*X2*X3 - 15.52*X3*X3。. 此外，用高通量Illumina测序技术对N. sylviforme HDFS4-26生物发酵培养第1d、3d、7d、9d、11d的菌丝体进行测序，构建了Illumina PE文库，通过Nr注释、GO（Gene ontology）注释、COG注释、KEGGp注释等生物信息学分析，筛选出了9条与菌株HDFS4-26紫杉醇生物合成相关的基因，分析了菌株HDFS4-26紫杉醇生物合成相关路径的差异表达分析。本研究为阐明内生真菌紫杉醇生物合成途径和解析其生物合成机制奠定了基础。