内生真菌紫杉醇的生物合成及机制解析

Supported by National Science Foundation of China (30970090), a cDNA library of taxol-producing fungus during taxol-synthesis phage subtracting non-synthesis phage, a high proportion full-length cDNA library and a highly efficient genetic transformation system were successfully constructed in the study. The mutants with changed yield of taxol were screened, and six taxol biosynthesis related genes have been isolated, including taxadiene-5α-hydroxylase gene, geranylgeranyl diphosphate synthase gene, taxane-10β-hydroxylase gene, taxadiene-5α-ethanol- acetyl-transferase gene, taxane-2α-hydroxylase and taxane-13α-hydroxylase. Based on the previous work, we will construct expression vector for the high taxol-producing fungus HDFS4-26 with the yield of 516.37μg/l, Agrobacterium tumefaciens mediated transformation (ATMT) will be used to screen the N. sylviforme HDFS4-26 regulatory genes related to taxol production; the gene cluster of taxol biosynthesis from the strain HDFS4-26 will be obtained using a chromosome walking method. This study will lay a foundation for the study of taxol biosynthesis pathway and understand the taxol biosynthetic mechanisms in the N. sylviforme taxol producing endophytic fungi.

在国家基金（30970090）资助下，构建了N. sylviforme紫杉醇产生菌紫杉醇合成期消减非合成期cDNA文库、高比例全长cDNA文库和高效遗传转化体系，分离到了6个内生真菌紫杉醇合成相关基因。本研究在以前工作基础上，构建N. sylviforme高产紫杉醇菌株HDFS4-26（产量516.37微克/升）紫杉醇生物合成相关基因打靶载体，并利用根癌农杆菌介导（ATMT）对前期已获得的侯选基因打靶，进一步筛选N. sylviforme紫杉醇产生菌紫杉醇生物合成相关基因；采用染色体步移获得高产紫杉醇菌株HDFS4-26紫杉醇合成的基因簇，并验证其功能。该研究将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基础。

在本项目资助下，构建了与高产紫杉醇菌株N. sylviforme HDFS4-26紫杉醇生物合成相关的侯选基因的打靶载体pBI121-43，并对pBI121-43稳定性和表达生物活性进行了检测；将pBI121-43导入根癌农杆菌 LBA4404，对菌株HDFS4-26进行遗传转化，根据转化子紫杉醇产量变化筛选菌株HDFS4-26紫杉醇生物合成相关基因，筛选到了4个相关基因，分别为紫杉烷-13α-羟化酶基因、GGPP合成酶基因、紫杉二烯5α-醇-乙酰基转运酶基因、紫杉二烯-5α-羟化酶基因。. 将pBI121-43分别与筛选到的生物合成相关基因连接，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，根据TLC和HPLC分析结果，初步推测这4个相关基因为N. sylviforme紫杉醇生物合成途径的关键酶。. 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（Cluster of orthologous groups of proteins）注释、KEGG （Kyoto encyclopedia of genes and genomes database）pathway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，其中，序列comp2568_c0_seq1属谷胱甘肽S-转移酶家族蛋白基因，comp145452_c0_seq1和comp30968_c0_seq1属谷胱甘肽S-转移酶基因，comp168754_c0_seq1和comp176327_c0_seq1属乙酰-CoA-酰基转移酶基因， comp2885_c0_seq1属GGPPS基因，comp6245_c0_seq11、comp6245_c0_seq2和comp6245_c0_seq7都属NADPH-细胞色素P450还原酶基因。本研究为阐明N. sylviforme紫杉醇生物合成途径和解析其生物合成机制奠定了基础。