基于转化相关重组克隆曲酸合成基因簇及其在黑曲霉中的异源整合表达

Transformation-associated recombination cloning and integrated expression of biosynthetic gene cluster for kojic acid in Aspergillus niger

摘要:

【背景】 黑曲霉(Aspergillus niger)是一种重要的丝状真菌底盘细胞，然而对其进行大片段DNA的遗传操作存在工具缺乏、效率低下的问题。海量基因组数据中存在大量未被鉴定的次级代谢产物合成基因簇，因相应DNA分子量大而难以克隆，大部分基因与产物分子之间的映射关系尚未被解析。【目的】 开发一种适配黑曲霉的大片段DNA分子直接克隆的方法。以直接克隆米曲霉(Aspergillus oryzae)来源的曲酸合成基因簇(约30 kb)在黑曲霉底盘细胞中表达，实现曲酸的异源发酵。【方法】 基于转化相关重组(transformation-associated recombination, TAR)克隆，构建适配黑曲霉转化的TAR捕获骨架载体pSEA-TAR；将曲酸合成基因簇上下游各1 kb同源DNA序列先后引入pSEA-TAR，中间以唯一的Xho Ⅰ相隔，得到曲酸合成基因簇捕获载体pSEA-KLR；随后，将Not Ⅰ/Sbf Ⅰ消化后含有完整曲酸合成基因簇及上下游同源序列的米曲霉基因组与经Xho Ⅰ线性化的pSEA-KLR共同转化酿酒酵母(Saccharomyces cerevisiae) VL6-48，通过营养缺陷筛选获得成功捕获曲酸合成基因簇的重组子，提取质粒，电转化大肠杆菌(Escherichia coli)扩增重组质粒；借助农杆菌(Agrobacterium tumefaciens)介导黑曲霉转化，对获得的含有曲酸合成基因簇的黑曲霉转化子进行曲酸发酵，检测曲酸合成水平。【结果】 获得了含有ARSH4/CEN6、TRP1、hph和LB/RB-T-DNA repeat的基于TAR克隆、适配黑曲霉表达的大片段DNA捕获载体pSEA-TAR。借助酿酒酵母高效的同源重组系统，通过同源重组获得含有曲酸合成基因簇的重组质粒pSEA-TAR-KA，捕获成功率为5.9%。对获得的含有曲酸合成基因簇的10株黑曲霉重组菌株KA1-KA10进行发酵分析发现均成功合成了曲酸，其中黑曲霉KA-2发酵7 d后曲酸水平最高，达到5.86 g/L。【结论】 构建了适配黑曲霉的TAR克隆体系，用于大片段DNA高效捕获、重构和表达大型生物合成基因簇。以来源于米曲霉的曲酸合成基因簇捕获及在黑曲霉中成功实现曲酸合成验证了其有效性，进一步丰富了黑曲霉作为底盘细胞在重要化合物绿色生物制造中的作用。

关键词:转化相关重组克隆;大片段DNA;黑曲霉;曲酸合成基因簇

Abstract:

[Background] Aspergillus niger is a commonly used filamentous fungal chassis, whereas the genetic manipulation of large DNA fragments of this fungus lacks tools and is inefficient. The massive genomic data include a large number of unidentified gene clusters for the synthesis of secondary metabolites, which are difficult to be cloned due to the large molecular weights. In addition, the relationships between most genes and products have not been resolved. [Objective] To develop a method for direct cloning of large DNA fragments for A. niger and achieve the heterologous production of kojic acid in A. niger by cloning and expressing the gene cluster (about 30 kb) for the biosynthesis of kojic acid from Aspergillus oryzae. [Methods] Transformation-associated recombination (TAR) cloning was employed to construct the TAR capture backbone vector pSEA-TAR for the transformation of A. niger. The homologous DNA sequences (about 1 kb) flanking the gene cluster for the biosynthesis of kojic acid were successively introduced into pSEA-TAR and separated by a unique digestion sequence of Xho Ⅰ. The obtained capture vector pSEA-KLR for the gene cluster was linearized by Xho Ⅰ and transformed into Saccharomyces cerevisiae VL6-48 together with the Not Ⅰ/Sbf Ⅰ digested A. oryzae genome containing the complete gene cluster as well as the flanking homologous sequences. The recombinants successfully capturing the gene cluster for the biosynthesis of kojic acid were screened by the medium lacking corresponding nutrients. The plasmid was extracted from the recombinant and electroporated into Escherichia coli for amplification. The Agrobacterium tumefaciens-mediated method was used for transformation of A. niger. The obtained transformants carrying the gene cluster for kojic acid biosynthesis were subjected to fermentation, and the kojic acid titer was measured. [Results] A large DNA fragment capture vector pSEA-TAR was obtained based on TAR cloning and adapted to the expression of A. niger with ARSH4/CEN6, TRP1, hph, and LB/RB-T-DNA repeats. pSEA-TAR-KA was obtained as a recombinant plasmid containing a gene cluster for the biosynthesis of kojic acid by an efficient homologous recombination system of S. cerevisiae, demonstrating a capture success rate of 5.9%. All the 10 A. niger recombinant strains (KA1-KA10) carrying the gene cluster produced kojic acid in fermentation, with the highest kojic acid titer (5.86 g/L) obtained by fermentation with the strain KA-2 for 7 d. [Conclusion] A TAR cloning system was constructed and adapted to A. niger for the efficient capture, recombination, and expression of large biosynthetic gene clusters. The performance of the system was verified by the capture of A. oryzae-derived gene cluster for the biosynthesis of kojic acid and the successful synthesis of kojic acid by A. niger. This study enriched the role of A. niger as a chassis in the green biomanufacturing of important compounds.

Key words:transformation-associated recombination (TAR) cloning;large DNA fragment;Aspergillus niger;biosynthetic gene cluster for kojic acid