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

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