Establishing an efficient gene-targeting system in an itaconic-acid producing Aspergillus terreus strain
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To develop an efficient gene-targeting platform in an excellent itaconic acid producing strain Aspergillus terreus CICC40205.
The frequency of homologous recombination was improved by deleting the ku80 gene. A nutritional transformation system based on the bidirectionally selectable marker, pyrGAn, was established in the ku80-/pyrG-double mutant which is convenient for following marker rescue. The modified Cre/loxP recombination system was applied for the excision of the pyrGAn marker by directly introducing Cre recombinase into the protoplasts.
This gene-targeting system is an efficient platform for sequential and multiple genetic modifications in A. terreus and is conducive to study biosynthesis mechanisms and to improve the production ability of itaconic acid and other products.
KeywordsAspergillus terreus Cre/loxP system Gene-targeting Itaconic acid Non-homologous end joining pyrG
Thanks to Dr. Xiaoming Tan for valuable discussions. This work was supported by National Natural Sciences Foundation of China (Nos. 31400080 and 31500042), National High Technology Research and Development Program of China (Nos. 2015AA021003), the Key Research Program of the Chinese Academy of Sciences (No. KSZD-EW-Z-016).
XL, XH and JL designed the experiments. XH, MC performed the experiments. XH, XL, JL and MC drafted the manuscript.
Supplementary Table 1—Primers used.
Supplementary Fig. 1—Construction of the plasmid pXH106
Supplementary Fig. 2—Characterization of the genotypes of At-∆ku80 mutant strain.
Supplementary Fig. 3—Genomic PCR analysis of the At-∆pyrG mutant strain.
Supplementary Fig. 4—Genomic PCR analysis of the At-loxP-pyrGAn mutants obtained in the complementation test of At-∆pyrG.
Supplementary Fig. 5—The DNA sequencing result of the PCR-amplified fragments from the pyrGAn marker region of At-loxP-∆pyrGAn mutant.
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Conflict of interests
The authors declare that they have no competing interests.