Applied Microbiology and Biotechnology

, Volume 103, Issue 19, pp 8105–8114 | Cite as

Development of a Cre-loxP-based genetic system in Aspergillus niger ATCC1015 and its application to construction of efficient organic acid-producing cell factories

  • Yongxue Xu
  • Lu Shan
  • Yutao Zhou
  • Zhoujie Xie
  • Andrew S. Ball
  • Wei CaoEmail author
  • Hao LiuEmail author
Applied genetics and molecular biotechnology


The filamentous fungus Aspergillus niger is widely used in the biotechnology industry for the production of chemicals and enzymes. Engineering of this valuable organism to improve its productivity is currently hampered by the lack of efficient genetic tools. Here, a Cre-loxP-based system for gene editing in A. niger was developed and its application in construction of A. niger cell factories to produce various organic acids was explored. Two established inducible systems, the xylanase A gene promoter Pxln and Tet-on system, were examined for driving cre expression and thus selection marker hyh deletion. Under inducing conditions, the efficiency of loxP site-specific recombination in the strain with cre driven by Pxln is about 2%, while cre driven by Tet-on system is about 34% which was used as the platform strain for further genetic engineering. As a proof of application of this system, strains containing different copies of oxaloacetate acetylhydrolase–encoding gene (oahA) were constructed, and the resultant strain S428 showed as high as 3.1-fold increase in oxalic acid production. Furthermore, an efficient malate-producing strain was generated through four-step genetic manipulation (oahA deletion, pyc, mdh3 and C4-dicarboxylate transporter gene c4t318 insertion). The resultant strain S575 achieved a titer 120.38 g/L malic acid with the flask culture, and a titer 201.24 g/L malic acid in fed-batch fermentation. These results demonstrated that this modified Cre-loxP system is a powerful tool for genetic engineering in A. niger, which has the potential to be genetically modified as a viable aciduric platform strain to produce high levels of various organic acids.


Aspergillus niger Cre-loxP system Tet-on system Organic acids Metabolic engineering 


Funding information

Funding for this research was provided by Tianjin Science and Technology Committee (18YFZCSY01360), the program of Distinguished Professor of Tianjin 2015 and Industrial Microbial Strain Selection and Fermentation Technology Public Service Platform Project (17PTGCCX00190).

Compliance with ethical standards

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the author.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

253_2019_10054_MOESM1_ESM.pdf (293 kb)
ESM 1 (PDF 293 kb)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.MOE Key Laboratory of Industrial Fermentation Microbiology, College of BiotechnologyTianjin University of Science & TechnologyTianjinChina
  2. 2.Tianjin Engineering Research Center of Microbial Metabolism and Fermentation Process ControlTianjin University of Science & TechnologyTianjinChina
  3. 3.Departmnet of Biosciences and Food TechnologyRMIT UniversityMelbourneAustralia

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