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Functional analysis of the mitochondrial alternative oxidase gene (aox1) from Aspergillus niger CGMCC 10142 and its effects on citric acid production

  • Li Hou
  • Ling Liu
  • Hongfei Zhang
  • Lin Zhang
  • Lan Zhang
  • Jian Zhang
  • Qiang Gao
  • Depei Wang
Applied Genetics and Molecular Biotechnology

Abstract

In this work, we constructed the aox1 disruption strains 3–4 and 4–10, as well as the aox1 overexpression strains 72 and 102 in Aspergillus niger. The energy metabolism, EMP, TCA pathways, and flux were investigated for the citric acid (CA) overproduction via the aox1 overexpression among them. As expected, the overexpression of the aox1 gene enabled a higher growth rate than that of the rate of its parent strain in medium with respiratory chain inhibitors. In liquefied corn starch medium supplemented with 0.2 μg/mL antimycin A, the CA production of the overexpression strain 102 reached up to 169.1 g/L, whereas the highest value of the parent strain was 158.9 g/L. For the perspective of the aox1 disruption strain 4–10, the yield of CA dropped to 125.6 g/L, and the loose mycelial pellets forming in the medium also revealed that the fundamentally important role of AOX in A. niger lies in the resistance to oxidative stress under fully aerobic conditions. Based on real-time qPCR gene expression analysis and measurement of intracellular ATP and NADH levels, we came to a conclusion that the higher NADH oxidation rate resulting from the overexpression of the aox1 gene mainly contributed to rate-limited step’s acceleration and strengthened metabolic flow via mycelia and led to the CA yield in these strains increased by 13.5 and 10.8%, respectively. Subsequently, it was found that overexpression strains had higher AOX relative content and more oxygen consumption at different fermentation stages, which fully confirmed the close relationship between aox1 gene and energy metabolism, and comprehensively revealed aox1 gene function through the combination with the above conclusions.

Keywords

Aspergillus niger Citric acid aox1 gene disruption and overexpression Respiratory inhibitors Energy metabolism Real-time qPCR 

Notes

Acknowledgements

This work was financially supported by RZBC Co., Ltd., Shandong, China; Shandong Provincial Key R&D Project of China (Fostering Talent Project) (No. 2016GRC3201); and the National Natural Science Foundation of China (31471725, 31370075).

Compliance with ethical standards

Ethical approval

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

Conflict of interest

The authors declare that they have no competing interest.

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Li Hou
    • 1
  • Ling Liu
    • 1
  • Hongfei Zhang
    • 1
  • Lin Zhang
    • 1
  • Lan Zhang
    • 1
  • Jian Zhang
    • 1
    • 2
    • 3
  • Qiang Gao
    • 1
    • 2
    • 3
  • Depei Wang
    • 1
    • 2
    • 3
  1. 1.Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin Key Laboratory of Industrial Fermentation MicrobiologyTianjinPeople’s Republic of China
  2. 2.National and Local United Engineering Lab of Metabolic Control Fermentation TechnologyTianjinPeople’s Republic of China
  3. 3.College of BiotechnologyTianjin University of Science and TechnologyTianjinPeople’s Republic of China

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