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

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.

Fine-tuning mitochondrial activity in Yarrowia lipolytica for citrate overproduction

“…Moreover, antimycin A compound used in the study is stated as an inhibitor of AOX protein, which is in contradiction with the effect sought by the overexpression of aox1. In the absence of contradicting our study, Hou et al (2018) confirm the hypothesis that AOX may contribute to clearing ROS species and improving cell survival. www.nature.com/scientificreports/…”

“…The proposed mechanism is in good agreement with the work of Kavšček et al, and Sabra et al, showing that the fine modulation of oxygen consumption enzymes in Y. lipolytica favors citrate overflow metabolism. A recent study of citrate production in Aspergillus niger by disruption or overexpression of aox1 gene, in addition to antimycin A during cell growth, showed an improved citrate production in overexpressed aox1 strains compared to the parental strain 55 . Up to 169.1 g/L citrate is reported for one of the overexpressed strains against 158.9 g/L for the parental stain.…”

Microbial citric acid: Production, properties, application, and future perspectives

“…Hou et al. (2018) modified A. niger strain CGMCC10142 by deleting and overexpressing mitochondrial AOX gene aox 1 and uncovered that the overexpression of aox 1 gene improve CA production, and cyanide‐resistant respiration (CRR) respiration was detected in mycelia of A. niger under CA production conditions. The presence of AOX thus alleviates the repression of PFK due to excess ATP and relieving the damage caused by ROS as well (Campos et al., 2015).…”

Citric acid and itaconic acid accumulation: variations of the same story?

“…These findings would imply that the operation of the alternative oxidase is essential for citric acid accumulation. However, Hou et al ( 2018 ) found that strains in which one of the alternative oxidase genes ( aoxA ) had been knocked out displayed only a 25% reduced citric acid yield in a high producing strain of A . niger .…”