Phenotypes of gene disruptants in relation to a putative mitochondrial malate–citrate shuttle protein in citric acid-producing Aspergillus niger

Abstract

The mitochondrial citrate transport protein (CTP) functions as a malate-citrate shuttle catalyzing the exchange of citrate plus a proton for malate between mitochondria and cytosol across the inner mitochondrial membrane in higher eukaryotic organisms. In this study, for functional analysis, we cloned the gene encoding putative CTP (ctpA) of citric acid-producing Aspergillus niger WU-2223L. The gene ctpA encodes a polypeptide consisting 296 amino acids conserved active residues required for citrate transport function. Only in early-log phase, the ctpA disruptant DCTPA-1 showed growth delay, and the amount of citric acid produced by strain DCTPA-1 was smaller than that by parental strain WU-2223L. These results indicate that the CTPA affects growth and thereby citric acid metabolism of A. niger changes, especially in early-log phase, but not citric acid-producing period. This is the first report showing that disruption of ctpA causes changes of phenotypes in relation to citric acid production in A. niger.

Mitochondrial citrate transporters CtpA and YhmA are involved in lysine biosynthesis in the white koji fungus,Aspergillus luchuensismut. kawachii

“…2A). This result agreed with a previous report indicating that the A. niger Δ ctpA strain is more sensitive to low-temperature stress (28). By contrast, the Δ yhmA strain exhibited smaller colony diameter than the control strain on M medium at all temperatures tested.…”

“…However, no phenotypic changes were observed in S. cerevisiae following disruption of the CTP1 gene (24), perhaps because other transport processes (e.g., involving mitochondrial succinate/fumarate transporter Acr1) control acetyl-CoA synthesis (25-27). A homolog to the CTP1 gene ( ctpA ) was recently characterized in A. niger , with a focus on the relationship between citrate transport and the organism’s high citrate production capability (28). Disruption of the ctpA gene led to reduced growth and citric acid production in A. niger only during the early logarithmic phase, indicating that CtpA is not a major mitochondrial citrate transporter in A. niger (28).…”

Comprehensive Improvement of Sample Preparation Methodologies Facilitates Dynamic Metabolomics ofAspergillus niger

“…In addition to an intermediate in the TCA cycle, malate plays a critical role in citrate transport. Malate is generated from oxaloacetate by malate dehydrogenase in the cytoplasm and imported into the mitochondria by the malate‐citrate shuttle transporter, which also exports citrate from mitochondria into the cytoplasm . In this study, the intracellular malate also showed a comparable correlation with the citrate production rate.…”

Aspergillus nigercitrate exporter revealed by comparison of two alternative citrate producing conditions

“…As initial screen, the shake flask experiments were not optimised for citrate production. To further understand the consequences of a citT deletion for citrate production, we used a medium with 120 g·L -1 glucose [29]. For citrate production, a pH below 3.0 is crucial [6, 30].…”

“…Growth experiments for determination of Δ citT c itrate production were performed in fermentors in triplicate. Pre-cultures of the N402 and a Δ citT strain were prepared with 1ˣ10 6 spores·mL -1 inoculated in 200 mL CM-SLZ, containing 1 g·L -1 casamino acid, 5 g·L -1 yeast extract, 3 g·L -1 (NH 4 ) 2 SO 4 , 1 g·L -1 K 2 HPO 4 , 1 g·L -1 KH 2 PO 4 , 0.5 g·L -1 MgSO 4 ·7H 2 O, 0.014 mg·L -1 MnSO 4 ·5H 2 O, 0.01 mg·L -1 FeCl 2 ·6H 2 O, 0.075 μg·L -1 , ZnCl 2 , 1 mL·L -1 and 10 g·L -1 glucose, adjusted to pH 5.5, and incubated at 30 °C, 200 rpm, for 18 h. Mycelium was harvested and then rinsed with equal amounts of water and 10 g (wet weight ~ 0.92 g CDW) of mycelium was transferred to 750 mL of SLZ medium, containing 3 g·L -1 (NH 4 ) 2 SO 4 , 1 g·L -1 K 2 HPO 4 , 1 g·L -1 , KH 2 PO 4 , 0.5 g·L -1 MgSO 4 ·7H 2 O, 0.014 mg·L -1 MnSO 4 ·5H 2 O, 0.01 mg·L -1 FeCl 2 ·6H 2 O, 0.075 μg·L -1 ZnCl 2 , and 120 g·L -1 glucose, and adjusted to pH 2.5 [29]. Methanol (2% (v/v)) was added as a promotive element for citrate production [43] and 30% (v/v) polypropylene glycol (ppg) was added as an anti-foaming agent.…”