Fig 1.
Production of citric acid is studied under up- and downregulation of the PPP via the G6PD encoding gene gsdA.
Glucose is uptaken by the fungal cell and metabolized via glycolysis (blue) to pyruvate that enters the TCA cycle (yellow) via acetyl-CoA. The metabolite citrate is produced and secreted as citric acid. Glycolysis is linked to the oxidative PPP (red) via the NADPH-generating reaction of G6PD and further downstream to the non-oxidative PPP (green). Abbreviations: GOX= glucose oxidase; GNL= gluconolactonase; GukA= gluconokinase; PPP= pentose phosphate pathway; P= phosphate; G6PD= Glucose-6-phosphate dehydrogenase; 6PGL= 6-phosphogluconolactonase; 6PDG= 6-phospholuconate dehydrogenase; CoA= Coenzyme A; TCA= Tricarboxylic acid. Dashed arrow means more reactions in between. Enzymes of the oxPPP are highlighted in blue. Created in BioRender. Fritsche, S. (2024) BioRender.com/c53c134.
Table 1.
List of plasmids used throughout the study.
Table 2.
List of primer sequences used throughout the study.
Table 3.
A. niger strains used or generated in this study.
Fig 2.
Secreted metabolites and G6PD activity produced by A. niger overexpressing gsdA.
108 conidia/L of the control strain, SF374 (blue bars), or the gsdA overexpression strain, SF369 (grey bars), were inoculated in Vogel’s medium supplemented with 0, 1 and 2.5 μg/mL doxycycline and cultivated in microtiter plates. Citric acid titers (A), citric acid yield (B), erythritol (C) and glycerol titers (D) of the cultivation supernatant were measured after 72 h of cultivation at 30°C. Cultivation was done with eight replicates and error bars indicate standard deviation. G6PD specific enzyme activity was determined in cell-free extracts from mycelia cultivated in shake flasks with Vogel’s medium supplemented with 0, 1 μg/mL doxycycline and harvested after 72 hours in biological triplicates. Significant differences (*p<0.05; **p<0.001; ***p<0.0001) were determined by t-test and highlighted by asterisks. Created in BioRender. Fritsche, S. (2024) BioRender.com/m29b218.
Fig 3.
Construction of a gsdA-regulated A. niger strain.
(A) The gsdA OE strain SF387 and ZWF1 OE strain SF388 harbor the ptet-on regulated expression cassette at the pyrG locus. 5’-pyrG and the truncated version of pyrG (pyrGm2,trunc) derived from respective transformation vectors for genomic integration via homologous recombination. Both strains carry an INDEL mutation in the pyrGm2,trunc sequence of the pyrG locus (dotted line) and were selected on plates supplemented with uridine and 5-FOA. (B) SF387 and SF388 were transformed with a Cas9/sgRNA containing plasmid pSF511 designed for a double-strand break event at the native gsdA locus (red line). The co-transformed disruption cassette was linearized from plasmid pSF2 and harbored the pyrG gene of A. nidulans with ~1 kb of its up- and downstream sequence. Flanks of the 5’ and 3’ end gsdA gene were provided for homologous directed repair of the DNA lesion. Transformation was done on plates without uridine and supplementation of doxycycline to induce gsdA expression at the pyrG locus. Primers P25 and 28 were used for PCR verification of the integration event. (C) After transformation only the gsdA OE strain SF387 was uridine prototroph, did not grow without doxycycline and was referred to strain SF395. (D) Gel electrophoresis shows the expected fragment of 3899 bp compared to the wild-type with 2563 bp using primers P25 and P28. Created in BioRender. Fritsche, S. (2024) BioRender.com/i92i049.
Fig 4.
Phenotypic characterization of A. niger with regulated gsdA expression.
Conidia of wild-type strain ATCC 1015 (WT) and the gsdA-regulated strain SF395 were grown on (A) minimal medium with 1% glucose to show dependency of growth on gsdA expression. (B) Strains were spotted on medium containing 20% glucose and CaCO3 to screen for acidification of the CaCO3. Increasing concentrations of doxycycline were applied to the media to regulate the expression of gsdA under the control of the tet-on promoter system in SF395. Growth plates were incubated at 30°C for 90h.
Fig 5.
Viability of A. niger on gluconate with regulation of gsdA expression levels.
The control strain SF374 and the gsdA-regulated strain SF395 were grown on a minimal medium with 1% Na-gluconate. Increasing concentrations of doxycycline were applied to the medium to regulate the expression of gsdA under the control of the ptet-on promoter system in SF395. Growth plates were incubated at 30°C for 90 h.
Fig 6.
A. niger with regulated gsdΑ expression cultivated in medium containing glucose.
109 conidia/L of the control strain SF374 or the gsdA-regulated strain SF395 were inoculated in Vogel’s medium with 0–2.5 μg/mL doxycycline to induce gsdA under the tet-on promoter system at the pyrG locus in SF395. (A) Citric acid titer and (B) yield on glucose, (C) erythritol and (D) glycerol levels were measured after 168 h of cultivation at 30°C and 200 rpm. Error bars indicate standard deviation of three biological replicates. Significant differences (*p<0.05; **p<0.001; ***p<0.0001) were determined by t-test and highlighted by asterisks.
Fig 7.
A. niger with regulated gsdA expression cultivated in medium containing glucose and gluconate.
109 conidia/L of the control strain SF374 or the gsdA-regulated strain SF395 were inoculated in Vogel’s medium with 1 and 2.5 μg/mL doxycycline (dox) to induce gsdA under the tet-on promoter system at the pyrG locus in SF395. Medium contained 20% (w/v) of a glucose/gluconate mixture with a ratio of either 80:20 or 60:40. (A) Citric acid titer and (B) citric acid yield on glucose/gluconate consumption measured in the supernatant of the culture during 168 h of cultivation at 30°C and 200 rpm. Error bars indicate standard deviation of three biological replicates. Asterisks in (B) mark significant differences (p < 0.05) of means from SF395 samples induced with 1 and 2.5 μg/mL doxycycline and were determined by t-test (n= 3).