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Aspergillus terreus, an acid tolerant fungus that grows over a wide range of pH from 2 to 8, was employed in this study. Submerged fermentation was conducted under low dissolved oxygen (10% DO) and pH (pH 2). The fermentation kinetics was investigated together with transcriptome to identify key genes associated with metabolic responses to such environmental stresses by high-throughput RNA-seq technology. It was clear that under low dissolved oxygen, glycolysis was uncoupled from oxidative phosphorylation due to limited ATP regeneration, as observed from the downregulation of genes in the mitochondrial respiratory chain complex and slow growth. On the contrary, the transcriptional levels of genes encoding ATPases and cation transporters remained unchanged at –1 ≤ log2 fold change ≤ 1, revealing that A. terreus possessed an excellent defense mechanism to low pH. In addition, genes associated with cell wall synthesis and repair, including chitin synthase and 1,3-beta-glucan synthase genes, and genes in the cluster for cell wall protection and repair were upregulated. This evidence clearly demonstrated that A. terreus was able to survive under the acidic environment. In summary, we comprehensively analyzed and discussed the transcriptome of A. terreus. The findings obtained here provide a theoretical basis for better understaning the molecular mechanism of the fungal response to the environmental factors, providing thereby an insight into the process of designing and optimizing the production of biological products of interest. This approach can be applicable to other metabolic pathways of interest in particular microorganisms.

The metabolic responses of Aspergillus terreus NRRL1960 to stress conditions (low dissolved oxygen and pH with limited nitrogen and phosphate) in the two-phase fermentation were investigated in this study. The fermentation kinetics suggested that itaconate production was suppressed under low dissolved oxygen (DO) concentrations. A slight change in pH caused a significant change in itaconate production. The transcriptomic data revealed that under low DO concentration, the glycolytic pathway was uncoupled from the oxidative phosphorylation, resulting in the activation of substrate-level phosphorylation as an alternative route for ATP regeneration. The downregulation of pdh genes, the genes encoding ATP synthase and succinate dehydrogenase, confirmed the observation of the uncoupling of the oxidative TCA cycle from glycolysis. It was found that the upregulation of pyc resulted in a large pool of oxaloacetate in the cytosol. This induced the conversion of oxaloacetate to malate. The upregulation of the gene encoding fumarate hydratase with the subsequent formation of fumarate was found to be responsible for the regeneration of NADPH and ATP under the condition of a low dissolved oxygen level. The large pool of oxaloacetate drove itaconic acid production also via the oxidative TCA cycle. Nevertheless, the downregulation of ATP synthase genes resulted in the deficiency of the proton-pumping H+ ATPase and the subsequent stress due to the failure to maintain the physiological pH. This resulted in itaconate production at a low titer. The fermentation kinetics and the transcriptomic data provided in this study can be used for further process optimization and control to improve itaconate production performance.

Aspergillus terreus was reported as the promising fungal strain for itaconic acid; however, the commercial production suffers from the low yield. Low production yield was claimed as the result of completing the tricarboxylic acid (TCA) cycle towards biomass synthesis while under limiting phosphate and nitrogen; TCA cycle was somewhat shunted and consequently, the metabolite fluxes move towards itaconic acid production route. By regulating enzymes in TCA cycle, it is believed that itaconic acid production can be improved. One of the key responsible enzymes involved in itaconic acid production was triggered in this study. Pyruvate carboxylase was allosterically inhibited by L-aspartate. The presence of 10 mM L-aspartate in the production medium directly repressed PC expression in the living A. terreus while the limited malate flux regulated the malate/citrate antiporters resulting in the increasing cis-aconitate decarboxylase activity to simultaneously convert cis-aconitate, citrate isomer, into itaconic acid. The transport of cis-aconitate via the antiporters induced citrate synthase and 6-phosphofructo-1-kinase activities in response to balance the fluxes of TCA intermediates. Successively, itaconic acid production yield and final concentration could be improved by 8.33 and 60.32 %, respectively, compared to those obtained from the control fermentation with the shortened lag time to produce itaconic acid during the production phase.

The objective of this study is to obtain new laccase and enzyme source with remarkable dye removal potential. Thirty isolates of white rot fungi were screened for extracellular laccase-production using 2,2-azinobis (3-ethylbenzothiazoline-6-sulfonate) (ABTS) assay as indicator. Among these, Polyporus pseudobetulinus strain WR77 exhibits the highestlaccase activity and its suitable enzyme production medium contains; 1% (w/v) rice chaff, 0.5 g/L di-ammonium tartrate, and 0.01 g/L peptone as the carbon; inorganic and organic nitrogen sources; respectively. The laccase was 60-fold concentrated (by ammonium sulphate precipitation, Q-sepharose anion-exchange chromatography, and Superdex G-75 gel filtration chromatography) and gave the specific activity of 617.12 U/mg. The MW of prepared enzyme is 75.2 kDa under SDS-PAGE determination. Empirical analyzing results indicate that the optimum pH and temperature of the enzyme are around 40°C and pH 4, respectively. Furthermore, this enzyme can resist to wide pH range (4.0-11.0) with more than 95% maximum activity remained. The enzyme’s Km and Vmax, with ABTS substrate, were 447.93 mM and 104.17 µmol/min/mg protein, respectively. The prepared enzyme was strongly inhibited by Hg2+ and Fe2+ but weakly (9.7%) stimulated by 10 mM Cu2+ ions. The strain WR77 shows good ability in decolorizing many synthetic dyes (200 mg/L initial concentration); Ambifix Blue H3R (98% in 8 days), Ambifix Yellow H3R (24% in 10 days) and Ambifix Red HE3B (50% in 18 days). The prepared laccase alone (5 U/ml)could decolorize Ambifix Blue H3R by 65% within 15 min and Malachite Green by 80% within 24 h. It can be concluded that new enzyme and source with satisfactory dye removal potential have been successfully achieved. Further studies should be attempted to evaluate their feasibility in industrial uses. Key words: Polyporus pseudobetulinus, white-rot fungi, Laccase, dye decolorization, synthetic dyes.