Proteomic Analysis Of Citric Acid Industrial Strain Aspergillus Niger

The intra-and extra-cellular proteomic analysis for Aspergillus niger wasperformed in this study. The strain used was an industrial citric acid producer offeredby the company COFCO.The industrial process of citric acid fermentation was mimiced by controling thedissolved oxygen, stirrer speed, aeration rate, pH and other parameters in a5-Llaboratory-scale bioreactor. The fungus morphology, carbon source and organic acidwere monitored during the fermentation process. And the time points for proteomicanalysis were determined accordingly. Intracellular protein samples were taken at16h,24h,40h,70h,80h and96h and extracellular were at24h,40h,70h and96h.Protein extraction process was optimized. Solution used for extracellular proteinprecipitation was precooled acetone solution (-20℃) containing0.07%ofβ-mercaptoethanol and10%of trichloroacetic acid (TCA). Extraction buffer forextracellular protein contains0.1M of Tris-HCl pH7.6,0.1M of dithiothreitol (DTT),1%of sodium dodecyl sulfonate (SDS)(w/V), and a tablet of cocktail for10mLbuffer. Extraction buffer for intracellular protein contains0.1M of Tris-HCl pH7.6,0.1M of DTT,4%of SDS (w/V), and a tablet of cocktail for10mL buffer.The iTRAQ (isobaric tag for relative and absolute quantitation) labelingtechnology combined with LC-MS/MS was firstly used for quantitive proteomicanalysis for A. niger during citric acid fermentation process.793groups ofintracellular proteins and55groups of extracellular proteins were identified. Proteinsof glycolysis and part of the TCA cycle pathways which directly participate in thecitric acid producion were up-regulated. Proteins involved in energy metabolism werealso up-regulated. In contrast, proteins involved in other pathways including the restpart of the TCA cycle, lipid metabolism, amino acid metabolism and geneticinformation processing pathways were significantly down-regulated in the rapidproduction phase, but reactivated when the production rate slowed down. Among allthe enzymes identified, the citrate synthase, aconitase and isocitrate dehydrogenaseseemed the key for citric acid production. Extracellular protein data showed that A.niger secreted different glycosidases, lipases and proteases to utilize different formsof carbon and nitrogen sources from the fermentation medium. Both process measurement and proteomic analysis indicated that the glyoxylateshunt was activated during citric acid production process. Itaconate and oil were usedto explore the the role of glyoxylate shunt in citric acid production. The respirationwas depressed when oil was added in the initial fermentation. Oil didn’t improvecitric acid production or the conversion rate from glucose to citric acid. Addition ofitaconate in the initial fermentation phase inhibited both the glyoxylate cycle and thefungus growth, therefore cutting down the citric acid production. However, additionof itaconate in the citric acid production preiod could improve the conversion ratefrom glucose to citric acid.