Eliminating manganese sensitivity and boosting citric acid production in Aspergillus niger through transporters manipulation.

BACKGROUND: Citric acid, a high-volume bioproduct, is manufactured by submerged fermentation using the filamentous fungus Aspergillus niger. Citric acid overflow requires Mn(II) ion concentrations not exceeding 5 µg L- 1. However, manganese easily leaches from media ingredients or stainless-steel bioreactors, necessitating costly, laborious removal. RESULTS: Aspergillus niger citrate fermentations were evaluated across varied manganese profiles, comparing a novel double transporter mutant against parental and single-mutant strains. This strain overexpresses cexA - the primary plasma membrane exporter whose activity overcomes a major production bottleneck - under the control of the strong glaA promoter in a background deficient in dmtA, which encodes the primary high-affinity NRAMP-family manganese transporter. Under high-manganese conditions (100-165 µg L- 1) simulating industrial substrate impurities and bioreactor leaching, the double transporter mutant outperformed the other strains. Despite abundant manganese that triggers high biomass (50 g L- 1) and low citrate yields in the parental strain NRRL2270, this cell factory maintained high-yield overflow metabolism. Driven by an accelerated maximal glucose consumption rate of 1.61 g L- 1 h- 1 and reduced biomass accumulation (9.1 g L- 1), it accumulated citric acid to levels comparable to those achieved under manganese deficiency. The double transporter mutant compressed the fermentation period by 17% (from 300 to 250 h), minimizing operational energy and utility costs and significantly enhancing overall productivity. To validate industrial scalability, the untreated, high-manganese process was replicated in 15-L stainless steel bioreactors - accounting for up to 30 µg L- 1 of manganese leaching - and successfully scaled up by an order of magnitude in a 150-L bioreactor. Carbon balance closures within ± 5% confirmed no appreciable by-product formation. CONCLUSIONS: By demonstrating the maintenance of high-yield overflow metabolism under manganese-replete conditions, alongside enhanced productivity through shortened fermentation times across different vessel scales, this study establishes this A. niger mutant as an attractive cell factory for industrial applications.