Genetic modification of Aspergillus niger for effective production of gluconic acid from renewable carbon sources

Aspergillus niger is a well-established industrial microorganism recognized as Generally Recognized As Safe (GRAS), widely used for the production of enzymes and organic acids. This fungus is capable of producing enzymes such as amylase, glucoamylase, and glucose oxidase, as well as organic acids including citric acid, oxalic acid, and gluconic acid. In A. niger, gluconic acid is converted from glucose via gluconate pathway, catalyzed by glucose oxidase. Although A. niger naturally secretes these compounds, strain development is necessary to enhance production for industrial applications. Key strategies include optimizing fermentation processes and applying genetic modifications. In this study, we focused on genetic modification by comparing two strategies. First, we examined the overexpression of glucose oxidase encoding gene (goxC) using strong constitutive promoters. Among the engineered strains, the PcitA strain exhibited the highest gluconic acid yield at 65.08 ± 3.39 g/L, representing a 12.87% increase over the wild type. The PenoA strain showed the highest glucose oxidase activity at 49.07 ± 6.93 g/L, while the PcoxA strain demonstrated continuous enzyme secretion beyond 96 hours. The second strategy involved disruption of the thioredoxin reductase-encoding gene (trxR) using the CRISPR system to target the specific domain of enzyme. However, the transformation was unsuccessful, likely due to incompatibility between the CRISPR plasmid and the A. niger strain. Despite this, both approaches will be further developed using homologous recombination for targeted gene overexpression or disruption. Additionally, cassava pulp was evaluated as an alternative carbon source for gluconic acid production. The PcoxA strain achieved a yield of 9.36 ± 0.21 g/L, equivalent to 0.37 g/g cassava pulp. This productivity is attributed to the strain’s ability to secrete amylase, which hydrolyzes starch into glucose. Overall, this study highlights the important of strain engineering in improving A. niger’s bioproduction capacity and supports the use of cassava pulp as a promising low-cost substrate for gluconic acid production.