Enhancing thermotolerance of Aspergillus niger for sodium gluconate production by combining metabolic engineering and...

Aspergillus niger (A. niger) is a leading industrial platform for sodium gluconate (SG) production. However, heat stress, arising from rapid catalytic reactions and neutralization, can impair the production rate and even compromise cell viability. To develop a thermotolerant cell factory, a pyrG* auxotrophic chassis using DNA-free CRISPR-Cas9 editing, thereby avoiding the 0.04 mol mol^-1 yield-loss from glucose to SG associated with gene editing at 37°C. Subsequently, yeast-derived thermotolerance-related genes, specifically sterol C-5 desaturase (erg3) and AMP deaminase (amd), were site-specifically overexpressed at the pyrG locus and amd-overexpressing strain (COE-AMD) exhibited superior thermotolerance compared to the erg3-overexpressing strain. Notably, COE-AMD achieved a 1.6-fold higher SG production rate than the wild type (WT), with an SG yield of 0.76 ± 0.03 mol mol^-1 at 45°C. To elucidate the underlying mechanism of thermotolerance in A. niger, integrated multi-omics analyses revealed that amd overexpression induced systematic metabolic reprogramming. In particular, enhanced intracellular NH₄⁺ assimilation during the late logarithmic phase contributed to the accelerated growth of COE-AMD at elevated temperature. Guided by these omics insights, supplementing the initial medium with 0.4 g L^-1 ammonium increased biomass, enzyme activity, and product formation rate by 3.0-, 4.0-, and 1.6-fold, respectively. In summary, the thermotolerant strain COE-AMD produced 311.5 ± 2.0 g L^-1 SG within 66 h at 45°C, achieving a yield of 0.78 ± 0.03 mol mol^-1. Overall, this work establishes a complete pipeline for SG process intensification, and the omics-guided bioprocess optimization provides fundamental insights into the global metabolic basis of A. niger thermotolerance.