Heterologous expression of a highly thermostable L-asparaginase from Thermococcus zilligii in Aspergillus niger for efficient reduction of acrylamide in French fries.

L-asparaginase (L-ASNase) can hydrolyze L-asparagine, a precursor to acrylamide, thereby reducing toxic acrylamide formation in fried foods. Currently, commercial L-ASNases are primarily produced by wild-type (WT) filamentous fungi; however, these enzymes often exhibit rapid activity loss during high-temperature processing due to limited thermal stability. In this study, we screened a thermostable L-ASNase gene from thermophile bacteria and expressed it in Aspergillus niger to reduce acrylamide content in French fries. Initially, four genes encoding thermostable L-ASNases were selected and integrated into the A. niger genome via non-homologous end joining. Among these, the L-ASNase gene tzi from Thermococcus zilligii was successfully expressed in A. niger, yielding an extracellular activity of 114 U·mg. The recombinant enzyme (An-Tzi) displayed the same optimal temperature and pH as its WT counterpart but exhibited superior catalytic efficiency, likely due to the efficient post-translational modifications in A. niger. To further enhance expression, the tzi gene was integrated into the amylase (amyA) locus of the A. niger genome using the CRISPR-Cas9 system, resulting in increased activity of 128 U·mg. Additionally, various lengths of the highly expressed glucoamylase (glaA) protein from A. niger AG11 were fused to the N-terminus of the Tzi. Notably, fusing the 500-amino-acid catalytic domain of glaA led to a substantial 3.3-fold increase in enzyme activity. Despite the metabolic stress induced by high-level expression of glaA, supplementing the culture medium with metal ions and sophorose resulted in an extracellular activity of 486.74 U·mg, the highest reported yield of L-ASNase in shake flasks. Finally, applying the An-Tzi to French fries achieved a 32 % greater reduction in acrylamide compared to the commercial enzyme. Overall, the recombinant A. niger strain expressing thermostable An-Tzi demonstrates significant potential for industrial applications targeting acrylamide reduction in fried and baked foods.