Construction of an Aspergillus oryzae △nptB△pyrG host for homologous expression of Aspergillus oryzae lipase and catalytic properties characterization of recombinant lipase

Abstract Aspergillus oryzae is an ideal cell factory for protein expression with powerful protein processing and secretion capabilities, and is suitable for expression of fungus-derived proteins. The current study aimed to explore the homologous expression of A. oryzae lipase AOL (GenBank: KP975533) by constructing an auxotrophic A. oryzae △pyrG△nptB, and subsequently characterizing the immobilization and catalytic properties of recombinant lipase. Initially, the pyrG gene knocked out in wild-type A. oryzae by homologous recombination of the transformed linearized fragments, followed by the creation of a uridine/uracil auxotroph transformation. Through this system, the protease gene nptB was precisely knocked out, leading to a substantial decrease in extracellular (39.04%) and intracellular (90.07%) protease activity. The A. oryzae △nptB△pyrG strain was used as host for homologous expression of lipase AOL. The engineered A. oryzae AOL-8 was screened out, which exhibiting extracellular and intracellular lipase activities of 1.75 U/mL and 46.4 U/g, respectively. Quantitative PCR analysis revealed that the lipase gene copy number in the engineered strain AOL-8 was 14. Subsequently, the production and immobilization of the recombinant lipase, via physical adsorption on macroporous resin, was achieved through submerged fermentation of the AOL-8 strain. The esterification catalytic properties of recombinant lipase with carboxylic acid and short-chain alcohols were characterized. The results indicated that the lipase exhibited optimal catalytic activity with lauric acid and methanol as substrates, a reaction temperature of 35°C, and n-hexane as the preferred solvent medium.