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L-asparaginase is an important enzyme in the pharmaceutical field used as treatment for acute lymphoblastic leukemia due to its ability to hydrolyze L-asparagine, an essential amino acid synthesized by normal cells, but not by neoplastic cells. Adverse effects of L-asparaginase formulations are associated with its glutaminase activity and bacterial origin; therefore, it is important to find new sources of L-asparaginase produced by eukaryotic microorganisms with low glutaminase activity. This work aimed to identify the L-asparaginase gene sequence from <i>Penicillium sizovae</i>, a filamentous fungus isolated from the Brazilian Savanna (Cerrado) soil with low glutaminase activity, and to biosynthesize higher yields of this enzyme in the yeast <i>Komagataella phaffii</i>. The L-asparaginase gene sequence of <i>P. sizovae</i> was identified by homology to L-asparaginases from species of <i>Penicillium</i> of the section <i>Citrina</i>: <i>P. citrinum</i> and <i>P. steckii</i>. Partial L-asparaginase from <i>P. sizovae</i>, lacking the periplasmic signaling sequence, was cloned, and expressed intracellularly with highest enzymatic activity achieved by a MUT⁺ clone cultured in BMM expression medium; a value 5-fold greater than that obtained by native L-asparaginase in <i>P. sizovae</i> cells. To the best of our knowledge, this is the first literature report of the heterologous production of an L-asparaginase from a filamentous fungus by a yeast.