Constitutive High Expression Level of A Synthetic Deleted Encoding Gene of <i>Talaromyces minioluteus</i> Endodextranase Variant (r-TmDEX49A-ΔSP-ΔN30) in <i>Komagataella phaffii</i> (<i>Pichia pastoris</i>)

Abstract

In the sugar industry, dextran generates difficulties in the manufacturing process. Crude dextranase (EC 3.2.1.11) to eliminate dextran in sugar is an effective practice. In this study, a synthetic dextranase encoding gene of the filamentous fungus Talaromyces minioluteus, lacking its putative native signal peptide (1-20 amino acids) and the next 30 amino acids (r-TmDEX49A-&amp;Delta;SP-&amp;Delta;N30), was fused to the Saccharomyces cerevisiae prepro -factor (MF-2) signal sequence and expressed in Komagataella phaffii under the constitutive GAP promoter. K. phaffii DEX49A-&amp;Delta;SP-&amp;Delta;N30, constitutively producing and secreting the truncated dextranase was obtained. The specific activity of the truncated variant resulted nearly the same in relation to the full-length mature enzyme (900-1000 U.mg-1 of protein). At shaker scale (100 mL) in YPG medium, the enzymatic activity was 273 U.mL-1. The highest production level was achieved in a fed-batch culture (30 h) at 5 L fermenter scale using the FM21-PTM1 culture medium. The enzymatic activity in the culture supernatant reached 1614 U.mL-1 and the productivity was 53800 U.L-1.h-1 (53.8 mg.L-1.h-1), the highest reported so far for a DEX49A variant. Dextran decreased r-TmDEX49A-&amp;Delta;SP-&amp;Delta;N30 mobility in affinity gel electrophoresis, providing evidence of carbohydrate-protein interactions. K. phaffii DEX49A-&amp;Delta;SP-&amp;Delta;N30 shows great potential as a methanol-free, commercial dextranase production system.