Constitutive High Expression Level of a Synthetic Deleted Encoding Gene of Talaromyces minioluteus Endodextranase Variant (r–TmDEX49A–ΔSP–ΔN30) in Komagataella phaffii (Pichia pastoris)

• Published in: Applied sciences 2022-08, Vol.12 (15), p.7562
• Main Authors: Arísticas Ribalta, Roberto Carlos, Valdés, Lisandra Martínez, Lafargue Gámez, Meinardo, Rodríguez Davydenko, Sonia, Dubreucq, Eric, Perrier, Veronique, Moreau, Benoît, Vidal, Reinaldo Fraga
• Format: Article
• Language: English
• Subjects: Amino acids; Bacteria; Batch culture; Carbohydrates; Dehydrogenases; DEX49A; Dextran; Dextranase; Dextrans; E coli; Electrophoresis; Electrophoretic mobility; Enzymatic activity; Enzymes; Fed-batch culture; Food engineering; GAP promoter; Gel electrophoresis; Glucose; Glycerol; Komagataella phaffii; Life Sciences; Manufacturing industry; Molecular weight; Peptides; Pichia pastoris; Process controls; Protein interaction; Proteins; Sugar industry; Sugarcane; Talaromyces; Viscosity; Yeast
• ISSN: 2076-3417; 2076-3417
• DOI: 10.3390/app12157562

In the sugar industry, dextran generates difficulties in the manufacturing process. Using 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–ΔSP–ΔN30), was fused to the Saccharomyces cerevisiae prepro α–factor (MFα–2) signal sequence and expressed in Komagataella phaffii under the constitutive GAP promoter. K. phaffii DEX49A–ΔSP–ΔN30, constitutively producing and secreting the truncated dextranase, was obtained. The specific activity of the truncated variant resulted in being nearly the same in relation to the full-length mature enzyme (900–1000 U·mg−1 of protein). At shaker scale (100 mL) in a 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 53,800 U·L−1·h−1 (53.8 mg·L−1·h−1), the highest reported thus far for a DEX49A variant. Dextran decreased r–TmDEX49A–ΔSP–ΔN30 mobility in affinity gel electrophoresis, providing evidence of carbohydrate–protein interactions. K. phaffii DEX49A–ΔSP–ΔN30 shows great potential as a methanol-free, commercial dextranase production system.