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

Abstract

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.

Key Proteomics Tools for Fundamental and Applied Microalgal Research

“…As for proteomics, heterologous expression consists of several steps that can only partly be automated, i.e., host selection, vector design and cloning, transformation of the host, screening of transformants, cultivation of the transformant, heterologous enzyme production, and finally protein recovery and purification. Several hosts can be used for heterologous expression such as Komagataella phaffii (formerly Pichia pastoris ) and Escherichia coli [ 140 , 141 , 142 ]. The yeast K. phaffii is commonly used because of its reliability, the possibility to achieve a high heterologous protein expression level, and the presence of the co-translational and post-translational processing necessary for eukaryotic proteins [ 140 ].…”