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

Featured Application The dextranase variant (r -TmDEX49A -Delta SP -Delta N30) can be used for dextran removal from sugar during the production process. 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-Delta SP-Delta N30), was fused to the Saccharomyces cerevisiae prepro alpha-factor (MF alpha-2) signal sequence and expressed in Komagataella phaffii under the constitutive GAP promoter. K. phaffii DEX49A-Delta SP-Delta N30, 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 center dot mg(-1) of protein). At shaker scale (100 mL) in a YPG medium, the enzymatic activity was 273 U center dot 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 center dot mL(-1), and the productivity was 53,800 U center dot L-1 center dot h(-1) (53.8 mg center dot L-1 center dot h(-1)), the highest reported thus far for a DEX49A variant. Dextran decreased r-TmDEX49A-Delta SP-Delta N30 mobility in affinity gel electrophoresis, providing evidence of carbohydrate-protein interactions. K. phaffii DEX49A-Delta SP-Delta N30 shows great potential as a methanol-free, commercial dextranase production system.

Automated summary

This study reveals the potential application of a novel dextranase variant in the sugar industry, which demonstrates advantages in both production yield and efficiency. It also provides evidence of carbohydrate-protein interactions.