Preprint Article Version 1 Preserved in Portico This version is not peer-reviewed

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

Version 1 : Received: 21 June 2022 / Approved: 22 June 2022 / Online: 22 June 2022 (10:02:33 CEST)

A peer-reviewed article of this Preprint also exists.

Arísticas Ribalta, R.C.; Martínez Valdés, L.; Lafargue Gámez, M.; Rodríguez Davydenko, S.; Dubreucq, E.; Perrier, V.; Moreau, B.; Vidal, R.F. Constitutive High Expression Level of a Synthetic Deleted Encoding Gene of Talaromyces minioluteus Endodextranase Variant (rTmDEX49A–ΔSP–ΔN30) in Komagataella phaffii (Pichia pastoris). Appl. Sci. 2022, 12, 7562. Arísticas Ribalta, R.C.; Martínez Valdés, L.; Lafargue Gámez, M.; Rodríguez Davydenko, S.; Dubreucq, E.; Perrier, V.; Moreau, B.; Vidal, R.F. Constitutive High Expression Level of a Synthetic Deleted Encoding Gene of Talaromyces minioluteus Endodextranase Variant (r–TmDEX49A–ΔSP–ΔN30) in Komagataella phaffii (Pichia pastoris). Appl. Sci. 2022, 12, 7562.

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-ΔSP-ΔN30), 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-ΔSP-ΔN30, 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-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.

Keywords

dextranase; DEX49A; Pichia pastoris; GAP promoter

Subject

Biology and Life Sciences, Biology and Biotechnology

Comments (0)

We encourage comments and feedback from a broad range of readers. See criteria for comments and our Diversity statement.

Leave a public comment
Send a private comment to the author(s)
* All users must log in before leaving a comment
Views 0
Downloads 0
Comments 0
Metrics 0


×
Alerts
Notify me about updates to this article or when a peer-reviewed version is published.
We use cookies on our website to ensure you get the best experience.
Read more about our cookies here.