HAL will be down for maintenance from Friday, June 10 at 4pm through Monday, June 13 at 9am. More information
Skip to Main content Skip to Navigation
Journal articles

Overproduction of pro-transglutaminase from Streptomyces hygroscopicus in Yarrowia lipolytica and its biochemical characterization

Abstract : BACKGROUND: Transglutaminases (TGase), synthesized as a zymogen (pro-TGase) in Streptomyces sp., are important enzymes in food industry. Due to the important applications of TGase in food industry, obtaining robust and food-safe TGase-producing strains has attracted much attention during the past decade. In this study, Streptomyces hygroscopicus pro-TGase was efficiently expressed and secreted by a food-grade host, Yarrowia lipolytica, without antibiotic markers. RESULTS: The pro-TGase gene was cloned into integrative vectors pINA1296 (monocopy) and pINA1297 (multicopy), and was used to transform the Y. lipolytica Po1g or Po1h strain, respectively. Expression was driven by a recombinant hp4d promoter and secretion obtained using a XPR2 pre-sequence as a signal peptide. The highest yield of extracellular pro-TGase produced by the recombinant Po1h strain corresponded to 5.3 U/mL of TGase, a level 8.8 fold higher than that obtained using the recombinant Po1g strain. Asparagines in two potential Asn-linked glycosylation sites (Asn160 and Asn355) from pro-TGase were mutated to glutamine individually or simultaneously, yielding the deglycosylated variants N160Q, N355Q, and N160Q/N355Q. The activities of N160Q, N355Q and N160Q/N355Q constructs were respectively 5.3 U/mL, 7.8 U/mL, and 3.0 U/mL, equivalent to 100 %, 147 %, and 57 % of that from wild-type pro-TGase. The TGase yield of N355Q variant was raised to 35.3 U/mL of by using a glycerol feeding strategy in a 3 L fermenter. The optimal pH and temperature of the activated pro-TGase, and of its deglycosylated variants, were in the range of 5.0-6.0 pH and 40-45 °C, respectively. The half-life of the recombinant wild-type pro-TGase at 37 °C reached 34.0 min, and those of the variants were from 24.2 min to 11.5 min. In contrast to the wild-type pro-TGase, all of the variants had decreased specific activities, and both the K m and k cat values of the variants decreased accordingly. CONCLUSIONS: This study constitutes the first report of the heterologous expression of a pro-TGase in Y. lipolytica, and provides new possibilities for the efficient production of TGases used in food processing.
Document type :
Journal articles
Complete list of metadata

Cited literature [34 references]  Display  Hide  Download

Contributor : Armelle Sielinou Connect in order to contact the contributor
Submitted on : Thursday, May 28, 2020 - 12:38:10 PM
Last modification on : Tuesday, August 3, 2021 - 12:14:06 PM


Publisher files allowed on an open archive



Song Liu, Dan Wan, Miao Wang, Catherine Madzak, Guocheng Du, et al.. Overproduction of pro-transglutaminase from Streptomyces hygroscopicus in Yarrowia lipolytica and its biochemical characterization. BMC Biotechnology, BioMed Central, 2015, 15, pp.75. ⟨10.1186/s12896-015-0193-1⟩. ⟨hal-01564918⟩



Record views


Files downloads