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Characterization and overproduction of cell-associated cholesterol oxidase ChoD from Streptomyces lavendulae YAKB-15.

ABSTRACT: Cholesterol oxidases are important enzymes with a wide range of applications from basic research to industry. In this study, we have discovered and described the first cell-associated cholesterol oxidase, ChoD, from Streptomyces lavendulae YAKB-15. This strain is a naturally high producer of ChoD, but only produces ChoD in a complex medium containing whole yeast cells. For characterization of ChoD, we acquired a draft genome sequence of S. lavendulae YAKB-15 and identified a gene product containing a flavin adenine dinucleotide binding motif, which could be responsible for the ChoD activity. The enzymatic activity was confirmed in vitro with histidine tagged ChoD produced in Escherichia coli TOP10, which lead to the determination of basic kinetic parameters with Km 15.9?µM and kcat 10.4/s. The optimum temperature and pH was 65?°C and 5, respectively. In order to increase the efficiency of production, we then expressed the cholesterol oxidase, choD, gene heterologously in Streptomyces lividans TK24 and Streptomyces albus J1074 using two different expression systems. In S. albus J1074, the ChoD activity was comparable to the wild type S. lavendulae YAKB-15, but importantly allowed production of ChoD without the presence of yeast cells.

SUBMITTER: Yamada K

PROVIDER: S-EPMC6694107 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Characterization and overproduction of cell-associated cholesterol oxidase ChoD from Streptomyces lavendulae YAKB-15.

Yamada Keith K Koroleva Arina A Laughlin Mitchell M Oksanen Niko N Akhgari Amir A Safronova Vera V Yakovleva Elena E Kolodyaznaya Vera V Buldakova Tatiana T Metsä-Ketelä Mikko M

Scientific reports 20190814 1

Cholesterol oxidases are important enzymes with a wide range of applications from basic research to industry. In this study, we have discovered and described the first cell-associated cholesterol oxidase, ChoD, from Streptomyces lavendulae YAKB-15. This strain is a naturally high producer of ChoD, but only produces ChoD in a complex medium containing whole yeast cells. For characterization of ChoD, we acquired a draft genome sequence of S. lavendulae YAKB-15 and identified a gene product contain ...[more]

PMID: 31413341

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Project description:BackgroundTransglutaminases (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.ResultsThe 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.ConclusionsThis 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.

Dataset Information

Characterization and overproduction of cell-associated cholesterol oxidase ChoD from Streptomyces lavendulae YAKB-15.

Publications

Characterization and overproduction of cell-associated cholesterol oxidase ChoD from Streptomyces lavendulae YAKB-15.

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