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 K m 15.9 µM and k cat 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.

Identification of cholesterol-assimilating actinomycetes strain and application of statistical modeling approaches for improvement of cholesterol oxidase production by Streptomyces anulatus strain NEAE-94

“…Cholesterol oxidase (EC 1.1.3.6) is a flavin adenine dinucleotide (FAD)-dependent enzyme that catalyzes cholesterol oxidation to cholestenone (4-cholesten-3one) and hydrogen peroxide [1]. Cholesterol oxidase of microbial origin exhibits a wide range of industrial applications besides to its clinical applications in order to determine food and serum cholesterol levels which are important in the diagnosis of cardiovascular disease, atherosclerosis and other lipid disorders [2,3].…”

Differential regulation of undecylprodigiosin biosynthesis in the yeast-scavenging Streptomyces strain MBK6

“…The initial aim was to find new Streptomyces strains that would be stimulated by yeast, like the cholesterol oxidase-producing Streptomyces lavendulae YAKB-15 (Yamada et al . 2019 ). Under co-culture conditions with yeast, the strain grows as a highly dispersed culture, the mycelium adheres to the yeast cells and the yeast is sequestered in the growing mycelial clumps (Fig.…”

“…We have previously noted that Streptomyces may respond to the presence of yeast by triggering production of extracellular cholesterol oxidase (Yamada et al . 2019 ). This study continues the characterization of Streptomyces –yeast interactions.…”

Cloning, expression, and in silico structural modeling of cholesterol oxidase of Acinetobacter sp. strain RAMD in E. coli

“…Typically, the cloning expression of CHOXs using the heterologous host E. coli , a species generally regarded as safe (GRAS), is considered the gold standard solution for avoiding these obstacles. In this regard, great attention has been paid in recent years toward the cloning and expression of CHOXs from a wide range of pathogenic and nonpathogenic bacteria [2,26–33], especially after the publication of a plethora of microbial genome sequences by the NCBI. Nevertheless, the cloning of CHOX‐encoding genes from their wild‐type producers is still considered a venture in its infancy.…”