Heterologous expression and functional characterization of the ligand-binding domain of oxysterol-binding protein from Aspergillus oryzae

Abstract

Oxysterol-binding proteins (OSBPs) comprise a family of sterol-binding proteins. In this study, we focused on AoOSBP1, one of the five OSBP proteins identified from the industrial fungus Aspergillus oryzae. The temporal expression pattern analysis showed that the expression of AoOSBP1, in both gene and protein levels, was stably expressed throughout the developmental stages, while was upregulated during the accelerated growth stage. The immunofluorescence observation revealed that AoOSBP1 protein was mainly distributed in the conidiophore, indicating its underlying role in spore formation. The ligand-binding domain of AoOSBP1, namely OSBP-related domain (ORD), was heterologously expressed in Escherichia coli and purified. The binding assay carried out using microscale thermophoresis showed that the recombinant AoORD protein exhibited binding affinity for ergosterol, and exhibited much higher affinity to oxysterols (25-hydroxycholesterol and 7-ketocholesterol) and phytosterols (βsitosterol and stigmasterol). By contrast, MBP tag as the negative control showed no binding affinity for sterols. The present work demonstrates that AoORD domain in AoOSBP1 is capable of binding sterols, plays an underlying role in sterols transportation, and may participate in spore formation.

Aspergillus oryzae accelerates the conversion of ergosterol to ergosterol peroxide by efficiently utilizing cholesterol

“…The saponification solution was prepared by adding 25 g potassium hydroxide in 35 ml ddH 2 O for dissolution, followed by the addition of absolute alcohol to a volume of 100 ml; the solution was shaken well and refrigerated at 4°C ( Wang et al, 2018 ). The methods of making the binding buffer, lysis buffer, and elution buffer were modified from Protein Purification Kits and described in Supplementary information ( Ma et al, 2019 ).…”

“…The next day, 2 ml culture was taken and diluted in 200 ml fresh medium and allowed to grow for 2 h to OD600 of 0.4–0.6, after which IPTG was added to a final concentration of 0.5 mM. The induction time for Osh3 was 10 h at 27°C, after which the cells were pelleted and stored at −80°C until further processing ( Ma et al, 2019 ).…”

“…OSBP and ORPs bind various sterols (e.g., 25hydroxycholesterol, 22-hydroxycholesterol, cholesterol, ergosterol) via the ORD domain and bind phospholipids via their PH domain (Kentala et al, 2015;Ma et al, 2019). OSBP and…”

“…This will increase soy sauce’s nutritional value, flavor, and taste, so it is considered a safe food-grade fermentation strain ( Sugiyama, 1984 ; Bechman et al, 2012 ). A. oryzae harbors five oxysterol-binding protein-related protein (ORP) family proteins, namely oxysterol binding proteins homologous 1 (Osh1), Osh3, Osh5, Osh7, and ORP8, which play essential roles in lipid metabolism and sterol transportation of A. oryzae ( Ma et al, 2019 ). Therefore, understanding the mechanism of cholesterol degradation by A. oryzae is important.…”

“…Especially at membrane contact sites (MCS) formed by the endoplasmic reticulum (ER) and other organelles (e.g., Golgi body, mitochondria, cell membrane, and lysosomes) ( Du et al, 2015 ). OSBP and ORPs bind various sterols (e.g., 25-hydroxycholesterol, 22-hydroxycholesterol, cholesterol, ergosterol) via the oxysterol-binding protein-related domain (ORD) and bind phospholipids via their pleckstrin homology (PH) domain ( Kentala et al, 2015 ; Ma et al, 2019 ). OSBP and ORP transport phospholipids formed on the plasma membrane or other organelles membranes to ER, which are hydrolyzed by Sac1 phosphatase enzyme, and reverse transport sterols from the ER to the corresponding membranes for metabolic activities ( Mesmin et al, 2017 ).…”