Thymoquinone, a monoterpene derived from the black seeds of Nigella sativa, has emerged as a promising pharmacological compound with diverse potential applications in treating various human diseases [1]. It is non-toxic to humans and has been studied for its efficacy in managing conditions such as diabetes and cancer. Additionally, it boasts numerous beneficial properties, including antioxidant, anti-inflammatory, antihyperglycemic, immunomodulatory, antihistaminic, and antitumor effects, suggesting its potential effectiveness in treating conditions such as asthma, arthritis, inflammation, gastrointestinal issues, and liver disorders [2]. Recent studies have also revealed its hepatoprotective, gastroprotective, neuroprotective, and nephroprotective effects [3], further enhancing its market potential. Thymol, another monoterpene structurally akin to thymoquinone, is approximately 100 times cheaper and possesses both antifungal and antibacterial properties [4]. However, it is generally considered less effective than thymoquinone in treating human diseases [5].

Previous studies have explored chemical methods for synthesizing thymoquinone from thymol, yielding approximately 18–31% conversion rates [6]. However, considering the environmental concerns and cost factors associated with chemical methods such as purification, we propose a bio-based whole-cell catalytic conversion method.

The biosynthetic pathways for thymoquinone in Origanum vulgare and thymol in Thymus vulgaris have recently been elucidated [7], revealing the involvement of cytochrome P450 enzymes (P450s) in this process. According to a previous study, thymol is converted to thymoquinone by the cytochrome P450 monooxygenases, CYP76S40 and CYP736A300. P450s, a superfamily of heme-thiolate proteins, are widely distributed across archaea, bacteria, animals, plants, and other eukaryotes and catalyze various oxidative reactions [8]. The functionality of P450s requires intricate electron transfer interactions with cytochrome P450 reductase (CPR), and the fact that most eukaryotic P450s are situated in the endoplasmic reticulum (ER) of cells presents a formidable challenge for enhancing their activity, which remains a complex issue [9]. Saccharomyces cerevisiae, also known as brewer’s or baker’s yeast, possesses an inherent ER, making it advantageous for expressing heterologous P450 proteins [10]. Additionally, it offers several advantages, such as cost-effective cultivation, which is generally regarded as safe (GRAS), and many molecular biological tools, including genome editing.

Therefore, in this study, we aimed to achieve one-step production of thymoquinone from thymol in S. cerevisiae by expressing the plant P450 enzyme CYP736A300 in S. cerevisiae. Furthermore, we aimed to increase thymoquinone production through increased enzyme activity by connecting P450 and CPR through a linker and expanding the ER space for the enzyme reaction. The primary goal of this study was to provide an effective alternative method for producing thymoquinone.