Flavonoids are prevalent and essential natural compounds found in plants. They play a significant role in transmitting cellular information and resisting external harsh environments[1]. Isoquercetin, also known as quercetin-3-O-β-D-glucopyranoside, is a natural flavonoid compound with numerous pharmacological activities. It has gained significant attention for its medicinal benefits to human health, such as antioxidant, antibacterial, and antiviral properties. Furthermore, isoquercetin could also be utilized to regulate lipid metabolism-related diseases and safeguard nerve cells [2], [3], [4]. While there are numerous methods for producing isoquercetin, the synthesis of isoquercetin using quercetin as a substrate is regarded as an environmentally friendly and convenient approach. In this process, quercetin undergoes glycosylation at the 3-OH position, catalyzed by flavonol 3-O-glucosyltransferase (F3GT) [5]. Compared to the quercetin, isoquercetin is more water-soluble and exhibits higher bioavailability [6]. However, there are few studies on modifying strains to increase isoquercetin production from quercetin or other feedstocks, as far as we know. The primary and most probable reason is that the biosynthesis of isoquercetin requires not only quercetin, but also uridine diphosphate glucose (UDPG), which is a crucial sugar donor. UDPG is expensive, thus posing a significant challenge to large-scale factory production of isoquercetin. In order to reduce the cost of UDPG, the incorporation of sucrose synthase could facilitate the self-recycling of UDPG [7], [8]. Sucrose synthase catalyzes the synthesis of UDP-glucose (UDPG) and fructose from sucrose and uridine diphosphate (UDP). Therefore, fructose would accumulate in the self-recycling pathway of UDPG. As a by-product of the self-recycling UDPG pathway, fructose has low economic value. Furthermore, fructose has high solubility in water, which may potentially affect the extraction of isoquercetin. Therefore, understanding how to effectively manage fructose is valuable for increasing product value and preventing product interference.

D-allulose is an isomer of D-fructose and has greater economic and practical value compared to D-fructose. Although D-allulose is only 70% as sweet as sucrose, it has a similar taste and properties to sucrose. In addition, the caloric value of D-allulose is 0.4 kcal/g, which is only 10% of sucrose [9]. Thus, D-allulose is considered a suitable sugar substitute and was recognized as a Generally Recognized as Safe (GRAS) food additive by the United States Food and Drug Administration (FDA) in 2011 [10]. In addition to being used as a food additive, D-allulose also demonstrates antioxidant, nerve-protecting, hypoglycemic, and hypolipidemic effects [11]. Therefore, D-allulose has a wide range of potential applications in food, dietary supplements, and pharmaceutical preparations. There are two primary methods for producing D-allulose: chemical synthesis and biocatalysis. Given the pressures of ecosystem protection and production costs, bio-catalysis has been recognized as a more efficient and eco-friendly method for producing D-allulose. D-allulose 3-epimerase (DAEase) is a crucial enzyme in the biocatalysis of D-allulose. It catalyzes the isomerization reactions of D-fructose into D-allulose [12]. According to previous reports [13], [14], DAEase has been successfully utilized to produce D-allulose from various substrates, including D-fructose, D-glucose, and inulin. However, the conversion efficiency will be less than optimal due to the influence of thermodynamic equilibrium, which resulting in high product costs. Therefore, a production technology with high efficiency or a catalyst with high catalytic performance is highly needed to enhance the industrial production of D-allulose.

In this study, we modified the standard principles in order to reduce the production costs of isoquercetin and D-allulose by co-producing them, thereby sharing the production costs. To achieve our objectives, we proposed the following research strategies: (1) Screening for the best isoquercetin synthase (also known as flavonol 3-O-glucosyltransferase, F3GT) for isoquercetin biosynthesis from quercetin. (2) Exogenous sucrose synthase (SUS) and sucrose permease (SUP) for self-recycling pathway of UDPG. (3) Exogenous D-allulose 3-epimerase (DAEase) to convert D-fructose to D-allulose. (4) Optimizing the conditions for one-pot system. In addition, this study presents, for the first time, the biosynthesis of isoquercetin from quercetin. It offers a valuable strategy for producing high-cost chemicals, in which two or more value-added chemicals are simultaneously produced to distribute the production costs.