A rare group of mono- or disaccharides and their derivatives that are found in small quantities in nature have garnered significant attention in recent years due to their potential health benefits [1]. These sugars differ from common forms such as glucose and fructose, and these rare versions have unique structures and metabolic pathways that lead to distinct physiological effects. The enzymatic production of rare sugars from natural resources has recently begun to attract a great deal of interest [2], [3], [4]. This process is referred to as an Izumoring strategy, and it enables the conversion of various rare monosaccharides through enzymatic isomerization and oxidization-reduction reactions [5], [6], [7]. While the Izumoring strategy is highly effective for producing rare monosaccharides, it does not offer an efficient approach for producing rare disaccharides and oligosaccharides.

As previously mentioned, isoprimeverose (α-d-xylopyranosyl-(1→6)-d-glucose) is a rare disaccharide that can be produced by enzymatically degrading xyloglucan [8]. Xyloglucan is a matrix polysaccharide found in plant cell walls and seeds [9]. It consists of a β-(1→4)-glucan backbone with α-(1→6)-linked xylopyranosyl side chains, and many of these xylopyranosyl side chains are further modified with β-(1→2)-linked galactopyranosyl moieties [10]. Additionally, xyloglucan is partially modified with fucose, arabinose, and other sugars, which adds to its structural complexity. Given these characteristics, enzymatic hydrolysis has recently emerged as an effective method for isoprimeverose production [11]. Although isoprimeverose can be synthesized from d-glucose and d-xylose via the Koenigs-Knorr reaction, enzymatic methods are superior in terms of yield and cost efficiency [12], [13].

Filamentous fungi are widely used for enzyme production due to their ability to efficiently secrete extracellular enzymes [14], [15], [16]. Among them, Aspergillus oryzae is particularly advantageous, as it naturally produces amylase, protease, and lipase, which makes it a key microorganism in Japanese fermented food production [17], [18]. Furthermore, A. oryzae is classified as ‘‘Generally Recognized as Safe (GRAS)’’ by the United States Food and Drug Administration [19], which reinforces its suitability for industrial applications. Given these advantages, we have previously examined the usefulness of A. oryzae as a host microorganism for bioproduction [20], [21], [22], [23], [24], [25], [26], and this includes the successful construction of a genetically engineered A. oryzae strains for the secretion of cellulases [21], [22], [24].

A. oryzae inherently possesses a wide array of carbohydrate-degrading enzymes (27). Thus, creating an enzyme cocktail using this organism is expected to be advantageous for the efficient degradation of plant-derived xyloglucans, which are often heavily modified. For instance, tamarind seed gum—commonly used in the food industry as a thickening agent—features xylose units that are frequently substituted with galactopyranose residues. These modifications cannot be removed by the isoprimeverose-producing enzyme (IpeA) alone, necessitating the presence of β-galactosidase activity for complete hydrolysis.　Moreover, to efficiently enzymatically process long-chain xyloglucans, endo-type enzymes are required to fragment the polymer. In the case of tamarind xyloglucan, treatment with endoglucanase (EG) targeting unsubstituted glucopyranose units in the backbone structure has proven effective for this purpose.

To achieve high-yield recovery of isoprimeverose, removal of by-products must also be considered. As described above, xyloglucans contain various sugar side chains, and enzymatic treatment inevitably generates sugars other than isoprimeverose as by-products. Several strategies can be employed for monosaccharide removal, including size exclusion chromatography, membrane separation, and microbial degradation. Among these, microbial degradation is particularly promising for large-scale reaction systems. Notably, Rodd et al. demonstrated the use of yeast for by-product removal in the production of xyloglucan oligosaccharides [27]. In light of this, microbial degradation is also considered a viable approach in the present study for the removal of by-products, especially with future large-scale isoprimeverose production in mind.

In the present study, we developed an efficient production process for isoprimeverose by constructing two genetically engineered A. oryzae strains: one producing IpeA and the other producing EG. Additionally, we established a simple and effective method for producing isoprimeverose from plant-derived xyloglucan in part via inspiration from traditional Sake fermentation whereby unwanted monosaccharides are efficiently removed from enzymatically degraded solutions.