Artificial Cascade Transformation Biosystem for One-Pot Biomanufacturing of Odd-Numbered Neoagarooligosaccharides and <scp>d</scp>-Tagatose through Wiser Agarose Utilization

The application of agarose oligosaccharides has garnered great attention, with their biological activities varying among different structures. However, it still meets a great bottleneck for the targeted production of odd-numbered neoagarooligosaccharides (NAOSs), such as neoagarotriose (NA3), due to the lack of one-step hydrolases. In this work, the α-agarase AgaA33 and β-galactosidase BgaD were synergistically used to prepare NA3 with agarose as a substrate. Additionally, an l-arabinose isomerase CaLAI from <i>Clostridium acetobutylicum</i> was characterized to valorize low-value byproducts (d-galactose) by forming d-tagatose, which exhibited good thermal stability without the need for additional metal ions. Under the optimal reaction conditions, the production of NA3 and d-galactose catalyzed by these three enzymes was 0.40 and 0.15 g/L, respectively. The artificial three-enzyme-based cascade transformation system not only achieved the highest production of NA3 until now but also allowed for the valorization of d-galactose, providing a wiser application route for agarose utilization.