Hongyu Zhang

Laminaribiose, a β-1,3-linked glucose disaccharide, possesses notable bioactivities with potential applications in food and health. Its industrial use is constrained by low yield and purity. Production strategies include natural extraction, chemical synthesis, and enzymatic synthesis. Natural extraction preserves bioactivity but is inefficient, chemical synthesis affords structural control but suffers from byproducts and toxicity, whereas enzymatic synthesis offers mild, efficient catalysis with improved yield. Enzymatic synthesis of laminaribiose has been established through systems ranging from single to multi-enzyme cascades. Single-enzyme approaches employ β-glucosidase or β-1,3-glucanase, while dual-enzyme systems use sucrose phosphorylase (SP) and laminaribiose phosphorylase (LBP) with sucrose and glucose. Tri-enzyme systems of α-glucan phosphorylase (αGP), α-glucosidase (αG), and LBP utilize starch. In contrast, four-enzyme cascades integrate αGP, LBP, isoamylase (IA), and 4-α-glucanotransferase (4GT) with maltodextrin and glucose, or combine cellobiohydrolase I (CBHI), cellodextrin phosphorylase (CDP), cellobiose phosphorylase (CBP), and LBP with cellulose. Advances in enzyme engineering, particularly for LBP, have further enhanced catalytic efficiency. This review critically summarizes recent advances in the biological functions, applications, and enzymatic synthesis of laminaribiose, with particular focus on multi-enzyme cascade strategies and their catalytic features. It aims to provide a conceptual framework for developing efficient, sustainable synthesis and advancing industrial applications.

Kojibiose, a rare disaccharide featuring a unique α-1,2-glycosidic linkage, exhibits functional properties such as significant sweetness, prebiotic activity, and iron absorption enhancement. It acts as an α-glucosidase I inhibitor, highlighting its potential in the food and pharmaceutical sectors. While natural extraction and chemical synthesis are limited by scalability and environmental concerns, enzymatic synthesis stands out as an alternative, offering high efficiency, specificity, and sustainability. Current enzymatic systems, including sucrose phosphorylase, kojibiose phosphorylase, and dextransucrase, combined with β-galactosidase or α-glucosidase, have shown considerable progress. Protein engineering of sucrose phosphorylase has further improved catalytic efficiency and thermostability. However, enzymatic synthesis still faces challenges, including low catalytic efficiency, instability, and high substrate costs, which impede large-scale production. Moreover, incomplete safety evaluations, unclear regulatory standards, and the absence of uniform quality specifications hinder compliant application. This review systematically outlines the structural features, physicochemical properties, and enzymatic synthesis strategies of kojibiose, with an emphasis on the role of protein engineering in scaling up production. It also evaluates its functional applications in health promotion, food quality improvement, and therapeutic uses. Finally, interdisciplinary innovations are proposed to establish technical standards and scalable bioprocesses, aiming to overcome current barriers and realize the commercial potential of sustainable kojibiose production.

Sucrose phosphorylase (SPase) is a member of the glycoside hydrolase family 13, catalyzing the reversible phosphorolysis of sucrose to produce α–glucose–1–phosphate and exhibiting transglycosylation activity toward multiple substrates. Its wide substrate specificity enables the synthesis of various glycosides, which are broadly applied in food, cosmetics, and pharmaceuticals. However, the industrial application of SPase is constrained by its poor thermostability and limited transglycosylation activity. Therefore, current research focuses on enhancing the thermostability and transglycosylation activity of SPase through efficient engineering strategies based on its crystal structure and catalytic mechanism. This paper systematically reviews the crystal structure and catalytic mechanism of SPase, outlines the application of protein engineering and immobilization strategies in improving the thermostability of SPase, and analyzes how modifications at key amino acid sites affect the synthesis of typical glycosylation products. It also summarizes the limitations of SPase engineering modification strategies and explores the potential of diversified approaches for SPase modification, highlighting its broad application prospects in industrial production and laying a solid foundation for further advancements in SPase engineering modification and its industrial application.

Sucrose phosphorylase (SPase), a member of the glycoside hydrolase GH13 family, possesses the ability to catalyze the hydrolysis of sucrose to generate α-glucose-1-phosphate and can also glycosylate diverse substrates, showcasing a wide substrate specificity. This enzyme has found extensive utility in the fields of food, medicine, and cosmetics, and has garnered significant attention as a focal point of research in transglycosylation enzymes. Nevertheless, SPase encounters numerous obstacles in industrial settings, including low enzyme yield, inadequate thermal stability, mixed regioselectivity, and limited transglycosylation activity. In-depth exploration of efficient expression strategies and molecular modifications based on the crystal structure and functional information of SPase is now a critical research priority. This paper systematically reviews the source microorganisms, crystal structure, and catalytic mechanism of SPase, summarizes diverse heterologous expression systems based on expression hosts and vectors, and examines the application and molecular modification progress of SPase in synthesizing typical glycosylated products. Additionally, it anticipates the broad application prospects of SPase in industrial production and related research fields, laying the groundwork for its engineering modification and industrial application.

AA10 family lytic polysaccharide monooxygenases (AA10 LPMOs) are mainly distributed in bacteria. Because of their characteristics of oxidative degradation of crystalline polysaccharides, such as cellulose and chitin, they have great application potential in industrial biomass conversion and have attracted wide attention. Efficient heterologous expression of LPMOs by recombinant engineering bacteria has become the main strategy for the industrial production of enzymes. The research progress of AA10 LPMOs’ heterologous expression systems was reviewed in this paper. The construction strategies of its diversified heterologous expression system were introduced based on the design and processing of the expression host, vector, and LPMOs gene. The effects of different expression systems on the soluble expression of LPMOs and the development direction of the construction of LPMOs’ heterologous expression systems were discussed. The broad application prospect of LPMOs in the biomass conversion and biofuel industry has been prospected.

The special structure of pentostatin causes it to possess a wide spectrum of biological and pharmacological properties, and it has been extensively employed to treat malignant tumors and is the first-line treatment for hairy cell leukemia. Pentostatin is mainly distributed in several actinomycetes and fungi species. However, its low titer in microbes is not able to meet medical needs. Here, we report a strain improvement strategy based on combined atmospheric and room-temperature plasma (ARTP) mutagenesis and ribosome engineering screening, as well as fermentation optimization, for enhanced pentostatin production. The original strain, Actinomadura sp. ATCC 39365, was treated with ARTP and screened by ribosome engineering to obtain one stable pentostatin high-yield mutant Actinomadura sp. S-15, which produced 86.35 mg/L pentostatin, representing a 33.79% increase compared to Actinomadura sp. ATCC 39365. qRT-PCR analysis revealed that pentostatin biosynthesis-related gene expression was significantly upregulated in Actinomadura sp. S-15. Then, to further enhance pentostatin production, the fermentation medium was optimized in flask culture and the pentostatin production of Actinomadura sp. S-15 reached 152.06 mg/L, which is the highest pentostatin production reported so far. These results demonstrate the effectiveness of combined ARTP mutation, ribosome engineering screening, and medium optimization for the enhancement of pentostatin production, and provide a methodology enabling the sustainable production of pentostatin on an industrial scale.

Pentostatin (PNT), a nucleoside antibiotic with a 1,3-diazo ring structure, is distributed in several actinomycetes and fungi species. Its special structure makes PNT possess a wide spectrum of biological and pharmacological properties, such as antibacterial, antitrypanosomal, anticancer, antiviral, herbicidal, insecticidal, and immunomodulatory effects. Because of the promising adenosine deaminase inhibitory activity of PNT, its extensive application in the clinical treatment of malignant tumors has been extensively studied. However, the fermentation level of microbial-derived PNT is low and cannot meet medical needs. Because the biosynthesis pathway of PNT is obscure, only high-yield mutant screening and optimization of medium components and fermentation processes have been conducted for enhancing its production. Recently, the biosynthesis pathways of PNT in actinomycetes and fungi hosts have been revealed successively, and the large-scale production of PNT by systematic metabolic engineering will become an inevitable trend. Therefore, this review covers all aspects of PNT research, in which major advances in understanding the resource microorganisms, mechanism of action, and biosynthesis pathway of PNT were achieved and diverse clinical applications of PNT were emphasized, and it will lay the foundation for commercial transformation and industrial technology of PNT based on systematic metabolic engineering.