Jiao Li

Bovine casein macropeptide (bCMP) is a glycosylated polypeptide generated by the chymosin hydrolysis of κ-casein. It exhibits various bioactivities, including antioxidant, anti-inflammatory, and gut microbiota-regulating effects. This study established a heterologous expression system for bCMP in Pichia pastoris and systematically optimized the promoter, signal peptide, and fermentation conditions to enhance its expression level. Initially, on the basis of the successful construction and expression verification of the bCMP-expressing strain, a fusion expression system of bCMP with green fluorescent protein (GFP) was developed, enabling real-time monitoring of target protein expression via fluorescence intensity. After identifying the optimal promoter pAOX1, the effects of the signal peptides α-factor, SCW, PHO1, SUC, and Cel5A on the secretion level of the fusion protein bCMP-GFP were analyzed and compared, with α-factor identified as the most effective signal peptide for promoting bCMP secretion. Furthermore, a synthetic promoter mutant library of pAOX1 was generated via error-prone PCR. After fluorescence-activated cell sorting (FACS), six mutants (pAOX1-1 to pAOX1-6) were isolated, which exhibited increases of 59%-160% in expression strength compared with the wild type. Finally, the fermentation parameters were optimized as 1.5% methanol, pH 7.5, initial OD₆₀₀ of 4.0, and 26 ℃, which led to a 240% increase in expression compared with the initial conditions. Ultimately, following the integration of the aforementioned optimization strategies, the production titer of bCMP by P. pastoris reached 1.02 g/L. This study successfully developed and optimized a heterologous expression system for bCMP in P. pastoris, yielding novel pAOX1 promoter variants with application potential and providing effective strategies and key regulatory elements for the efficient heterologous expression of other mammalian-derived functional proteins in P. pastoris.

Glycyrrhetic acid 3-O-mono-β-d-glucuronide (GAMG), a rare and innovative compound in licorice, exhibits high-potency sweetness and improved physiological activities. However, low amounts of GAMG from plants cannot meet the demands of growing markets. In this study, an efficient one-pot multienzyme cascade reaction for GAMG biosynthesis was constructed using a coupled catalysis of glycosyltransferase and uridine 5′-diphosphate (UDP) glucuronic acid (GlcA) regeneration system. The Glycyrrhiza uralensis glycosyltransferase UGT73F15 was expressed in Escherichia coli BL21 (DE3). The optimal reaction conditions of UGT73F15 were found to be pH 7.5 and 35 °C. The catalytic efficiency (k_cat/K_m) for glycyrrhetic acid (GA) was 2.14 min⁻¹ mM⁻¹ when using UDP-GlcA as sugar donor. To regenerate costly UDP-GlcA, the one-pot multienzyme cascade reaction including UGT73F15, sucrose synthase, UDP-glucose dehydrogenase, and lactate dehydrogenase was adopted to synthesize GAMG from GA on the basis of the UDP-GlcA regeneration system. By optimizing the cascade reaction conditions, the GAMG production successfully achieved 226.38 mg/L. Our study developed an economical and efficient one-pot multienzyme cascade method for facile synthesis of GAMG and other bioactive glucuronosides.

Cucurbitacins, a group of diverse tetracyclic triterpenes, display a variety of biological effects. Glycosylation mediated by glycosyltransferases (UGTs) plays a vital role in structural and functional diversity of natural products and influences their biological activities. In this study, GT-SM, a mutant of UGT74AC1 from Siraitia grosvenorii, was chosen as a potential catalyst in glycosylation of cucurbitacins, and its optimal pH, temperature, and divalent metal ions were detected. This enzyme showed high activity (k_cat/K_m, 120 s⁻¹ µM⁻¹) toward cucurbitacin F 25-O-acetate (CA-F₂₅) and only produced CA-F₂₅ 2-O-β-d-glucose which was isolated and confirmed by ¹D and ²D nuclear magnetic resonance. A pathway for uridine diphosphate glucose (UDP-Glc) regeneration and cucurbitacin glycoside synthesis was constructed by combing GT-SM and sucrose synthase to cut down the costly UDP-Glc. The molar conversion of CA-F₂₅ was 80.4% in cascade reaction. Molecular docking and dynamics simulations showed that CA-F₂₅ was stabilized by hydrophobic interactions, and the C2-OH of CA-F₂₅ showed more favorable catalytic conformation than that of C3-OH, explaining the high regioselectivity toward the C2-OH rather than the ortho-C3-OH of CA-F₂₅. This work proved the important potential application of UGT74AC1 in cucurbitacins and provided an understanding of glycosylation of cucurbitacins.

Glycyrrhetinic acid (GA) and glycyrrhizin (GA-3-O-[β-d-glucuronopyranosyl-(1→2)-β-d-glucuronopyranoside], GL) are the major bioactive components of Glycyrrhiza uralensis and possess multifarious notable biological activities. UDP-glycosyltransferase (UGT)–catalyzed glycosylation remarkably extends the structural and functional diversification of GA-glycoside derivatives. In this study, six glucosides (1–6) of GA and GL were synthesized using a Bacillus subtilis 168–originated flexible UDP-glycosyltransferase Bs-YjiC. Bs-YjiC could transfer a glucosyl moiety from UDP-glucose to the free C3 hydroxyl and/or C30 carboxyl groups of GA and GL and further elongate the C30 glucosyl chain via a β-1-2-glycosidic bond. Glycosylation significantly increased the water solubility of these novel glucosides by 4–90 folds. In vitro assays showed that GA monoglucosides (1 and 2) showed stronger antiproliferative activity against human liver cancer cells HepG2 and breast cancer cells MCF-7 than that of GL and GL glucosides. These findings provide significant insights into the important role of promiscuous UGTs for the enzymatic synthesis of novel bioactive GA derivatives.