Optimized anchor and cell wall engineering boost UDP-glycosyltransferase display for glycyrrhetinic acid glycosylation in Komagataella phaffii

TY - JOUR

T1 - Optimized anchor and cell wall engineering boost UDP-glycosyltransferase display for glycyrrhetinic acid glycosylation in Komagataella phaffii

AU - Cao, Weijie

AU - Wang, Jing

AU - Feng, Xudong

AU - Liu, Hu

AU - Li, Chun

N1 - Publisher Copyright: © 2025 The Chemical Industry and Engineering Society of China, and Chemical Industry Press Co., Ltd.

PY - 2026/3

Y1 - 2026/3

N2 - The glycosylation of natural products like glycyrrhetinic acid (GA) is critical for improving their pharmaceutical properties. However, the industrial application of uridine diphosphate-glycosyltransferases (UGTs) is hindered by their low stability and difficult recovery. Microbial surface display addresses these issues but faces a key bottleneck in achieving high display efficiency and robustness, primarily dependent on the anchoring system. This study presents a systematic engineering approach to optimize the display of UGT on the surface of Komagataella phaffii (K. phaffii) for enhanced biocatalytic performance in GA glycosylation. Initial attempts using the K. phaffii-derived anchored protein GCW51 revealed low display efficiency, with only 2852 enzymes per cell and 8.1 μmol·L−1 GA-3-O-Glc production, which severely limited its industrial application potential. To address this, we expanded screening to five additional anchor proteins, and structural characterization via fluorescence microscope, flow cytometry, and TEM confirmed successful surface localization of UGT in all engineered strains, with distinct differences in display efficiency. Among them, the GPI-anchored Agα demonstrated superior performance, achieving 8379 enzymes per cell (2.9-fold higher than GCW51) and 26.8 μmol·L−1 GA-3-O-Glc production (3.3-fold improvement over GCW51). All displayed systems exhibited significantly improved thermal stability, with Agα-mediated display showing 3.0-fold longer half-life at 50 °C than free enzyme and 1.2-fold longer than GCW51. To further enhance performance, we implemented β-glucan biosynthesis pathway engineering, yielding strain UGT-A-OE-3 with 11369 enzymes per cell (4.0-fold increase over GCW51) and 53% higher β-glucan content. The optimized system achieved 34.9 μmol·L−1 GA-3-O-Glc production, representing a 4.3-fold improvement over the initial GCW51 system and 30% greater activity than the parental Agα strain. This work establishes an effective dual-engineering strategy combining anchor optimization with metabolic pathway enhancement, significantly elevating display efficiency. The developed platform shows significant potential for industrial biocatalysis applications, particularly in glycosylation reactions requiring robust and reusable enzyme systems.

AB - The glycosylation of natural products like glycyrrhetinic acid (GA) is critical for improving their pharmaceutical properties. However, the industrial application of uridine diphosphate-glycosyltransferases (UGTs) is hindered by their low stability and difficult recovery. Microbial surface display addresses these issues but faces a key bottleneck in achieving high display efficiency and robustness, primarily dependent on the anchoring system. This study presents a systematic engineering approach to optimize the display of UGT on the surface of Komagataella phaffii (K. phaffii) for enhanced biocatalytic performance in GA glycosylation. Initial attempts using the K. phaffii-derived anchored protein GCW51 revealed low display efficiency, with only 2852 enzymes per cell and 8.1 μmol·L−1 GA-3-O-Glc production, which severely limited its industrial application potential. To address this, we expanded screening to five additional anchor proteins, and structural characterization via fluorescence microscope, flow cytometry, and TEM confirmed successful surface localization of UGT in all engineered strains, with distinct differences in display efficiency. Among them, the GPI-anchored Agα demonstrated superior performance, achieving 8379 enzymes per cell (2.9-fold higher than GCW51) and 26.8 μmol·L−1 GA-3-O-Glc production (3.3-fold improvement over GCW51). All displayed systems exhibited significantly improved thermal stability, with Agα-mediated display showing 3.0-fold longer half-life at 50 °C than free enzyme and 1.2-fold longer than GCW51. To further enhance performance, we implemented β-glucan biosynthesis pathway engineering, yielding strain UGT-A-OE-3 with 11369 enzymes per cell (4.0-fold increase over GCW51) and 53% higher β-glucan content. The optimized system achieved 34.9 μmol·L−1 GA-3-O-Glc production, representing a 4.3-fold improvement over the initial GCW51 system and 30% greater activity than the parental Agα strain. This work establishes an effective dual-engineering strategy combining anchor optimization with metabolic pathway enhancement, significantly elevating display efficiency. The developed platform shows significant potential for industrial biocatalysis applications, particularly in glycosylation reactions requiring robust and reusable enzyme systems.

KW - Biocatalysis

KW - Catalyst support

KW - Immobilization

KW - Protein stability

KW - Synthetic biology

KW - Yeast surface display

UR - https://www.scopus.com/pages/publications/105031426687

U2 - 10.1016/j.cjche.2025.10.017

DO - 10.1016/j.cjche.2025.10.017

M3 - Article

AN - SCOPUS:105031426687

SN - 1004-9541

VL - 91

SP - 60

EP - 68

JO - Chinese Journal of Chemical Engineering

JF - Chinese Journal of Chemical Engineering

ER -