TY - JOUR
T1 - Phase Separation-Mediated Multienzyme Assembly In Vivo
AU - Chen, Yao
AU - Shi, Yi
AU - Li, Mengyu
AU - Ming, Dengming
AU - Liu, Wei
AU - Xu, Xian
AU - Jiang, Ling
N1 - Publisher Copyright:
© 2025 American Chemical Society.
PY - 2025/4/2
Y1 - 2025/4/2
N2 - Multienzyme assembly catalysis is a cornerstone of synthetic biology. In this study, we integrated the phase-separated protein RGGRGG with the ReverseTag/ReverseCatcher tagging system to investigate its potential for multienzyme complex formation. Initially, we utilized coarse-grained simulations to explore the interactions among RGGRGG domains, revealing their ability to form condensates and demonstrating how these condensates can interface with the ReverseTag/ReverseCatcher system for the assembly of multienzyme complexes. The ReverseTagged-proteins effectively self-assembled within ReverseCatcher_RGGRGG condensates. In cellular systems, we overexpressed six enzymes involved in lycopene biosynthesis and immobilized ReverseTag_Dxr, ReverseTag_Dxs, and ReverseTag_Idi by forming ester bonds with ReverseCatcher_RGGRGG. This approach resulted in a remarkable 5.4-fold enhancement in lycopene production compared to the control groups. This multienzyme assembly strategy holds significant promise for advancing the field of multienzyme catalysis, both in vivo and in vitro, and is expected to stimulate further research in this area.
AB - Multienzyme assembly catalysis is a cornerstone of synthetic biology. In this study, we integrated the phase-separated protein RGGRGG with the ReverseTag/ReverseCatcher tagging system to investigate its potential for multienzyme complex formation. Initially, we utilized coarse-grained simulations to explore the interactions among RGGRGG domains, revealing their ability to form condensates and demonstrating how these condensates can interface with the ReverseTag/ReverseCatcher system for the assembly of multienzyme complexes. The ReverseTagged-proteins effectively self-assembled within ReverseCatcher_RGGRGG condensates. In cellular systems, we overexpressed six enzymes involved in lycopene biosynthesis and immobilized ReverseTag_Dxr, ReverseTag_Dxs, and ReverseTag_Idi by forming ester bonds with ReverseCatcher_RGGRGG. This approach resulted in a remarkable 5.4-fold enhancement in lycopene production compared to the control groups. This multienzyme assembly strategy holds significant promise for advancing the field of multienzyme catalysis, both in vivo and in vitro, and is expected to stimulate further research in this area.
KW - RGGRGG
KW - ReverseTag/ReverseCacther
KW - multienzyme assembly
UR - http://www.scopus.com/inward/record.url?scp=105000243213&partnerID=8YFLogxK
U2 - 10.1021/acs.jafc.4c09585
DO - 10.1021/acs.jafc.4c09585
M3 - 文章
AN - SCOPUS:105000243213
SN - 0021-8561
VL - 73
SP - 7867
EP - 7876
JO - Journal of Agricultural and Food Chemistry
JF - Journal of Agricultural and Food Chemistry
IS - 13
ER -