Geranylgeranyl diphosphate synthase (GGPPS) is the crucial
bottleneck
in carotenoid biosynthesis. However, low activity limits the broad
application of GGPPS. In this study, OsGGPPS1 in rice was engineered
based on ancestral sequence reconstruction (ASR) and semirational
design to improve the catalytic performances of existing GGPPS. The
better mutant of A22R/A26P with improved enzyme activity was generated
based on ASR. Additionally, the improved enzyme activity of mutants
as V162A/M218S/F227Y was designed using a semirational design. The
combinatorial assembly of the d-OsGGPPS1 mutant (A22R/A26P/V162A/M218S/F227Y)
exhibited higher conversion of IPP and each cosubstrate of DMAPP for
9.8-fold in GPP production, GPP for 6.4-fold in FPP production, and
FPP for 1.4-fold in GGPP production relative to wild-type OsGGPPS1
at 25 °C, which showed higher conversion than wild-type OsGGPPS1
at temperatures as high as 50 °C. The successful design of OsGGPPS1
was representative of protein engineering, which will shed new light
on GGPPS engineering and active plant pigment resource utilization.