Enhancing the Catalytic Activity of Geranylgeranyl Diphosphate Synthase through Ancestral Sequence Reconstruction and Semirational Design.

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.

Overexpression of geranyl diphosphate synthase 1 (NnGGPPS1) from Nelumbo nucifera enhances carotenoid and chlorophyll content and biomass.

As the traditional herb with pharmacological compounds in China, the key genes related with terpenoid biosynthesis are still unveiled in Nelumbo nucifera. Geranylgeranyl pyrophosphate synthase (GGPPS) is one of the key enzymes in terpenoids biosynthesis, synthesizing the common precursor of GGPP for downstream enzymes for generating various terpenoids. In this study, four NnGGPPS genes were isolated from N. nucifera. Sequence and phylogenetic analyses indicate that NnGGPPS1 and NnGGPPS2 belong to large subunit (LSU). Whereas NnGGPPS3 and NnGGPPS4 are classified as small subunit (SSU) of SSU Ⅱ and SSU I, respectively. Among four NnGGPPSs, only NnGGPPS1 and NnGGPPS2 can produce GGPP in bacterial pigment complementation assay. Combination analysis of subcellular localization and gene co-expression analysis (GCN) illustrates that NnGGPPS1 is the main transcript related with methylerythritol phosphate (MEP) pathway, abscisic acid (ABA) biosynthesis, carotenoid and chlorophyll biosynthesis and degradation. Overexpression of NnGGPPS1 improves the growth of transgenic tobacco, and increases carotenoids and chlorophyll contents. Moreover, NnGGPPS1 transgenic tobacco exhibits improved photosynthesis efficiency and ROS scavenging ability. The up-regulated expression of the key genes in MEP pathway, carotenoid biosynthesis and chlorophyll biosynthesis, result in the increase of metabolic flux in NnGGPPS1 transgenic lines. Furthermore, the elevated MEP-derived primary metabolites of carotenoid and chlorophyll was attributed to enhancement of plant biomass of NnGGPPS1 transgenic lines. Therefore, NnGGPPS1 plays a vital role in biosynthesis of carotenoid and chlorophyll.

Melatonin-induced myeloblastosis viral oncogene homologs alleviate fresh-cut lotus root browning during storage by attenuating flavonoid biosynthesis and reactive oxygen species.

BACKGROUND: Lotus roots (Nelumbo nucifera Gaertn.) are rich in nutrients and have ornamental and food value. However, browning has caused huge economic losses and security risks during the storage and harvesting of fresh-cut lotus. This study investigated the role of melatonin in inhibiting lotus browning, and illustrates its molecular mechanism. RESULTS: The application of melatonin effectively retarded the process of lotus browning, enhanced reactive oxygen species (ROS) scavenging enzyme activity, and inhibited the activity of polyphenol oxidase (PPO), and peroxidase (POD). Melatonin reduced flavonoid content, and decreased enzymatic activity in flavonoid biosynthesis. Transcriptome Sequencing (RNA-seq) was used to screen the genes regulated by exogenous melatonin when defending against fresh-cut lotus browning. Gene co-expression analysis (GCN) indicated that the transcription factors MYB5, MYB6, and MYB308, activated by melatonin, were negatively related to the expression of PPO and the genes related to flavonoid and phenylpropanoid biosynthesis. These myeloblastosis viral oncogene homologs (MYBs) were positively related to the expression of genes encoding the enzymes in glutathione metabolism. CONCLUSION: Melatonin retarded lotus browning by transcriptional suppression of key genes associated with flavonoid and phenylpropanoid biosynthesis through the stimulation of MYB5, MYB6, and MYB308. © 2023 Society of Chemical Industry.