Synthetic biology is leading the third biotechnology revolution. As the underlying technology, it will drive innovative development in many fields, including medicine, food, agriculture, materials, environment and even information storage. Synthetic biology is a highly cross-cutting frontier research field of biological engineering, including several different research levels: understanding life, transforming life and creating life; in order to achieve its ultimate goal, it is necessary to explore the nature of life and the development of related technologies. Continuous innovation and application continue to deepen. We will keep up with the cutting-edge research progress in the field of synthetic biology, and interpret the latest scientific research results in this field for you.
In this issue, we will share new methods for the study of plant enzyme functions. The in-depth understanding of enzyme functions will provide an important basis for the next step of heterologous design of cell factories. The research results come from a research paper entitled “Glycosides-specific metabolomics combined with precursor isotopic labeling for characterizing plant glycosyltransferases” published on Molecular Plant by Zhao Qiao’s research group from the Synthetic Genomics Research Center of Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences[1] , to introduce a method that combines glycosides-specific metabolomics (GSM) and isotope-labeled precursor compound tracking (precursor isotopic labeling, PIL), which can efficiently and accurately identify glycosyl transfer The products of enzymes (glycosyltransferases, GTs) in plants, and the role of GTs in specific metabolic pathways are analyzed.
This method greatly narrows the scope of target compounds, and has greatly improved the qualitative and method reliability of glycosyl compounds compared with traditional biochemical methods or non-targeted methods, and provides a new method for the functional analysis of plant glycosyltransferases.
“Plant secondary metabolites are diverse and highly modified, among which glycosylation modification not only provides the structural basis but also plays an important role in its diverse biological functions. In order to effectively identify the glycosylation process, it is necessary to use high-resolution Mass spectrometry for non-targeted specific metabolomics research, combined with isotope labeling to track the tracking results of different glycoside metabolites in mutants to analyze the function of UGTs, and then comprehensively characterize the secondary metabolites of plant glycosylation modification , providing a basis for expanding the efficient biosynthesis of natural compounds.”
Plants are rich in secondary metabolites, with more than 400,000 species. Glycosylation is a common modification method that endows compounds with complex and diverse structures and forms a wide variety of glycosylation products. Glycosylation modification can change the catalytic activity, solubility, stability and localization of the corresponding aglycone, and play an important role in regulating the homeostasis of hormones, detoxifying exogenous harmful substances, and resisting biotic and abiotic stresses. important role. At the same time, glycosylation modification can change the pharmacological activity and bioavailability of natural products, and these glycoside compounds are an important source of natural medicines.
Plant UGTs (UDP glycosyltransferases) exist as multigene families capable of glycosylation of a wide variety of small plant molecules using different sugar donors. Most of the current research focuses on the determination of biochemical functions. UGTs have substrate heterogeneity and catalytic heterogeneity. The same UGT can catalyze substrates with different structures in vitro, and different UGTs can recognize the same substrate. In addition, due to the availability of substrates and the special and complex and changeable cellular environment in plants, these research results on the activity and physiological functions of UGTs through biochemical methods often cannot reflect the true functions of UGTs in plants.
Non-target-specific metabolomics (GSM):
A non-target-specific metabolomics approach based on the neutral mass loss model of endogenous collision-induced dissociation (ISCID) for targeted analysis of glycosylated secondary metabolites. This GSM approach narrows the range of metabolites affected by UDP glycosyltransferases (UGT72Es as an example) from 1000 to 100.
Isotopically Labeled Precursor Compound Tracing (PIL, Flux):
Using isotope-labeled phenylalanine precursors to trace the role of UGT72E in specific phenylalanine metabolic pathways further narrowed down the list of target products to 22.
Through the GSM-PIL method, not only two published lignin monomer glycosylation products could be identified, but also the UGT72E family was found to be involved in the glycosyl modification of 15 other compounds in the plant phenylpropane pathway.
The glycosylation of these compounds by UGT72Es was further confirmed by the in vitro enzyme activity analysis of UGT72Es, plant endogenous gene overexpression and genetic complementation experiments, which verified the reliability of the GSM-PIL method.
At the same time, the study also found that UGT72Es glycosylated coumarin in plants, and then played an important role in the plant alkaline iron deficiency stress environment.
Finally, the general applicability of the GSM-PIL method is demonstrated through the functional analysis of UGT78D2.
In order to ensure the efficient detection of glycosylated secondary metabolites and isotope tracer compounds, this study used the Agilent 6546 QTOF LCMS system for data acquisition; further combined with MassHunter and Profinder data processing software for metabolome and isotope tracer data Efficient extraction and parsing.
In summary, the GSM-PIL method based on liquid phase-high resolution mass spectrometry can efficiently analyze the functions of UGTs in plants. Compared with the traditional one-to-one “fishing” method to explore the functions of UGTs, the GSM-PIL method can catch all the products of UGTs in a “fishing” way, comprehensively identify unknown substrates or glycosylation products, and analyze the unknown functions of UGTs in plants. Physiological functions, revealing that the glycosylation network in plants is more complex than we imagined. At the same time, this method can be used to explore other metabolic pathways, help people to further understand and then use plant synthesis pathways, and provide a basis for expanding the efficient biosynthesis of natural compounds.