Suqing Chen , Guocui Zhu , Zhijie Wang , Bowen Fu , Mengjuan Sun , Jie Liu , Chengjia Xie , Xian Xu
{"title":"番茄红素合成无细胞代谢工程系统的构建与优化","authors":"Suqing Chen , Guocui Zhu , Zhijie Wang , Bowen Fu , Mengjuan Sun , Jie Liu , Chengjia Xie , Xian Xu","doi":"10.1016/j.bej.2025.109849","DOIUrl":null,"url":null,"abstract":"<div><div>In this study, a cell-free metabolic engineering system was designed for the synthesis of lycopene in vitro. Initially, comprehensive bioinformatic analysis was performed on three key enzymes from <em>Deinococcus wulumuqiensis</em> R12, geranylgeranyl pyrophosphate synthase, phytoene synthase and phytoene desaturase to assess their phylogenetic relationships and characterize functional domains. Subsequently, heterologous expression systems were constructed to enable soluble overexpression of these enzymes in recombinant <em>Escherichia coli</em>. The cell-free metabolic engineering synthesis system was constructed and optimized, based on the purified enzymes and crude extracts from recombinant <em>Escherichia coli</em> for lycopene synthesis. After comparing the two different platforms and optimizing various key parameters, including enzyme concentration, substrate concentration, enzyme ratio, cofactors, and metal ions, a lycopene titer of 14.06 mg/L was achieved in the cell-free system after 24 h. Finally, the synthetic system was employed to validate a rapid enzyme screen and to validate lycopene synthesis in vivo. This construction strategy demonstrates three major applications of CFME in rapid optimization of cell-free systems based on purified enzymes, the optimization of cell-free systems based on crude cell extracts and the rapid enzyme screening and provides a referable research strategy and synthesis platform for the design and optimization of multienzyme pathways for the synthesis of complex compounds such as carotenoids.</div></div>","PeriodicalId":8766,"journal":{"name":"Biochemical Engineering Journal","volume":"222 ","pages":"Article 109849"},"PeriodicalIF":3.7000,"publicationDate":"2025-07-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Construction and optimization of a cell-free metabolic engineering system for lycopene synthesis\",\"authors\":\"Suqing Chen , Guocui Zhu , Zhijie Wang , Bowen Fu , Mengjuan Sun , Jie Liu , Chengjia Xie , Xian Xu\",\"doi\":\"10.1016/j.bej.2025.109849\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><div>In this study, a cell-free metabolic engineering system was designed for the synthesis of lycopene in vitro. Initially, comprehensive bioinformatic analysis was performed on three key enzymes from <em>Deinococcus wulumuqiensis</em> R12, geranylgeranyl pyrophosphate synthase, phytoene synthase and phytoene desaturase to assess their phylogenetic relationships and characterize functional domains. Subsequently, heterologous expression systems were constructed to enable soluble overexpression of these enzymes in recombinant <em>Escherichia coli</em>. The cell-free metabolic engineering synthesis system was constructed and optimized, based on the purified enzymes and crude extracts from recombinant <em>Escherichia coli</em> for lycopene synthesis. After comparing the two different platforms and optimizing various key parameters, including enzyme concentration, substrate concentration, enzyme ratio, cofactors, and metal ions, a lycopene titer of 14.06 mg/L was achieved in the cell-free system after 24 h. Finally, the synthetic system was employed to validate a rapid enzyme screen and to validate lycopene synthesis in vivo. This construction strategy demonstrates three major applications of CFME in rapid optimization of cell-free systems based on purified enzymes, the optimization of cell-free systems based on crude cell extracts and the rapid enzyme screening and provides a referable research strategy and synthesis platform for the design and optimization of multienzyme pathways for the synthesis of complex compounds such as carotenoids.</div></div>\",\"PeriodicalId\":8766,\"journal\":{\"name\":\"Biochemical Engineering Journal\",\"volume\":\"222 \",\"pages\":\"Article 109849\"},\"PeriodicalIF\":3.7000,\"publicationDate\":\"2025-07-03\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Biochemical Engineering Journal\",\"FirstCategoryId\":\"5\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S1369703X25002232\",\"RegionNum\":3,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"BIOTECHNOLOGY & APPLIED MICROBIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Biochemical Engineering Journal","FirstCategoryId":"5","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S1369703X25002232","RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}
Construction and optimization of a cell-free metabolic engineering system for lycopene synthesis
In this study, a cell-free metabolic engineering system was designed for the synthesis of lycopene in vitro. Initially, comprehensive bioinformatic analysis was performed on three key enzymes from Deinococcus wulumuqiensis R12, geranylgeranyl pyrophosphate synthase, phytoene synthase and phytoene desaturase to assess their phylogenetic relationships and characterize functional domains. Subsequently, heterologous expression systems were constructed to enable soluble overexpression of these enzymes in recombinant Escherichia coli. The cell-free metabolic engineering synthesis system was constructed and optimized, based on the purified enzymes and crude extracts from recombinant Escherichia coli for lycopene synthesis. After comparing the two different platforms and optimizing various key parameters, including enzyme concentration, substrate concentration, enzyme ratio, cofactors, and metal ions, a lycopene titer of 14.06 mg/L was achieved in the cell-free system after 24 h. Finally, the synthetic system was employed to validate a rapid enzyme screen and to validate lycopene synthesis in vivo. This construction strategy demonstrates three major applications of CFME in rapid optimization of cell-free systems based on purified enzymes, the optimization of cell-free systems based on crude cell extracts and the rapid enzyme screening and provides a referable research strategy and synthesis platform for the design and optimization of multienzyme pathways for the synthesis of complex compounds such as carotenoids.
期刊介绍:
The Biochemical Engineering Journal aims to promote progress in the crucial chemical engineering aspects of the development of biological processes associated with everything from raw materials preparation to product recovery relevant to industries as diverse as medical/healthcare, industrial biotechnology, and environmental biotechnology.
The Journal welcomes full length original research papers, short communications, and review papers* in the following research fields:
Biocatalysis (enzyme or microbial) and biotransformations, including immobilized biocatalyst preparation and kinetics
Biosensors and Biodevices including biofabrication and novel fuel cell development
Bioseparations including scale-up and protein refolding/renaturation
Environmental Bioengineering including bioconversion, bioremediation, and microbial fuel cells
Bioreactor Systems including characterization, optimization and scale-up
Bioresources and Biorefinery Engineering including biomass conversion, biofuels, bioenergy, and optimization
Industrial Biotechnology including specialty chemicals, platform chemicals and neutraceuticals
Biomaterials and Tissue Engineering including bioartificial organs, cell encapsulation, and controlled release
Cell Culture Engineering (plant, animal or insect cells) including viral vectors, monoclonal antibodies, recombinant proteins, vaccines, and secondary metabolites
Cell Therapies and Stem Cells including pluripotent, mesenchymal and hematopoietic stem cells; immunotherapies; tissue-specific differentiation; and cryopreservation
Metabolic Engineering, Systems and Synthetic Biology including OMICS, bioinformatics, in silico biology, and metabolic flux analysis
Protein Engineering including enzyme engineering and directed evolution.