Metabolic engineering of Saccharomyces cerevisiae for gram-scale diosgenin production |
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| Affiliation: | 1. School of Life Science, Division of Life Sciences and Medicine, University of Science and Technology of China, No.443, Huangshan Road, Shushan District, Hefei, Anhui, 230027, P.R. China;2. Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 West 7th Avenue, Tianjin Airport Economic Area, Tianjin, 300308, P.R. China;3. Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, P.R. China;4. National Innovation Center for Synthetic Biotechnology, Tianjin, 300308, P.R. China;1. Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China;2. National Technology Innovation Center of Synthetic Biology, Tianjin, 300308, China;3. Beijing Advanced Innovation Center for Soft Matter Science and Engineering, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing, 100029, China;4. Department of Biology and Biological Engineering, Chalmers University of Technology, SE 412 96, Gothenburg, Sweden;1. State Key Laboratory of Biocatalysis and Enzyme Engineering, Environmental Microbial Technology Center of Hubei Province, Hubei Province Key Laboratory of Biotechnology of Chinese Traditional Medicine, National & Local Joint Engineering Research Center of High-throughput Drug Screening Technology, School of Life Sciences, Hubei University, Wuhan, 430062, PR China;2. School of Chemistry and Chemical Engineering, Wuhan University of Science and Technology, Wuhan, 430081, PR China;1. Agricultural Biotechnology Department, Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran;2. Agronomy and Plant Breeding Department, Faculty of Agriculture, Shahrood University of Technology, Semnan, Iran;3. Department of Entomology and Plant Pathology, College of Aburaihan, University of Tehran, Tehran, Iran;4. Genetics and Plant Breeding Department, Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran;1. College of Light Industry, Textile and Food Engineering and Healthy Food Evaluation Research Center, Sichuan University, Chengdu 610065, PR China;2. The Key Laboratory of Food Science and Technology of Ministry of Education of Sichuan Province, Sichuan University, Chengdu 610065, PR China;3. School of Food Science and Engineering, South China University of Technology, Guangzhou 510641, PR China;1. Key Laboratory for Green Processing of Chemical Engineering of Xinjiang Bingtuan, School of Chemistry and Chemical Engineering, Shihezi University, Shihezi, 832003, PR China;2. Key Lab for Industrial Biocatalysis, Ministry of Education, Department of Chemical Engineering, Tsinghua University, Beijing, 100084, PR China;3. Key Laboratory of Medical Molecule Science and Pharmaceutics Engineering, Ministry of Industry and Information Technology, Institute of Biochemical Engineering, School of Chemistry and Chemical Engineering, Beijing Institute of Technology, Beijing, 100081, PR China;1. National Engineering Research Center of Cereal Fermentation and Food Biomanufacturing, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu, 214122, China;2. Jiangsu Provincial Research Center for Bioactive Product Processing Technology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu, 214122, China |
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| Abstract: | Diosgenin (DSG) is a naturally occurring steroidal saponin with a variety of biological activities that is also an important precursor for the synthesis of various steroidal drugs. The traditional industrial production of DSG is based on natural plant extraction and chemical processing. However, the whole process is time-consuming, laborious, and accompanied by severe environmental pollution. Therefore, it is necessary to develop a more convenient and environmentally-friendly process to realize the green production of DSG. In our previous work, we achieved de novo synthesis of DSG in Saccharomyces cerevisiae using glucose as the carbon source. However, DSG production was only at the milligram level, which is too low for industrial production. In this work, we further developed yeast strains for DSG overproduction by optimizing the synthesis pathway, fine-tuning pathway gene expression, and eliminating competing pathways. Cholesterol 22-hydroxylase was used to construct the DSG biosynthesis pathway. The optimal ratio of cytochrome P450 (CYP) to cytochrome P450 reductase (CPR) associated with DSG synthesis was screened to increase DSG production. Weakening the expression of the ERG6 gene further increased DSG synthesis and reduced the formation of by-products. In addition, we investigated the impact of DSG accumulation on yeast cell physiology and growth by transcriptome analysis and found that the multidrug transporter PDR5 and the sterol-binding protein PRY1 contributed to DSG production. Finally, we obtained a DSG titer of 2.03 g/L after 288 h of high-cell-density fed-batch fermentation using the engineered strain LP118, which represents the highest DSG titer reported to date for a yeast de novo synthesis system. |
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| Keywords: | Diosgenin Cholesterol Cholesterol 22-hydroxylase ERG6 Steroid transport |
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