Heterologous production of the epoxycarotenoid violaxanthin in Saccharomyces cerevisiae |
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| Affiliation: | 1. Department of Agricultural and Biological Engineering, University of Illinois at Urbana-Champaign, Urbana, IL, USA;2. Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, Urbana, IL, USA;3. Bioenergy Research Unit, National Center for Agricultural Utilization Research, USDA-ARS, Peoria, IL 61604, USA;1. Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, PR China;2. Key Laboratory of Biomass Chemical Engineering of Ministry of Education, Zhejiang University, Hangzhou 310027, PR China;1. Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, PR China;2. Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, PR China;3. College of Biological Sciences and Technology, Beijing Forestry University, Beijing 100083, PR China;1. Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 plus program), Institute for the BioCentury, Korea Advanced Institute of Science and Technology (KAIST), 34141 Daejeon, Republic of Korea;2. Systems Metabolic Engineering and Systems Healthcare Cross-Generation Collaborative Laboratory, KAIST, Daejeon 34141, Republic of Korea;3. BioProcess Engineering Research Center and BioInformatics Research Center, KAIST, 34141 Daejeon, Republic of Korea |
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| Abstract: | Microbial production of carotenoids has mainly focused towards a few products, such as β-carotene, lycopene and astaxanthin. However, other less explored carotenoids, like violaxanthin, have also shown unique properties and promissory applications. Violaxanthin is a plant-derived epoxidated carotenoid with strong antioxidant activity and a key precursor of valuable compounds, such as fucoxanthin and β-damascenone. In this study, we report for the first time the heterologous production of epoxycarotenoids in yeast. We engineered the yeast Saccharomyces cerevisiae following multi-level strategies for the efficient accumulation of violaxanthin. Starting from a β-carotenogenic yeast strain, we first evaluated the performance of several β-carotene hydroxylases (CrtZ), and zeaxanthin epoxidases (ZEP) from different species, together with their respective N-terminal truncated variants. The combined expression of CrtZ from Pantoea ananatis and truncated ZEP of Haematococcus lacustris showed the best performance and led to a yield of 1.6 mg/gDCW of violaxanthin. Further improvement of the epoxidase activity was achieved by promoting the transfer of reducing equivalents to ZEP by expressing several redox partner systems. The co-expression of the plant truncated ferredoxin-3, and truncated root ferredoxin oxidoreductase-1 resulted in a 2.2-fold increase in violaxanthin yield (3.2 mg/gDCW). Finally, increasing gene copy number of carotenogenic genes enabled reaching a final production of 7.3 mg/gDCW in shake flask cultures and batch bioreactors, which is the highest yield of microbially produced violaxanthin reported to date. |
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| Keywords: | Carotenoid Xanthophyll Epoxycarotenoid Violaxanthin Metabolic engineering |
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