Young-Kyoung Park, Lara Sellés Vidal, David Bell, Jure Zabret, Mladen Soldat, Martin Kavšček, Rodrigo Ledesma-Amaro
{"title":"通过组合工程策略在脂溶性亚罗酵母中高效合成柠檬烯。","authors":"Young-Kyoung Park, Lara Sellés Vidal, David Bell, Jure Zabret, Mladen Soldat, Martin Kavšček, Rodrigo Ledesma-Amaro","doi":"10.1186/s13068-024-02535-z","DOIUrl":null,"url":null,"abstract":"<div><h3>Background</h3><p>Limonene has a variety of applications in the foods, cosmetics, pharmaceuticals, biomaterials, and biofuels industries. In order to meet the growing demand for sustainable production of limonene at industry scale, it is essential to find an alternative production system to traditional plant extraction. A promising and eco-friendly alternative is the use of microbes as cell factories for the synthesis of limonene.</p><h3>Results</h3><p>In this study, the oleaginous yeast <i>Yarrowia lipolytica</i> has been engineered to produce <span>d</span>- and <span>l</span>-limonene. Four target genes, <i><span>l</span></i>- or <i><span>d</span></i>-<i>LS</i> (limonene synthase), <i>HMG</i> (HMG-CoA reductase), <i>ERG20</i> (geranyl diphosphate synthase), and <i>NDPS1</i> (neryl diphosphate) were expressed individually or fused together to find the optimal combination for higher limonene production. The strain expressing HMGR and the fusion protein ERG20-LS was the best limonene producer and, therefore, selected for further improvement. By increasing the expression of target genes and optimizing initial OD, 29.4 mg/L of <span>l</span>-limonene and 24.8 mg/L of <span>d</span>-limonene were obtained. We also studied whether peroxisomal compartmentalization of the synthesis pathway was beneficial for limonene production. The introduction of <span>d</span>-LS and ERG20 within the peroxisome improved limonene titers over cytosolic expression. Then, the entire MVA pathway was targeted to the peroxisome to improve precursor supply, which increased <span>d</span>-limonene production to 47.8 mg/L. Finally, through the optimization of fermentation conditions, <span>d</span>-limonene production titer reached 69.3 mg/L.</p><h3>Conclusions</h3><p>In this work, <i>Y. lipolytica</i> was successfully engineered to produce limonene. Our results showed that higher production of limonene was achieved when the synthesis pathway was targeted to the peroxisome, which indicates that this organelle can favor the bioproduction of terpenes in yeasts. This study opens new avenues for the efficient synthesis of valuable monoterpenes in <i>Y. lipolytica</i>.</p></div>","PeriodicalId":494,"journal":{"name":"Biotechnology for Biofuels","volume":null,"pages":null},"PeriodicalIF":6.1000,"publicationDate":"2024-07-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://biotechnologyforbiofuels.biomedcentral.com/counter/pdf/10.1186/s13068-024-02535-z","citationCount":"0","resultStr":"{\"title\":\"Efficient synthesis of limonene production in Yarrowia lipolytica by combinatorial engineering strategies\",\"authors\":\"Young-Kyoung Park, Lara Sellés Vidal, David Bell, Jure Zabret, Mladen Soldat, Martin Kavšček, Rodrigo Ledesma-Amaro\",\"doi\":\"10.1186/s13068-024-02535-z\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><h3>Background</h3><p>Limonene has a variety of applications in the foods, cosmetics, pharmaceuticals, biomaterials, and biofuels industries. In order to meet the growing demand for sustainable production of limonene at industry scale, it is essential to find an alternative production system to traditional plant extraction. A promising and eco-friendly alternative is the use of microbes as cell factories for the synthesis of limonene.</p><h3>Results</h3><p>In this study, the oleaginous yeast <i>Yarrowia lipolytica</i> has been engineered to produce <span>d</span>- and <span>l</span>-limonene. Four target genes, <i><span>l</span></i>- or <i><span>d</span></i>-<i>LS</i> (limonene synthase), <i>HMG</i> (HMG-CoA reductase), <i>ERG20</i> (geranyl diphosphate synthase), and <i>NDPS1</i> (neryl diphosphate) were expressed individually or fused together to find the optimal combination for higher limonene production. The strain expressing HMGR and the fusion protein ERG20-LS was the best limonene producer and, therefore, selected for further improvement. By increasing the expression of target genes and optimizing initial OD, 29.4 mg/L of <span>l</span>-limonene and 24.8 mg/L of <span>d</span>-limonene were obtained. We also studied whether peroxisomal compartmentalization of the synthesis pathway was beneficial for limonene production. The introduction of <span>d</span>-LS and ERG20 within the peroxisome improved limonene titers over cytosolic expression. Then, the entire MVA pathway was targeted to the peroxisome to improve precursor supply, which increased <span>d</span>-limonene production to 47.8 mg/L. Finally, through the optimization of fermentation conditions, <span>d</span>-limonene production titer reached 69.3 mg/L.</p><h3>Conclusions</h3><p>In this work, <i>Y. lipolytica</i> was successfully engineered to produce limonene. 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This study opens new avenues for the efficient synthesis of valuable monoterpenes in <i>Y. lipolytica</i>.</p></div>\",\"PeriodicalId\":494,\"journal\":{\"name\":\"Biotechnology for Biofuels\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":6.1000,\"publicationDate\":\"2024-07-03\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://biotechnologyforbiofuels.biomedcentral.com/counter/pdf/10.1186/s13068-024-02535-z\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Biotechnology for Biofuels\",\"FirstCategoryId\":\"5\",\"ListUrlMain\":\"https://link.springer.com/article/10.1186/s13068-024-02535-z\",\"RegionNum\":1,\"RegionCategory\":\"工程技术\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"BIOTECHNOLOGY & APPLIED MICROBIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Biotechnology for Biofuels","FirstCategoryId":"5","ListUrlMain":"https://link.springer.com/article/10.1186/s13068-024-02535-z","RegionNum":1,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}
Efficient synthesis of limonene production in Yarrowia lipolytica by combinatorial engineering strategies
Background
Limonene has a variety of applications in the foods, cosmetics, pharmaceuticals, biomaterials, and biofuels industries. In order to meet the growing demand for sustainable production of limonene at industry scale, it is essential to find an alternative production system to traditional plant extraction. A promising and eco-friendly alternative is the use of microbes as cell factories for the synthesis of limonene.
Results
In this study, the oleaginous yeast Yarrowia lipolytica has been engineered to produce d- and l-limonene. Four target genes, l- or d-LS (limonene synthase), HMG (HMG-CoA reductase), ERG20 (geranyl diphosphate synthase), and NDPS1 (neryl diphosphate) were expressed individually or fused together to find the optimal combination for higher limonene production. The strain expressing HMGR and the fusion protein ERG20-LS was the best limonene producer and, therefore, selected for further improvement. By increasing the expression of target genes and optimizing initial OD, 29.4 mg/L of l-limonene and 24.8 mg/L of d-limonene were obtained. We also studied whether peroxisomal compartmentalization of the synthesis pathway was beneficial for limonene production. The introduction of d-LS and ERG20 within the peroxisome improved limonene titers over cytosolic expression. Then, the entire MVA pathway was targeted to the peroxisome to improve precursor supply, which increased d-limonene production to 47.8 mg/L. Finally, through the optimization of fermentation conditions, d-limonene production titer reached 69.3 mg/L.
Conclusions
In this work, Y. lipolytica was successfully engineered to produce limonene. Our results showed that higher production of limonene was achieved when the synthesis pathway was targeted to the peroxisome, which indicates that this organelle can favor the bioproduction of terpenes in yeasts. This study opens new avenues for the efficient synthesis of valuable monoterpenes in Y. lipolytica.
期刊介绍：
Biotechnology for Biofuels is an open access peer-reviewed journal featuring high-quality studies describing technological and operational advances in the production of biofuels, chemicals and other bioproducts. The journal emphasizes understanding and advancing the application of biotechnology and synergistic operations to improve plants and biological conversion systems for the biological production of these products from biomass, intermediates derived from biomass, or CO2, as well as upstream or downstream operations that are integral to biological conversion of biomass.
Biotechnology for Biofuels focuses on the following areas:
• Development of terrestrial plant feedstocks
• Development of algal feedstocks
• Biomass pretreatment, fractionation and extraction for biological conversion
• Enzyme engineering, production and analysis
• Bacterial genetics, physiology and metabolic engineering
• Fungal/yeast genetics, physiology and metabolic engineering
• Fermentation, biocatalytic conversion and reaction dynamics
• Biological production of chemicals and bioproducts from biomass
• Anaerobic digestion, biohydrogen and bioelectricity
• Bioprocess integration, techno-economic analysis, modelling and policy
• Life cycle assessment and environmental impact analysis
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