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Abstract：
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.
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.
摘要：
背景：柠檬烯在食品中具有多种应用，化妆品,制药，生物材料,和生物燃料工业。为了满足工业规模不断增长的柠檬烯可持续生产需求，找到一种替代传统植物提取的生产系统是至关重要的。一种有前途的生态友好的替代方案是使用微生物作为细胞工厂来合成柠檬烯。
结果：在这项研究中，含油酵母Yarrowialipolytica已被设计为生产D-和L-柠檬烯。四个靶基因，l-或d-LS（柠檬烯合酶），HMG（HMG-CoA还原酶），ERG20（香叶二磷酸合成酶），和NDPS1（橙基二磷酸）分别表达或融合在一起，以找到更高的柠檬烯产量的最佳组合。表达HMGR和融合蛋白ERG20-LS的菌株是最好的柠檬烯生产者，因此,选择进一步改进。通过增加靶基因的表达和优化初始OD，得到29.4mg/L的L-柠檬烯和24.8mg/L的D-柠檬烯。我们还研究了合成途径的过氧化物酶体隔室化对柠檬烯生产是否有益。过氧化物酶体内D-LS和ERG20的引入提高了柠檬烯的滴度超过了胞浆表达。然后，整个MVA途径靶向过氧化物酶体以改善前体供应，将D-柠檬烯的产量提高到47.8mg/L。最后,通过优化发酵条件,D-柠檬烯生产效价达到69.3mg/L。
结论：在这项工作中，Y.Lipolytica被成功地设计为生产柠檬烯。我们的结果表明，当合成途径靶向过氧化物酶体时，柠檬烯的产量更高，这表明该细胞器可以促进酵母中萜烯的生物生产。这项研究为Y.lipolytica中有效合成有价值的单萜开辟了新途径。
结果：在这项研究中，含油酵母Yarrowialipolytica已被设计为生产D-和L-柠檬烯。四个靶基因，l-或d-LS（柠檬烯合酶），HMG（HMG-CoA还原酶），ERG20（香叶二磷酸合成酶），和NDPS1（橙基二磷酸）分别表达或融合在一起，以找到更高的柠檬烯产量的最佳组合。表达HMGR和融合蛋白ERG20-LS的菌株是最好的柠檬烯生产者，因此,选择进一步改进。通过增加靶基因的表达和优化初始OD，得到29.4mg/L的L-柠檬烯和24.8mg/L的D-柠檬烯。我们还研究了合成途径的过氧化物酶体隔室化对柠檬烯生产是否有益。过氧化物酶体内D-LS和ERG20的引入提高了柠檬烯的滴度超过了胞浆表达。然后，整个MVA途径靶向过氧化物酶体以改善前体供应，将D-柠檬烯的产量提高到47.8mg/L。最后,通过优化发酵条件,D-柠檬烯生产效价达到69.3mg/L。
结论：在这项工作中，Y.Lipolytica被成功地设计为生产柠檬烯。我们的结果表明，当合成途径靶向过氧化物酶体时，柠檬烯的产量更高，这表明该细胞器可以促进酵母中萜烯的生物生产。这项研究为Y.lipolytica中有效合成有价值的单萜开辟了新途径。
