Park Young-Kyoung, Sellés Vidal Lara, Bell David, Zabret Jure, Soldat Mladen, Kavšček Martin, Ledesma-Amaro Rodrigo
Department of Bioengineering and Centre for Synthetic Biology, Imperial College London, London, SW72AZ, UK.
INRAE, AgroParisTech, Micalis Institute, Université Paris-Saclay, 78350, Jouy-en-Josas, France.
Biotechnol Biofuels Bioprod. 2024 Jul 3;17(1):94. doi: 10.1186/s13068-024-02535-z.
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.
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.
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.
柠檬烯在食品、化妆品、制药、生物材料和生物燃料行业有多种应用。为了满足工业规模可持续生产柠檬烯不断增长的需求，找到一种替代传统植物提取的生产系统至关重要。一种有前景且环保的替代方法是利用微生物作为细胞工厂来合成柠檬烯。
在本研究中，已对产油酵母解脂耶氏酵母进行工程改造以生产D - 和L - 柠檬烯。四个目标基因，l - 或d - LS（柠檬烯合酶）、HMG（HMG - CoA还原酶）、ERG20（香叶基二磷酸合酶）和NDPS1（橙花基二磷酸）被单独表达或融合在一起，以找到实现更高柠檬烯产量的最佳组合。表达HMGR和融合蛋白ERG20 - LS的菌株是最佳的柠檬烯生产者，因此被选用于进一步改良。通过增加目标基因的表达并优化初始OD，获得了29.4 mg/L的L - 柠檬烯和24.8 mg/L的D - 柠檬烯。我们还研究了合成途径的过氧化物酶体区室化是否有利于柠檬烯生产。在过氧化物酶体内引入D - LS和ERG20比胞质表达提高了柠檬烯滴度。然后，将整个MVA途径靶向过氧化物酶体以改善前体供应，这将D - 柠檬烯产量提高到47.8 mg/L。最后，通过优化发酵条件，D - 柠檬烯生产滴度达到69.3 mg/L。
在这项工作中，解脂耶氏酵母被成功工程改造以生产柠檬烯。我们的结果表明，当合成途径靶向过氧化物酶体时，柠檬烯产量更高，这表明该细胞器有利于酵母中萜类化合物的生物生产。本研究为在解脂耶氏酵母中高效合成有价值的单萜类化合物开辟了新途径。
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