Peroxisome compartmentalization of a toxic enzyme improves alkaloid production
Peroxisome compartmentalization of a toxic enzyme improves alkaloid production
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DOI：
10.1038/s41589-020-00668-4
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发表时间：
2020-10-12
影响因子：
14.8
通讯作者：
Dueber, John E.
中科院分区：
文献类型：
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作者：
Grewal, Parbir S.;Samson, Jennifer A.;Dueber, John E.
真核细胞在细胞器中划分代谢途径以实现最佳反应条件并避免与胞质因子的串扰。我们发现去甲乌药碱合成酶（NCS）的胞质表达对酵母菌具有毒性，从而限制了（S）-牛心果碱的产生。NCS是苄基异喹啉生物碱生物合成中催化第一个关键反应的酶。我们开发了一种区室化策略，该策略在降低NCS毒性的同时促进（S）-牛心果碱滴度的增加。该策略是通过将有毒NCS有效靶向过氧化物酶体而实现的，同时至关重要的是，利用代谢物底物和产物穿过过氧化物酶体膜的自由流动。我们表明，工程转录因子的表达可以模拟油酸响应较大的过氧化物酶体，进一步增加苄基异喹啉生物碱滴度，而不需要过氧化物酶体诱导与脂肪酸。这项工作专门解决了与有毒的NCS表达相关的挑战，更广泛地说，突出了潜在的工程细胞器所需的特性代谢engineering.Increased生产的（S）-牛心果碱和其他生物碱是通过减轻去甲乌药碱合酶毒性靶向酶的过氧化物酶体加上扩大过氧化物酶体的表达工程转录因子。
Eukaryotic cells compartmentalize metabolic pathways in organelles to achieve optimal reaction conditions and avoid crosstalk with cytosolic factors. We found that cytosolic expression of norcoclaurine synthase (NCS), the enzyme that catalyzes the first committed reaction in benzylisoquinoline alkaloid biosynthesis, is toxic inSaccharomyces cerevisiaeand, consequently, restricts (S)-reticuline production. We developed a compartmentalization strategy that alleviates NCS toxicity while promoting increased (S)-reticuline titer. This strategy is achieved through efficient targeting of toxic NCS to the peroxisome while, crucially, taking advantage of the free flow of metabolite substrates and products across the peroxisome membrane. We demonstrate that expression of engineered transcription factors can mimic the oleate response for larger peroxisomes, further increasing benzylisoquinoline alkaloid titer without the requirement for peroxisome induction with fatty acids. This work specifically addresses the challenges associated with toxic NCS expression and, more broadly, highlights the potential for engineering organelles with desired characteristics for metabolic engineering.Increased production of (S)-reticuline and other alkaloids is achieved through alleviating norcoclaurine synthase toxicity by targeting the enzyme to the peroxisome plus enlarging peroxisomes by expression of engineered transcription factors.