Biosynthesis of tetrahydropapaverine and semisynthesis of papaverine in yeast.
Biosynthesis of tetrahydropapaverine and semisynthesis of papaverine in yeast.
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DOI：
10.1073/pnas.2205848119
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发表时间：
2022-08-16
影响因子：
11.1
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中科院分区：
文献类型：
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我们报道了(S)-四氢罂粟碱(THP)的微生物从头合成和利用微生物合成的THP半合成罂粟碱。我们使用蛋白质工程开发了两种酶的变体，它们在途径中间体上具有更高的非天然活性。THP的生物合成表明，在异源生物合成途径中，可以使用蛋白质同源物和蛋白质工程来取代未知酶的活性。我们通过敲除两个酵母多药耐药(MDR)转运蛋白来改善途径通量，这减少了途径中间产物的出口。MDR基因敲除可用于增加通过其他异源途径的通量。这项工作中的菌株工程提供了基于发酵生产临床上重要的分子THP和罂粟碱的示范，这两种分子最近经历了供应链短缺。四氢罂粟碱(THP)和罂粟碱是具有重要临床意义的植物天然产物。THP是生产药物阿曲库铵和顺式阿曲库铵的前体，罂粟碱在血管手术中用作解痉剂。近年来，代谢工程的进展使得通过在酵母中异源表达途径酶来生产天然产物成为可能。异源生物合成THP和罂粟碱可能在确保这些具有临床意义的产品的稳定供应方面发挥作用。到目前为止，THP和罂粟碱的生物合成还没有实现，部分原因是多途径酶还没有被阐明。在这里，我们描述了一种用于从头合成THP的工程酵母菌株的开发。THP的产生是通过在非天然底物上异源表达两种具有活性的酶变体来实现的。通过蛋白质工程，我们开发了一种N-甲基去甲黄嘌呤羟基酶的变异体，具有对去甲黄碱的活性，使去甲肾上腺素的从头合成成为可能。类似地，我们开发了一种Soulerine 9-O-甲基转移酶的变体，能够在3‘位对1-苄基异喹啉生物碱进行O-甲基化，从而能够从头合成THP。通过敲除酵母多药耐药转运蛋白和优化培养条件，提高了通过异源途径的通量。总体而言，菌株工程将生物合成的THP浓度提高了600倍，达到121µg/L。最后，我们展示了一种以过氧化氢为氧化剂的罂粟碱半合成策略。通过对pH、温度、反应时间和氧化剂浓度的优化，证明了生物合成的THP氧化生成半合成罂粟碱的能力。
We report the de novo microbial biosynthesis of (S)-tetrahydropapaverine (THP) and the semisynthesis of papaverine using microbially biosynthesized THP. We used protein engineering to develop variants of two enzymes with improved nonnative activity on pathway intermediates. The biosynthesis of THP demonstrates the ability to use protein homologs and protein engineering to replace the activity of unknown enzymes in heterologous biosynthetic pathways. We improved pathway flux by knocking out two yeast multidrug resistance (MDR) transporters, which reduces the export of pathway intermediates. MDR knockouts may be applied to increase flux through other heterologous pathways. The strain engineering in this work provides a demonstration of fermentation-based production of the clinically significant molecules THP and papaverine, which have experienced recent supply chain shortages. Tetrahydropapaverine (THP) and papaverine are plant natural products with clinically significant roles. THP is a precursor in the production of the drugs atracurium and cisatracurium, and papaverine is used as an antispasmodic during vascular surgery. In recent years, metabolic engineering advances have enabled the production of natural products through heterologous expression of pathway enzymes in yeast. Heterologous biosynthesis of THP and papaverine could play a role in ensuring a stable supply of these clinically significant products. Biosynthesis of THP and papaverine has not been achieved to date, in part because multiple pathway enzymes have not been elucidated. Here, we describe the development of an engineered yeast strain for de novo biosynthesis of THP. The production of THP is achieved through heterologous expression of two enzyme variants with activity on nonnative substrates. Through protein engineering, we developed a variant of N-methylcoclaurine hydroxylase with activity on coclaurine, enabling de novo norreticuline biosynthesis. Similarly, we developed a variant of scoulerine 9-O-methyltransferase capable of O-methylating 1-benzylisoquinoline alkaloids at the 3′ position, enabling de novo THP biosynthesis. Flux through the heterologous pathway was improved by knocking out yeast multidrug resistance transporters and optimization of media conditions. Overall, strain engineering increased the concentration of biosynthesized THP 600-fold to 121 µg/L. Finally, we demonstrate a strategy for papaverine semisynthesis using hydrogen peroxide as an oxidizing agent. Through optimizing pH, temperature, reaction time, and oxidizing agent concentration, we demonstrated the ability to produce semisynthesized papaverine through oxidation of biosynthesized THP.
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