Nan Wu, Mingdong Yao, Wenhai Xiao, Ying Wang, Ying-Jin Yuan
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引用次数: 0
摘要
麦角酸(LA)是生物合成各种具有重要药用价值的麦角生物碱的基本前体。在微生物中异源生物合成 LA 是一种很有前途的方法,可以减少工业对高成本、高毒性的作物致病麦角菌的依赖。然而,由于缺乏有效途径和异源生产性能低下,LA 的生物合成效率仍不令人满意。在这项工作中,通过应用高效生产 LA 的代谢工程策略,构建了微生物宿主,特别是酿酒酵母菌株。SCH9基因敲除被认为是提高LA产量的关键因素。转录分析表明,SCH9 基因缺失可显著增强过氧物酶体代谢和细胞翻译。因此,设计了量身定制的方法来优化 SCH9 基因缺失菌株中两种限速酶 EasC 和 CloA 的活性。将 EasC 移至过氧化物酶体并结合 PEX34 的过表达,明显提高了这两种酶的催化活性,使 LA 的产量增加了 2.31 倍。此外,还对内质网上的细胞色素 P450 CloA 及其还原酶进行了空间定向,提高了电子传递效率,从而使 LA 产量提高了 36.8%。这些工程策略最终使 LA 滴度提高了 17.4 倍。最终的工程菌株在 50 升饲料批量发酵条件下产生了 509.8 mg L-1 的 LA,是目前报道的异源宿主最高滴度。这些研究结果表明,目前基于麦角的发酵路线是一种绿色替代方案,为可持续、大规模发酵药用麦角生物碱提供了一个多功能平台。
Highly efficient synthesis of lysergic acid using engineered budding yeast
Lysergic acid (LA) is the basic precursor for the biosynthesis of various ergot alkaloids of pharmaceutical importance. The heterologous biosynthesis of LA in microbes is a promising method to reduce industrial dependence on highly costly and toxic crop-pathogenic ergot fungi. However, the biosynthetic efficiency of LA remains unsatisfactory because of the lack of effective pathways and low heterologous production performance. In this work, a microbial host, specifically a Saccharomyces cerevisiae strain, was constructed by applying a metabolic engineering strategy for efficient LA production. The SCH9 knockout was identified as a key factor for enhancing LA yield. Transcriptional analysis revealed that SCH9 deletion significantly enhanced peroxisomal metabolism and cellular translation. Accordingly, tailored approaches were designed to optimize the activities of two rate-limiting enzymes, EasC and CloA, in SCH9 deletion strains. The relocation of EasC to peroxisomes combined with PEX34 overexpression clearly increased the catalytic activity of these enzymes, increasing LA production by 2.31-fold. Moreover, spatial reorientation of the cytochrome P450 CloA and its reductase on the endoplasmic reticulum was performed, which improved electron transfer efficiency, resulting in a 36.8% improvement in LA production. These engineering strategies finally led to a 17.4-fold increase in the LA titre. The final engineered strain produced 509.8 mg L−1 LA under 50 L fed-batch fermentation, yielding the highest reported titre for heterologous hosts. These findings demonstrated a green alternative to the current ergot-based routes, offering a versatile platform for the sustainable, large-scale fermentation of pharmaceutical ergot alkaloids.
期刊介绍:
Green Chemistry is a journal that provides a unique forum for the publication of innovative research on the development of alternative green and sustainable technologies. The scope of Green Chemistry is based on the definition proposed by Anastas and Warner (Green Chemistry: Theory and Practice, P T Anastas and J C Warner, Oxford University Press, Oxford, 1998), which defines green chemistry as the utilisation of a set of principles that reduces or eliminates the use or generation of hazardous substances in the design, manufacture and application of chemical products. Green Chemistry aims to reduce the environmental impact of the chemical enterprise by developing a technology base that is inherently non-toxic to living things and the environment. The journal welcomes submissions on all aspects of research relating to this endeavor and publishes original and significant cutting-edge research that is likely to be of wide general appeal. For a work to be published, it must present a significant advance in green chemistry, including a comparison with existing methods and a demonstration of advantages over those methods.