Metabolic engineering of Pichia pastoris for overproduction of cis-trans nepetalactol

IF 6.8 1区 生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Cuifang Ye , Mengxin Li , Jucan Gao , Yimeng Zuo , Feng Xiao , Xiaojing Jiang , Jintao Cheng , Lei Huang , Zhinan Xu , Jiazhang Lian
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Abstract

Monoterpene indole alkaloids (MIAs) are a group of plant-derived natural products with high-value medicinal properties. However, their availability for clinical application is limited due to challenges in plant extraction. Microbial production has emerged as a promising strategy to meet the clinical demands for MIAs. The biosynthetic pathway of cis-trans nepetalactol, which serves as the universal iridoid scaffold for all MIAs, has been successfully identified and reconstituted. However, bottlenecks and challenges remain to construct a high-yielding platform strain for cis-trans nepetalactol production, which is vital for subsequent MIAs biosynthesis. In the present study, we focused on engineering of Pichia pastoris cell factories to enhance the production of geraniol, 8-hydroxygeraniol, and cis-trans nepetalactol. By targeting the biosynthetic pathway from acetyl-CoA to geraniol in both peroxisomes and cytoplasm, we achieved comparable geraniol titers in both compartments. Through protein engineering, we found that either G8H or CPR truncation increased the production of 8-hydroxygeraniol, with a 47.8-fold and 14.0-fold increase in the peroxisomal and cytosolic pathway strain, respectively. Furthermore, through a combination of dynamical control of ERG20, precursor and cofactor supply engineering, diploid engineering, and dual subcellular compartmentalization engineering, we achieved the highest ever reported production of cis-trans nepetalactol, with a titer of 4429.4 mg/L using fed-batch fermentation in a 5-L bioreactor. We anticipate our systematic metabolic engineering strategies to facilitate the development of P. pastoris cell factories for sustainable production of MIAs and other plant natural products.

过量生产顺式-反式新内酯的 Pichia pastoris 代谢工程。
单萜吲哚生物碱(MIAs)是一类从植物中提取的天然产品,具有很高的药用价值。然而,由于植物提取方面的挑战,它们的临床应用受到限制。为满足临床对 MIAs 的需求,微生物生产已成为一种前景广阔的策略。顺式-反式新内酯是所有 MIAs 的通用铱类支架,其生物合成途径已被成功鉴定和重组。然而,在构建顺式反式内酯生产的高产平台菌株方面仍存在瓶颈和挑战,而顺式反式内酯对后续的 MIAs 生物合成至关重要。在本研究中,我们重点对 Pichia pastoris 细胞工厂进行工程改造,以提高香叶醇、8-羟基香叶醇和顺式-反式萘内酯的产量。通过锁定过氧物酶体和细胞质中从乙酰-CoA 到香叶醇的生物合成途径,我们在两个区室中都获得了相当的香叶醇滴度。通过蛋白质工程,我们发现 G8H 或 CPR 截断都能增加 8-羟基香叶醇的产生,过氧物酶体和细胞质途径菌株的产生量分别增加了 47.8 倍和 14.0 倍。此外,通过对 ERG20 的动态控制、前体和辅助因子供应工程、二倍体工程和双亚细胞区隔工程的结合,我们在 5 升生物反应器中采用喂料批量发酵的方法,获得了有报道以来最高的顺式-反式炔内酯产量,滴度为 4429.4 mg/L。我们预计,我们的系统代谢工程策略将促进P. pastoris细胞工厂的发展,从而实现MIAs和其他植物天然产品的可持续生产。
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来源期刊
Metabolic engineering
Metabolic engineering 工程技术-生物工程与应用微生物
CiteScore
15.60
自引率
6.00%
发文量
140
审稿时长
44 days
期刊介绍: Metabolic Engineering (MBE) is a journal that focuses on publishing original research papers on the directed modulation of metabolic pathways for metabolite overproduction or the enhancement of cellular properties. It welcomes papers that describe the engineering of native pathways and the synthesis of heterologous pathways to convert microorganisms into microbial cell factories. The journal covers experimental, computational, and modeling approaches for understanding metabolic pathways and manipulating them through genetic, media, or environmental means. Effective exploration of metabolic pathways necessitates the use of molecular biology and biochemistry methods, as well as engineering techniques for modeling and data analysis. MBE serves as a platform for interdisciplinary research in fields such as biochemistry, molecular biology, applied microbiology, cellular physiology, cellular nutrition in health and disease, and biochemical engineering. The journal publishes various types of papers, including original research papers and review papers. It is indexed and abstracted in databases such as Scopus, Embase, EMBiology, Current Contents - Life Sciences and Clinical Medicine, Science Citation Index, PubMed/Medline, CAS and Biotechnology Citation Index.
文献相关原料
公司名称 产品信息 采购帮参考价格
上海源叶 Geraniol
¥25.00~¥11313.00
希恩思 Isopropyl myristate
¥16.00~¥7448.68
希恩思 Isopropyl myristate
上海源叶 geraniol
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