Genome-wide host-pathway interactions affecting cis-cis-muconic acid production in yeast

IF 6.8 1区 生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Paul Cachera , Nikolaj Can Kurt , Andreas Røpke , Tomas Strucko , Uffe H. Mortensen , Michael K. Jensen
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引用次数: 0

Abstract

The success of forward metabolic engineering depends on a thorough understanding of the behaviour of a heterologous metabolic pathway within its host. We have recently described CRI-SPA, a high-throughput gene editing method enabling the delivery of a metabolic pathway to all strains of the Saccharomyces cerevisiae knock-out library. CRI-SPA systematically quantifies the effect of each modified gene present in the library on product synthesis, providing a complete map of host:pathway interactions. In its first version, CRI-SPA relied on the colour of the product betaxanthins to quantify strains synthesis ability. However, only a few compounds produce a visible or fluorescent phenotype limiting the scope of our approach. Here, we adapt CRI-SPA to onboard a biosensor reporting the interactions between host genes and the synthesis of the colourless product cis-cis-muconic acid (CCM). We phenotype >9,000 genotypes, including both gene knock-out and overexpression, by quantifying the fluorescence of yeast colonies growing in high-density agar arrays. We identify novel metabolic targets belonging to a broad range of cellular functions and confirm their positive impact on CCM biosynthesis. In particular, our data suggests a new interplay between CCM biosynthesis and cytosolic redox through their common interaction with the oxidative pentose phosphate pathway. Our genome-wide exploration of host:pathway interaction opens novel strategies for improved production of CCM in yeast cell factories.

影响酵母中顺式-顺式粘液酸产生的全基因组宿主-途径相互作用。
前向代谢工程的成功取决于对异源代谢途径在宿主体内行为的透彻理解。我们最近介绍了 CRI-SPA,这是一种高通量基因编辑方法,可将代谢途径传递到所有菌株的酿酒酵母基因敲除库中。CRI-SPA 系统地量化了文库中每个修饰基因对产物合成的影响,提供了宿主与途径相互作用的完整图谱。在第一个版本中,CRI-SPA 依靠产品甜菜黄素的颜色来量化菌株的合成能力。然而,只有少数化合物能产生可见或荧光表型,这限制了我们的方法范围。在这里,我们对 CRI-SPA 进行了调整,使其能够搭载生物传感器,报告宿主基因与无色产品顺式-顺式-粘液酸(CCM)合成之间的相互作用。通过量化在高密度琼脂阵列中生长的酵母菌落的荧光,我们对超过 9,000 种基因型进行了表型,包括基因敲除和过表达。我们发现了属于多种细胞功能的新的代谢靶标,并证实了它们对 CCM 生物合成的积极影响。特别是,我们的数据表明,通过与氧化磷酸戊糖途径的共同作用,CCM 生物合成与细胞膜氧化还原之间存在新的相互作用。我们对宿主与途径相互作用的全基因组探索为酵母细胞工厂改进 CCM 的生产开辟了新策略。
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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.
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