Chemical evolution of synthetic bacterial cells by reprograming protein translation with non-canonical amino acids

通过使用非规范氨基酸重新编程蛋白质翻译来合成细菌细胞的化学进化

• 批准号: RGPIN-2020-05669
• 负责人: Budisa, Nediljko
• 金额: $ 3.64万
• 依托单位: University of Manitoba
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=717910
• 关键词: Chemical; evolution; synthetic; bacterial; cells

The genetic code is one of the earliest manifestations of life. It is ubiquitous, unites all living organisms, and provides a shared biochemical basis for the entire biosphere. The long-term goal of my research is to understand the evolution and origin of the code, and to manipulate and expand the scope of protein biosynthesis. My experimental approach is directed towards construction of proteins with new and unique chemical functions, and manipulation of the chemistry of life as the result. This should provide an experimental ground for examination of evolutionary theories, and will enable construction of synthetic living cells. These goals are a shared endeavor of the worldwide community of scientists that extends beyond the borders of individual countries or scientific disciplines. The rationale of the research proposed under these two specific objectives is to answer fundamental scientific and technological questions and fill important gaps in the current state of knowledge. In the short term we have two major goals: I. Adaptive Laboratory Evolution (ALE) of Escherichia coli and Bacillus subtilis We will establish ALE as a method to propagate chemical changes in microbial populations. Specifically, ALE will be performed to introduce 3,4-dihydroxyphenylalanine (Dopa) globally into bacterial proteomes and single proteins in E. coli and B. subtilis species. Evolved strains will be used to elucidate biochemical mechanisms behind the adaptation. Final strains will be utilized for production of proteins endowed with unique features, such as adhesive capabilities or metal-binding. II. Tailor-made modular scaffolds designed by an expanded genetic code We will establish production of Dopa-containing proteins in microbial strains. An expanded genetic code approach will be used in order to bypass the posttranslational modification that is not readily available in prokaryotic microbial production strains. Dopa-containing proteins will be engineered based on modular scaffolds combining adhesive, elastic, and antimicrobial features. Their molecular properties such as metal coordinating, red-ox, acid-base, will be studied, and involvement of the Dopa residues in these features will be investigated. My newly established lab provides a state-of-the-art environment for trainees to learn the discipline of synthetic biology with opportunities for international and interdisciplinary collaboration with faculty in Chemistry, Microbiology and Bioengineering. Early-career researchers trained in multidisciplinary and multi-sectors research will acquire advanced skills for careers in industry, government, and academia. The proposed research will provide an experimental ground for the examination of evolutionary theories, and will enable construction of synthetic living cells for ultimate use in advanced biomaterials and technologies based on mussel adhesion proteins and lantibiotics (as a potential scaffold for the development of anti-infective agents).

遗传密码是生命中最早的表现之一。它无处不在，将所有生物体统一，并为整个生物圈提供共同的生化基础。 我的研究的长期目标是了解法规的演变和起源，并操纵和扩大蛋白质生物合成的范围。我的实验方法是针对具有新的和独特的化学功能的蛋白质的构建，结果是对生命的化学作用。这应该为研究进化理论提供一个实验基础，并能够构建合成活细胞。这些目标是全球科学家社区的共同努力，它超出了各个国家或科学学科的边界。 根据这两个特定的研究的理由 目标是回答基本的科学和技术 问题并填补当前知识状态的重要空白。 在短期内，我们有两个主要目标： I.大肠杆菌和枯草芽孢杆菌的自适应实验室进化（AL） 我们将建立啤酒作为一种传播微生物种群化学变化的方法。具体而言，将在全球范围内引入3,4-二羟基苯丙氨酸（DOPA）中的3,4-二羟基苯基丙氨酸（DOPA），并在大肠杆菌和枯草芽孢杆菌中引入细菌蛋白质组和单蛋白。 进化的菌株将用于阐明适应后面的生化机制。最终菌株将用于生产具有独特特征的蛋白质，例如粘合功能或金属结合。 ii。量身定制的模块化脚手架由扩展的遗传密码设计 我们将在微生物菌株中建立含DOPA的蛋白质的产生。将使用扩展的遗传密码方法，以绕过原核生物微生物生产菌株中不容易获得的翻译后修饰。含DOPA的蛋白质将基于结合粘合剂，弹性和抗菌特征的模块化支架进行设计。将研究它们的分子特性，例如金属配位，红氧，酸碱，并研究DOPA残基参与这些特征。 我新成立的实验室为学员提供了一个最先进的环境，可以通过与化学，微生物学和生物工程学领域的国际和跨学科合作的机会学习合成生物学学科。接受多学科和多部门研究培训的早期研究人员将获得行业，政府和学术界职业的高级技能。 拟议的研究将为检验进化论提供一个实验基础，并能够基于贻贝粘附蛋白和耐脂蛋白（作为抗抗染剂开发的潜在支架）来构建合成活细胞的最终使用。