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

遗传密码是生命最早的表现形式之一。它无处不在，将所有生物体联合起来，并为整个生物圈提供了共享的生化基础。 我研究的长期目标是了解密码的进化和起源，并操纵和扩大蛋白质生物合成的范围。我的实验方法旨在构建具有新的、独特的化学功能的蛋白质，并由此操纵生命的化学过程。这将为检验进化理论提供一个实验基础，并将使得合成活细胞的构建成为可能。这些目标是全世界科学家界的共同努力，超越了单个国家或科学学科的边界。 根据这两个具体问题提出的研究的基本原理 目标是回答基础科学技术问题 问题并填补当前知识状况的重要空白。 短期内我们有两个主要目标： 一、大肠杆菌和枯草芽孢杆菌的适应性实验室进化（ALE） 我们将建立 ALE 作为一种在微生物种群中传播化学变化的方法。具体来说，将进行 ALE 将 3,4-二羟基苯丙氨酸 (Dopa) 全面引入细菌蛋白质组以及大肠杆菌和枯草芽孢杆菌物种中的单一蛋白质中。 进化的菌株将用于阐明适应背后的生化机制。最终菌株将用于生产具有独特功能的蛋白质，例如粘附能力或金属结合能力。 二.由扩展的遗传密码设计的定制模块化支架 我们将在微生物菌株中生产含多巴的蛋白质。将使用扩展的遗传密码方法来绕过原核微生物生产菌株中不易获得的翻译后修饰。含多巴的蛋白质将基于结合粘合、弹性和抗菌特性的模块化支架进行工程设计。将研究它们的分子特性，例如金属配位、氧化还原、酸碱，并且将研究多巴残基在这些特性中的参与。 我新建立的实验室为学员提供了最先进的环境，让他们学习合成生物学学科，并有机会与化学、微生物学和生物工程教师进行国际和跨学科合作。接受过多学科和多部门研究培训的早期职业研究人员将获得工业界、政府和学术界职业所需的高级技能。 拟议的研究将为检验进化理论提供一个实验基础，并将能够构建合成活细胞，最终用于基于贻贝粘附蛋白和羊毛硫抗生素（作为开发抗感染剂的潜在支架）的先进生物材料和技术。

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).