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之一。它无处不在，将所有生物联系在一起，并为整个生物圈提供共同的生化基础。我研究的长期目标是了解编码的进化和起源，并操纵和扩大蛋白质生物合成的范围。我的实验方法是针对具有新的和独特的化学功能的蛋白质的构建，并作为生命的化学操作的结果。这将为检验进化理论提供一个实验基础，并将使合成活细胞的构建成为可能。这些目标是超越个别国家或科学学科边界的全球科学家共同努力的结果。在这两个具体目标下提出的研究的基本原理是回答基本的科学和技术问题，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：1 .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作为一种方法来传播微生物种群的化学变化。具体来说，ALE将在大肠杆菌和枯草芽孢杆菌的细菌蛋白质组和单个蛋白质中全面引入3,4-二羟基苯丙氨酸（Dopa）。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，如粘附能力或金属结合能力。2。我们将在微生物菌株中建立含多巴蛋白的生产。一种扩展的遗传密码方法将被用来绕过翻译后修饰，这在原核微生物生产菌株中是不容易获得的。含有多巴的蛋白质将被设计成结合了粘合、弹性和抗菌特性的模块化支架。将研究它们的分子性质，如金属配位、红牛、酸碱等，并研究多巴残基在这些特征中的作用。我新建立的实验室为学员提供了一个最先进的环境来学习合成生物学，并有机会与化学、微生物学和生物工程的教师进行国际和跨学科的合作。接受多学科和多部门研究培训的早期职业研究人员将获得在工业、政府和学术界工作的高级技能。拟议的研究将为检验进化理论提供实验基础，并将使合成活细胞最终用于基于贻贝粘附蛋白和l抗生素的高级生物材料和技术（作为开发抗感染药物的潜在支架）。

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).