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"Exploiting the syntegron technology platform for assembly and optimisation of complex genetic ensembles"

“利用 Syntegron 技术平台来组装和优化复杂的遗传集合体”

基本信息

批准号：
EP/K034359/1
负责人：
Susan Rosser
金额：
$ 181.78万
依托单位：
University of Edinburgh
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2014
资助国家：
英国
起止时间：
2014 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=EP%2FK034359%2F1
关键词：
Exploiting syntegron technology platform assembly

项目摘要

Previously we developed an innovative, versatile technology for enzymatically assembling and dynamically rearranging DNA modules. This "Syntegron" platform enables rapid assembly of multiple standardized DNA modules into large assemblies such as metabolic pathways and to exchange individual parts of assemblies (e.g., regulatory elements) to allow variation and optimization. We propose to build on these core technologies to develop a comprehensive experimental and computational toolkit enabling the rapid generation of important biomolecules:Objective 1: Develop Syntegron "parts" enabling the discovery and production of diverse, valuable biomolecules. An important source for drug leads has been plant natural products but many of these drugs are produced in miniscule amounts in their native hosts, making the drugs expensive and environmentally taxing to harvest. Organic chemistry methodologies have been widely used to synthesize pharmaceuticals but many natural pharmaceutically-relevant scaffolds cannot be achieved by these methods. An alternative is the use of enzymes to enable the production of drug candidates from inexpensive, green starting materials. The goal of this research is to synthesize a variety of natural product variants e.g. terpenes using the Syntegron technology platform constructed in the first phase of funding, and to manufacture these natural product variants for drug discovery applications. We will 1) Take advantage of the recently described phenomena of plant metabolic gene clusters to identify genes encoding enzymes involved in plant secondary metabolite pathways via the development of novel bioinformatic and data mining tools. 2) Use the Syntegron platform to assemble the synthetic metabolic pathways for natural products e.g. the triterpenes and enzymes that will decorate them with functional groups to synthesize a variety of natural product variants. 3) Develop new Syntegron host strains for metabolic engineering 4) Engineer in vivo biosensors for natural products and their variants and use these sensors in high throughput screens allowing dynamic optimization of metabolic pathways.Objective 2: Develop analytical tools, methods, and conceptual insights enabling the optimisation of diverse multigenic functions using Syntegron technology. Cell-free technology is useful for decoupling intracellular biochemical transformations from confounding experimental factors including cellular toxicity and mass transfer limitations. This has proven useful for quantitatively characterizing fundamental synthetic biology "parts". The relationship between cell-free parts characterization and performance in vivo remains unclear, and remains a fundamentally important scientific question. We propose to extend cell-free approaches to the high-throughput characterization of multi-genic assemblies constructed using the Syntegron platform, and to compare them with performance in vivo. The development of the Syntegron platform into a true technology for the synthetic biology community requires the development of novel computational modelling frameworks and quantitative tools for analysing and performing Syntegron-based directed evolution. We will therefore 1) develop a quantitative computational modelling framework describing Syntegron-based diversification and selection. 2) Optimise Syntegron-based directed evolution using in silico simulations paired with experiments. The ultimate test of the combined experimental and computational Syntegron platform will be to perform, analyse, and ultimately guide Syntegron-based directed evolution of a model metabolic pathway. We will initially pair in silico simulation with computational optimization to explore the influence of tuneable experimental parameters on the predicted chances of (a) generating sufficient genetic diversity to sample many potentially functional syntegron configurations, and (b) successfully selecting for variants that exhibit optimised biosynthesis.

此前，我们开发了一种创新的多功能技术，用于酶促组装和动态重排 DNA 模块。该“Syntegron”平台能够将多个标准化 DNA 模块快速组装成大型组件，例如代谢途径，并交换组件的各个部分（例如调控元件）以允许变化和优化。我们建议在这些核心技术的基础上开发一个全面的实验和计算工具包，从而能够快速生成重要的生物分子：目标 1：开发 Syntegron“部件”，从而能够发现和生产多种有价值的生物分子。先导药物的一个重要来源是植物天然产物，但许多这些药物在其天然宿主中产生的量微乎其微，这使得这些药物的收获成本昂贵且对环境造成负担。有机化学方法已广泛用于合成药物，但许多天然药物相关支架无法通过这些方法获得。另一种方法是使用酶，利用廉价的绿色原材料生产候选药物。这项研究的目标是合成各种天然产物变体，例如使用在第一阶段融资中构建的 Syntegron 技术平台来生产萜烯，并生产这些用于药物发现应用的天然产物变体。我们将1）利用最近描述的植物代谢基因簇现象，通过开发新型生物信息学和数据挖掘工具来识别编码参与植物次生代谢途径的酶的基因。 2）使用Syntegron平台组装天然产物的合成代谢途径，例如：三萜和酶将用官能团装饰它们以合成各种天然产物变体。 3) 开发用于代谢工程的新 Syntegron 宿主菌株 4) 设计天然产物及其变体的体内生物传感器，并在高通量筛选中使用这些传感器，从而动态优化代谢途径。目标 2：开发分析工具、方法和概念见解，从而能够使用 Syntegron 技术优化多种多基因功能。无细胞技术可用于将细胞内生化转化与混杂的实验因素（包括细胞毒性和传质限制）分开。事实证明，这对于定量表征基本合成生物学“部件”非常有用。无细胞部分的表征与体内性能之间的关系仍不清楚，并且仍然是一个根本上重要的科学问题。我们建议将无细胞方法扩展到使用 Syntegron 平台构建的多基因组件的高通量表征，并将它们与体内性能进行比较。将 Syntegron 平台发展为合成生物学界的真正技术需要开发新颖的计算建模框架和定量工具来分析和执行基于 Syntegron 的定向进化。因此，我们将 1) 开发一个定量计算建模框架，描述基于 Syntegron 的多样化和选择。 2) 使用计算机模拟与实验相结合来优化基于 Syntegron 的定向进化。实验和计算相结合的 Syntegron 平台的最终测试将是执行、分析并最终指导基于 Syntegron 的模型代谢途径的定向进化。我们首先将计算机模拟与计算优化配对，以探索可调实验参数对以下预测机会的影响：（a）产生足够的遗传多样性以采样许多潜在的功能性合成子配置，以及（b）成功选择表现出优化生物合成的变体。

项目成果

期刊论文数量（10）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

gDesigner: computational design of synthetic gRNAs for Cas12a-based transcriptional repression in mammalian cells.

DOI：
10.1038/s41540-022-00241-w
发表时间：
2022-09-16
期刊：
NPJ systems biology and applications
影响因子：
4
作者：
通讯作者：

Rapid metabolic pathway assembly and modification using serine integrase site-specific recombination.

DOI：
10.1093/nar/gkt1101
发表时间：
2014-02
期刊：
Nucleic acids research
影响因子：
14.9
作者：
Colloms SD;Merrick CA;Olorunniji FJ;Stark WM;Smith MC;Osbourn A;Keasling JD;Rosser SJ
通讯作者：
Rosser SJ

A forward-design approach to increase the production of poly-3-hydroxybutyrate in genetically engineered Escherichia coli.

DOI：
10.1371/journal.pone.0117202
发表时间：
2015
期刊：
PloS one
影响因子：
3.7
作者：
Kelwick R;Kopniczky M;Bower I;Chi W;Chin MH;Fan S;Pilcher J;Strutt J;Webb AJ;Jensen K;Stan GB;Kitney R;Freemont P
通讯作者：
Freemont P

New applications for phage integrases.