Using synthetic biology to forward engineer naphthenic acid catabolic pathways in Pseudomonads for use as biotechnological tools in the bioremediation

利用合成生物学对假单胞菌中的环烷酸分解代谢途径进行正向工程设计，用作生物修复中的生物技术工具

• 批准号: 1711579
• 金额: --
• 依托单位: University of Essex
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2015
• 资助国家: 英国
• 起止时间: 2015 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-1711579
• 关键词: Using; synthetic; biology; forward; engineer

Currently, >50% of global oil reserves are found in vast oil sand deposits. During refining, large quantities of oil sands process wastewaters (OSPW) are generated which contain mixtures of carboxylic acids known as naphthenic acids (NAs). NAs are highly toxic to many organisms and have to be stored >10 yrs until their toxicity is reduced to acceptable levels. Over the next 15-20 yrs, it is estimated that there will be ~1 billion m3 of contaminated OSPW, causing a significant economic and environmental concern. Current information on NA biodegradation mechanisms is limited; however, we have identified microbes capable of degrading NAs. We recently have obtained detailed proteomics data using Pseudomonas fluorescens Pf-5, which has identified candidate proteins (specifically those involved in amino acid metabolism/ transport) that were up-regulated during NA biodegradation. We hypothesise that specific proteins when selectively up-regulated will enhance NA degradation rates in P. fluorescens Pf-5. Overall Aim: to use synthetic biology to forward engineer NA catabolic pathways in P. fluorescens Pf-5 and apply the resulting novel strains as biotechnological tools for the rapid removal of NAs from OSPW. Specific Objectives: i) use bioinformatic tools to identify candidate proteins involved in NA biodegradation. ii) recombinantly over express protein targets to test whether this increases the ability of the organism to degrade NAs; Generate knockouts for confirmation.iii) correlate expression levels of the proteins with in vivo NA degradation rates. iii) use pathway assembly tools to construct NA catabolic pathways. iv) purify and characterize the target proteins. v) test engineered strains (& purified proteins) to remove NAs from different OSPW & more complex model NAs. Approach: The project involves emerging synthetic biology approaches to incorporate engineering into classical biotechnology. Candidate proteins (with likely functions to allow reactivity with NAs or related functions e.g. transport or export) will be selected using existing proteomics data & bioinformatics tools e.g. sequence homology, enzyme pathway assignment, protein data bank. Genes encoding target proteins will be commercially synthesized & ligated into a suitable plasmid vector for homologous overexpression. For the engineered cells, NA degradation and metabolite production will be measured and catabolic pathways assembled. Overexpressed proteins will be purified and enzyme function in the context of the NA degradation pathways will be determined. The engineered strains will be tested as biotechnological tools for rapid removal of NAs from different OSPW process feeds (via the CASE partner Oil Plus Ltd-OPL). The successful candidate will be based at the University of Essex but with periods at the CASE partners (OPL) to test engineered strains with different OSPW process feeds and two shorter periods in the Diaz labs at CISC, Spain to learn the genetic and catabolic pathway assembly tools that are well established by these world leaders in the field. This iCASE studentship provides a unique opportunity for training in bioinformatics, molecular biology, biochemistry, environmental microbiology and analytical chemistry methods.

目前，全球石油储量的50%以上存在于巨大的油砂矿床中。在精炼过程中，产生大量的油砂工艺废水（OSPW），其含有被称为环烷酸（NAs）的羧酸的混合物。NA对许多生物体具有高毒性，必须储存>10年，直到其毒性降低到可接受的水平。在未来15-20年内，估计将有约10亿立方米的OSPW被污染，造成重大的经济和环境问题。目前NA生物降解机制的信息是有限的，但是，我们已经确定了微生物能够降解NA。我们最近已经获得了详细的蛋白质组学数据使用荧光假单胞菌Pf-5，它已经确定了候选蛋白（特别是那些参与氨基酸代谢/运输），在NA生物降解过程中上调。我们假设特异性蛋白质在选择性上调时将增强荧光假单胞菌Pf-5中的NA降解速率。总体目标：使用合成生物学来正向工程化荧光假单胞菌Pf-5中的NA分解代谢途径，并将所得的新菌株用作从OSPW中快速去除NA的生物技术工具。具体目标：i）使用生物信息学工具来鉴定参与NA生物降解的候选蛋白质。ii）重组过表达蛋白质靶以测试这是否增加生物体降解NA的能力;产生敲除用于确认。iii）将蛋白质的表达水平与体内NA降解速率相关联。iii）使用途径组装工具构建NA分解代谢途径。iv）纯化和表征靶蛋白。v）测试工程菌株（和纯化的蛋白质）以从不同的OSPW和更复杂的模型NA中去除NA。方法：该项目涉及新兴的合成生物学方法，将工程纳入经典生物技术。将使用现有的蛋白质组学数据和生物信息学工具（例如序列同源性、酶途径分配、蛋白质数据库）选择候选蛋白质（具有允许与NA反应的可能功能或相关功能，例如运输或输出）。编码靶蛋白的基因将商业合成并连接到合适的质粒载体中用于同源过表达。对于工程化细胞，将测量NA降解和代谢产物产生，并组装分解代谢途径。将纯化过表达的蛋白质，并确定NA降解途径背景下的酶功能。工程菌株将作为生物技术工具进行测试，用于从不同的OSPW工艺进料中快速去除NA（通过CASE合作伙伴Oil Plus Ltd-OPL）。成功的候选人将在埃塞克斯大学工作，但在CASE合作伙伴（OPL）工作一段时间，以测试不同OSPW工艺进料的工程菌株，并在西班牙CISC的迪亚兹实验室工作两个较短的时间，以学习该领域的世界领导者所建立的遗传和分解代谢途径组装工具。这个iCASE学生奖学金提供了一个独特的机会，在生物信息学，分子生物学，生物化学，环境微生物学和分析化学方法的培训。