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

目前，全球50%的石油储量是在巨大的油砂矿藏中发现的。在炼油过程中，会产生大量的油砂处理废水，其中含有被称为环烷酸(Nas)的羧酸混合物。NAS对许多生物具有高度毒性，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10亿立方米的受污染的OSPW，造成重大的经济和环境问题。目前关于NA生物降解机制的信息有限；然而，我们已经确定了能够降解NA的微生物。我们最近利用荧光假单胞菌PF-5获得了详细的蛋白质组学数据，它已经确定了在NA生物降解过程中上调的候选蛋白质(特别是那些参与氨基酸代谢/运输的蛋白质)。我们假设，当选择性上调特定蛋白时，将提高荧光假单胞菌PF-5中NA的降解率。总体目标：利用合成生物学在荧光假单胞菌PF-5中正向工程NA分解代谢途径，并应用所产生的新菌株作为生物技术工具从OSPW中快速去除Nas。具体目标：i)使用生物信息学工具确定与NA生物降解有关的候选蛋白质。Ii)重组过表达蛋白质靶标，以测试这是否增加了有机体降解NA的能力；生成用于确认的敲除。iii)将蛋白质的表达水平与体内NA降解率相关联。Iii)使用途径组装工具构建NA分解代谢途径。Iv)纯化和鉴定目的蛋白。V)测试工程菌株(和纯化蛋白)以从不同的OSPW和更复杂的模型NAS中去除NAS。方法：该项目涉及新兴的合成生物学方法，将工程学融入经典生物技术。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(可能具有允许与NAS反应或相关功能，例如运输或输出的功能)。编码目标蛋白的基因将被商业化合成，并连接到合适的质粒载体上进行同源过度表达。对于工程细胞，将测量NA降解和代谢物产生，并组装分解代谢途径。过表达的蛋白质将被提纯，NA降解途径中的酶功能将被确定。工程菌株将作为生物技术工具进行测试，以从不同的OSPW工艺饲料中快速去除NAS(通过Case Partner Oil Plus Ltd-OPL)。成功的候选人将在埃塞克斯大学工作，但需要在Case Partners(OPL)用不同的OSPW工艺饲料测试工程菌株，并在西班牙CISC的Diaz实验室学习这些世界领先者在该领域建立的遗传和分解代谢途径组装工具。该iCASE项目为生物信息学、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提供了一个独特的机会。

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.