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

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.