A Toolkit to Enable Hydrocarbon Conversion in Aqueous Environments

Summary

A sustainable auto regulating bacterial system for the remediation of oil pollutions was designed using standard interchangeable DNA parts (BioBricks). An engineered E. coli strain was used to degrade alkanes via β-oxidation in toxic aqueous environments. The respective enzymes from different species showed alkane degradation activity. Additionally, an increased tolerance to n-hexane was achieved by introducing genes from alkane-tolerant bacteria.

Abstract

This work puts forward a toolkit that enables the conversion of alkanes by Escherichia coli and presents a proof of principle of its applicability. The toolkit consists of multiple standard interchangeable parts (BioBricks)⁹ addressing the conversion of alkanes, regulation of gene expression and survival in toxic hydrocarbon-rich environments.

A three-step pathway for alkane degradation was implemented in E. coli to enable the conversion of medium- and long-chain alkanes to their respective alkanols, alkanals and ultimately alkanoic-acids. The latter were metabolized via the native β-oxidation pathway. To facilitate the oxidation of medium-chain alkanes (C5-C13) and cycloalkanes (C5-C8), four genes (alkB2, rubA3, rubA4and rubB) of the alkane hydroxylase system from Gordonia sp. TF6^8,21 were transformed into E. coli. For the conversion of long-chain alkanes (C15-C36), theladA gene from Geobacillus thermodenitrificans was implemented. For the required further steps of the degradation process, ADH and ALDH (originating from G. thermodenitrificans) were introduced^10,11. The activity was measured by resting cell assays. For each oxidative step, enzyme activity was observed.

To optimize the process efficiency, the expression was only induced under low glucose conditions: a substrate-regulated promoter, pCaiF, was used. pCaiF is present in E. coli K12 and regulates the expression of the genes involved in the degradation of non-glucose carbon sources.

The last part of the toolkit - targeting survival - was implemented using solvent tolerance genes, PhPFDα and β, both from Pyrococcus horikoshii OT3. Organic solvents can induce cell stress and decreased survivability by negatively affecting protein folding. As chaperones, PhPFDα and β improve the protein folding process e.g. under the presence of alkanes. The expression of these genes led to an improved hydrocarbon tolerance shown by an increased growth rate (up to 50%) in the presences of 10% n-hexane in the culture medium were observed.

Summarizing, the results indicate that the toolkit enables E. coli to convert and tolerate hydrocarbons in aqueous environments. As such, it represents an initial step towards a sustainable solution for oil-remediation using a synthetic biology approach.

Introduction

Oil pollution is among the most serious causes of environmental contamination, and greatly affects ecosystems, businesses and communities ³. Solutions are for example required to battle the continuous oil pollution originating from the oil sands tailing waters in Alberta, Canada. During the process of oil extraction from oil sands, bitumen, a semi-solid oxidized form of oil, is removed using thermal recovery techniques that consume about 3.1 barrels of water per single barrel of oil ¹. Oil contaminated process water, mainly originating from a local river, is stored in tailing ponds after bitumen extraction. A more effective recycling of process w....

Protocol

1. BioBrick Assembly

1. BioBricks from the Registry of Standard Biological parts are provided by iGEM headquarters. To construct a new BioBrick from existing BioBricks, digest the donor BioBrick (up to 1.0 μg) with the enzymes EcoRI and SpeI for positioning the donor part downstream of the acceptor part. Digest with XbaI and PstI for positioning the donor part upstream of the acceptor part. Add a third appropriate restriction enzyme that cuts in the backbone of the donor. Perform the digestions in a total volume of 20-25 ml with the appropriate buffer, according to the supplier (final concentration 1x). Use 5 units/μg DNA for the restriction enzymes.

Results

Alkane conversion

The activity of the three oxidation steps from the alkane to the respective fatty acid was evaluated using resting cell assays and enzyme activity measurements. The results are presented following the pathway reactions (1) alkane hydroxylase, (2) alcohol dehydrogenase and (3) aldehyde dehydrogenase.
For the first step, different plasmids were constructed for medium and long-chain alkanes. The plasmid BBa_K3.......

Discussion

The BioBrick principle is used to construct a chassis for the degradation of alkanes and a proof of principle for the single components of the toolkit was obtained. Several assays are proposed to measure the in vivo and in vitro activity of alkane degrading pathway enzymes. The presented work successfully demonstrates a number of methods that can be used to determine enzyme activities and expression in the host organism E. coli after implementation of suitable BioBricks. Furthermore, it is show.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
E. coli K12	New England Biolabs	C2523H
Octane	Fluka	74822
Hexadecane	Fluka	52209
octanol-1	Fluka	95446
dodecanol-1	Sigma-Aldrich	126799
Hexane	Sigma-Aldrich	296090
NADH	Sigma-Aldrich	N4505
FMN	Sigma-Aldrich	F2253
MgSO4	J.T. Baker Casno	7487 889
Triton X-100	Sigma-Aldrich	T8787
T4 ligase	New England Biolabs	M0202L
Gas chromatograph
Cell disrupter	LA Biosystems	CD-019
Spectrophotometer	Amersham pharmacia	spec 2000
Plate reader	Tecan Group Ltd.	Magellan v7.0
Incubator	Innova, 44
BioBrickTM K398014: BBa_J23100-BBa_J61100-alkB2-BBa_J61100-rubA3-BBa_J61100-rubA4- BBa_J61100-rubB	Delft University of Technology at the department of Biotechnology or Registry of Standard Biological Parts	BBa_K398014	Alkane Hydroxylase System Resistance: Chloramphenicol
BioBrickTM K398027: BBa_R0040-BBa_B0034-ladA	Delft University of Technology at the department of Biotechnology or Registry of Standard Biological Parts	BBa_K398027	ladA Protein Generator Resistance: Chloramphenicol
BioBrickTM K398018: BBa_J23100-BBa_J61101-ADH	Delft University of Technology at the department of Biotechnology or Registry of Standard Biological Parts	BBa_K398018	ADH generator Resistance: Chloramphenicol
BioBrickTM K398030: BBa_R0040-BBa_B0034-ALDH	Delft University of Technology at the department of Biotechnology or Registry of Standard Biological Parts	BBa_K398030	ALDH generator Resistance: Chloramphenicol
BioBrickTM K398326: pCaiF	Delft University of Technology at the department of Biotechnology or Registry of Standard Biological Parts	BBa_K398326	pCaiF promoter Resistance: Chloramphenicol
BioBrickTM K398331: pCaiF-BBa_B0032-BBa_I13401	Delft University of Technology at the department of Biotechnology or Registry of Standard Biological Parts	BBa_K398331	pCaiF measurement device Resistance: Chloramphenicol
BioBrickTM K398406: BBa_J23002-BBa_J61107-phPFDα-BBa_J61107-	Delft University of Technology at the department of Biotechnology or Registry of Standard Biological Parts	BBa_K398406	Solvent tolerance cluster Resistance: Chloramphenicol