The findings presented in this work emerge from the discovery of a multidrug efflux
system Enterobacter lignolyticus, a bacterium isolated from Puerto Rican cloud
forest soil. This system, which consists of the inner membrane transporter EilA and
its cognate repressor EilR, confers tolerance to imidazolium-based ionic liquids. In
my research, I characterized these genes using molecular genetics, improved a
method of microbial biofuel production using synthetic biology techniques, and
demonstrated new biotechnological applications by both decoupling the genes from
their natural context and engineering the regulatory elements.
Chapter 1 targets the removal of a bottleneck in the microbial conversion of
lignocellulose to biofuels or chemicals. In this process, pretreatment of plant biomass
is necessary due to the inherent recalcitrance of lignocellulose. Certain ionic liquids
(ILs) are solvents remarkably effective in solubilizing cellulosic biomass. By
dissociating lignin from hemicellulose and cellulose in cell walls, enzymatic
hydrolysis to fermentable sugars can be achieved. However, ILs are toxic to most
microbes, inhibiting growth and subsequent fermentation of sugars to fuels. In
searching for a solution to this problem, I discovered a novel molecular system for
bacterial resistance to IL toxicity by screening the genome of the IL-tolerant
bacterium E. lignolyticus. A single gene was identified that promotes growth in the
presence of IL, namely, an multidrug transporter, EilA, which acts to export IL from
the cell. In response to changes in external IL levels, expression of the transporter is
controlled by a repressor, EilR, providing a self regulating system that maintains cell
viability. The gene pair encoding EilA and EilR remains functional when transferred
to an Escherichia coli strain that expresses a biosynthetic pathway to produce a
terpene-based biofuel. In this host organism, the auto-regulatory efflux system
enables growth and biofuel production in a previously toxic environment.
Chapter 2 focuses on the EilR protein and how it performs its task as a
transcriptional regulator. Identification of the DNA binding site, the eil-operator,
provided the basis to develop a sensitive EilR-regulated promoter that drives
expression of a reporter gene in E. coli. Using this cellular biosensor, I identified a
range of cationic dyes with high affinity to the EilR repressor. These anthropogenic
ligands are unrelated to ILs, and some of them can induce the biosensor at
2
nanomolar concentrations – up to five orders of magnitude lower than ILs. Activitybased
assays with cell extracts indicated a possible way of identifying metabolites as
natural inducers. In addition, experiments with homologous repressors and a
transporter provided further insights on bacterial regulation of efflux.
The strong binding affinity of EilR to its operator and to the readily available ligands
motivated me to use this mechanism to develop an inducible system for gene
expression (Chapter 3). The three approaches taken to achieve this goal comprise
1) targeted modifications in the native promoter region, 2) refinements of the
biosensor promoter and, 3) insertion of operator sites into early promoters from
bacteriophages. Using this approach, I generated a set of tightly repressible
promoters that are – upon addition of the characterized effector molecules –
inducible over more than four orders of magnitude, reaching expression levels
comparable to those of the strongest characterized expression systems. Besides
Escherichia coli, these promoters are functional in other distantly related bacteria,
such as Pseudomonas putida and Sinorhizobium meliloti. I then introduced the EilRguided
regulatory mechanism into the yeast Saccharomyces cerevisiae to show how
this bacterial repressor can control the activity of a modified yeast promoter
containing EilR-binding sites. The EilR-based molecular switch can therefore serve
as a tool for independent gene regulation in prokaryotic and eukaryotic organisms.