Abstract
BackgroundThe tolerance of cells toward different stresses is very important for industrial strains of microbes, but difficult to improve by the manipulation of single genes. Traditional methods for enhancing cellular tolerances are inefficient and time-consuming. Recently, approaches employing global transcriptional or translational engineering methods have been increasingly explored. We found that an exogenous global regulator, irrE from an extremely radiation-resistant bacterium, Deinococcus radiodurans, has the potential to act as a global regulator in Escherichia coli, and that laboratory-evolution might be applied to alter this regulator to elicit different phenotypes for E. coli.Methodology/Principal FindingsTo extend the methodology for strain improvement and to obtain higher tolerances toward different stresses, we here describe an approach of engineering irrE gene in E. coli. An irrE library was constructed by randomly mutating the gene, and this library was then selected for tolerance to ethanol, butanol and acetate stresses. Several mutants showing significant tolerances were obtained and characterized. The tolerances of E. coli cells containing these mutants were enhanced 2 to 50-fold, based on cell growth tests using different concentrations of alcohols or acetate, and enhanced 10 to 100-fold based on ethanol or butanol shock experiments. Intracellular reactive oxygen species (ROS) assays showed that intracellular ROS levels were sharply reduced for cells containing the irrE mutants. Sequence analysis of the mutants revealed that the mutations distribute cross all three domains of the protein.ConclusionsTo our knowledge, this is the first time that an exogenous global regulator has been artificially evolved to suit its new host. The successes suggest the possibility of improving tolerances of industrial strains by introducing and engineering exogenous global regulators, such as those from extremophiles. This new approach can be applied alone or in combination with other global methods, such as global transcriptional machinery engineering (gTME) for strain improvements.
Highlights
In recent years, more and more attention has been attached to the production of fuels and chemicals through native or engineered microbes [1,2], and synthetic biology has accelerated the development of industrial strains [3,4]
The successes suggest the possibility of improving tolerances of industrial strains by introducing and engineering exogenous global regulators, such as those from extremophiles
Present methods used for the improvement of strain tolerance include traditional chemical and physical mutagenesis followed by selection under challenging conditions [6], genome shuffling [7,8], reprogramming of gene transcription using artificial transcription factors [9,10], and introduction or over-expression of proteins of specific functions, such as heat shock proteins (HSPs), or enzymes related to the production of protective substances or stress signal transduction [11,12]
Summary
More and more attention has been attached to the production of fuels and chemicals through native or engineered microbes [1,2], and synthetic biology has accelerated the development of industrial strains [3,4] Tolerance of these microorganisms toward stresses (including harmful substrates, toxic contaminants, metabolic products and byproducts at high concentrations) is often crucial for the economics of a bioprocess. We found that an exogenous global regulator, irrE from an extremely radiation-resistant bacterium, Deinococcus radiodurans, has the potential to act as a global regulator in Escherichia coli, and that laboratory-evolution might be applied to alter this regulator to elicit different phenotypes for E. coli
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