Engineering butanol‐tolerance in escherichia coli with artificial transcription factor libraries

Abstract

Escherichia coli has been explored as a host for butanol production because of its many advantages such as a fast growth and easy genetic manipulation. Butanol toxicity, however, is a major concern in the biobutanol production with E. coli. In particular, E. coli growth is severely inhibited by butanol, being almost completely stopped by 1% (vol/vol) butanol. Here we developed a new method to increase the butanol-tolerance of E. coli with artificial transcription factor (ATF) libraries which consist of zinc finger (ZF) DNA-binding proteins and an E. coli cyclic AMP receptor protein (CRP). Using these ATFs, we selected a butanol-tolerant E. coli which can tolerate up to 1.5% (vol/vol) butanol, with a concomitant increase in heat resistance. We also identified genes of E. coli that are associated with the butanol-tolerance. These results show that E. coli can be engineered as a promising host for high-yield butanol production.

Extending CRISPR-Cas9 Technology from Genome Editing to Transcriptional Engineering in the Genus Clostridium

“…Hence, implementing genome engineering with more capacities, such as genome editing and transcriptional regulation, can be useful in developing complex phenotypes (22)(23)(24). Similar to the state of genome editing, transcriptional modulation techniques in Clostridium are generally lacking despite significant advancement of sophisticated trans-acting elements, such as transcription activator-like effectors (TALEs) and zinc finger (ZF) DNA-binding domains fused to various activator and repressor domains in eukaryotes and, more recently, in prokaryotes (3,22,(25)(26)(27). Antisense RNA (asRNA) technology (3,23), in which an RNA molecule complementary to a target mRNA is transcribed for in vivo hybridization with the target mRNA (28), remains the most prevalent transcriptional silencing mechanism employed in Clostridium, particularly for metabolic engineering applications (3).…”

Biosensor-driven adaptive laboratory evolution of l-valine production in Corynebacterium glutamicum

“…Simple serial cultivation strategies have been successfully applied to increase the growth rate (Cheng et al, 2014;Fong et al, 2005) as well as tolerance of certain growth substrates (Lee et al, 2013(Lee et al, , 2011 or stress conditions immediately linked to product/byproduct formation (Reyes et al, 2014). Especially, in the case of growth-linked production processes, an increased growth rate immediately coincides with an improved production rate.…”

“…Short generation times and a natural mutation frequency of 10 À 10 to 10 À 9 mutations per base pair per replication cycle enable the selection of beneficial phenotypical traits from high genetic diversity (Barrick and Lenski, 2013). During the last few years, laboratory evolution strategies went more and more into the focus to adapt industrial producer strains to detrimental growth conditions such as oxidative and thermal stress (Lee et al, 2013;Oide et al, 2015;Sandberg et al, 2014;Tenaillon et al, 2012), to improve product formation (Raman et al, 2014;Reyes et al, 2014;Xie et al, 2015) or solvent tolerance (Atsumi et al, 2010;Lee et al, 2011;Oide et al, 2015) (for reviews discussing the use of adaptive evolution approaches in metabolic engineering, see Abatemarco et al, 2013;Portnoy et al, 2011).…”

Genome Replication Engineering Assisted Continuous Evolution (GREACE) to Improve Microbial Tolerance for Biofuels Production

“…However, n-butanol tolerance of E. coli is a bottleneck hampering further increase of n-butanol titer, as n-butanol is highly toxic to microbial cells. Many efforts have been made to improve and analyze butanol tolerance of E. coli [13,[27][28][29][30]. We then wanted to test if GREACE can be used to improve n-butanol tolerance of E. coli, a tougher physiological trait comparing with kanamycin resistance.…”

“…Instead, such complex phenotypes can be more effectively improved by evolutionary engineering approaches [6]. Examples of evolutionary engineering include successive passage for metabolic evolution [7][8][9], physical and chemical mutagenesis [10], global transcription machinery engineering [2,11], artificial transcription factors engineering [12,13], and ribosome engineering [14]. All these methods use "Mutagenesis followed-by Selection" as core principle, meaning that firstly introducing genetic diversity by spontaneous mutations, exogenous mutagens, or genetic perturbations, followed by selection of desired phenotypes [6].…”