Combinatorial Strategies for Improving Multiple-Stress Resistance in Industrially Relevant Escherichia coli Strains

Abstract

High-cell-density fermentation for industrial production of chemicals can impose numerous stresses on cells due to high substrate, product, and by-product concentrations; high osmolarity; reactive oxygen species; and elevated temperatures. There is a need to develop platform strains of industrial microorganisms that are more tolerant toward these typical processing conditions. In this study, the growth of six industrially relevant strains of Escherichia coli was characterized under eight stress conditions representative of fed-batch fermentation, and strains W and BL21(DE3) were selected as platforms for transposon (Tn) mutagenesis due to favorable resistance characteristics. Selection experiments, followed by either targeted or genome-wide nextgeneration-sequencing-based Tn insertion site determination, were performed to identify mutants with improved growth properties under a subset of three stress conditions and two combinations of individual stresses. A subset of the identified loss-offunction mutants were selected for a combinatorial approach, where strains with combinations of two and three gene deletions were systematically constructed and tested for single and multistress resistance. These approaches allowed identification of (i) strain-background-specific stress resistance phenotypes, (ii) novel gene deletion mutants in E. coli that confer single and multistress resistance in a strain-background-dependent manner, and (iii) synergistic effects of multiple gene deletions that confer improved resistance over single deletions. The results of this study underscore the suboptimality and strain-specific variability of the genetic network regulating growth under stressful conditions and suggest that further exploration of the combinatorial gene deletion space in multiple strain backgrounds is needed for optimizing strains for microbial bioprocessing applications.

Genome‐wide identification of tolerance mechanisms toward p‐coumaric acid in Pseudomonas putida

“…Sequencing of the junctions between the transposon and the genome was achieved by generating an enriched library as previously described (Lennen & Herrgård, 2014). A pUT‐Km transposon specific primer, BioTEG‐Tn5‐fw, was designed for the enrichment of the junctions (Table 2).…”

“…gDNA libraries were prepared as previously described (Lennen & Herrgård, 2014). P. putida gDNA was extracted using the PureLink genomic DNA kit (Invitrogen, Thermo Fisher, Waltham, MA) and 3 µg of total gDNA was sheared in 300‐bp fragment size using a Covaris E220 ultrasonicator.…”

“…Transposon insertion sequencing (Tn‐seq) (Figure 1a), enables high‐throughput identification of genomic regions involved in the survival of cells when exposed to various conditions (Barquist, Boinett, & Cain, 2013; van Opijnen & Camilli, 2013). A number of organisms have been investigated using this technique, thereby successfully identifying genes important for growth under different stress conditions, essentially by simultaneously investigating the fitness of all single mutants (Gawronski, Wong, Giannoukos, Ward, & Akerley, 2009; Langridge et al, 2009; Lennen & Herrgård, 2014; Santiago et al, 2015). So far, a Tn‐seq method has, however, not been developed for P. putida .…”

Multi-omics Quantification of Species Variation of Escherichia coli Links Molecular Features with Strain Phenotypes

“…For example, E. coli strain DH5a is often used in cloning applications due to an endA1 mutation that inactivates an intracellular endonuclease (Taylor et al, 1993) and BL21 is well established in recombinant protein production due to a lack of the Lon and OmpT proteases (Ratelade et al, 2009). Thus, aspects such as transformation efficiency (Liu et al, 2014), phage resistance (Furukawa and Mizushima, 1982), product tolerance (Lennen and Herrgard, 2014), and other traits must also be considered. Furthermore, maximizing theoretical yield does not necessarily lead to increases in titre or productivity.…”

PprM, a Cold Shock Domain-Containing Protein from Deinococcus radiodurans, Confers Oxidative Stress Tolerance to Escherichia coli

“…In an effort to improve the robustness of E. coli as an industrial host, many strategies have been used, including stress-induced mutagenesis, combinatorial gene deletion, engineering of transcriptional regulators, and synthetic biology (Zhu et al, 2012; Lin et al, 2013; Lennen and Herrgård, 2014; Zhu et al, 2014). The introduction or overexpression of specific proteins, such as heat shock proteins (HSPs), also enhances resistance at the cellular level (Jia et al, 2014).…”