Localization of Insertion Sequences in Plasmids for L-Cysteine Production in E. coli

Abstract

Insertion sequence elements (ISE) are often found to be responsible for the collapse of production in synthetically engineered Escherichia coli. By the transposition of ISE into the open reading frame of the synthetic pathway, E. coli cells gain selection advantage over cells expressing the metabolic burdensome production genes. Here, we present the exact entry sites of insertion sequence (IS) families 3 and 5 within plasmids for l-cysteine production in evolved E. coli populations. Furthermore, we identified an uncommon occurrence of an 8-bp direct repeat of IS5 which is atypical for this particular family, potentially indicating a new IS5 target site.

Keywords: E. coli; insertion sequence elements; l-cysteine; metabolic engineering; plasmid deep sequencing; target site duplications.

Figure 1

Localization of IS entry sites in plasmids from late W3110 populations. The entry sites of families 3 (indicated by red dots) and 5 (indicated by green dots) were identified and quantified through target site duplications resulting from transposition events in pCYS (A), pCYS_i (B) and pCYS_m (C). The analysis was performed using the Genome ARTIST (Artificial Transposon Insertion Site Tracker), version 2.0 software [10].

Figure 2

Potential effects on intracellular sulphate and l-cysteine level due to disruption of the synthetic l-cysteine pathway. Case A describes how intracellular sulphate level would increase when cysM is disrupted. O-acetylserine (OAS) and thiosulphate cannot be converted to S-sulphocysteine anymore. Case B highlights the effect of a disrupted l-cysteine exporter. EamA cannot transport l-cysteine anymore, which results in an increase in intracellular l-cysteine level. Both cases would be beneficial for the sulphur- and l-cysteine homeostasis of the cell.