Metabolic stress constrains microbial L-cysteine production in Escherichia coli by accelerating transposition through mobile genetic elements

Abstract

AbstractBackgroundL-cysteine is an essential chemical building block in the pharmaceutical-, cosmetic-, food and agricultural sector. Conventionally, L-cysteine production relies on the conversion of keratinous biomass mediated by hydrochloric acid. Today, fermentative production based on recombinantE. coli, where L-cysteine production is streamlined and facilitated by synthetic plasmid constructs, is an alternative process at industrial scale. However, metabolic stress and the resulting production escape mechanisms in evolving populations are severely limiting factors during industrial biomanufacturing. We emulate high generation numbers typically reached in industrial fermentation processes withEscherichia coliharbouring L-cysteine production plasmid constructs.So far no genotypic and phenotypic alterations in early and late L-cysteine producingE. colipopulations have been studied.ResultsIn a comparative experimental design, theE. coliK12 production strain W3110 and the reduced genome strain MDS42, almost free of insertion sequences, were used as hosts. Data indicates that W3110 populations acquire growth fitness at the expense of L-cysteine productivity within 60 generations, while production in MDS42 populations remains stable. For the first time, the negative impact of predominantly insertion sequence family 3 and 5 transposases on L-cysteine production is reported, by combining differential transcriptome analysis with NGS based deep plasmid sequencing. Furthermore, metabolic clustering of differentially expressed genes supports the hypothesis, that metabolic stress induces rapid propagation of plasmid rearrangements, leading to reduced L-cysteine yields in evolving populations over industrial fermentation time scales.ConclusionThe results of this study implicate how selective deletion of insertion sequence families could be a new route for improving industrial L-cysteine or even general amino acid production using recombinantE. colihosts. Instead of using minimal genome strains, a selective deletion of certain IS families could offer the benefits of adaptive laboratory evolution (ALE) while maintaining enhanced L-cysteine production stability.