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

Abstract

Background L-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 recombinant E. 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 with Escherichia coli harbouring L-cysteine production plasmid constructs. So far no genotypic and phenotypic alterations in early and late L-cysteine producing E. coli populations have been studied. Results In a comparative experimental design, the E. coli K12 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. Conclusion The 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 recombinant E. coli hosts. 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.

Genomic and metabolic instability during long-term fermentation of an industrial Saccharomyces cerevisiae strain engineered for C5 sugar utilization.

“…The genetic and phenotypic stability of microorganisms designed for the production of value-added products from carbonaceous biomass is a crucial issue in industrial biotechnology and one that is the subject of a growing body of work. Initial work on this subject focused on the stability of production systems in bacteria, where it was clearly shown that the use of plasmids carrying these production pathways is not advisable (see for instance [3]). Work on biomolecules-producing yeasts is less advanced, with a few works also revealing the problem of the stability of producing strains when the metabolic system is plasmid-based expressed [4, 5].…”

“…In Escherichia coli , it was reported that the rise of frequencies of mobile element insertions in chromosomally-inserted transgenes is the main factor that interrupts production in mevalonic acid-producing cells [1]. Similarly, it has been shown that the metabolic stress induced by L-cysteine production in E. coli triggers insertion sequence (IS) transposition, resulting in structural genetic rearrangements in production plasmids that cause L-cysteine production capacities to drop by up to 65–85% within 60 generations [3]. Consequently, selective deletion of IS and their corresponding transposases could prove useful for stabilizing industrially production strains [3].…”

“…Similarly, it has been shown that the metabolic stress induced by L-cysteine production in E. coli triggers insertion sequence (IS) transposition, resulting in structural genetic rearrangements in production plasmids that cause L-cysteine production capacities to drop by up to 65–85% within 60 generations [3]. Consequently, selective deletion of IS and their corresponding transposases could prove useful for stabilizing industrially production strains [3]. In Saccharomyces cerevisiae , homologous recombination (HR) has been recently reported as an important genetic process leading to the loss of production capacity [4], as illustrated in the case of vanillin-β-glucoside [5].…”

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

“…The often metabolically burdensome production drives populations to escape mechanisms. Within 60-70 generations, mutations can accumulate in the genome, giving cells a fitness advantage at the expense of production [4,5]. Thereby, mutations did not involve single-nucleotide polymorphisms but rather insertions of ISE into critical regions of synthetic plasmid constructs [4][5][6][7].…”

“…Within 60-70 generations, mutations can accumulate in the genome, giving cells a fitness advantage at the expense of production [4,5]. Thereby, mutations did not involve single-nucleotide polymorphisms but rather insertions of ISE into critical regions of synthetic plasmid constructs [4][5][6][7]. Insertions within genes can lead to mutations that result in loss-of-function.…”