BackgroundThe biotechnology industry has extensively exploited Escherichia coli for producing recombinant proteins, biofuels etc. However, high growth rate aerobic E. coli cultivations are accompanied by acetate excretion i.e. overflow metabolism which is harmful as it inhibits growth, diverts valuable carbon from biomass formation and is detrimental for target product synthesis. Although overflow metabolism has been studied for decades, its regulation mechanisms still remain unclear.ResultsIn the current work, growth rate dependent acetate overflow metabolism of E. coli was continuously monitored using advanced continuous cultivation methods (A-stat and D-stat). The first step in acetate overflow switch (at μ = 0.27 ± 0.02 h-1) is the repression of acetyl-CoA synthethase (Acs) activity triggered by carbon catabolite repression resulting in decreased assimilation of acetate produced by phosphotransacetylase (Pta), and disruption of the PTA-ACS node. This was indicated by acetate synthesis pathways PTA-ACKA and POXB component expression down-regulation before the overflow switch at μ = 0.27 ± 0.02 h-1 with concurrent 5-fold stronger repression of acetate-consuming Acs. This in turn suggests insufficient Acs activity for consuming all the acetate produced by Pta, leading to disruption of the acetate cycling process in PTA-ACS node where constant acetyl phosphate or acetate regeneration is essential for E. coli chemotaxis, proteolysis, pathogenesis etc. regulation. In addition, two-substrate A-stat and D-stat experiments showed that acetate consumption capability of E. coli decreased drastically, just as Acs expression, before the start of overflow metabolism. The second step in overflow switch is the sharp decline in cAMP production at μ = 0.45 h-1 leading to total Acs inhibition and fast accumulation of acetate.ConclusionThis study is an example of how a systems biology approach allowed to propose a new regulation mechanism for overflow metabolism in E. coli shown by proteomic, transcriptomic and metabolomic levels coupled to two-phase acetate accumulation: acetate overflow metabolism in E. coli is triggered by Acs down-regulation resulting in decreased assimilation of acetic acid produced by Pta, and disruption of the PTA-ACS node.

Regulation levels of the gene expression cascade controlling protein levels and metabolic fluxes for cells to achieve faster growth have not been elaborated in acceptable detail. Furthermore, there is need for specific growth rate (μ) dependent absolute quantitative transcriptome and proteome data to understand the molecular relationships for enabling cells to modify μ. We address these questions, for the first time, by presenting regulatory strategies for more efficient metabolism of Escherichia coli at higher μ by statistical covariance analysis of genome-wide intracellular mRNA and protein concentrations coupled to metabolic flux analysis in the steady state range of μ = 0.11-0.49 h(-1). Our analyses show dominating post-transcriptional control of protein abundances and post-translational control of flux rates. On average, E. coli achieved five-times faster growth through 3.7-fold increase of apparent catalytic rates of enzymes (kapp) and 2.5-fold increased translation rates, demonstrating the relevance of post-translational regulation for increasing flux throughput. Interestingly, pathways carrying the highest flux showed both high protein abundance and kapp values. Furthermore, co-regulation analysis of enzymatic capacities revealed tightly coupled regulatory dependencies of protein synthesis and RNA precursor synthesis, substrate utilization, biosynthetic and energy generation pathways carrying the highest flux. We also observed metabolic pathway and COG specific protein and metabolic flux control levels, protein expression costs and genome-wide principles for translation efficiency and transcription unit polarity. This work contributes to the much needed quantitative understanding of coordinated gene expression regulation and metabolic flux control. Our findings will also advance modeling and metabolic engineering of industrial strains.

Cells usually respond to changing growth conditions with a change in the specific growth rate (μ) and adjustment of their proteome to adapt and maintain metabolic efficiency. Description of the principles behind proteome resource allocation is important for understanding metabolic regulation in response to changing μ. Thus, we analysed the proteome resource allocation dynamics of Escherichia coli into different metabolic processes in response to changing μ. E. coli was grown on minimal and defined rich media in steady state continuous cultures at different μ and characterised combining two LC-MS/MS-based proteomics methods: stable isotope labelling by amino acids in cell culture (SILAC) and intensity based label-free absolute quantification. We detected slowly growing cells investing more proteome resources in energy generation and carbohydrate transport and metabolism whereas for achieving faster growth cells needed to devote most resources to translation and processes closely related to the protein synthesis pipeline. Furthermore, down-regulation of energy generation and carbohydrate metabolism proteins with faster growth displayed very similar expression dynamics with the global transcriptional regulator CRP (cyclic AMP receptor protein), pointing to a dominant protein resource allocating role of this protein. Our data also suggest that acetate overflow may be the result of global proteome resource optimisation as cells saved proteome resources by switching from fully respiratory to respiro-fermentative growth. The presented results give a quantitative overview of how E. coli adjusts its proteome to achieve faster growth and in future could contribute to the design of more efficient cell factories through proteome optimisation.