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.

Protein turnover plays an important role in cell metabolism by regulating metabolic fluxes. Furthermore, the energy costs for protein turnover have been estimated to account for up to a third of the total energy production during cell replication and hence may represent a major limiting factor in achieving either higher biomass or production yields. This work aimed to measure the specific growth rate (m)-dependent abundance and turnover rate of individual proteins, estimate the ATP cost for protein production and turnover, and compare this with the total energy balance and other maintenance costs. The lactic acid bacteria model organism Lactococcus lactis was used to measure protein turnover rates at m50.1 and 0.5 h "1 in chemostat experiments.Individual turnover rates were measured for~75 % of the total proteome. On average, protein turnover increased by sevenfold with a fivefold increase in growth rate, whilst biomass yield increased by 35 %. The median turnover rates found were higher than the specific growth rate of the bacterium, which suggests relatively high energy consumption for protein turnover. We found that protein turnover costs alone account for 38 and 47 % of the total energy produced at m50.1 and 0.5 h "1 , respectively, and gene ontology groups Energy metabolism and Translation dominated synthesis costs at both growth rates studied. These results reflect the complexity of metabolic changes that occur in response to changes in environmental conditions, and signify the trade-off between biomass yield and the need to produce ATP for maintenance processes. INTRODUCTIONRecently, protein turnover rates have been recognized as an important factor that provides growth advantage both in terms of higher maximal specific growth rate (m) and biomass yield between different strains (Hong et al., 2012), and in laboratory evolution experiments (González-Ramos et al., 2013). In addition, protein synthesis and degradation are firmly related and therefore represent an important control factor for metabolic regulation (Schwanhäusser et al., 2013). Individual protein turnover rates have been determined in mammalian cells, yeast and bacteria by measuring the incorporation rate of fluorescent tags (Khmelinskii et al., 2012), affinity tags (Belle et al., 2006) or isotopic labels into the proteome (methods reviewed by Hughes & Krijgsveld, 2012; Trötschel et al., 2013). The metabolic incorporation of isotopic labels is currently the most widely used method for cell cultures and conducted using either labelled ammonium (Helbig et al., 2011;Martin et al., 2012), carbon (Cargile et al., 2004) or amino acids (Gerth et al., 2008;Maier et al., 2011; Schwanhäusser et al., 2011). Protein turnover rates are predominantly measured during the logarithmic or stationary phase of batch growth and are assumed to be constant during this phase of growth.Only a few studies report protein degradation rates determined from a steady physiological state (Helbig et al., 2011; Pratt et al., 2002). Although overall protein turnover has been...

Graphical AbstractHighlights d Dataset of protein turnover rate and expression along the mice intestinal tract d Protein turnover rate is slower in colon than in small intestine d Median protein half-life is 1 day longer in germ-free mice SUMMARYThe gastrointestinal tract is covered by a single layer of epithelial cells that, together with the mucus layers, protect the underlying tissue from microbial invasion. The epithelium has one of the highest turnover rates in the body. Using stable isotope labeling, high-resolution mass spectrometry, and computational analysis, we report a comprehensive dataset of the turnover of more than 3,000 and the expression of more than 5,000 intestinal epithelial cell proteins, analyzed under conventional and germ-free conditions across five different segments in mouse intestine. The median protein half-life is shorter in the small intestine than in the colon. Differences in protein turnover rates along the intestinal tract can be explained by distinct physiological and immune-related functions between the small and large intestine. An absence of microbiota results in an approximately 1 day longer protein half-life in germfree animals.

Lactic acid bacteria are extensively used in food technology and for the production of various compounds, but they are fastidious in nutrient requirements. In order to elucidate the role of each component precisely, defined multicomponent media are required. This study focuses on determining nutrient auxotrophies and minimizing media components (amino acids, vitamins, metal ions, buffers and additional compounds) for the cultivation of Lactococcus lactis subsp. lactis IL1403, using microtitre plates and test tubes. It was shown that glutamine and asparagine were the most important media components for achieving higher biomass yields while the branched-chain amino acids were necessary to increase specific growth rate. The amino acid and glucose ratio was reduced to achieve minimal residual concentration of amino acids in the medium after the growth of cells, whereas the specific growth rate and biomass yield of cells were not considerably affected. As the percentage of each consumed amino acid compared to initial amount is larger than measurement error, these optimized media are important for achieving more precise data about amino acid utilization and metabolism.

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