Amino acids are key substrates to Escherichia coli BW25113 for achieving high specific growth rate.

Studying substrate consumption in nutrient-rich conditions is challenging because often the growth medium includes undefined components like yeast extract or peptone. For clear and consistent results, it is necessary to use defined medium, where substrate utilization can be followed. In the present work, Escherichia coli BW25113 batch growth in a medium supplemented with 20 proteinogenic amino acids and glucose was studied. Focus was on the quantitative differences in substrate consumption and proteome composition between minimal and nutrient-rich medium. In the latter, 72% of carbon used for biomass growth came from amino acids and 28% from glucose. Serine was identified as the most consumed substrate with 41% of total carbon consumption. Proteome comparison between nutrient-rich and minimal medium revealed changes in TCA cycle and acetate producing enzymes that together with extracellular metabolite data pointed to serine being consumed mainly for energy generation purposes. Serine removal from the growth medium decreased specific growth rate by 22%. In addition, proteome comparison between media revealed a large shift in amino acid synthesis and translation related proteins. Overall, this work describes in quantitative terms the batch growth carbon uptake profile and proteome allocation of E. coli BW25113 in minimal and nutrient-rich medium.

Avoiding amino acid depletion in a complex medium results in improved Escherichia coli BW25113 growth.

We studied Escherichia coli BW25113 growth in a complex medium with emphasis on amino acid consumption. The aim was to profile amino acid utilization in acid-hydrolysed casein and a defined nutrient-rich medium and based on these measurements modify the medium for better growth performance. Amino acid depletions in both media caused apparent biomass growth stops that prolonged growth duration. Obtained amino acid consumption values enabled a new defined medium to be formulated, where no growth stops were observed, the specific growth rate was constant, and the provided substrates were fully utilized. Similarly, we modified the acid-hydrolysed casein medium by adding pure amino acids that removed the apparent biomass growth stops. Key to our results was the combination of growth medium analysis and process monitoring data, specifically oxygen partial pressure and produced carbon dioxide that were used to track growth changes. Our findings showed the deficiencies of the nutrient-rich medium and how rational medium design, based on consumption values, removed these shortcomings. The resulting balanced medium gives a high specific growth rate and is suitable for studying E. coli physiology at fast growth.

Proteome reallocation in Escherichia coli with increasing specific growth rate.

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.