BackgroundThis study aimed at exploring the molecular physiological consequences of a major redirection of carbon flow in so-called cyanobacterial cell factories: quantitative whole-cell proteomics analyses were carried out on two 14N-labelled Synechocystis mutant strains, relative to their 15N-labelled wild-type counterpart. Each mutant strain overproduced one specific commodity product, i.e. ethanol or lactic acid, to such an extent that the majority of the incoming CO2 in the organism was directly converted into the product.ResultsIn total, 267 proteins have been identified with a significantly up- or down-regulated expression level. In the ethanol-producing mutant, which had the highest relative direct flux of carbon-to-product (>65%), significant up-regulation of several components involved in the initial stages of CO2 fixation for cellular metabolism was detected. Also a general decrease in abundance of the protein synthesizing machinery of the cells and a specific induction of an oxidative stress response were observed in this mutant. In the lactic acid overproducing mutant, that expresses part of the heterologous l-lactate dehydrogenase from a self-replicating plasmid, specific activation of two CRISPR associated proteins, encoded on the endogenous pSYSA plasmid, was observed. RT-qPCR was used to measure, of nine of the genes identified in the proteomics studies, also the adjustment of the corresponding mRNA level.ConclusionThe most striking adjustments detected in the proteome of the engineered cells were dependent on the specific product formed, with, e.g. more stress caused by lactic acid- than by ethanol production. Up-regulation of the total capacity for CO2 fixation in the ethanol-producing strain was due to hierarchical- rather than metabolic regulation. Furthermore, plasmid-based expression of heterologous gene(s) may induce genetic instability. For selected, limited, number of genes a striking correlation between the respective mRNA- and the corresponding protein expression level was observed, suggesting that for the expression of these genes regulation takes place primarily at the level of gene transcription.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0294-z) contains supplementary material, which is available to authorized users.

Time-series transcript- and protein-profiles were measured upon initiation of carbon catabolite repression in Escherichia coli, in order to investigate the extent of post-transcriptional control in this prototypical response. A glucose-limited chemostat culture was used as the CCR-free reference condition. Stopping the pump and simultaneously adding a pulse of glucose, that saturated the cells for at least 1h, was used to initiate the glucose response. Samples were collected and subjected to quantitative time-series analysis of both the transcriptome (using microarray analysis) and the proteome (through a combination of 15N-metabolic labeling and mass spectrometry). Changes in the transcriptome and corresponding proteome were analyzed using statistical procedures designed specifically for time-series data. By comparison of the two sets of data, a total of 96 genes were identified that are post-transcriptionally regulated. This gene list provides candidates for future in-depth investigation of the molecular mechanisms involved in post-transcriptional regulation during carbon catabolite repression in E. coli, like the involvement of small RNAs.

We report on the use of the chemostat as an optimal device to create time-invariant conditions that allow accurate sampling for various omics assays in Escherichia coli, in combination with recording of the dynamics of the physiological transition in the organism under study that accompany the initiation of glucose repression. E. coli cells respond to the addition of glucose not only with the well-known transcriptional response, as was revealed through quantitative PCR analysis of the transcript levels of key genes from the CRP (cAMP receptor protein) regulon, but also with an increased growth rate and a transient decrease in the efficiency of its aerobic catabolism. Less than half of a doubling time is required for the organism to recover to maximal values of growth rate and efficiency. Furthermore, calculations based on our results show that the specific glucose uptake rate (q s ) and the H + /e " ratio increase proportionally, up to a growth rate of 0.4 h -1 , whilst biomass yield on glucose (Y x/s ) drops during the first 15 min, followed by a gradual recovery. Surprisingly, the growth yields after the recovery phase show values even higher than the maximum theoretical yield. Possible explanations for these high yields are discussed.