Proteomic Profiling of Escherichiacoli Proteins under High Cell Density Fed‐Batch Cultivation with Overexpression of Phosphogluconolactonase

Abstract

In this study, we used proteomics to better understand the growth on glucose of Escherichia coli in high cell density, fed-batch cultures and the response to overexpression of plasmid-encoded 6-phosphogluconolactonase (PGL). Using liquid chromatography coupled to electrospray mass spectrometry, at least 300 proteins were identified in the cytosolic fraction of the six time points used to monitor the fermentation. The relative abundance changes of selected proteins were obtained by comparing the peak area of the corresponding peptides at a particular m/z (mass over charge ratio) value. During the time course of samples collected during the rapid growth achieved under batch and fed-batch conditions, both the control and recombinant E. coli strains showed up-regulation of proteins participating in the tricarboxylic acid (TCA) cycle, particularly acetyl-CoA synthetase (AcCoAS), malate dehydrogenase (MDH), and succinyl-CoA synthetase (SuccCoAS). In the recombinant strain culture, fumarase was up-regulated until 35 h after inoculation but was not in the control strain culture. In addition, the proteomic measurement detected up-regulation of three well-characterized binding transport proteins in both control and recombinant strains. The up-regulation of TCA cycle enzymes is consistent with the increase in growth rate observed in the cell culture. In addition, up-regulation of these proteins demonstrated the importance of both the pentose-phosphate shunt and TCA cycle to the increased biosynthetic activity required by a high level protein synthesis. This study shows the potential of proteomics using shotgun sequencing (LC/MS of tryptic digests) to measure global changes in protein abundance during a fermentation process and will facilitate the development of robust manufacturing systems.

Suppressing Posttranslational Gluconoylation of Heterologous Proteins by Metabolic Engineering of Escherichia coli

“…coli was grown under fed-batch culture conditions as described by Wang et al (23), except that it was induced with IPTG for production of an 18-kDa protein. Also, the expression of either an LXR␣/RXR␣ heterodimer or elongin C was IPTG inducible, but E. coli was simply grown in batch cultures (see the supplemental material).…”

“…In the light of our results, there is complete agreement with the idea that the role of the PGL enzyme is to facilitate the flow of metabolic intermediates through the PP shunt. The increases in the specific growth rate and biomass yield in the E. coli BL21(DE3) strain are associated with a faster and efficient supply by the PP pathway of aromatic amino acids and nucleic acids, as well as NADPH, for biomass synthesis (23). Our studies showed that when PGL is overexpressed, it increases the biomass yield, as well as the yield of a heterologous product (e.g., the 18-kDa protein), in high-cell-density cultures.…”

“…Another important product of the PP oxidative branch is NADPH, which participates mostly in anabolic pathways (e.g., protein biosynthesis). Under the current conditions, where extra synthesis of nucleotides is required to maintain a multicopy plasmid and extra synthesis of amino acids is required to express a recombinant protein, by increasing the flux through the oxidative branch of the PP pathway it is possible to satisfy some of the extra requirements for production of a recombinant protein, as well as to increase the biomass yield and specific growth rate of the E. coli BL21(DE3) host (23). Expression of plasmid-encoded genes in bacteria is widely used for the production of recombinant proteins in biotechnological processes.…”

“…Other researchers have shown that there is interference with crystallization of proteins (13). Due to the observation that E. coli host strain BL21(DE3) exhibits low endogenous PGL activity in vitro (23) and significant product gluconoylation, we hypothesized that overexpression of a PGL enzyme could suppress spontaneous gluconoylation. In addition, we reported that constitutive PGL overexpression increased the biomass yield, as well as the specific growth rate, of E. coli strain BL21(DE3) grown in glucose minimal medium and in fedbatch cultures (23).…”

“…Due to the observation that E. coli host strain BL21(DE3) exhibits low endogenous PGL activity in vitro (23) and significant product gluconoylation, we hypothesized that overexpression of a PGL enzyme could suppress spontaneous gluconoylation. In addition, we reported that constitutive PGL overexpression increased the biomass yield, as well as the specific growth rate, of E. coli strain BL21(DE3) grown in glucose minimal medium and in fedbatch cultures (23). Furthermore, in the present work we de-scribe the effect of overexpression of the PGL enzyme on the specific growth rate, the biomass yield, and protein productivity, as well as application of this approach to three unrelated heterologous proteins that exhibit gluconoylation adducts when they are expressed in E. coli BL21(DE3).…”