ABSTRACT
BACKGROUND: The modification of glucose import capacity is an engineering strategy that has been shown to improve the characteristics of Escherichia coli as a microbial factory. A reduction in glucose import capacity can have a positive effect on production strain performance, however, this is not always the case. In this study, E. coli W3110 and a group of four isogenic derivative strains, harboring single or multiple deletions of genes encoding phosphoenolpyruvate:sugar phosphotransferase system (PTS)-dependent transporters as well as non-PTS transporters were characterized by determining their transcriptomic response to reduced glucose import capacity. RESULTS: These strains were grown in bioreactors with M9 mineral salts medium containing 20 g/L of glucose, where they displayed specific growth rates ranging from 0.67 to 0.27 h-1, and specific glucose consumption rates (qs) ranging from 1.78 to 0.37 g/g h. RNA-seq analysis revealed a transcriptional response consistent with carbon source limitation among all the mutant strains, involving functions related to transport and metabolism of alternate carbon sources and characterized by a decrease in genes encoding glycolytic enzymes and an increase in gluconeogenic functions. A total of 107 and 185 genes displayed positive and negative correlations with qs, respectively. Functions displaying positive correlation included energy generation, amino acid biosynthesis, and sugar import. CONCLUSION: Changes in gene expression of E. coli strains with impaired glucose import capacity could be correlated with qs values and this allowed an inference of the physiological state of each mutant. In strains with lower qs values, a gene expression pattern is consistent with energy limitation and entry into the stationary phase. This physiological state could explain why these strains display a lower capacity to produce recombinant protein, even when they show very low rates of acetate production. The comparison of the transcriptomes of the engineered strains employed as microbial factories is an effective approach for identifying favorable phenotypes with the potential to improve the synthesis of biotechnological products.
Subject(s)
Escherichia coli Proteins , Escherichia coli , Carbon/metabolism , Escherichia coli/metabolism , Escherichia coli Proteins/metabolism , Gene Expression Profiling , Glucose/metabolism , Sugars/metabolismABSTRACT
The aromatic compound p-coumaric acid (p-CA) is a secondary metabolite produced by plants. This aromatic acid and derived compounds have positive effects on human health, so there is interest in producing them in biotechnological processes with recombinant Escherichia coli strains. To determine the physiologic response of E. coli W3110 to p-CA, dynamic expression analysis of selected genes fused to a fluorescent protein reporter as well as RNA-seq and RT-qPCR were performed. The observed transcriptional profile revealed the induction of genes involved in functions related to p-CA active export, synthesis of cell wall and membrane components, synthesis of amino acids, detoxification of formaldehyde, phosphate limitation, acid stress, protein folding and degradation. Downregulation of genes encoding proteins involved in energy production, carbohydrate import and metabolism, as well as several outer and plasma membrane proteins was detected. This response is indicative of cell envelope damage causing the leakage of intracellular components including amino acids and phosphate-containing compounds. The cellular functions responding to p-CA that were identified in this study will help in defining targets for production strains improvement.
Subject(s)
Escherichia coli , Transcriptome , Amino Acids/metabolism , Coumaric Acids , Escherichia coli/genetics , Escherichia coli/metabolism , Humans , Phosphates/metabolismABSTRACT
BACKGROUND: Escherichia coli W3110 and a group of six isogenic derivatives, each displaying distinct specific rates of glucose consumption were characterized to determine levels of GFP production and population heterogeneity. These strains have single or combinatory deletions in genes encoding phosphoenolpyruvate:sugar phosphotransferase system (PTS) permeases as PtsG and ManX, as well as common components EI, Hpr protein and EIIA, also the non-PTS Mgl galactose/glucose ABC transporter. They have been transformed for expressing GFP based on a lac-based expression vector, which is subject to bistability. RESULTS: These strains displayed specific glucose consumption and growth rates ranging from 1.75 to 0.45 g/g h and 0.54 to 0.16 h-1, respectively. The rate of acetate production was strongly reduced in all mutant strains when compared with W3110/pV21. In bioreactor cultures, wild type W3110/pV21 produced 50.51 mg/L GFP, whereas strains WG/pV21 with inactive PTS IICBGlc and WGM/pV21 with the additional inactivation of PTS IIABMan showed the highest titers of GFP, corresponding to 342 and 438 mg/L, respectively. Moreover, we showed experimentally that bistable expression systems, as lac-based ones, induce strong phenotypic segregation among microbial populations. CONCLUSIONS: We have demonstrated that reduction on glucose consumption rate in E. coli leads to an improvement of GFP production. Furthermore, from the perspective of phenotypic heterogeneity, we observed in this case that heterogeneous systems are also the ones leading to the highest performance. This observation suggests reconsidering the generally accepted proposition stating that phenotypic heterogeneity is generally unwanted in bioprocess applications.
Subject(s)
Escherichia coli/genetics , Glucose/metabolism , Metabolic Engineering/methods , Acetates/metabolism , Biological Transport , Bioreactors , Escherichia coli/growth & development , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Flow Cytometry , Glucose Transport Proteins, Facilitative/metabolism , Green Fluorescent Proteins/analysis , Kinetics , Microfluidic Analytical TechniquesABSTRACT
Azotobacter vinelandii, a strict aerobic, nitrogen fixing bacterium in the Pseudomonadaceae family, exhibits a preferential use of acetate over glucose as a carbon source. In this study, we show that GluP (Avin04150), annotated as an H+-coupled glucose-galactose symporter, is the glucose transporter in A. vinelandii. This protein, which is widely distributed in bacteria and archaea, is uncommon in Pseudomonas species. We found that expression of gluP was under catabolite repression control thorugh the CbrA/CbrB and Crc/Hfq regulatory systems, which were functionally conserved between A. vinelandii and Pseudomonas species. While the histidine kinase CbrA was essential for glucose utilization, over-expression of the Crc protein arrested cell growth when glucose was the sole carbon source. Crc and Hfq proteins from either A. vinelandii or P. putida could form a stable complex with an RNA A-rich Hfq-binding motif present in the leader region of gluP mRNA. Moreover, in P. putida, the gluP A-rich Hfq-binding motif was functional and promoted translational inhibition of a lacZ reporter gene. The fact that gluP is not widely distributed in the Pseudomonas genus but is under control of the CbrA/CbrB and Crc/Hfq systems demonstrates the relevance of these systems in regulating metabolism in the Pseudomonadaceae family.