Global Analyses of Transcriptomes and Proteomes of a Parent Strain and an l -Threonine-Overproducing Mutant Strain

Abstract

We compared the transcriptome, proteome, and nucleotide sequences between the parent strain Escherichia coli W3110 and the L-threonine-overproducing mutant E. coli TF5015. DNA macroarrays were used to measure mRNA levels for all of the genes of E. coli, and two-dimensional gel electrophoresis was used to compare protein levels. It was observed that only 54 of 4,290 genes (1.3%) exhibited differential expression profiles. Typically, genes such as aceA, aceB, icdA, gltA, glnA, leu operon, proA, thrA, thrC, and yigJ, which are involved in the glyoxylate shunt, the tricarboxylic acid cycle, and amino acid biosynthesis (L-glutamine, L-leucine, proline, and L-threonine), were significantly upregulated, whereas the genes dadAX, hdeA, hdeB, ompF, oppA, oppB, oppF, yfiD, and many ribosomal protein genes were downregulated in TF5015 compared to W3110. The differential expression such as upregulation of thr operon and expression of yigJ would result in an accumulation of L-threonine in TF5015. Furthermore, two significant mutations, thrA345 and ilvA97, which are essential for overproduction of L-threonine, were identified in TF5015 by the sequence analysis. In particular, expression of the mutated thrABC (pATF92) in W3110 resulted in a significant incremental effect on L-threonine production. Upregulation of aceBA and downregulation of b1795, hdeAB, oppA, and yfiD seem to be linked to a low accumulation of acetate in TF5015. Such comprehensive analyses provide information regarding the regulatory mechanism of L-threonine production and the physiological consequences in the mutant stain.In recent years, the completion of the genome project on numerous organisms has accelerated the development of very powerful tools for functional genomics such as DNA arrays (6) and two-dimensional gel electrophoresis (31). Comparative analysis of the gene expression profiles has provided extensive biological information on a genome scale regarding response to stress and/or environmental change, dissection of regulatory circuitry, drug target characterization or identification, cellular response to bacterial infection, and other information for many organisms, including Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, and human cells (1,6,22,25,38). In addition to studies of transcription levels, proteome analysis is important in the understanding of global regulatory processes in living organisms (13,14,17,19,31) since the gene expression profiles often do not directly relate to protein expression levels (28). In this sense, functional genomic techniques, along with genomic information, may enable us to unravel the global regulatory processes or complex metabolic networks in living organisms (18), consequently offering a comprehensive blueprint of the physiology of the bacterium (17,19,22,38).Amino acids have been the prominent target metabolites from microorganisms in bioindustry due to large commercial demands for flavor enhancers, animal feed, sweeteners, and therapeutic agents. Of them, L-threonine, one of the essential am...

The Acetate Switch

“…Intriguingly, peptidoglycan-free L-forms up-regulate YfiD, which appears as a phosphoprotein (141). In contrast, cells entrapped in gels down-regulate YfiD (344), as do those that overproduce threonine and excrete less acetate (262) or lack the global regulator FlhDC or the aerotaxis receptor Aer (356). Since YfiD is induced in acidic and/or microaerobic environments, it has been proposed to prevent the accumulation of acidic fermentation products by facilitating carbon flux through PFL (476).…”

Integrating multiple ‘omics’ analysis for microbial biology: application and methodologies

“…When a good correlation or consensus at both RNA and protein level is found, conclusions can then be made with high confidence (Corbin et al, 2003;Lee et al, 2003;Yoon et al, 2003;Nunez et al, 2006).…”

TheEscherichia coliProteome: Past, Present, and Future Prospects

“…Survival under extremely acidic conditions may be associated with the viability of pathogenic bacteria in the stomach (195), so an improved understanding of pH sensors in virulence may lead to the development of therapeutic strategies targeting these functions. Proteomic analysis has been used to directly monitor cellular changes occurring during the production of heterologous proteins in E. coli and develop efficient strains for the enhanced production of bioproducts (3,37,38,97,98,114,115,116,135,138,160,162,235,306) and biodegradable polymers (99,139). Furthermore, many of these proteomic studies have been performed in large-scale processes employing E. coli and recombinant E. coli for industrial applications (3,37,38,97,114,115,116,135,138,139,145,241,245,325).…”