Global transcriptomic analysis of an engineered Escherichia coli strain lacking the phosphoenolpyruvate: carbohydrate phosphotransferase system during shikimic acid production in rich culture medium

Abstract

BackgroundEfficient production of SA in Escherichia coli has been achieved by modifying key genes of the central carbon metabolism and SA pathway, resulting in overproducing strains grown in batch- or fed-batch-fermentor cultures using a complex broth including glucose and YE. In this study, we performed a GTA to identify those genes significantly upregulated in an engineered E. coli strain, PB12.SA22, in mid EXP (5 h), early STA (STA1, 9 h), and late STA (STA2, 44 h) phases, grown in complex fermentation broth in batch-fermentor cultures.ResultsGrowth of E. coli PB12.SA22 in complex fermentation broth for SA production resulted in an EXP growth during the first 9 h of cultivation depending of supernatant available aromatic amino acids provided by YE because, when tryptophan was totally consumed, cells entered into a second, low-growth phase (even in the presence of glucose) until 26 h of cultivation. At this point, glucose was completely consumed but SA production continued until the end of the fermentation (50 h) achieving the highest accumulation (7.63 g/L of SA). GTA between EXP/STA1, EXP/STA2 and STA1/STA2 comparisons showed no significant differences in the regulation of genes encoding enzymes of central carbon metabolism as in SA pathway, but those genes encoding enzymes involved in sugar, amino acid, nucleotide/nucleoside, iron and sulfur transport; amino acid catabolism and biosynthesis; nucleotide/nucleoside salvage; acid stress response and modification of IM and OM were upregulated between comparisons.ConclusionsGTA during SA production in batch-fermentor cultures of strain PB12.SA22 grown in complex fermentation broth during the EXP, STA1 and STA2 phases was studied. Significantly, upregulated genes during the EXP and STA1 phases were associated with transport, amino acid catabolism, biosynthesis, and nucleotide/nucleoside salvage. In STA2, upregulation of genes encoding transporters and enzymes involved in the synthesis and catabolism of Arg suggests that this amino acid could have a key role in the fuelling of carbon toward SA synthesis, whereas upregulation of genes involved in pH stress response, such as membrane modifications, suggests a possible response to environmental conditions imposed on the cell at the end of the fermentation.

Engineering

“…Finally, a reverse engineered strain was constructed, which gave a titer of 902 mg/L and an L-TYR yield on glucose of 0.18 g/g on a genetically-defined background [101]. Other works have also characterized the global transcriptional response to the presence of high levels of L-PHE or SHK in simple and complex media [120,121] or starvation conditions [122], revealing metabolic information that can be used for further improvement of the strains and cultivation conditions. Along with data obtained by high-throughput systems, modeling of metabolism by mathematical approaches has become an important tool for analyzing cell responses and unravel the metabolic regulation between the cell information/control systems [111].…”

The Role of the <b><i>ydiB</i></b> Gene, Which Encodes Quinate/Shikimate Dehydrogenase, in the Production of Quinic, Dehydroshikimic and Shikimic Acids in a PTS^- Strain of <b><i>Escherichia coli</i></b>

“…This result is consistent with the highest glucose consumption and product formation observed during the early STA stage. The growth of strain PB12.SA22 was associated with low glucose consumption in the complex fermentation broth, including 25 g/l of glucose and 15 g/l of YE, which has been attributed to the consumption of available nutrients in YE [Cortés-Tolalpa et al, 2014]. It was determined that the YE included in the fermentation broth primarily supports growth during the EXP phase; when aromatic amino acids, specifically L -tryptophan present in YE are completely consumed, the cells enter the STA stage, despite the high residual glucose available in the fermentor [Cortés-Tolalpa et al, 2014].…”

“…The growth of strain PB12.SA22 was associated with low glucose consumption in the complex fermentation broth, including 25 g/l of glucose and 15 g/l of YE, which has been attributed to the consumption of available nutrients in YE [Cortés-Tolalpa et al, 2014]. It was determined that the YE included in the fermentation broth primarily supports growth during the EXP phase; when aromatic amino acids, specifically L -tryptophan present in YE are completely consumed, the cells enter the STA stage, despite the high residual glucose available in the fermentor [Cortés-Tolalpa et al, 2014]. This twostage growth behavior associated with glucose consumption and SA production has also been observed in other SA-overproducing strains (AR36) derived from E. coli PB12, even in the presence of a higher glucose concentration (100 g/l) in the fermentation broth [Rodriguez et al, 2013].…”

“…Cultures were incubated overnight in a shaker (New Brunswick Scientific, Edison, USA) at 37 ° C and 300 rpm. An aliquot of 150 μl from each culture was used to inoculate a 250-ml baffled flask with 50 ml of fermentation medium, whose composition has been previously reported for the production of SA [Chandran et al, 2003], modified as reported previously [Cortés-Tolalpa et al, 2014], and grown as described above. This medium contained a mineral base composition, 25 g/l of glucose, 15 g/l of YE and the required antibiotics.…”

“…As previous studies have suggested that ydiB plays the key role, over aroE , during the hydroaromatic intracellular equilibrium in cultures of engineered E. coli strains for the production of SA, the aim of the present study was to assess the effect of the inactivation of ydiB (ydiB -) or its overexpression (plasmid cloned ydiB; ydiB + ) on SA and the production of DHS and QA in the engineered E. coli strain PB12.SA22 (PTS-glucose + ) used previously for SA production purposes [Escalante et al, 2010;Cortés-Tolalpa, 2014] in batch fermentor cultures using a complex culture production broth.…”