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.
Highlights
Efficient production of Shikimic acid (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 Yeast extract (YE)
Growth and SA pathway intermediate production Based on growth and glucose consumption profiles, strain PB12.SA22 showed its characteristic two-phase growth behavior [10], with an initial Exponential growth phase (EXP) growth phase during the first 8 h of cultivation reaching an OD600 nm = 13.37 with a μ = 0.47 ± 0.002 h−1 and a Glucose consumption rate (qS) = 3.34 ± 0.29 mmol glucose g Dry weight (DW)−1 h−1
Upregulation of genes involved in sugar transport Genes encoding sugar transporters that were found to be upregulated in the EXP/STA1 comparison included the sugar porin LamB, the periplasmic binding protein, the membrane subunit and the ATP binding subunit components of the ABC maltose transport system [17] and the glycerol-3-P (G3P) MFS transporter (GlpT) glycerol 3-P MFS transporter, whereas in the EXP/STA2 comparison, the upregulation of the nonspecific Outer membrane porin (OMP) ompC (OmpC), a general porin and the periplasmic binding protein of the maltose ABC transporter was observed
Summary
Efficient 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. 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. In Escherichia coli, the first step in this pathway is the condensation of the CCM intermediates PEP and E4P, into DAHP by the DAHP synthase isoenzymes AroF, AroG, and AroH, which are encoded by the aroF, aroG, and aroH genes, respectively (Figure 1). DHQ dehydratase, encoded by aroD, converts DHQ into DHS, and this compound is transformed to SA by shikimate dehydrogenase, which is encoded by aroE. CHA is the common building block for the formation of aromatic amino acids and compounds such as quinone, menaquinone and enterobactin [3,4,5]
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