Dataset Information

Metabolic engineering for the production of shikimic acid in an evolved Escherichia coli strain lacking the phosphoenolpyruvate: carbohydrate phosphotransferase system.

ABSTRACT:

Background

Shikimic acid (SA) is utilized in the synthesis of oseltamivir-phosphate, an anti-influenza drug. In this work, metabolic engineering approaches were employed to produce SA in Escherichia coli strains derived from an evolved strain (PB12) lacking the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS-) but with capacity to grow on glucose. Derivatives of PB12 strain were constructed to determine the effects of inactivating aroK, aroL, pykF or pykA and the expression of plasmid-coded genes aroGfbr, tktA, aroB and aroE, on SA synthesis.

Results

Batch cultures were performed to evaluate the effects of genetic modifications on growth, glucose consumption, and aromatic intermediate production. All derivatives showed a two-phase growth behavior with initial high specific growth rate (mu) and specific glucose consumption rate (qs), but low level production of aromatic intermediates. During the second growth phase the mu decreased, whereas aromatic intermediate production reached its maximum. The double aroK- aroL- mutant expressing plasmid-coded genes (strain PB12.SA22) accumulated SA up to 7 g/L with a yield of SA on glucose of 0.29 mol/mol and a total aromatic compound yield (TACY) of 0.38 mol/mol. Single inactivation of pykF or pykA was performed in PB12.SA22 strain. Inactivation of pykF caused a decrease in mu, qs, SA production, and yield; whereas TACY increased by 33% (0.5 mol/mol).

Conclusions

The effect of increased availability of carbon metabolites, their channeling into the synthesis of aromatic intermediates, and disruption of the SA pathway on SA production was studied. Inactivation of both aroK and aroL, and transformation with plasmid-coded genes resulted in the accumulation of SA up to 7 g/L with a yield on glucose of 0.29 mol/mol PB12.SA22, which represents the highest reported yield. The pykF and pykA genes were inactivated in strain PB12.SA22 to increase the production of aromatic compounds in the PTS- background. Results indicate differential roles of Pyk isoenzymes on growth and aromatic compound production. This study demonstrated for the first time the simultaneous inactivation of PTS and pykF as part of a strategy to improve SA production and its aromatic precursors in E. coli, with a resulting high yield of aromatic compounds on glucose of 0.5 mol/mol.

SUBMITTER: Escalante A

PROVIDER: S-EPMC2873404 | biostudies-literature | 2010 Apr

REPOSITORIES: biostudies-literature

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Publications

Metabolic engineering for the production of shikimic acid in an evolved Escherichia coli strain lacking the phosphoenolpyruvate: carbohydrate phosphotransferase system.

Escalante Adelfo A Calderón Rocío R Valdivia Araceli A de Anda Ramón R Hernández Georgina G Ramírez Octavio T OT Gosset Guillermo G Bolívar Francisco F

Microbial cell factories 20100412

<h4>Background</h4>Shikimic acid (SA) is utilized in the synthesis of oseltamivir-phosphate, an anti-influenza drug. In this work, metabolic engineering approaches were employed to produce SA in Escherichia coli strains derived from an evolved strain (PB12) lacking the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS-) but with capacity to grow on glucose. Derivatives of PB12 strain were constructed to determine the effects of inactivating aroK, aroL, pykF or pykA and the expressi ...[more]

PMID: 20385022

Similar Datasets

Project description: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.

Project description:Numerous gram-negative and gram-positive bacteria take up carbohydrates through the phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS). This system transports and phosphorylates carbohydrates at the expense of PEP and is the subject of this review. The PTS consists of two general proteins, enzyme I and HPr, and a number of carbohydrate-specific enzymes, the enzymes II. PTS proteins are phosphoproteins in which the phospho group is attached to either a histidine residue or, in a number of cases, a cysteine residue. After phosphorylation of enzyme I by PEP, the phospho group is transferred to HPr. The enzymes II are required for the transport of the carbohydrates across the membrane and the transfer of the phospho group from phospho-HPr to the carbohydrates. Biochemical, structural, and molecular genetic studies have shown that the various enzymes II have the same basic structure. Each enzyme II consists of domains for specific functions, e.g., binding of the carbohydrate or phosphorylation. Each enzyme II complex can consist of one to four different polypeptides. The enzymes II can be placed into at least four classes on the basis of sequence similarity. The genetics of the PTS is complex, and the expression of PTS proteins is intricately regulated because of the central roles of these proteins in nutrient acquisition. In addition to classical induction-repression mechanisms involving repressor and activator proteins, other types of regulation, such as antitermination, have been observed in some PTSs. Apart from their role in carbohydrate transport, PTS proteins are involved in chemotaxis toward PTS carbohydrates. Furthermore, the IIAGlc protein, part of the glucose-specific PTS, is a central regulatory protein which in its nonphosphorylated form can bind to and inhibit several non-PTS uptake systems and thus prevent entry of inducers. In its phosphorylated form, P-IIAGlc is involved in the activation of adenylate cyclase and thus in the regulation of gene expression. By sensing the presence of PTS carbohydrates in the medium and adjusting the phosphorylation state of IIAGlc, cells can adapt quickly to changing conditions in the environment. In gram-positive bacteria, it has been demonstrated that HPr can be phosphorylated by ATP on a serine residue and this modification may perform a regulatory function.

Project description:BackgroundShikimic acid (SA) produced from the seeds of Chinese star anise (Illicium verum) is a key intermediate for the synthesis of neuraminidase inhibitors such as oseltamivir (Tamiflu®), an anti-influenza drug. However, plants cannot deliver a stable supply of SA. To avoid the resulting shortages and price fluctuations, a stable source of affordable SA is required. Although recent achievements in metabolic engineering of Escherichia coli strains have significantly increased SA productivity, commonly-used plasmid-based expression systems are prone to genetic instability and require constant selective pressure to ensure plasmid maintenance. Cofactors also play an important role in the biosynthesis of different fermentation products. In this study, we first constructed an E. coli SA production strain that carries no plasmid or antibiotic marker. We then investigated the effect of endogenous NADPH availability on SA production.ResultsThe pps and csrB genes were first overexpressed by replacing their native promoter and integrating an additional copy of the genes in a double gene knockout (aroK and aroL) of E. coli. The aroG(fbr), aroB, aroE and tktA gene cluster was integrated into the above E. coli chromosome by direct transformation. The gene copy number was then evolved to the desired value by triclosan induction. The resulting strain, E. coli SA110, produced 8.9-fold more SA than did the parental strain E. coli (ΔaroKΔaroL). Following qRT-PCR analysis, another copy of the tktA gene under the control of the 5P(tac) promoter was inserted into the chromosome of E. coli SA110 to obtain the more productive strain E. coli SA110. Next, the NADPH availability was increased by overexpressing the pntAB or nadK genes, which further enhanced SA production. The final strain, E. coli SA116, produced 3.12 g/L of SA with a yield on glucose substrate of 0.33 mol/mol.ConclusionAn SA-producing E. coli strain that carries neither a plasmid nor an antibiotic marker was constructed by triclosan-induced chromosomal evolution. We present the first demonstration that increasing NADPH availability by overexpressing the pntAB or nadK genes significantly enhances SA production.

Project description:BackgroundClassic metabolic engineering strategies often induce significant flux imbalances to microbial metabolism, causing undesirable outcomes such as suboptimal conversion of substrates to products. Several mathematical frameworks have been developed to understand the physiological and metabolic state of production strains and to identify genetic modification targets for improved bioproduct formation. In this work, a modeling approach was applied to describe the physiological behavior and the metabolic fluxes of a shikimic acid overproducing Escherichia coli strain lacking the major glucose transport system, grown on complex media.ResultsThe obtained flux distributions indicate the presence of high fluxes through the pentose phosphate and Entner-Doudoroff pathways, which could limit the availability of erythrose-4-phosphate for shikimic acid production even with high flux redirection through the pentose phosphate pathway. In addition, highly active glyoxylate shunt fluxes and a pyruvate/acetate cycle are indicators of overflow glycolytic metabolism in the tested conditions. The analysis of the combined physiological and flux response surfaces, enabled zone allocation for different physiological outputs within variant substrate conditions. This information was then used for an improved fed-batch process designed to preserve the metabolic conditions that were found to enhance shikimic acid productivity. This resulted in a 40% increase in the shikimic acid titer (60 g/L) and 70% increase in volumetric productivity (2.45 gSA/L*h), while preserving yields, compared to the batch process.ConclusionsThe combination of dynamic metabolic modeling and experimental parameter response surfaces was a successful approach to understand and predict the behavior of a shikimic acid producing strain under variable substrate concentrations. Response surfaces were useful for allocating different physiological behavior zones with different preferential product outcomes. Both model sets provided information that could be applied to enhance shikimic acid production on an engineered shikimic acid overproducing Escherichia coli strain.

Dataset Information

Metabolic engineering for the production of shikimic acid in an evolved Escherichia coli strain lacking the phosphoenolpyruvate: carbohydrate phosphotransferase system.

Background

Results

Conclusions

Publications

Metabolic engineering for the production of shikimic acid in an evolved Escherichia coli strain lacking the phosphoenolpyruvate: carbohydrate phosphotransferase system.

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