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Shikimic Acid Production in Escherichia coli: From Classical Metabolic Engineering Strategies to Omics Applied to Improve Its Production.

Martínez JA, Bolívar F, Escalante A - Front Bioeng Biotechnol (2015)

Bottom Line: Global transcriptomic analyses have been performed in SA-producing strains, resulting in the identification of possible key target genes for the design of a rational strain improvement strategy.Because possible target genes are involved in the transport, catabolism, and interconversion of different carbon sources and metabolic intermediates outside the central carbon metabolism and SA pathways, as genes involved in diverse cellular stress responses, the development of rational cellular strain improvement strategies based on omics data constitutes a challenging task to improve SA production in currently overproducing engineered strains.In this review, we discuss the main metabolic engineering strategies that have been applied for the development of efficient SA-producing strains, as the perspective of omics analysis has focused on further strain improvement for the production of this valuable aromatic intermediate.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma de México , Cuernavaca , Mexico.

ABSTRACT
Shikimic acid (SA) is an intermediate of the SA pathway that is present in bacteria and plants. SA has gained great interest because it is a precursor in the synthesis of the drug oseltamivir phosphate (OSF), an efficient inhibitor of the neuraminidase enzyme of diverse seasonal influenza viruses, the avian influenza virus H5N1, and the human influenza virus H1N1. For the purposes of OSF production, SA is extracted from the pods of Chinese star anise plants (Illicium spp.), yielding up to 17% of SA (dry basis content). The high demand for OSF necessary to manage a major influenza outbreak is not adequately met by industrial production using SA from plants sources. As the SA pathway is present in the model bacteria Escherichia coli, several "intuitive" metabolically engineered strains have been applied for its successful overproduction by biotechnological processes, resulting in strains producing up to 71 g/L of SA, with high conversion yields of up to 0.42 (mol SA/mol Glc), in both batch and fed-batch cultures using complex fermentation broths, including glucose as a carbon source and yeast extract. Global transcriptomic analyses have been performed in SA-producing strains, resulting in the identification of possible key target genes for the design of a rational strain improvement strategy. Because possible target genes are involved in the transport, catabolism, and interconversion of different carbon sources and metabolic intermediates outside the central carbon metabolism and SA pathways, as genes involved in diverse cellular stress responses, the development of rational cellular strain improvement strategies based on omics data constitutes a challenging task to improve SA production in currently overproducing engineered strains. In this review, we discuss the main metabolic engineering strategies that have been applied for the development of efficient SA-producing strains, as the perspective of omics analysis has focused on further strain improvement for the production of this valuable aromatic intermediate.

No MeSH data available.


Related in: MedlinePlus

Identification of possible key genes involved in carbon supply for SA synthesis as determined by global transcriptomic analysis in E. coli PB12.SA22 in batch culture using complex fermentation broth. Global transcriptomic analysis (GTA) showed no changes in expression profile in comparisons between EXP/STA1 (A) and STA1/STA2 (B) stages of those genes coding for enzymes of CCM and SA pathways but differential overexpression of diverse genes involved the transport, catabolism and interconversion of amino acids was observed (in red color) [(A,B), lower panels]. During STA1/STA2 comparison, genes coding for l-arginine, l-lysine, l-glutamic acid, and l-ornithine transporters were upregulated. These amino acids are probably converted to succinate fueling carbon to TCA. Additionally diverse genes coding for stress response proteins to pH and osmotic pressure were overexpressed. Blue arrows in upper panels showed samples from fermentor culture analyzed for GTA. Growth (•), glucose consumption (▪), and SA production (▴). Adapted from Escalante et al. (2010), Keseler et al. (2013), and Cortés-Tolalpa et al. (2014).
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Figure 5: Identification of possible key genes involved in carbon supply for SA synthesis as determined by global transcriptomic analysis in E. coli PB12.SA22 in batch culture using complex fermentation broth. Global transcriptomic analysis (GTA) showed no changes in expression profile in comparisons between EXP/STA1 (A) and STA1/STA2 (B) stages of those genes coding for enzymes of CCM and SA pathways but differential overexpression of diverse genes involved the transport, catabolism and interconversion of amino acids was observed (in red color) [(A,B), lower panels]. During STA1/STA2 comparison, genes coding for l-arginine, l-lysine, l-glutamic acid, and l-ornithine transporters were upregulated. These amino acids are probably converted to succinate fueling carbon to TCA. Additionally diverse genes coding for stress response proteins to pH and osmotic pressure were overexpressed. Blue arrows in upper panels showed samples from fermentor culture analyzed for GTA. Growth (•), glucose consumption (▪), and SA production (▴). Adapted from Escalante et al. (2010), Keseler et al. (2013), and Cortés-Tolalpa et al. (2014).

Mentions: GTA was performed to corroborate this hypothesis during SA production in batch fermentation cultures using complex fermentation broth (Chandran et al., 2003; Escalante et al., 2010; Rodriguez et al., 2013) by comparing global expression profiling between the mid-exponential growth phase (EXP, 5 h of cultivation), the early stationary phase (STA1, 9 h) and the late STA phase (44 h); EXP/STA1, EXP/STA2, and STA1/STA2 comparisons were conducted (Cortés-Tolalpa et al., 2014) (Figure 5).


Shikimic Acid Production in Escherichia coli: From Classical Metabolic Engineering Strategies to Omics Applied to Improve Its Production.

Martínez JA, Bolívar F, Escalante A - Front Bioeng Biotechnol (2015)

Identification of possible key genes involved in carbon supply for SA synthesis as determined by global transcriptomic analysis in E. coli PB12.SA22 in batch culture using complex fermentation broth. Global transcriptomic analysis (GTA) showed no changes in expression profile in comparisons between EXP/STA1 (A) and STA1/STA2 (B) stages of those genes coding for enzymes of CCM and SA pathways but differential overexpression of diverse genes involved the transport, catabolism and interconversion of amino acids was observed (in red color) [(A,B), lower panels]. During STA1/STA2 comparison, genes coding for l-arginine, l-lysine, l-glutamic acid, and l-ornithine transporters were upregulated. These amino acids are probably converted to succinate fueling carbon to TCA. Additionally diverse genes coding for stress response proteins to pH and osmotic pressure were overexpressed. Blue arrows in upper panels showed samples from fermentor culture analyzed for GTA. Growth (•), glucose consumption (▪), and SA production (▴). Adapted from Escalante et al. (2010), Keseler et al. (2013), and Cortés-Tolalpa et al. (2014).
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Figure 5: Identification of possible key genes involved in carbon supply for SA synthesis as determined by global transcriptomic analysis in E. coli PB12.SA22 in batch culture using complex fermentation broth. Global transcriptomic analysis (GTA) showed no changes in expression profile in comparisons between EXP/STA1 (A) and STA1/STA2 (B) stages of those genes coding for enzymes of CCM and SA pathways but differential overexpression of diverse genes involved the transport, catabolism and interconversion of amino acids was observed (in red color) [(A,B), lower panels]. During STA1/STA2 comparison, genes coding for l-arginine, l-lysine, l-glutamic acid, and l-ornithine transporters were upregulated. These amino acids are probably converted to succinate fueling carbon to TCA. Additionally diverse genes coding for stress response proteins to pH and osmotic pressure were overexpressed. Blue arrows in upper panels showed samples from fermentor culture analyzed for GTA. Growth (•), glucose consumption (▪), and SA production (▴). Adapted from Escalante et al. (2010), Keseler et al. (2013), and Cortés-Tolalpa et al. (2014).
Mentions: GTA was performed to corroborate this hypothesis during SA production in batch fermentation cultures using complex fermentation broth (Chandran et al., 2003; Escalante et al., 2010; Rodriguez et al., 2013) by comparing global expression profiling between the mid-exponential growth phase (EXP, 5 h of cultivation), the early stationary phase (STA1, 9 h) and the late STA phase (44 h); EXP/STA1, EXP/STA2, and STA1/STA2 comparisons were conducted (Cortés-Tolalpa et al., 2014) (Figure 5).

Bottom Line: Global transcriptomic analyses have been performed in SA-producing strains, resulting in the identification of possible key target genes for the design of a rational strain improvement strategy.Because possible target genes are involved in the transport, catabolism, and interconversion of different carbon sources and metabolic intermediates outside the central carbon metabolism and SA pathways, as genes involved in diverse cellular stress responses, the development of rational cellular strain improvement strategies based on omics data constitutes a challenging task to improve SA production in currently overproducing engineered strains.In this review, we discuss the main metabolic engineering strategies that have been applied for the development of efficient SA-producing strains, as the perspective of omics analysis has focused on further strain improvement for the production of this valuable aromatic intermediate.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma de México , Cuernavaca , Mexico.

ABSTRACT
Shikimic acid (SA) is an intermediate of the SA pathway that is present in bacteria and plants. SA has gained great interest because it is a precursor in the synthesis of the drug oseltamivir phosphate (OSF), an efficient inhibitor of the neuraminidase enzyme of diverse seasonal influenza viruses, the avian influenza virus H5N1, and the human influenza virus H1N1. For the purposes of OSF production, SA is extracted from the pods of Chinese star anise plants (Illicium spp.), yielding up to 17% of SA (dry basis content). The high demand for OSF necessary to manage a major influenza outbreak is not adequately met by industrial production using SA from plants sources. As the SA pathway is present in the model bacteria Escherichia coli, several "intuitive" metabolically engineered strains have been applied for its successful overproduction by biotechnological processes, resulting in strains producing up to 71 g/L of SA, with high conversion yields of up to 0.42 (mol SA/mol Glc), in both batch and fed-batch cultures using complex fermentation broths, including glucose as a carbon source and yeast extract. Global transcriptomic analyses have been performed in SA-producing strains, resulting in the identification of possible key target genes for the design of a rational strain improvement strategy. Because possible target genes are involved in the transport, catabolism, and interconversion of different carbon sources and metabolic intermediates outside the central carbon metabolism and SA pathways, as genes involved in diverse cellular stress responses, the development of rational cellular strain improvement strategies based on omics data constitutes a challenging task to improve SA production in currently overproducing engineered strains. In this review, we discuss the main metabolic engineering strategies that have been applied for the development of efficient SA-producing strains, as the perspective of omics analysis has focused on further strain improvement for the production of this valuable aromatic intermediate.

No MeSH data available.


Related in: MedlinePlus