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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

Relevant engineered E. coli strains for SA production. Metabolic traits of E. coli derivative strains engineered for SA production resulting in highest SA titer and yield from glucose. The figure illustrates alterative glucose transporter GalP (galactose permease) selected by the cell after laboratory adaptive evolution process of a PTS− mutant (Flores et al., 1996, 2007; Aguilar et al., 2012). Glf (glucose facilitator) and Glk (glucokinase) from Z. mobilis (plasmid cloned). Resultant characteristics of engineered strains are shown for pSC6.090B (Chandran et al., 2003), PB12.SA22 (Escalante et al., 2010), AR36 (Rodriguez et al., 2013), and SA116 strain (Cui et al., 2014). CCM key intermediates and protein encoding genes: TCA, tricarboxylic acid pathway; E4P, erythrose-4-P; PGNL, 6-phospho d-glucono-1,5-lactone; PEP, phosphoenolpyruvate; PYR, pyruvate; ACoA, acetyl-CoA; CIT, citrate; OAA, oxaloacetate; zwf, glucose 6-phosphate-1-dehydrogenase; tktA, transketolase I; pykA, pyruvate kinase II; lpdA, aceE and aceF, coding for PYR dehydrogenase subunits; gltA, citrate synthase; pck, PEP carboxykinase; ppc PEP carboxylase; ppsA, PEP synthetase. SA pathway intermediates and genes: DAHP, 3-deoxy-d-arabino-heptulosonate-7-phosphate; DHQ, 3-dehydroquinate; DHS, 3-dehydroshikimate; SA, shikimic acid. Continuous arrows indicate single enzymatic reactions; dashed arrows show several enzymatic reactions. Bold arrows show improved carbon flux. Black squares in plasmids/operons indicate gene interruption; c, chromosomal gene interruption or integration; p, plasmid-cloned genes.
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Figure 3: Relevant engineered E. coli strains for SA production. Metabolic traits of E. coli derivative strains engineered for SA production resulting in highest SA titer and yield from glucose. The figure illustrates alterative glucose transporter GalP (galactose permease) selected by the cell after laboratory adaptive evolution process of a PTS− mutant (Flores et al., 1996, 2007; Aguilar et al., 2012). Glf (glucose facilitator) and Glk (glucokinase) from Z. mobilis (plasmid cloned). Resultant characteristics of engineered strains are shown for pSC6.090B (Chandran et al., 2003), PB12.SA22 (Escalante et al., 2010), AR36 (Rodriguez et al., 2013), and SA116 strain (Cui et al., 2014). CCM key intermediates and protein encoding genes: TCA, tricarboxylic acid pathway; E4P, erythrose-4-P; PGNL, 6-phospho d-glucono-1,5-lactone; PEP, phosphoenolpyruvate; PYR, pyruvate; ACoA, acetyl-CoA; CIT, citrate; OAA, oxaloacetate; zwf, glucose 6-phosphate-1-dehydrogenase; tktA, transketolase I; pykA, pyruvate kinase II; lpdA, aceE and aceF, coding for PYR dehydrogenase subunits; gltA, citrate synthase; pck, PEP carboxykinase; ppc PEP carboxylase; ppsA, PEP synthetase. SA pathway intermediates and genes: DAHP, 3-deoxy-d-arabino-heptulosonate-7-phosphate; DHQ, 3-dehydroquinate; DHS, 3-dehydroshikimate; SA, shikimic acid. Continuous arrows indicate single enzymatic reactions; dashed arrows show several enzymatic reactions. Bold arrows show improved carbon flux. Black squares in plasmids/operons indicate gene interruption; c, chromosomal gene interruption or integration; p, plasmid-cloned genes.

Mentions: Finally, with precursors known to induce redirection, deregulation, and overexpression of the SA pathway genes aroB, aroD, and aroE have been achieved, resulting in an efficient carbon flux from CCM to the SA pathway. The highest production to date corresponds to the SP1.1pts-/pSC6.090B strain, a PTS− derivative strain with a plasmid containing two tac promoters, the first of which controls expression of the glf, glk, aroFfbr, and tktA genes and the second of which controls expression of the aroE and serA genes (Chandran et al., 2003). The reasoning behind this construction was to increase the PEP pool by deleting PTS, to recuperate glucose consumption by overexpressing glf and glk, to assure E4P pool enhancement by overexpressing tktA and to induce a deregulated pull toward the AA pathway via an aroFfbr, as discussed before. The second promotor in the plasmid was designed to overexpress aroE, allowing continuous flux of the SA pathway; additionally, a second copy of aroB was introduced into the chromosome instead of reintroducing the serine production-related gene serA to the cell via the plasmid and was used as a selection marker for plasmid retention. This approximation, along with the deletion of genes related to SA consumption (aroK and aroL), allowed SA accumulation, achieving a production capacity of 87 g/L SA, with a yield of 36% (mol/mol) and a productivity of ~5.3 g/L h when a 10 L glucose-fed batch was cultured. This strain has the highest titer accumulation recorded in the literature to date (Table 1; Figure 3).


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)

Relevant engineered E. coli strains for SA production. Metabolic traits of E. coli derivative strains engineered for SA production resulting in highest SA titer and yield from glucose. The figure illustrates alterative glucose transporter GalP (galactose permease) selected by the cell after laboratory adaptive evolution process of a PTS− mutant (Flores et al., 1996, 2007; Aguilar et al., 2012). Glf (glucose facilitator) and Glk (glucokinase) from Z. mobilis (plasmid cloned). Resultant characteristics of engineered strains are shown for pSC6.090B (Chandran et al., 2003), PB12.SA22 (Escalante et al., 2010), AR36 (Rodriguez et al., 2013), and SA116 strain (Cui et al., 2014). CCM key intermediates and protein encoding genes: TCA, tricarboxylic acid pathway; E4P, erythrose-4-P; PGNL, 6-phospho d-glucono-1,5-lactone; PEP, phosphoenolpyruvate; PYR, pyruvate; ACoA, acetyl-CoA; CIT, citrate; OAA, oxaloacetate; zwf, glucose 6-phosphate-1-dehydrogenase; tktA, transketolase I; pykA, pyruvate kinase II; lpdA, aceE and aceF, coding for PYR dehydrogenase subunits; gltA, citrate synthase; pck, PEP carboxykinase; ppc PEP carboxylase; ppsA, PEP synthetase. SA pathway intermediates and genes: DAHP, 3-deoxy-d-arabino-heptulosonate-7-phosphate; DHQ, 3-dehydroquinate; DHS, 3-dehydroshikimate; SA, shikimic acid. Continuous arrows indicate single enzymatic reactions; dashed arrows show several enzymatic reactions. Bold arrows show improved carbon flux. Black squares in plasmids/operons indicate gene interruption; c, chromosomal gene interruption or integration; p, plasmid-cloned genes.
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Related In: Results  -  Collection

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Figure 3: Relevant engineered E. coli strains for SA production. Metabolic traits of E. coli derivative strains engineered for SA production resulting in highest SA titer and yield from glucose. The figure illustrates alterative glucose transporter GalP (galactose permease) selected by the cell after laboratory adaptive evolution process of a PTS− mutant (Flores et al., 1996, 2007; Aguilar et al., 2012). Glf (glucose facilitator) and Glk (glucokinase) from Z. mobilis (plasmid cloned). Resultant characteristics of engineered strains are shown for pSC6.090B (Chandran et al., 2003), PB12.SA22 (Escalante et al., 2010), AR36 (Rodriguez et al., 2013), and SA116 strain (Cui et al., 2014). CCM key intermediates and protein encoding genes: TCA, tricarboxylic acid pathway; E4P, erythrose-4-P; PGNL, 6-phospho d-glucono-1,5-lactone; PEP, phosphoenolpyruvate; PYR, pyruvate; ACoA, acetyl-CoA; CIT, citrate; OAA, oxaloacetate; zwf, glucose 6-phosphate-1-dehydrogenase; tktA, transketolase I; pykA, pyruvate kinase II; lpdA, aceE and aceF, coding for PYR dehydrogenase subunits; gltA, citrate synthase; pck, PEP carboxykinase; ppc PEP carboxylase; ppsA, PEP synthetase. SA pathway intermediates and genes: DAHP, 3-deoxy-d-arabino-heptulosonate-7-phosphate; DHQ, 3-dehydroquinate; DHS, 3-dehydroshikimate; SA, shikimic acid. Continuous arrows indicate single enzymatic reactions; dashed arrows show several enzymatic reactions. Bold arrows show improved carbon flux. Black squares in plasmids/operons indicate gene interruption; c, chromosomal gene interruption or integration; p, plasmid-cloned genes.
Mentions: Finally, with precursors known to induce redirection, deregulation, and overexpression of the SA pathway genes aroB, aroD, and aroE have been achieved, resulting in an efficient carbon flux from CCM to the SA pathway. The highest production to date corresponds to the SP1.1pts-/pSC6.090B strain, a PTS− derivative strain with a plasmid containing two tac promoters, the first of which controls expression of the glf, glk, aroFfbr, and tktA genes and the second of which controls expression of the aroE and serA genes (Chandran et al., 2003). The reasoning behind this construction was to increase the PEP pool by deleting PTS, to recuperate glucose consumption by overexpressing glf and glk, to assure E4P pool enhancement by overexpressing tktA and to induce a deregulated pull toward the AA pathway via an aroFfbr, as discussed before. The second promotor in the plasmid was designed to overexpress aroE, allowing continuous flux of the SA pathway; additionally, a second copy of aroB was introduced into the chromosome instead of reintroducing the serine production-related gene serA to the cell via the plasmid and was used as a selection marker for plasmid retention. This approximation, along with the deletion of genes related to SA consumption (aroK and aroL), allowed SA accumulation, achieving a production capacity of 87 g/L SA, with a yield of 36% (mol/mol) and a productivity of ~5.3 g/L h when a 10 L glucose-fed batch was cultured. This strain has the highest titer accumulation recorded in the literature to date (Table 1; Figure 3).

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