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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 key genes of the SA pathway involved in the biosynthesis of aromatic byproducts QA and DHS from SA as determined by global transcriptomic analysis in E. coli W3110.shik1. Overexpression of ydiB, aroD, and ydiN genes allowed proposing that under carbon limiting growth conditions, SA is intracellularly accumulated as consequence of an inefficient export to periplasmic space or as consequence of its back transport to the cytoplasm as consequence of extracellular accumulation. YdiN, a putative transporter coded by ydiN was proposed to be involved in SA back import. Backflow of SA to DHS was possibly catalyzed by YdiB, whereas synthesis of DHQ from DHS was performed by AroD enzyme and finally, YdiB performed synthesis of QA from DHQ. Adapted from Johansson and Lidén (2006).
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Figure 4: Identification of key genes of the SA pathway involved in the biosynthesis of aromatic byproducts QA and DHS from SA as determined by global transcriptomic analysis in E. coli W3110.shik1. Overexpression of ydiB, aroD, and ydiN genes allowed proposing that under carbon limiting growth conditions, SA is intracellularly accumulated as consequence of an inefficient export to periplasmic space or as consequence of its back transport to the cytoplasm as consequence of extracellular accumulation. YdiN, a putative transporter coded by ydiN was proposed to be involved in SA back import. Backflow of SA to DHS was possibly catalyzed by YdiB, whereas synthesis of DHQ from DHS was performed by AroD enzyme and finally, YdiB performed synthesis of QA from DHQ. Adapted from Johansson and Lidén (2006).

Mentions: The strain W3110.shik1 (ΔaroL, aroGfbr, trpEfbr, and tnaA) engineered for SA production growing in low glucose (high phosphate) or glucose-rich (low phosphate) conditions resulted in the production of SA in cultures with mineral broth, as the single inactivation of shikimate kinase II (aroL) allows carbon flux to CHA through shikimate kinase I (aroK), resulting in the synthesis of aromatic AAs. However, under carbon-limited conditions, SA production decreased by 59%, and the byproducts DHS, DHQ, gallic acid (GA), and QA were detected in the culture supernatant with respect to phosphate limiting culture conditions (Johansson et al., 2005). Global transcriptomic analysis (GTA) of the strain W3110.shik1 in chemostatic culture conditions, comparing between glucose and phosphate limiting conditions, allowed identification of the significantly upregulated genes ydiB (coding for shikimate dehydrogenase/quinate dehydrogenase), aroD, and ydiN, which encodes a putative transporter, in carbon limiting conditions. The upregulation of these genes, particularly ydiB (10× with respect to its paralogs, aroE), was proposed to increase the YdiB level, which uses DHQ and SA as substrates, as this enzyme has a lower Km for SA in the presence of NAD+ (Keseler et al., 2013). Additionally, the intracellular concentration of NAD+ is reported to be 40-fold higher than that of NADH+, suggesting that the dehydrogenase activity on SA to produce DHS is favored by YdiB in vivo (Johansson and Lidén, 2006). These results suggests that byproduct formation during SA production was associated with the reversal of the biosynthetic pathway from (1) SA + NAD(P)+ ↔ DHS + NAD(P)H + H+ and (2) DHS + NAD(P)H + H+ ↔ QA + NAD(P)+ by YdiB or (3) DHS + H2O ↔ DHQ by AroD (Figure 4). The presence of a large amount of intracellular SA was proposed to drive the reversal of the pathway, whereas YdiN was proposed to be the exporter of the aromatic byproducts (Johansson and Lidén, 2006).


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 key genes of the SA pathway involved in the biosynthesis of aromatic byproducts QA and DHS from SA as determined by global transcriptomic analysis in E. coli W3110.shik1. Overexpression of ydiB, aroD, and ydiN genes allowed proposing that under carbon limiting growth conditions, SA is intracellularly accumulated as consequence of an inefficient export to periplasmic space or as consequence of its back transport to the cytoplasm as consequence of extracellular accumulation. YdiN, a putative transporter coded by ydiN was proposed to be involved in SA back import. Backflow of SA to DHS was possibly catalyzed by YdiB, whereas synthesis of DHQ from DHS was performed by AroD enzyme and finally, YdiB performed synthesis of QA from DHQ. Adapted from Johansson and Lidén (2006).
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Figure 4: Identification of key genes of the SA pathway involved in the biosynthesis of aromatic byproducts QA and DHS from SA as determined by global transcriptomic analysis in E. coli W3110.shik1. Overexpression of ydiB, aroD, and ydiN genes allowed proposing that under carbon limiting growth conditions, SA is intracellularly accumulated as consequence of an inefficient export to periplasmic space or as consequence of its back transport to the cytoplasm as consequence of extracellular accumulation. YdiN, a putative transporter coded by ydiN was proposed to be involved in SA back import. Backflow of SA to DHS was possibly catalyzed by YdiB, whereas synthesis of DHQ from DHS was performed by AroD enzyme and finally, YdiB performed synthesis of QA from DHQ. Adapted from Johansson and Lidén (2006).
Mentions: The strain W3110.shik1 (ΔaroL, aroGfbr, trpEfbr, and tnaA) engineered for SA production growing in low glucose (high phosphate) or glucose-rich (low phosphate) conditions resulted in the production of SA in cultures with mineral broth, as the single inactivation of shikimate kinase II (aroL) allows carbon flux to CHA through shikimate kinase I (aroK), resulting in the synthesis of aromatic AAs. However, under carbon-limited conditions, SA production decreased by 59%, and the byproducts DHS, DHQ, gallic acid (GA), and QA were detected in the culture supernatant with respect to phosphate limiting culture conditions (Johansson et al., 2005). Global transcriptomic analysis (GTA) of the strain W3110.shik1 in chemostatic culture conditions, comparing between glucose and phosphate limiting conditions, allowed identification of the significantly upregulated genes ydiB (coding for shikimate dehydrogenase/quinate dehydrogenase), aroD, and ydiN, which encodes a putative transporter, in carbon limiting conditions. The upregulation of these genes, particularly ydiB (10× with respect to its paralogs, aroE), was proposed to increase the YdiB level, which uses DHQ and SA as substrates, as this enzyme has a lower Km for SA in the presence of NAD+ (Keseler et al., 2013). Additionally, the intracellular concentration of NAD+ is reported to be 40-fold higher than that of NADH+, suggesting that the dehydrogenase activity on SA to produce DHS is favored by YdiB in vivo (Johansson and Lidén, 2006). These results suggests that byproduct formation during SA production was associated with the reversal of the biosynthetic pathway from (1) SA + NAD(P)+ ↔ DHS + NAD(P)H + H+ and (2) DHS + NAD(P)H + H+ ↔ QA + NAD(P)+ by YdiB or (3) DHS + H2O ↔ DHQ by AroD (Figure 4). The presence of a large amount of intracellular SA was proposed to drive the reversal of the pathway, whereas YdiN was proposed to be the exporter of the aromatic byproducts (Johansson and Lidén, 2006).

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