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

Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma de México (UNAM), Av, Universidad 2001, Col, Chamilpa, Cuernavaca, Morelos, 62210, México.
Microbial Cell Factories (Impact Factor: 4.22). 04/2010; 9(1). DOI: 10.1186/1475-2859-9-21
Source: DOAJ

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.

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 (μ) and specific glucose consumption rate (qs), but low level production of aromatic intermediates. During the second growth phase the μ 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 μ, qs, SA production, and yield; whereas TACY increased by 33% (0.5 mol/mol).

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.

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Available from: Ramon De Anda, Sep 26, 2015
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    • "Using galactose permease system to replace phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS) to transport glucose has been reported to increase the concentration of phosphoenolpyruvate (PEP) and G3P (Escalante et al., 2010; Zhang et al., 2013) _ENREF_28. Other targets (except the gdhA deletion) are directly related with G3P, PEP, or pyruvate metabolism. "
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    Metabolic Engineering 06/2015; 31. DOI:10.1016/j.ymben.2015.06.006 · 6.77 Impact Factor
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    • "In addition, the reaction catalyzed by the enzyme quinate/shikimate dehydrogenase (coded by ydiB) was also reported as limiting in the development of L-TYR production strains [58]. Either the overexpression of some of these genes by plasmid-cloned copies [28,66], their co-expression in a modular operon under control of diverse promoters [50,58,67], or their expression by chromosomal integration of additional gene copies and promoter engineering by chromosomal evolution [68], have relieved to a great extent these rate-limiting steps typically encountered during the development of SHK and AAA overproducing strains (Table 1). To date, genetically modified E. coli strains can overproduce SHK from glucose with yields in the range of 0.08 to 0.42 mol SHK / mol glucose under diverse culture conditions [28,50,68-70]. "
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    ABSTRACT: The production of aromatic amino acids using fermentation processes with recombinant microorganisms can be an advantageous approach to reach their global demands. In addition, a large array of compounds with alimentary and pharmaceutical applications can potentially be synthesized from intermediates of this metabolic pathway. However, contrary to other amino acids and primary metabolites, the artificial channelling of building blocks from central metabolism towards the aromatic amino acid pathway is complicated to achieve in an efficient manner. The length and complex regulation of this pathway have progressively called for the employment of more integral approaches, promoting the merge of complementary tools and techniques in order to surpass metabolic and regulatory bottlenecks. As a result, relevant insights on the subject have been obtained during the last years, especially with genetically modified strains of Escherichia coli. By combining metabolic engineering strategies with developments in synthetic biology, systems biology and bioprocess engineering, notable advances were achieved regarding the generation, characterization and optimization of E. coli strains for the overproduction of aromatic amino acids, some of their precursors and related compounds. In this paper we review and compare recent successful reports dealing with the modification of metabolic traits to attain these objectives.
    Microbial Cell Factories 12/2014; 13(126). DOI:10.1186/s12934-014-0126-z · 4.22 Impact Factor
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    • "The resulting strains were transformed with plasmid pJLBaroG fbr tktA, which harbors the aroG fbr gene encoding a feedback inhibition resistant mutant of DAHP synthase. It also contains the tktA gene in order to avoid a limitation for E4P (Balderas-Hernández et al., 2009; Escalante et al., 2010). All primers used in this study are listed in Supplementary file 2. "
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