Metabolically engineered Escherichia coli for efficient production of glycosylated natural products.
Bottom Line: Significant achievements in polyketide gene expression have made Escherichia coli one of the most promising hosts for the heterologous production of pharmacologically important polyketides.However, attempts to produce glycosylated polyketides, by the expression of heterologous sugar pathways, have been hampered until now by the low levels of glycosylated compounds produced by the recombinant hosts.By carrying out metabolic engineering of three endogenous pathways that lead to the synthesis of TDP sugars in E. coli, we have greatly improved the intracellular levels of the common deoxysugar intermediate TDP-4-keto-6-deoxyglucose resulting in increased production of the heterologous sugars TDP-L-mycarose and TDP-D-desosamine, both components of medically important polyketides.
Affiliation: Microbiology Division, IBR (Instituto de Biología Molecular y Celular de Rosario), Consejo Nacional de Investigaciones Científicas y Técnicas, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina.Show MeSH
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Mentions: Expression of the TDP‐d‐desosamine pathway was tested in K207‐3 and LB14b transformed with pKOS506‐72B. This plasmid harbours all the genes required for the synthesis of TDP‐d‐desosamine from TKDG, the glycosyltransferases megCII/megCIII and the erythromycin resistance gene ermE (Table 1). The resulting strains were next transformed with plasmid pGro7, which overproduces GroES/EL chaperones, shown to improve the solubility of heterologously expressed proteins (Peiru et al., 2005). TDP‐sugar analysis of the cell‐free extracts obtained of both strains showed a peak with a parent/daughter pair of m/z 558/321, which corresponds to TDP‐d‐desosamine (Fig. 3A and B). The relative peak height corresponding to the intracellular accumulation of TDP‐d‐desosamine observed in LB14b extracts exhibited a 50‐fold increase over the K207‐3 first‐generation strain, confirming that the increase of TKDG pools is essential to achieve larger amounts of heterologous TDP‐sugars in E. coli. This was further confirmed through the production of a second TDP‐sugar from plasmid pLB353, which encodes the complete TDP‐l‐mycarose biosynthetic pathway (Table 1). The expression of this pathway in LB14b resulted in a similar increase of TDP‐sugar production when compared with the parental strain, as observed with TDP‐d‐desosamine (data not shown), validating this new host for the improved production of heterologous TDP‐sugars.
Affiliation: Microbiology Division, IBR (Instituto de Biología Molecular y Celular de Rosario), Consejo Nacional de Investigaciones Científicas y Técnicas, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina.