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: The main goal in the construction of these metabolically engineered strains was to obtain an improved system for polyketide glycosylation. For this reason, once we demonstrated that LB14b was able to accumulate high levels of intracellular TDP‐d‐desosamine and TDP‐l‐mycarose, we tested the capacity of this strain to yield high levels of glycosylated polyketides through bioconversion experiments (Fig. 4A). These experiments were performed in flask cultures of strains K207‐3 and LB14b transformed with pGro7 and pKOS506‐72B. After induction with IPTG, MEB was added to the cultures to a final concentration of 100 mg l−1 and incubated for 24 h at 22°C. LC/MS analysis of extracts obtained from these cultures showed a dramatic increase in the conversion of MEB to EryD by LB14b compared with the parental K207‐3 strain (Fig. 4B and C). The concentration of EryD was estimated through a Micrococcus luteus inhibition assay using an EryA standard as reference, and considering the activity of EryD against this microorganism as four times lower than that of EryA (Kibwage et al., 1985). The estimated production of EryD was 3 mg l−1 in K207‐3 and 80 mg l−1 in LB14b cultures (about 2% and 60% conversion of the total MEB fed respectively; Fig. 5). This represents a > 25‐fold increase in the production of EryD in LB14b compared with its parental strain K207‐3, validating the effectiveness of our metabolic engineering approach to enhance the heterologous glycosylation of polyketides in E. coli.
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.