Engineering Actinobacteria to Enhance Natural Product Biosynthesis

Enhancement of antibiotic fermentation yield has traditionally involved combining random mutagenesis with feedstock optimisation. However, recent advances in genetics and an improved understanding of the link between the primary and secondary metabolism have unlocked the ability to improve productivity by targeted genetic modification of specific metabolic regulators. Building on work in Mycobacterium and Corynebacterium, it was hypothesised that genetic engineering of the metabolic regulator GarA in antibiotic-producing Actinobacteria could enhance antibiotic yield, thus rendering these strains more aBractive for novel antibiotic development. To investigate this hypothesis, erythromycin-producing Saccharopolyspora erythraea was engineered to express a second copy of garA under the control of a constitutive promoter. An additional strain was created in which this second copy lacks regulatory phosphorylation sites and should be constitutively active towards TCA cycle enzymes. The concentration of erythromycin produced by these strains was assessed by Liquid Chromatography coupled with Mass Spectrometry. In addition, the level of amino acids was measured to provide an understanding of how GarA affects metabolism. The findings of this study indicate that GarA was present throughout fermentation, with a truncated form predominating during the antibiotic production phase. Expression of phosphoablative GarA had an effect on erythromycin yield and mutiple phenotypes: flaviolin production, growth in nutrient limited conditions and sporulation. The strain with phosphoablative GarA had a 2-fold increase in erythromycin yield compared to the control strain and was also found to have higher concentrations of amino acids. This enhancement in antibiotic production observed for the strain with an additional phosphoablative GarA is believed to be due to changes in the primary metabolism of S. erythraea and it seems a promising method for yield enhancement for pharmaceutically important natural products.