Haakuria, VM; (2015) An Integrated Approach to Studying the Regulation of Erythromycin Biosynthesis in Saccharopolyspora erythraea. Doctoral thesis , UCL (University College London).

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Abstract

S. erythraea is the main producing organism of erythromycin, an important broad spectrum antibiotic. Its yield is low because the carbon source utilisation, product kinetics and the regulatory pathways are not yet fully understood. This work provides a detailed characterisation of the carbon source metabolism of S. erythraea. The organism was cultured under various conditions and the growth and product kinetics investigated. In batch bioreactor fermentations, S. erythraea was cultured on both glucose and gluconate as sole carbon and energy sources. The culture was subsequently evaluated in bolus feed addition fermentations using phosphoenolpyruvate, oxaloacetate, propionate and methyl oleate with glucose as main carbon source. On both glucose and gluconate, erythromycin production depended on the nature of the carbon source and the growth rate. On glucose, growth was fast with erythromycin production commencing after the growth phase. On gluconate, growth was subdued and erythromycin production was growth-related. Two phases were distinguished for both carbon sources: 1) erythromycin synthesis and 2) precursor accumulation phase. Erythromycin production was enhanced by increased activity of the pentose phosphate and the anaplerotic pathway. Growth rate and the carbon source uptake rate were found to have a major effect on erythromycin production. The split in pathway activities at key branch points were found to be dependent on the growth rate and the rate of carbon source uptake. During bolus feed addition fermentation in shake flasks, erythromycin levels were increased by the addition of oxaloacetate, propionate and methyl oleate respectively. Then effect of metabolite supplementation on erythromycin levels depended on the phase during which feeding is done. In bioreactor culture, levels of erythromycin were enhanced following addition of PEP or methyl oleate. The pyruvate metabolite node was found to be flexible responding to supplementation and the nature of the sole carbon and energy source. Carbon allocation to pyruvate was 1.7 % and 5.4 % for growth on glucose and gluconate respectively. This metabolite node is, therefore, critical to improving the biosynthesis of erythromycin. However, the phosphoenolpyruvate node appears rigid and addition of PEP was excreted as 7-Orhamnosyl flaviolin (red pigment). The ratio of the rate of carbon source consumption and oxygen uptake is concluded to be a critical parameter in erythromycin biosynthesis.

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