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Streptomyces clavuligerus is a filamentous Gram-positive bacterial producer of the β-lactamase inhibitor clavulanic acid. Antibiotics biosynthesis in the Streptomyces genus is usually triggered by nutritional and environmental perturbations. In this work, a new genome scale metabolic network of Streptomyces clavuligerus was reconstructed and used to study the experimentally observed effect of oxygen and phosphate concentrations on clavulanic acid biosynthesis under high and low shear stress. A flux balance analysis based on experimental evidence revealed that clavulanic acid biosynthetic reaction fluxes are favored in conditions of phosphate limitation, and this is correlated with enhanced activity of central and amino acid metabolism, as well as with enhanced oxygen uptake. In silico and experimental results show a possible slowing down of tricarboxylic acid (TCA) due to reduced oxygen availability in low shear stress conditions. In contrast, high shear stress conditions are connected with high intracellular oxygen availability favoring TCA activity, precursors availability and clavulanic acid (CA) production.

Streptomyces clavuligerus is a gram-positive filamentous bacterium notable for producing clavulanic acid (CA), an inhibitor of β-lactamase enzymes, which confers resistance to bacteria against several antibiotics. Here we present a comparative analysis of the morphological and metabolic response of S. clavuligerus linked to the CA production under low and high shear stress conditions in a 2D rocking-motion single-use bioreactor (CELL-tainer ®) and stirred tank bioreactor (STR), respectively. The CELL-tainer® guarantees high turbulence and enhanced volumetric mass transfer at low shear stress, which (in contrast to bubble columns) allows the investigation of the impact of shear stress without oxygen limitation. The results indicate that high shear forces do not compromise the viability of S. clavuligerus cells; even higher specific growth rate, biomass, and specific CA production rate were observed in the STR. Under low shear forces in the CELL-tainer® the mycelial diameter increased considerably (average diameter 2.27 in CELL-tainer® vs. 1.44 µm in STR). This suggests that CA production may be affected by a lower surface-to-volume ratio which would lead to lower diffusion and transport of nutrients, oxygen, and product. The present study shows that there is a strong correlation between macromorphology and CA production, which should be an important aspect to consider in industrial production of CA.

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