A Genome-Scale Insight into the Effect of Shear Stress During the Fed-Batch Production of Clavulanic Acid by Streptomyces Clavuligerus.

David Gómez-Ríos,Rigoberto Ríos-Estepa,Stefan Junne,Victor A López-Agudelo,Silvia Ochoa,Peter Neubauer,Howard Ramírez-Malule

doi:10.3390/microorganisms8091255

Abstract

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.

Highlights

Streptomyces clavuligerus (S. clavuligerus) is a biotechnologically important Gram-positive filamentous bacterium for the production of β-lactam antibiotic compounds such as clavulanic acid (CA) and cephamycin C
It is likely that phosphate limitation and oxygen supply play an important role in the activation of secondary metabolism and CA biosynthesis in S. clavuligerus [5,6]
The in silico and experimental results of this study provide insights regarding the nutritional regulation of CA biosynthesis and the effects of intracellular oxygen availability in S. clavuligerus metabolism, when cultivated at high and low shear stress conditions

Summary

Introduction

Streptomyces clavuligerus (S. clavuligerus) is a biotechnologically important Gram-positive filamentous bacterium for the production of β-lactam antibiotic compounds such as clavulanic acid (CA) and cephamycin C. It is likely that phosphate limitation and oxygen supply play an important role in the activation of secondary metabolism and CA biosynthesis in S. clavuligerus [5,6]. The metabolic response of S. clavuligerus to different environmental and nutritional factors and its relationship with CA biosynthesis is not completely understood. In this regard, a strong correlation between macromorphology and CA production under low and high shear stress conditions in submerged cultures of S. clavuligerus was recently reported [7]. The study describes that high shear forces, as they occur in stirred tank bioreactors at typical stirrer speeds for sufficient oxygen supply at elevated biomass concentrations, do not compromise the viability of S. clavuligerus cells, as comparably high specific growth and CA production rates were achieved. It was assumed that CA production might be affected by a lower surface-to-volume ratio at thicker mycelia evolving under low-shear cultivation conditions, which would impact negatively upon the diffusion capacity and, the transport of nutrients, oxygen and product secretion

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