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

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.

Clavulanic Acid Production by Streptomyces clavuligerus: Insights from Systems Biology, Strain Engineering, and Downstream Processing

“…As a β-lactamase inhibitor, CA may be part of a self-resistance mechanism involving the synthesis of β-lactam antibiotics, β-lactamase enzymes, and β-lactamase inhibitors; possibly developed by Streptomyces species to defend themselves from the effect of the antimicrobials [ 10 ]. CA biosynthesis is induced during phosphate limited conditions; recently it has been proposed that CA production may be a consequence of a homeostatic response aimed to compensate for the ATP deficit under phosphate depletion [ 19 ]. This mechanism would trigger a strong activation of oxidative and amino acid metabolism, producing reduced cofactors and ATP and favoring the antibiotic biosynthesis as a feasible mechanism to adjust the ATP generation [ 20 , 21 ].…”

“…This mechanism would trigger a strong activation of oxidative and amino acid metabolism, producing reduced cofactors and ATP and favoring the antibiotic biosynthesis as a feasible mechanism to adjust the ATP generation [ 20 , 21 ]. Moreover, the simultaneous secretion of penicillin and cephalosporins antibiotics along with β-lactamase enzymes and β-lactamase inhibitors would tightly control the energetic impact of an ATP-consuming futile cycle of polymerization/degradation of the cell wall caused by the β-lactam antibiotics accumulation [ 19 , 22 , 23 ].…”

“…Application and use of genome-scale metabolic modeling of Streptomyces bacteria is currently an active field of study [ 138 , 145 , 162 , 163 , 164 ], including the modeling of S. clavuligerus metabolism ( Figure 4 ) [ 19 , 43 , 122 , 165 , 166 ].…”

“…Finally, in 2020, a new and independent reconstruction was published [ 19 ]. The topologically and thermodynamically curated iDG1237 GEM encompasses 2177 reactions, 1518 metabolites, and 1237 genes.…”

Robustifying Experimental Tracer Design for13C-Metabolic Flux Analysis

“…A 13 C-MFA network model of the core central metabolism of S. clavuligerus was formulated based on previous work (Medema et al, 2010;Ramirez-Malule et al, 2016, 2018Gómez-Ríos et al, 2020). Reactions of CA biosynthesis via the clavam pathway were added (Ramirez-Malule et al, 2018).…”