Screening of medium constituents for clavulanic acid production by Streptomyces clavuligerus

Abstract

Clavulanic acid is a β-lactam compound with potent inhibitory activity against β-lactamases. Studies have shown that certain amino acids play essential roles in CA biosynthesis. However, quantitative evaluations of the effects of these amino acids are still needed in order to improve CA production. Here, we report a study of the nutritional requirements of Streptomyces clavuligerus for CA production. Firstly, the influence of the primary nitrogen source and the salts composition was investigated. Subsequently, soybean protein isolate was supplemented with arginine (0.0–3.20 g L−1), threonine (0.0–1.44 g L−1), ornithine (0.0–4.08 g L−1), and glutamate (0.0–8.16 g L−1), according to a two-level central composite rotatable design. A medium containing ferrous sulfate yielded CA production of 437 mg L−1, while a formulation without this salt produced only 41 mg L−1 of CA. This substantial difference suggested that Fe2+ is important for CA biosynthesis. The experimental design showed that glutamate and ornithine negatively influenced CA production while arginine and threonine had no influence. The soybean protein isolate provided sufficient C5 precursor for CA biosynthesis, so that supplementation was unnecessary. Screening of medium components, together with experimental design tools, could be a valuable way of enhancing CA titers and reducing the process costs.

Production of β-Lactamase Inhibitors by Streptomyces Species

“…According to Viana-Marques et al [ 69 ], certain nutrients present in the culture medium (such as C, N, P, S sources and salts) and compounds produced in the biosynthetic steps can enhance CA production. Additionally, there are many ways to increase CA production such as optimization of bioreactor operation, conditions of agitation and aeration, and medium composition [ 70 ].…”

“…After the discovery of β-lactamase inhibitors with antibacterial activity reported by Brown et al [ 87 ], many studies have been carried out in recent decades regarding the production processes of β-lactamases inhibitors [ 70 ]. Usually, the industrial production of CA is almost entirely based on S. clavuligerus cultivation in complex medium [ 91 ].…”

“…Because of the clinical importance of this compound, the increase in CA production has been the focus of several studies. Strategies to enhance CA production include optimization of batch or fed-batch operation [ 92 , 93 ], temperature [ 94 , 95 , 96 ], agitation and aeration [ 60 , 93 ], and medium composition [ 70 , 94 , 95 ], as well as the selection of new microbial strains [ 97 ].…”

“…The selection of the most suitable medium composition is of primary importance to increase the productivity and decrease the cost of any bioprocess [ 7 ]. It is well known that extracellular microbial CA production is greatly influenced by medium components, especially carbon and nitrogen sources [ 70 , 94 ], salts composition [ 70 , 91 , 98 ], and pH [ 93 , 94 , 99 ]. However, no single medium has been established to optimize CA production by different strains, because each organism requires different conditions for maximum production, which is mainly controlled by intracellular effectors [ 100 ].…”

“…Several carbon sources (glycerol, starch, sucrose, and lipid) have been used to produce CA [ 70 , 95 , 101 , 102 , 103 ]. Glycerol plays an important role in CA production as the C3 precursor of the molecule [ 104 ].…”