## 2 Overview of CA Biosynthesis in _S. clavuligerus_  
The _Streptomyces_ genus produces a wide variety of secondary metabolites with antimicrobial activity (approximately two-thirds of which occur naturally) ([PERSON], 1971). In 1971, [PERSON] et al. (1971) reported a new Streptomyceset species as producer of two cephalosporporin compounds. This new species was then named and described as _S. clavuligerus_ by [PERSON] and [PERSON], also in 1971 ([PERSON], 1976). Later, in 1976, [PERSON] and [PERSON] (1976) described the CA chemical structure, which was elucidated via spectroscopic and X-ray analyses and reported as a novel fused \(\beta\)-lactam compound with a significant inhibitory activity of \(\beta\)-lactamases. In 1977, [PERSON] and [PERSON] described the cultivation conditions of _S. clavuligerus_ to produce CA and the spectrometric method for CA detection ([PERSON], 1971). In 1941, the biochemist [PERSON] described the most accepted definition of \"antibiotic\" as a small molecule made by microorganisms that inhibits the growth of another microorganism. During the \"golden age\" of antibiotics, approximately 70-80% of the antibiotics discovered came from Streptomyceset, but the evolutionary reason for the development of antibiotic biosynthetic capacity of soil bacteria and its ecological role are still unknown. Soil bacteria are not very efficient at up-taking nutrients, and their growth rate is considerably low in comparison with other bacteria and fungi. A plausible hypothesis that iswidely accepted implies that antibiotics secretion allows the producer to control the organisms competing for the same nutritional resources in a hostile multispecies environment ([PERSON], 1971; [PERSON], 1971). This is consistent with the secondary nature of antibiotics secretion under nutritional restriction. However, the antibiotic compounds at very low concentrations can modulate the transcriptional profiles of target bacteria and the products of resistance genes would silence those messages, opposing the theory of the development of attack and defense mechanisms ([PERSON], 1971). Mathematical estimations point out that Streptomyceset are able to produce around 100,000 antibiotics and only 3% of them have been discovered ([PERSON], 1971). Similarly, the existence of cryptic biosynthetic gene clusters (BGCs) suggests a higher capacity of antibiotic production in those organisms ([PERSON], 1971; [PERSON], 1971; [PERSON], 1971). CA is secreted as a secondary metabolite, also referred as a specialized metabolite, by _S. clavuligerus_ under nutritional restriction. The role of CA in the physiology and adaptation of _S. clavuligerus_ is also unknown. As a \(\beta\)-lactamase inhibitor, CA may be part of a self-resistance mechanism involving the synthesis of \(\beta\)-lactam antibiotics, \(\beta\)-lactamase  
Figure 2: Holistic overview of the CA production process. enzymes, and \(\beta\)-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]. Moreover, the simultaneous secretion of penicillin and cephalosporitisotis along with \(\beta\)-lactamase enzymes and \(\beta\)-lactamase inhibitors would tightly control the energetic impact of an ATP-consuming futile cycle of polymerization/degradation of the cell wall caused by the \(\beta\)-lactam antibiotics accumulation [19; 22; 23]. CA is a product of the clavam pathway, which proceeds towards two branches: the CA and the clavams 5S biosynthesis [6]. The clavam pathway contains two sets of reactions: the so-called \"early\" and \"late\" steps.