## 4 Regulation of the CA Production in _S. clavuligerus_
Upregulation of _cas1_ improved the CA yield in _S. clavuligerus_ OR, leading to CA concentration of 4.95 g\(\cdot\)L\({}^{-1}\), approximately 25% more compared to the control _S. clavuligerus_ OR. Additionally, the overexpression of _claR_ _and_ _ccaR_ in the same mutant strain increased the CA accumulation up to 5.66 g\(\cdot\)L\({}^{-1}\) in shake flask cultivations. Nevertheless, a co-expression of _claR_, _ccaR_, and _cas1_ did not improve the CA titers. Batch cultivation of _S. clavuligerus_ OR with overexpressed ccaR and claR in a 7 L bioreactor accumulated 6.010 g\(\cdot\)L\({}^{-1}\) of CA. In addition to _claR and ccaR_, the regulatory pathway that controls CA production contains other important regulators. The CcaR formation is modulated by other regulatory proteins like AreB and ScaR, both \(\gamma\)-butyrolactone receptor proteins. AreB is an autoregulation binding protein that belongs to the transcriptional regulators family termed lcR and is located upstream of the _caR_ gene; it binds to the sequences (ARE, autoregulatory elements) located upstream of target genes [24]. [PERSON] and colleagues [98] found that AreB modulates the assimilation and biosynthesis of lysine. The mutant _AarB_ of _S. clavuligerus_ induces an increase in CA and Cephamycin C levels; highlighting an important role of _areB_ in connecting secondary and primary metabolism. ScaR (Brp, \(\gamma\)-butyrolactone receptor protein) is an autoregulator induced by \(\gamma\)-butyrolactone that controls directly _ccaR_ and indirectly _claR_ via the AdpA signaling pathway. The AdpA transcriptional regulator is downregulated by ScaR (in _scar_ mutants _adpA_ is upregulated), and regulates positively the CA production, as it was demonstrated by the mutant \(\Delta\)_adpA_ strain [99]. In the _Streptomyces_ genus, an important type of global regulators is the two-component system (TCS), which usually regulates both antibiotic production and morphological development. The TCS consists of a histidine kinase bound to the extracellular membrane, which senses external perturbations, and a response regulator, which triggers the transcription of target genes by undergoing a signal transduction pathway [100; 101]. [PERSON] and colleagues [102; 103] found that CagRS is a pleiotropic regulatory TCS that has a positive effect on CA production. Transcriptome analysis of the mutant \(\Delta\)_cagRS_ versus wild type shown that CagRS TCS induces the up regulation of the CA biosynthetic gene pathway and also that CagRS positively regulates glyceraldehyde 3-phosphate but negatively regulates arginine biosynthesis [102]. Similarly, other TCS have been identified for _S. clavuligerus_. [PERSON] and colleagues [104] found that the transcription of the transcriptional activator _cvm7p_ is controlled by a TCS formed by _snk_ and _res2_. Mutants of _res2_ and _snk_ were incapable to synthesize metabolites of the 5S clavams pathway [104]. Another important two-component regulatory system is associated with the phosphate (Pho) regulon, the unique known regulatory mechanism in response to phosphate starvation in bacteria [105]. The hypothesis of a Pho regulon present in _S. clavuligerus_ is supported by the high production of CA under phosphate limitations. [PERSON] et al. [106] predicted the existence of this regulon by the identification of PHO binding sequences in promoter regions of 31 genes of _S. clavuligerus_. Likewise, other TCS is the AfsK-AfsR serine/threonine kinase system, which induces the production of secondary metabolism gene clusters in _S. lividans_ and _S. coelicolor_[107]. The _asfR-p_ is a homologue of _asfR_, the substrate of AfsK from the AfsK-AfsR. [PERSON] and colleagues [108] demonstrated the potential of this pleiotropic regulation by overexpressing _asfR-p_ in distinct _Streptomyces_ bacteria, including _S. clavuligerus_, which showed a CA production of 0.065 g\(\cdot\)L\({}^{-1}\). Recently, it was found that CepRS (cephalicyn regulator/sensor) is a TCS regulator with a key role in the cephalosporic C biosynthesis [109].