## 4 Regulation of the CA Production in _S. clavuligerus_  
Successful secondary metabolite biosynthesis by the _Streptomyces_ genus undergoes complex regulation at many levels, involving transcriptional regulators (pleiotropic regulators and pathway-specific regulators), sigma-factors, and small non-coding RNAs [85]. Therefore, the system-level understanding of the complex regulatory interactions and signaling pathways may lead to novel improvement strategies in the CA production. Usually, antibiotic biosynthetic clusters are regulated by two types of transcriptional regulators: pathway-specific regulators (PSRs) and pleiotropic regulators [86]. At a lower level, PSRs are located inside the antibiotic biosynthetic cluster and these proteins can bind directly to the promoters of genes in the same biosynthetic pathway [87; 88]. Similarly, at the top level, global regulators are known to control the production of antibiotics in the _Streptomyces_ genus [87; 88]. The _claR_ is a PSR that belongs to the LysR family and triggers the late genes (_cyp_, _oppA1_, and _cad_) of the CA biosynthetic pathway, specifically those participating in the reactions of CA and clavaminic acid biosynthesis [85; 89]. Furthermore, the _ccaR_ is a PSR located in the adjacency of the cephalospor C cluster, and triggers the synthesis of both CA and cephalospor C. The last compound is induced by the _ccaR_ through the regulation of the early genes _cas2_, _bls_, _pah_, and _orf2_[24; 85; 90]. The evidence suggests that both _ccaR_ and _claR_ form a regulatory system that controls the CA biosynthesis [91]. Recently, by means of a comparative transcriptomic analysis, [PERSON] and colleagues [92] identified that CA cluster genes were up regulated, including both _ccaR_ and _claR_, when _S. clavuligerus_ was cultured in complex media. As the CA titers in cultivations using the wild type strain are still low, engineered strains of _S. clavuligerus_ perturbing these regulators have posed as attractive alternatives for improving the CA production. [PERSON] and co-workers [93] found that a deletion mutant of _ccaR_ is unable to synthesize CA and cephamycin C; besides, an amplification of this gene led to 2-3-fold rise in the CA and cephalospor C biosynthesis. Furthermore, [PERSON] and [PERSON] [94] investigated the role of the CcaR protein in the cephamycin C gene cluster in _S. clavuligerus_. The _ccaR_ gene was also considered as essential in the synthesis of CA, cephamycin C, and non-CA clavams [94]. The authors also concluded that the lack of cephamycin C production in _ccaR_ mutants was mainly due to the absence of some enzymes in the early and middle steps in the cephamycin C pathway. [PERSON] et al. [89] observed that high replication of the _claR_ gene in multicopy plasmids led to 3-fold and 5-6-fold increases in the CA and alanylcavam production, respectively, with a consequent reduction of cephamycin C production. Furthermore, [PERSON] and co-workers [95] engineered a CA-overproducer (_S. clavuligerus_ IDG3) strain yielding CA titers up to 6.690 g\(\cdot\)L\({}^{-1}\). In that study, the authors overexpressed clavaminic acid synthase (_cas2_ gene) and the genes _claR_ and _ccaR_ independently using diverse promoters that were introduced into the industrial strain _S. clavuligerus_ DEPA. The overexpression of _ccaR_ under the control of a _glpF_ promoter, introduced into _S. clavuligerus_ DEPA, led to 25.9-fold CA production compared to its vector control. [PERSON] et al. [96] fused the _neo_ gene (a kanamycin resistance gene without promoters) with _claR_, generating the _S. clavuligerus_ (NEO) strain able to produce CA titers up to 3.260 g\(\cdot\)L\({}^{-1}\). Subsequently, and after a series of treatments and screening, the authors obtained a new engineered strain (M3-19), which produced up to 4.330 g\(\cdot\)L\({}^{-1}\) of CA, an increase of 33.8% compared to the NEO strain. Recently, [PERSON] et al. [97] overexpressed the genes _ccaR_, _cas1_, and _claR_ in the mutant strain _S. clavuligerus_ OR.