## 4 Regulation of the CA Production in _S. clavuligerus_
Although this TCS did not affect the production of CA, this mutant strain (\(\Delta\)_cepRS_) in combination with the overexpression of CA specific regulators may be an interesting alternative for improving CA production. Expression of secondary metabolism in bacteria is also regulated by sigma factors. Sigma factors are proteins that recognize the RNA polymerase and allow the proper union to the gene promoter, leading to the synthesis of a mRNA molecule [110]. Similarly, an anti-sigma factor inhibits the transcription, as they will bind to the RNA polymerase, thus preventing that sigma factors from mediating the binding to the promoter site [110]. The sigma factor gene, _orf21_, is located downstream of the CA biosynthesis cluster in _S. clavuligerus_. [PERSON] et al. [111] disrupted and overexpressed this gene in a _S. clavuligerus_ NRRL3585 and found a reduction 10-15% in the levels of CA in the \(\Delta\)_orf21_ mutant; while in the same conditions, overexpression of _orf21_ exhibited a 1.45-fold increase in CA yields during fermentation. _S. clavuligerus_ also encodes _bldG_, an anti-anti-sigma factor, which controlsgenes (regulated by sigma factors) involved in morphological development and secondary metabolite biosynthesis. [PERSON] and colleagues [90] found that _blindG_ regulates CA biosynthesis by modulating the expression of _caR_, and recently, this regulator has been associated with a direct regulatory role in the tunicamycin biosynthesis [112]. The stringent response is a stress response phenomenon that occurs in bacteria under substrate starvation, e.g., amino acid deprivation, iron restriction, and other stringent conditions [113]. This stress mechanism involves the RelA/SpoT protein system (ppGpp synthase (RelA) and (p)ppGpp synthase/hydrolase (SpoT)), which mediates the synthesis of phosphorylated nucleotides like (p)ppGpp [24; 85]. This nucleotide exerts a regulatory role in ribosomal genes via RNA Polymerase binding it is also involved in the morphological differentiation of _S. clavuligerus_[114]. Likewise, a _relA_ mutant of _S. clavuligerus_ showed reduced capability to yield both CA and cephamycin C [115]. In addition to the strain design based on overexpression or mutation of regulatory genes, some research efforts have been focused in targeting the primary metabolism. [PERSON] and [PERSON] [116] doubled CA production by deleting the _gap1_ gene in a mutant of _S. clavuligerus_ NRRL3585, increasing the glyceraldehyde 3-phosphate pool for CA biosynthesis. [PERSON] et al. [117] used the last approach along with the overexpression of caR and claR to create an overproducer engineered strain. Recently, overexpression of _lysA_ in _S. clavuligerus_ NRRL 3585, a gene involved in L-lysine biosynthesis (coding for a diaminopimelic acid decarboxylase), showed improvements in the production of CA, cephamycin C and tunicamycin. In this decade, it has been also described engineered strains of _S. clavuligerus_ perturbing random genes through mutagenesis techniques. [PERSON] and colleagues [118] engineered an industrial strain (DS48802) through an iterative process of mutagenesis and screening of _S. clavuligerus_ ATCC 27064. This mutant strain produced CA levels 100 times more than the wild type. Likewise, an improvement in CA yields using mutagenesis induced by ultraviolet on _Streptomyces sp. NRC77_ was reported [119]. The authors found a mutant strain after screening (_Streptomyces sp. MUI-NRC77_) that was able to produce 0.272 g\(\cdot\)L\({}^{-1}\) of CA in the production medium, while the addition of H\({}_{2}\)O\({}_{2}\) and plant charcoal increased the production up to 0.649 and 0.683 g\(\cdot\)L\({}^{-1}\), respectively. Recently, [PERSON] et al. [120] also used UV mutagenesis for enhanced CA titer by _S. clavuligerus_. The authors obtained mutants of _S. clavuligerus_ ATCC 27064 via UV radiation, and one of them achieved a CA concentration of 0.500 g\(\cdot\)L\({}^{-1}\).