## 5 Advances in Systems Level Understanding of the CA Production in _S. clavuligerus_
The authors observed that cebphamycin C and CA biosynthetic genes were negatively regulated in the _caR_ mutant, a gene expression pattern previously identified [128]. Likewise, _blip_ and _blp_, two genes encoding \(\beta\)-lactamase inhibitory proteins, were downregulated. The most interesting metabolic phenotype was the overexpression of all the genes involved in holomycin biosynthesis, in agreement with previous studies reporting high holomycin production in _caaR_ mutants [129]. Similar results were found in a _claR_ mutant; genes like _hlml_ and _hlmH_ were upregulated during the entire fermentation [130]. In contrast, poor regulation of genes in the initial and late steps of CA biosynthesis, and thus no CA production were detected [130]. Recently, the _oppA2_ gene (oligopeptide binding protein permease) was identified as essential for CA production [131]. Transcriptional analysis of _oppA2_ mutant compared to the wild type strain also allowed identifying upregulation of holomycin genes and no significant expression of cephalospor C and CA biosynthesis clusters. However, the most interesting result was that _oppA2_ mutant secreted N-acetylglyglygly-clavaminic acid, an intermediate of the CA biosynthesis. The evidence suggests that this peptide probably is bound to the _oppA2_ protein forming a multi-enzymatic complex in the final stages of CA biosynthesis [131]. A proteomic study of the same _oppA2_ mutant identified high level abundance of rhodanese-like enzyme (_rhLA_, thiosulfate sulfur transferase) [132]. Deletion of this gene showed impairment of holomycin (90%) and lower CA and cephalospor C production. Overexpression of non-native transcriptional regulators into _S. clavuligerus_ could become an interesting approach for improving antibiotic production in the _Streptomyces_ genus. In 2019, [PERSON] et al. [133] overexpressed _pimM_ in _S. clavuligerus_ ATCC 27064, a PAS-LuxR activator of the pimaricin biosynthetic cluster present in _Streptomyces nataliensis_. A comparative transcriptome analysis indicated that overexpression of _pimM_ triggers the production of both CA and cephalospor C, by affecting the expression of secondary metabolite gene clusters like SMC10, SMC13, and SMCp5, including the upregulation of CA biosynthesis and cephalospor C clusters [133]. Untargeted metabolic profiling of the _pimM_ expression identified antifungal compounds similar to tunicamycin. A proteomic analysis comparing the wild type strain of _S. clavuligerus_ and two mutants lacking the global regulators _bldA_ and _bldG_ (_bldA_ regulates the morphological development and _bldg_ regulates CA and cephalospor C production) was conducted [134]. The authors showed that the _bldA_ mutant reduced the CA production by 80%, while in the _bldG_ mutant the CA levels were not detected compared to the wild type [134]. The _bldG_ mutant exhibited a large percentage of differentially expressed proteins; those classified in functional categories like metabolism, cellular processes, regulation, translation, and energetics. When compared to the wild type, the _bldA_ mutant showed a lower percentage of differential expressed proteins in the same categories [134]. The most interesting result of this whole-cell proteomic analysis was the detection of protein regulators (such as BldG, RshA, SigH, BldD, AdsA, BldC, AdpA, Afsa, AbsB, DraK, Afsk, and PhoP) involved in the regulatory cascade of CA biosynthesis [134]. Another whole-cell proteome analysis was developed by [PERSON] et al. (2017). They performed a proteome-wide study in an industrial CA overproducer strain (_S. clavuligerus_ DEPA) [135]. In that case, three CA biosynthetic enzymes were overexpressed: (i) \(N^{2}\)-(2-carboxyethyl)-arginine synthase (CEAS2), (ii) clavaldehyde dehydrogenase (Car) (also known as clavulanic acid dehydrogenase (CAD)), and (iii) carboxylethyl-arginine \(\beta\)-lactam synthase (BLS2). The authors identified 33 different ORFs that were underexpressed and 60 ORFs that were overexpressed.