## 2 Overview of CA Biosynthesis in _S. clavuligerus_
The early steps start with the condensation of the glycolysis intermediate glyceraldehyde-3-phosphate and the amino acid L-arginine, producing the \(N^{2}\)-(2-carboxyethyl)-arginine. This condensation is catalyzed by the \(N^{2}\)-(2-carboxyethyl)-arginine synthase (CEAS). The early steps comprise five well-known reactions leading to the (3S, 5S)-clavaminic acid. The formation of the clavaminic acid is considered a bifurcation point of the carbon flowing through the pathway. The CA biosynthesis late steps consider the reactions leading to CA, as well as the reactions forming the clavam 5S compounds after the clavaminic acid bifurcation [24; 25; 26]. The known reactions of the clavam pathway are summarized in Figure 3. The \(\beta\)-lactam compound deoxyguanidinoproclavaminic acid is synthesized from the \(N^{2}\)-(2-carboxyethyl)-arginine in the second reaction of the clavam pathway, catalyzed by the \(\beta\)-lactam synthetase (BLS) [27; 28; 29]. The hydroxylation of the deoxyguanidinoproclavaminic acid takes place to form the guanidinoproclavaminic acid; a reaction mediated by clavaminate synthase (CAS) [29; 30; 31; 32]. Subsequently, the proclavaminic acid is produced by removing the amidino group from the arginine residue on the guanidinoproclavaminic acid, by action of proclavaminate amidino hydrolase (PAH) [33; 34]. The dihydrolacvaminic acid is obtained from the proclavaminic acid through oxidative cyclization and desaturation, both catalyzed by CAS, leading to the (3S, 5S)-clavaminic acid [29; 35]. At this point, the late steps start with the carbon flux bifurcation in two branches: one branch leading to the clavulanate-9-aldehyde and further the CA, and the other branch forming clavam 5S compounds. It is important to highlight that the 3S, 5S stereochemistry of clavaminic acid is conserved in the synthesis of all clavam 5S compounds. In the case of the CA, a transition from 3S, 5S configuration to 3R, 5R is required. Some authors have suggested that this stereochemical inversion may occur via the N-glycyl-clavaminic acid intermediate to form the clavulanate-9-aldehyde with a 3R, 5R configuration, and therefore the CA [36; 37]. However, more experimental evidence is required for the complete elucidation of intermediate reactions connecting the clavaminic acid with the clavulanate-9-aldehyde [36; 37], which is lastly reduced to CA by the action of clavulanate dehydrogenase (CAD) [38]. Recently, [PERSON] et al. [39] proposed six reaction steps for the 3S, 5S to the 3R, 5R transition, by using a computational approach. In addition to the CA, other metabolites have been identified as side compounds of this biosynthetic pathway, namely, N-glycyl-clavaminic acid, N-acetylglycyl-clavaminic acid, and N-acetyl-clavaminic acid. It has been suggested that those compounds result from the intermediate steps involved in the transition of the 3S, 5S stereochemistry into the 3R, 5R of CA [36]. Additionally, several metabolites (2-hydroxymethylclavam, 2-formyloxymethylclavam, clavamam-2-carboxylic acid, and alanylclavam) have been identified and grouped as clavam 5S compounds due to their 3S, 5S stereochemistry [37; 40; 41]. Despite their structural similarity and common precursors, only the clavam compounds with a bicyclic nucleus formed by the \(\beta\)-lactam ring and an oxazolidine ring with 3R, 5R stereochemistry can effectively inhibit the \(\beta\)-lactamases [10; 40]. Glycerol is the main substrate in the CA production by _S. clavuligerus_ as it has direct incorporation into the glycolytic pathway by forming glyceraldehyde 3-phosphate, the first C-3 precursor of CA [42; 43]. Then, the carbon flux splits into three pathways, two of them belonging to the primary metabolism: glycolysis and gluconeogenesis, and one belonging to the secondary metabolism: the clavam pathway [43; 44; 45]. In addition to glyceraldehyde 3-phosphate, the need for a C-5 precursor implies the constant demand for L-arginine.