Production
Penicillin is produced by the fermentation of various types of sugar by the fungus penicillium rubens. The fermentation process produces penicillin as a secondary metabolite when the growth of the fungus is inhibited by stress. It is not produced during active growth. Production is also limited by feedback in the synthesis pathway of penicillin. α-ketoglutarate + AcCoA → homocitrate → L-α-aminoadipic acid → L-lysine + β-lactamThe by-product, l-lysine, inhibits the production of homocitrate, so the presence of exogenous lysine should be avoided in penicillin production. The Penicillium cells are grown using a technique called fed-batch culture, in which the cells are constantly subject to stress, which is required for induction of penicillin production. The available carbon sources are also important: glucose inhibits penicillin production, whereas lactose does not. The pH and the levels of nitrogen, lysine, phosphate, and oxygen of the batches must also be carefully controlled.The biotechnological method of directed evolution has been applied to produce by mutation a large number of Penicillium strains. These techniques include error-prone PCR, DNA shuffling, ITCHY, and strand-overlap PCR. Semisynthetic penicillins are prepared to start from the penicillin nucleus 6-APA. Biosynthesis
Overall, there are three main and important steps to the biosynthesis of penicillin G (benzylpenicillin). The first step is the condensation of three amino acids—L-α-aminoadipic acid, L-cysteine, L-valine into a tripeptide. Before condensing into the tripeptide, the amino acid L-valine must undergo epimerization to become D-valine. The condensed tripeptide is named δ-(L-α-aminoadipyl)-L-cysteine-D-valine (ACV).
For example, resistance to vancomycin in S. aureus is due to additional peptidoglycan synthesis that makes the cell wall much thicker preventing effective penicillin entry. Resistance in Gram-negative bacteria is due to mutational variations in the structure and number of porins. In bacteria like Pseudomonas aeruginosa, there is reduced number of porins; whereas in bacteria like Enterobacter species, Escherichia coli and Klebsiella pneumoniae, there are modified porins such as non-specific porins (such as OmpC and OmpF groups) that cannot transport penicillin.Resistance due to PBP alterations is highly varied. A common case is found in Streptococcus pneumoniae where there is mutation in the gene for PBP, and the mutant PBPs have decreased binding affinity for penicillins. There are six mutant PBPs in S. pneumoniae, of which PBP1a, PBP2b, PBP2x and sometimes PBP2a are responsible for reduced binding affinity. S. aureus can activate a hidden gene that produces a different PBP, PBD2, which has low binding affinity for penicillins. There is a different strain of S. aureus named methicillin-resistant S. aureus (MRSA) which is resistant not only to penicllin and other β-lactams, but also to most antibiotics. The bacterial strain developed after introduction of methicillin in 1959. In MRSA, mutations in the genes (mec system) for PBP produce a variant protein called PBP2a (also termed PBP2), while making four normal PBPs. PBP2a has poor binding affinity for penicillin and also lacks glycosyltransferase activity required for complete peptidoglycan synthesis (which is carried out by the four normal PBPs).