Question

Explain how a rational design approach was used to create semi synthetic aminoglycosides

Which classical approach was used to increase the production of penicillin?

Answer 1

The structural information of biomacromolecule-aminoglycoside complexes, a series of kanamycin B analogues were rationally designed and synthesized.

Most synthetic analogues exhibited good to excellent antibiotic activity against some typical drug-resistant bacteria. The disclosed results suggested that the C4'-position of aminoglycosides

Aminoglycosides are a group of natural antibiotics from Streptomyces that have been used in clinical practice for more than 50 years [4]. Their potent bactericidal activity relies upon binding specifically to the 16S rRNA of the 30S ribosomal subunit.

RNA recognition by natural aminoglycoside antibiotics depends on the 2-deoxystreptamine (2-DOS) scaffold which participates in specific hydrogen bonds with the ribosomal decoding-site target. Three-dimensional structure information has been used for the design of azepane-monoglycosides, building blocks for novel antibiotics in which 2-DOS is replaced by a heterocyclic scaffold. Azepane-glycosides showed target binding and translation inhibition in the low micromolar range and inhibited growth of Staphylococcus aureus, including aminoglycoside-resistant strains.

The discovery of penicillin by Alexander Fleming , classical strain improvement has been the main method to improve penicillin production.

Intense classical strain improvement has yielded industrial Penicillium chrysogenum strains that produce high titers of penicillin. These strains contain multiple copies of the penicillin biosynthesis cluster encoding the three key enzymes: δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine synthetase (ACVS), isopenicillin N synthase (IPNS), and isopenicillin N acyltransferase (IAT). The phenylacetic acid coenzyme A (CoA) ligase (PCL) gene encoding the enzyme responsible for the activation of the side chain precursor phenylacetic acid is localized elsewhere in the genome in a single copy. Since the protein level of IAT already saturates at low cluster copy numbers, IAT might catalyze a limiting step in high-yielding strains. Here, we show that penicillin production in high-yielding strains can be further improved by the overexpression of IAT while at very high levels of IAT the precursor 6-aminopenicillic acid (6-APA) accumulates. Overproduction of PCL only marginally stimulates penicillin production. These data demonstrate that in high-yielding strains IAT is the limiting factor and that this limitation can be alleviated by a balanced overproduction of this enzyme.

INTRODUCTION

Since the discovery of penicillin by Alexander Fleming classical strain improvement has been the main method to improve penicillin production. One of the most important phenomena in high-yielding Penicillium chrysogenum strains is the amplification of the penicillin biosynthetic gene cluster between tandem repeats (5, 18, 21). Other changes are the upregulation of genes involved in side chain activation, α-aminoadipic acid, and valine and cysteine biosynthesis . The penicillin biosynthesis cluster in P. chrysogenum consists of three genes, pcbAB, pcbC, and penDE , encoding the enzymes that catalyze the key biosynthetic conversions for penicillin production

A) Penicillin biosynthesis cluster: pcbAB coding for ACVS, pcbC coding for IPNS, and penDEcoding for IAT. Schematic representation of the penicillin biosynthetic pathway; the names of the enzymes and their corresponding genes are indicated next ...

Penicillin biosynthesis starts with the condensation of three amino acids, l-α-aminoadipic acid, l-cysteine, and l-valine, into the tripeptide δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine (LLD-ACV). This step is catalyzed by the nonribosomal peptide synthetase δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine synthetase (ACVS) encoded by pcbAB (1, 11–13). Next, the β-lactam ring is formed by isopenicillin N synthase (IPNS) encoded by the pcbC gene After isopenicillin N enters the microbody, the l-α-aminoadipic acid side chain is replaced by an activated phenyl- or phenoxyacetyl group yielding penicillin G or V, respectively. This conversion is catalyzed by acyl-coenzyme A: isopenicillin N acyltransferase (IAT), encoded by penDE(3, 17, 22, 25). IAT is capable of substituting l-α-aminoadipic acid with phenylacetic acid (PAA) or phenoxyacetic acid (POA) only when these precursors are activated to their coenzyme A (CoA) thioesters. One of the main enzymes capable of carrying out this reaction is phenylacetic acid CoA ligase (PCL) (14, 16). The phl (Pc22g14900) gene is not part of the penicillin biosynthetic gene cluster (for a review, see reference 24), but the PCL protein also localizes to the microbody lumen that contains the IAT enzyme.

Penicillin production by Penicillium chrysogenumis not only commercially important but arguably the most intensively investigated secondary-metabolic pathway in fungi. Isolation of the structural genes encoding the three main penicillin-biosynthetic enzymes has stimulated the use of molecular approaches to optimize yield and permitted genetic analysis of current production strains, which are themselves the products of 50 years of strain and process improvement. Parallel studies on the penicillin-producing genetic model organism Aspergillus nidulans are now addressing questions about the genetic regulation of primary and secondary metabolism,