Question

2. (a) Penicillin N 2 is converted into 7-ADCA 5 in Acremonium chrysogenum by the pathway shown below. Briefly explain why 7-ADCA 5 is a valuable compound in the pharmaceutical industry. (2 points) I ZI HH ES IZ od NH3 ż- NH3 cete áo Boo penicillin N 2 DAOC 3 'N 2 D-amino acid - CO2, -NH3 oxidase, O21 -HO HNS H2O glutaryl acylase Y CHE INCH CH3 7-ADCA 5 0 (b) Wild type strains of Acremonium chrysogenum normally produce deacetylcephalosporin C 6 rather than DAOC 3 because they express an enzyme (cefEF) that catalyses ring-expansion and an oxidation reaction. Briefly explain how you would obtain a strain of Acremonium chrysogenum that can only produce DAOC 3 given that there is an enzyme in Streptomyces clavuligerus (cefE) that catalyses only the ring-expansion reaction. (4 points) o Oo HWS- 0 Y T Hнн T X © AND 04 NH3 cefEF NH3 N LOH penicillin N 2 deacetylcephalosporin C6 (c) A strong promoter was needed on the gene encoding Streptomyces clavuligerus cefE to ensure large amounts of 7-ADCA were made by the engineered strain of Acremonium chrysogenum. Briefly explain this observation. (4 points)

Answer 1

2 a.

Cephalosporins are much more expensive than penicillins. One reason is that some cephalosporins (e.g. cephalexin) are made from penicillins by a number of chemical conversions. Another reason is that, so far, only cephalosporins with a D-5-amino-5-carboxypentanoyl side chain could be fermented. Cephalosporin C, by far the most important starting material in this respect, is very soluble in water at any pH, thus implying lengthy and costly isolation processes using cumbersome and expensive column technology. Cephalosporin C obtained in this way has to be converted into therapeutically used cephalosporins by a number of chemical and enzymatic conversions.

The methods currently favoured in industry to prepare the intermediate 7-ADCA involve complex chemical steps leading to the expansion and derivatization of penicillin G. One of the necessary chemical steps to produce 7-ADCA involves the expansion of the 5-membered penicillin ring structure to a 6-membered cephalosporin ring structure. This complex chemical processing is both expensive and noxious to the environment.

B.

There is a great desire to replace such chemical processes with enzymatical reactions such as enzymatic catalysis, preferably during fermentation. A key to the replacement of the chemical expansion process by a biological process is the central enzyme in the cephalosporin biosynthetic pathway, deacetoxycephalosporin C synthetase, or expandase.

The expandase enzyme from the bacterium Streptomyces clavuligerus was found to carry out, in some cases, penicillin ring expansions. When introduced into P. chrysogenum, it can convert the penicillin ring structure into the cephalosporin ring structure. The expandase enzyme has been well characterized both biochemically and functionally, as has its corresponding gene.

Another source for a ring expansion enzyme is the bacterium Nocardia lactamdurans (formerly Streptomyces lactamdurans). Both the biochemical properties of the enzyme and the DNA sequence of the gene have been described

Since the expandase catalyses the expansion of the 5-membered thiazolidine ring of penicillin N to the 6-membered dihydrothiazine ring of deacetoxycephalosporine C this enzyme would of course replace the ring expansion steps of the chemical process. Unfortunately, the enzyme works on the penicillin N intermediate of the cephalosporin biosynthetic pathway, but not on the readily available inexpensive penicillins as produced by P.chrysogenum, including penicillin G. Penicillin N is commercially not available and even when expanded, its D-aminoadipyl side chain cannot be easily removed by penicillin acylases.