LONDON - Current industrial processes for manufacturing derivatives of cephalosporin - a range of antibiotics with an important role in modern medicine - are expensive and involve the use of toxic chemicals. Many biotechnology companies have been investigating how to manipulate antibiotic-producing strains of fungi in order to simplify the process, but have had little success. Until now.
In the August edition of Nature Biotechnology, Jose-Luis Barredo and colleagues at the biotechnology company Antibioticos, in Leon, Spain, report that they have used genetic engineering to create a novel strain of fungus. The transformed strain produces a compound that easily can be converted into a chemical that forms the basis for manufacture of the antibiotic derivatives of cephalosporin.
Medically useful cephalosporin antibiotics are made from either 7-aminodeacetoxycephalosporanic acid (7-ADCA) or 7-aminocephalosporanic acid (7-ACA). In their article, titled "Environmentally safe production of 7-aminodeacetoxycephalosporanic acid (7-ADCA) using recombinant strains of Acremonium chrysogenum," Barredo and his colleagues write that current methods of producing 7-ADCA involve "a multistep, expensive and polluting chemical ring expansion of penicillin G to phenylacetyl-7-ADCA, from which the aromatic side chain is removed using a penicillin acylase."
It would be better, they added, to develop a process that makes use of enzymes or fermentation to produce large amounts of deacetoxycephalosporin C (DAOC), which could be deacylated to 7-ADCA, as this would be cheaper and more environmentally friendly.
That is what they set out to do. Barredo told BioWorld International, "We inactivated a gene called cefEF in a strain of A. chrysogenum, which is involved in the expansion of the five-member ring of penicillin into the six-member ring of the cephalosporins. This led to transformed strains of A. chrysogenum able to produce large amounts of penicillin N. We then inserted another gene into these transformed strains, called cefE, which was from Streptomyces clavuligerus."
The result was a strain able to convert penicillin N naturally into DAOC. In their conclusion, Barredo and his colleagues wrote: "DAOC constitutes the starting material for 7-ADCA production using a two-step enyzmatic bioconversion with D-amino acid oxidase and glutaryl acylase. Thus the entire synthesis of 7-ADCA is carried out using a green route that avoids chemical steps and constitutes a very efficient bioprocess from both industrial and environmental points of view."
"In this method," Barredo said, "A. chrysogenum produces lower quantities of cephalosporin than Penicillium produces of penicillin, but practically all the antibiotic produced is cephalosporin. There is no gross contamination with penicillin."
Antibioticos has applied for European and American patents on the process. Barredo said the traditional process involves high costs in recovering solvents and eliminating byproducts. "We hope that in the near future, the process we have developed should be competitive to the chemical one."
He and his colleagues now are working on improving the transformed strains to ensure high production levels. Barredo said, "The work described in Nature Biotechnology is just at fermentation flask level, but we are now developing scale-up of fermentation, in parallel with the strain improvement program. We hope to get better production in two ways - one, by obtaining new mutants by random mutagenesis of the recombinant microorganisms, and two, by trying to improve expression of the bacterial gene in the recombinant strains."