title: Modified Deacetylcephalosporin C Synthase for the Biotransformation of Semisynthetic Cephalosporins.
abstract: Deacetylcephalosporin C synthase (DACS), a 2-oxoglutarate-dependent oxygenase synthesized by Streptomyces clavuligerus, transforms an inert methyl group of deacetoxycephalosporin C (DAOC) into an active hydroxyl group of deacetylcephalosporin C (DAC) during the biosynthesis of cephalosporin. It is a step which is chemically difficult to accomplish, but its development by use of an enzymatic method with DACS can facilitate a cost-effective technology for the manufacture of semisynthetic cephalosporin intermediates such as 7-amino-cephalosporanic acid (7ACA) and hydroxymethyl-7-amino-cephalosporanic acid (HACA) from cephalosporin G. As the native enzyme showed negligible activity toward cephalosporin G, an unnatural and less expensive substrate analogue, directed-evolution strategies such as random, semirational, rational, and computational methods were used for systematic engineering of DACS for improved activity. In comparison to the native enzyme, several variants with improved catalytic efficiency were found. The enzyme was stable for several days and is expressed in soluble form at high levels with significantly higher kcat/Km values. The efficacy and industrial scalability of one of the selected variants, CefFGOS, were demonstrated in a process showing complete bioconversion of 18 g/liter of cephalosporin G into deacetylcephalosporin G (DAG) in about 80 min and showed reproducible results at higher substrate concentrations as well. DAG could be converted completely into HACA in about 30 min by a subsequent reaction, thus facilitating scalability toward commercialization. The experimental findings with several mutants were also used to rationalize the functional conformation deduced from homology modeling, and this led to the disclosure of critical regions involved in the catalysis of DACS.
  
  
  
  7ACA and HACA serve as core intermediates for the manufacture of several semisynthetic cephalosporins. As they are expensive, a cost-effective enzyme technology for the manufacture of these intermediates is required. Deacetylcephalosporin C synthase (DACS) was identified as a candidate enzyme for the development of technology from cephalosporin G in this study. Directed-evolution strategies were employed to enhance the catalytic efficiency of deacetylcephalosporin C synthase. One of the selected mutants of deacetylcephalosporin C synthase could convert high concentrations of cephalosporin G into DAG, which subsequently could be converted into HACA completely. As cephalosporin G is inexpensive and readily available, the technology would lead to a substantial reduction in the cost for these intermediates upon commercialization.
authors.affiliation: Biotechnology Division, R&D Centre, Orchid Chemicals and Pharmaceuticals Ltd., Chennai, India.
authors.forename: Nataraj
authors.initials: N
authors.lastname: Balakrishnan
authors.affiliation: Biotechnology Division, R&D Centre, Orchid Chemicals and Pharmaceuticals Ltd., Chennai, India.
authors.forename: Sadhasivam
authors.initials: S
authors.lastname: Ganesan
authors.affiliation: Biotechnology Division, R&D Centre, Orchid Chemicals and Pharmaceuticals Ltd., Chennai, India.
authors.forename: Padma
authors.initials: P
authors.lastname: Rajasekaran
authors.affiliation: Biotechnology Division, R&D Centre, Orchid Chemicals and Pharmaceuticals Ltd., Chennai, India.
authors.forename: Lingeshwaran
authors.initials: L
authors.lastname: Rajendran
authors.affiliation: Biotechnology Division, R&D Centre, Orchid Chemicals and Pharmaceuticals Ltd., Chennai, India.
authors.forename: Sivaprasad
authors.initials: S
authors.lastname: Teddu
authors.affiliation: Biotechnology Division, R&D Centre, Orchid Chemicals and Pharmaceuticals Ltd., Chennai, India durairaj@orchidpharma.com.
authors.forename: Micheal
authors.initials: M
authors.lastname: Durairaaj
mesh_terms: D000900:Anti-Bacterial Agents / Q000378:metabolism*; D001711:Biotransformation; D002511:Cephalosporins / Q000378:metabolism*; D010105:Oxygenases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D013302:Streptomyces / Q000201:enzymology*
chemical_list: D000900:Anti-Bacterial Agents; D002511:Cephalosporins; D010105:Oxygenases; C045288:deacetoxycephalosporin C hydroxylase