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 k cat /K m values. The efficacy and industrial scalability of one of the selected variants, CefF GOS , 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. IMPORTANCE7ACA 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. Directedevolution 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. S emisynthetic cephalosporins, a class of ␤-lactam antibiotics, have shown remarkable effectiveness in the treatment of infectious diseases. Together with penicillins, they comprise nearly 65% of anti-infectives used worldwide. Their high specificity and low toxicity, coupled with the evolvability of newer generations of antibiotics, have led to ␤-lactams being by far the most fr...

Roles of 2-oxoglutarate oxygenases and isopenicillin N synthase in β-lactam biosynthesis

“…In order to enable production of cephalosporins with hydrophobic (or, potentially clinically useful side-chains) which are easier to purify, approaches guided by structural studies or directed evolution to engineer the natural cephalosporin biosynthesis pathway are being employed. 112 , 115 – 131 One approach involves using mutagenesis to alter the selectivity of DAOCS such that it efficiently accepts penicillins with hydrophobic side-chain ( e.g. penicillin G) rather than the natural δ-( d -α-aminoadipoyl) side chain.…”

“…The main focus of recent studies has been on modifications in the C -terminal region of DAOCS, which is involved in substrate recognition. 118 , 123 , 131 …”

Whole‐Cell Biotransformation of Penicillin G by a Three‐Enzyme Co‐expression System with Engineered Deacetoxycephalosporin C Synthase

“…Along this line, the first study on characterization and biocatalytic application of DAOCS was reported in 1970s [6] . Since then, great efforts have been explored, [2,7] including identifying different gene sources, [2] exploiting substrates specificity, [6] resolving crystal structures, [7c] analyzing heterologous expression [7a,b] as well as investigating the catalytic mechanisms [7d–i] . These studies revealed that the DAOCS‐catalyzed reactions depend on Fe 2+ , O 2 and the cofactor 2‐oxoglutarate (2OG), and the natural DAOCSs display a narrow substrate scope only toward the penicillin analogs.…”