The structure of the capsular polysaccharide of Escherichia coli K5 is identical to that of N-acetyl-heparosan, a nonsulfated precursor of heparin, which makes this E. coli antigen an attractive starting point for the chemical synthesis of analogs of low-molecular-weight heparin. This polysaccharide is synthesized as a high-molecular-weight molecule that can be depolymerized by an enzyme displaying endo-␤-eliminase activity. The eliminase-encoding gene, designated elmA, has been cloned from E. coli K5 by expression in E. coli K-12. The K-12 genome is devoid of the elmA sequence. The elmA gene product is 820 amino acids long. Active recombinant eliminase is produced by K-12 cells in both cell-bound and secreted forms. Deletion analyses have shown that the C terminus and the N terminus are required for activity and secretion, respectively.The K5-specific capsular polysaccharide of Escherichia coli (K5 antigen) is composed of regular repeats of 4-␤-glucuronyl-(1-4)-␣-N-acetylglucosaminyl-1 (8). This structure is identical to that of N-acetyl-heparosan, a nonsulfated precursor of heparin. This similarity makes the K5 polysaccharide an attractive molecule to be used as the starting point for the chemical synthesis of pharmacologically active analogs of low-molecular-weight (LMW) heparin. N-Acetyl-heparosan is released into the medium by E. coli K5 as long polymers of LMW, ranging in size from 100 to 200 kDa (unpublished data). Under certain culture conditions, e.g., growth in glucose-containing complex medium (our results to be published elsewhere), E. coli K5 produces an enzyme which degrades extracellular Nacetyl-heparosan into LMW species by an endo-␤-elimination reaction. Like the N-acetyl-heparosan lyase of the K5-specific phage (2), this bacterial lyase can be used to fragment highmolecular-weight (HMW) K5 antigen in vitro. However, the bacterial lyase yields oligosaccharides of about 5 kDa as major end products, compared to up to 1,000-kDa fragments obtained with the phage enzyme (2). Since the desirable size of the starting material for synthesis of the heparin analog precisely corresponds to a 5-kDa oligosaccharide, the bacterial enzyme is of special interest for the preparation of LMW N-acetyl-heparosan. However, it is produced by E. coli K5 only at a low level. We have therefore isolated the corresponding gene by screening E. coli K-12 colonies capable of producing N-acetyl-heparosan eliminase after transformation with an E. coli K5 genomic DNA bank, and we have thus constructed K-12 strains that produced extracellular eliminase at high levels. MATERIALS AND METHODSBacterial strains and plasmids. The E. coli strains were K5 SEBR 3282 (O10:K5:H4) and K-12 RR1 (Boehringer GmbH, Mannheim, Germany). The N-acetyl-heparosan lyase-encoding gene was cloned from a library consisting of fragments obtained from partial Sau3A digestion of SEBR 3282 genomic DNA cloned into the BamHI site of pUC18. The ligation mix was used to transform RR1 cells to ampicillin resistance. About 7,000 colonies were obtained. The mean siz...

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