Microbial production of N-acetylneuraminic acid by genetically engineered Escherichia coli

Previously we described the production of N-acetylneuraminic acid (NeuAc) from N-acetylglucosamine (GlcNAc) in a system combining recombinant Escherichia colt expressing GlcNAc 2-epimerase (slr1975) E coli expressing NeuAc synthetase (neuB) and Corynebacterium ammoniagenes However this system was unsuitable for large-scale production because of its complexity and low productivity To overcome these problems we constructed a recombinant E coli simultaneously overexpressing slr1975 and neuB This recombinant E coli produced 81 mM (25 g/L) NeuAc in 22 h without the addition of C ammoniagenes cells For manufacturing on an industrial scale it is preferable to use unconcentrated culture broth as the source of enzymes and therefore a high-density cell culture is required An acetate-resistant mutant strain of E coli (HN0074) was selected as the host strain because of its ability to grow to a high cell density The NeuAc aldolase gene of E cob HN0074 was disrupted by homologous recombination yielding E coli N18-14 which cannot degrade NeuAc After a 22 h reaction with 540 mM (120 g/L) GlcNAc in a 5 L jar fermenter the culture broth of E coli N18-14 overexpressing slr1975 and neuB contained 172 mM (53 g/L) NeuAc (C) 2010 Elsevier Ltd All rights reserved