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 coli 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 81mM (25g/L) NeuAc in 22h 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. coli HN0074 was disrupted by homologous recombination yielding E. coli N18-14, which cannot degrade NeuAc. After a 22h reaction with 540mM (120g/L) GlcNAc in a 5L jar fermenter, the culture broth of E. coli N18-14 overexpressing slr1975 and neuB contained 172mM (53g/L) NeuAc.
Bioprocess Development Center, Kyowa Hakko Bio Co., Ltd, 2 Miyukigaoka, Tsukuba, Ibaraki 305-0841, Japan.
Mari
M
Ishikawa
Satoshi
S
Koizumi
D002238:Carbohydrate Epimerases / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D005818:Genetic Engineering / Q000379:methods*; D008954:Models, Biological; D019158:N-Acetylneuraminic Acid / Q000096:biosynthesis*; D010957:Plasmids
D002352:Carrier Proteins; D002238:Carbohydrate Epimerases; C024519:N-acyl-D-glucosamine 2-epimerase; D019158:N-Acetylneuraminic Acid