Recombinant synthesis of hyaluronan by Agrobacterium sp.
Hyaluronan (HA) is a sugar polymer of a repeating disaccharide, beta1-3 D-N-acetylglucosamine (GlcNAc) beta1-4 D-glucuronic acid (GlcA). It finds applications in numerous biomedical procedures such as ophthalmic surgery and osteoarthritis treatment. Until recently, the only commercial sources were extraction of rooster combs and from fermentation of pathogenic Streptococcus. In this work, we demonstrate that metabolic engineering strategies enable the recombinant synthesis of hyaluronan in a safe microorganism. Agrobacterium sp. ATCC 31749 is a commercial production strain for a food polymer, Curdlan. A broad host range expression vector was successfully developed to express the 3 kb HA synthase gene from Pasteurella multocida, along with a kfiD gene encoding UDP-glucose dehydrogenase from Escherichia coli K5 strain. Coexpression of these two heterologous enzymes enables Agrobacterium to produce HA. Hyaluronan was accumulated up to 0.3 g/L in shaker flask cultivation. The molecular weight of the polymer from various Agrobacterium strains is in the range of 0.7-2 MD. To our knowledge, this is the first successful recombinant hyaluronan synthesis in a Gram-negative bacterium that naturally produces a food product. The ease of genetic modifications provides future opportunities to tailor properties of polymers for specific applications.
Mao
Zichao
Z
School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, 311 Ferst Drive, Atlanta, Georgia 30332-0100, USA.
Chen
Rachel Ruizhen
RR
D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D014453:Glucuronosyltransferase / Q000235:genetics / Q000378:metabolism*; D000076002:Hyaluronan Synthases; D006820:Hyaluronic Acid / Q000235:genetics / Q000378:metabolism*; D016979:Pasteurella multocida / Q000201:enzymology* / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000378:metabolism; D012231:Rhizobium / Q000235:genetics / Q000378:metabolism*; D014533:Uridine Diphosphate Glucose Dehydrogenase / Q000235:genetics / Q000378:metabolism*
D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D006820:Hyaluronic Acid; C097522:KfiD protein, E coli; D014533:Uridine Diphosphate Glucose Dehydrogenase; D014453:Glucuronosyltransferase; D000076002:Hyaluronan Synthases