title: Metabolic engineering of Escherichia coli for biosynthesis of hyaluronic acid.
abstract: Engineering of hyaluronic acid (HA) biosynthetic pathway in recombinant Escherichia coli as production host is reported in this work. A hyaluronic acid synthase (HAS) gene, sphasA, from Sreptococcus pyogenes with the start codon gtg to atg mutant, was expressed in recombinant E. coli with or without the genes ugd, galF and glmU, which are analogs of hasB, hasC and hasD from Streptococcus, respectively, encoding UDP-glucose 6-dehygrogenase, Glucose-1-P uridyltransferase, and N-acetyl glucosamine uridyltransferase enzymes in the HA biosynthetic pathway. The single, double and triple organized artificial operons of sphasA, ugd, galF and glmU were designed and constructed using the inducible plasmid backbone of pMBAD. Only the triple expression recombinant, Top10/pMBAD-spABC, generated a relatively high titer of HA (approximately 48 mg/l at 48 h), indicating that both of the enzymes encoded by ugd and galF are essential for HA biosynthesis. A new gene of ssehasA with identical protein sequence of seHAS from Streptococcus equisimilis, was artificially synthesized after substituting all of the rare codons in the natural sehasA. The HA titer at 24 h flask culture increased to approximately 190 mg/l in sseAB and 160 mg/l in sseABC, respectively. Sorbitol could be used as another carbon source for HA accumulation, and the metabolic pathway for HA synthesis in a recombinant E. coli was presented. The concentration of Mg(2+) cofactor of HA synthase was optimized and a cell growth inhibition phenomenon was observed during HA accumulation. Molecular weight (MW) measurements revealed that the mean MW of HA produced from the recombinant E. coli under different conditions ranges from approximately 3.5x10(5) to 1.9x10(6)Da, indicating that the recombinant E. coli can be used as a potential host candidate for industrial production of HA.
authors.affiliation: Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Huimin
authors.initials: H
authors.lastname: Yu
authors.forename: Gregory
authors.initials: G
authors.lastname: Stephanopoulos
mesh_terms: D002850:Chromatography, Gel; D003062:Codon / Q000235:genetics; D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D005818:Genetic Engineering; D005822:Genetic Vectors; D014453:Glucuronosyltransferase / Q000737:chemistry / Q000235:genetics; D000076002:Hyaluronan Synthases; D006820:Hyaluronic Acid / Q000096:biosynthesis*; D008970:Molecular Weight; D009876:Operon / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013297:Streptococcus pyogenes / Q000201:enzymology / Q000235:genetics
chemical_list: D003062:Codon; D017931:DNA Primers; D004269:DNA, Bacterial; D006820:Hyaluronic Acid; D014453:Glucuronosyltransferase; D000076002:Hyaluronan Synthases