title: Metabolic engineering of Corynebacterium glutamicum for shikimate overproduction by growth-arrested cell reaction.
abstract: Corynebacterium glutamicum with the ability to simultaneously utilize glucose/pentose mixed sugars was metabolically engineered to overproduce shikimate, a valuable hydroaromatic compound used as a starting material for the synthesis of the anti-influenza drug oseltamivir. To achieve this, the shikimate kinase and other potential metabolic activities for the consumption of shikimate and its precursor dehydroshikimate were inactivated. Carbon flux toward shikimate synthesis was enhanced by overexpression of genes for the shikimate pathway and the non-oxidative pentose phosphate pathway. Subsequently, to improve the availability of the key aromatics precursor phosphoenolpyruvate (PEP) toward shikimate synthesis, the PEP: sugar phosphotransferase system (PTS) was inactivated and an endogenous myo-inositol transporter IolT1 and glucokinases were overexpressed. Unexpectedly, the resultant non-PTS strain accumulated 1,3-dihydroxyacetone (DHA) and glycerol as major byproducts. This observation and metabolome analysis identified glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-catalyzed reaction as a limiting step in glycolysis. Consistently, overexpression of GAPDH significantly stimulated both glucose consumption and shikimate production. Blockage of the DHA synthesis further improved shikimate yield. We applied an aerobic, growth-arrested and high-density cell reaction to the shikimate production by the resulting strain and notably achieved the highest shikimate titer (141g/l) and a yield (51% (mol/mol)) from glucose reported to date after 48h in minimal medium lacking nutrients required for cell growth. Moreover, comparable shikimate productivity could be attained through simultaneous utilization of glucose, xylose, and arabinose, enabling efficient shikimate production from lignocellulosic feedstocks. These findings demonstrate that C. glutamicum has significant potential for the production of shikimate and derived aromatic compounds.
authors.affiliation: Research Institute of Innovative Technology for the Earth, 9-2, Kizugawadai, Kizugawa, Kyoto 619-0292, Japan.
authors.forename: Takahisa
authors.initials: T
authors.lastname: Kogure
authors.affiliation: Research Institute of Innovative Technology for the Earth, 9-2, Kizugawadai, Kizugawa, Kyoto 619-0292, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Kubota
authors.affiliation: Research Institute of Innovative Technology for the Earth, 9-2, Kizugawadai, Kizugawa, Kyoto 619-0292, Japan.
authors.forename: Masako
authors.initials: M
authors.lastname: Suda
authors.affiliation: Research Institute of Innovative Technology for the Earth, 9-2, Kizugawadai, Kizugawa, Kyoto 619-0292, Japan.
authors.forename: Kazumi
authors.initials: K
authors.lastname: Hiraga
authors.affiliation: Research Institute of Innovative Technology for the Earth, 9-2, Kizugawadai, Kizugawa, Kyoto 619-0292, Japan; Graduate School of Biological Sciences, Nara Institute of Science and Technology, Nara, Japan. Electronic address: inui@rite.or.jp.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Inui
mesh_terms: D049109:Cell Proliferation / Q000502:physiology*; D048230:Corynebacterium glutamicum / Q000166:cytology / Q000502:physiology*; D004926:Escherichia coli / Q000235:genetics; D024861:Genetic Enhancement / Q000379:methods*; D005947:Glucose / Q000378:metabolism; D060847:Metabolic Engineering / Q000379:methods*; D053858:Metabolic Networks and Pathways / Q000502:physiology; D010429:Pentoses / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012765:Shikimic Acid / Q000378:metabolism*; D000073893:Sugars / Q000378:metabolism*; D015854:Up-Regulation / Q000502:physiology
chemical_list: D010429:Pentoses; D011994:Recombinant Proteins; D000073893:Sugars; D012765:Shikimic Acid; D017853:Phosphotransferases (Alcohol Group Acceptor); C022642:shikimate kinase; D005947:Glucose
keywords: Corynebacterium glutamicum; Metabolic engineering; Oseltamivir; Phosphoenolpyruvate; Shikimate pathway; Shikimic acid