title: Site-specific integration and constitutive expression of key genes into Escherichia coli chromosome increases shikimic acid yields.
abstract: As the key starting material for the chemical synthesis of Oseltamivir, shikimic acid (SA) has captured worldwide attention. Many researchers have tried to improve SA production by metabolic engineering, yet expression plasmids were used generally. In recent years, site-specific integration of key genes into chromosome to increase the yield of metabolites showed considerable advantages. The genes could maintain stably and express constitutively without induction. Herein, crucial genes aroG, aroB, tktA, aroE (encoding 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase, dehydroquinate synthase, transketolase and shikimate dehydrogenase, respectively) of SA pathway and glk, galP (encoding glucokinase and galactose permease) were integrated into the locus of ptsHIcrr (phosphoenolpyruvate: carbohydrate phosphotransferase system operon) in a shikimate kinase genetic defect strain Escherichia coli BW25113 (ΔaroL/aroK, DE3). Furthermore, another key gene ppsA (encoding phosphoenolpyruvate synthase) was integrated into tyrR (encoding Tyr regulator protein). As a result, SA production of the recombinant (SA5/pGBAE) reached to 4.14 g/L in shake flask and 27.41 g/L in a 5-L bioreactor. These data suggested that integration of key genes increased SA yields effectively. This strategy is environmentally friendly for no antibiotic is added, simple to handle without induction, and suitable for industrial production.
authors.affiliation: State Key Laboratory of New Drug and Pharmaceutical Process, Shanghai Institute of Pharmaceutical Industry, China State Institute of Pharmaceutical Industry, Shanghai 201203, China.
authors.forename: Xianglei
authors.initials: X
authors.lastname: Liu
authors.affiliation: State Key Laboratory of New Drug and Pharmaceutical Process, Shanghai Institute of Pharmaceutical Industry, China State Institute of Pharmaceutical Industry, Shanghai 201203, China. Electronic address: junlin621@126.com.
authors.forename: Jun
authors.initials: J
authors.lastname: Lin
authors.affiliation: State Key Laboratory of New Drug and Pharmaceutical Process, Shanghai Institute of Pharmaceutical Industry, China State Institute of Pharmaceutical Industry, Shanghai 201203, China.
authors.forename: Haifeng
authors.initials: H
authors.lastname: Hu
authors.affiliation: State Key Laboratory of New Drug and Pharmaceutical Process, Shanghai Institute of Pharmaceutical Industry, China State Institute of Pharmaceutical Industry, Shanghai 201203, China.
authors.forename: Bin
authors.initials: B
authors.lastname: Zhou
authors.affiliation: State Key Laboratory of New Drug and Pharmaceutical Process, Shanghai Institute of Pharmaceutical Industry, China State Institute of Pharmaceutical Industry, Shanghai 201203, China. Electronic address: baoquanzhu@126.com.
authors.forename: Baoquan
authors.initials: B
authors.lastname: Zhu
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D019149:Bioreactors; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics*; D003001:Cloning, Molecular; D004798:Enzymes / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D007218:Industrial Microbiology / Q000379:methods*; D060847:Metabolic Engineering / Q000379:methods*; D016254:Mutagenesis, Insertional; D053139:Oseltamivir / Q000302:isolation & purification; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D012765:Shikimic Acid / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D004798:Enzymes; D011994:Recombinant Proteins; D053139:Oseltamivir; D012765:Shikimic Acid; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System
keywords: Constitutive expression; E. coli; Shikimic acid; Site-specific integration