title: Aerobic biosynthesis of hydrocinnamic acids in Escherichia coli with a strictly oxygen-sensitive enoate reductase.
abstract: 3-Phenylpropionic acid (3PPA) and 3-(4-hydroxyphenyl) propionic acid (HPPA) are important commodity aromatic acids widely used in food, pharmaceutical and chemical industries. Currently, 3PPA and HPPA are mainly manufactured through chemical synthesis, which contains multiple steps involving toxic solvents and catalysts harmful to environment. Therefore, replacement of such existing petroleum-derived approaches with simple and environmentally friendly biological processes is highly desirable for manufacture of these chemicals. Here, for the first time we demonstrated the de novo biosynthesis of 3PPA and HPPA using simple carbon sources in E. coli by extending the cinnamic acids biosynthesis pathways through biological hydrogenation. We first screened 11 2-enoate reductases (ER) from nine microorganisms, leading to efficient conversion of cinnamic acid and p-coumaric acid to 3PPA and HPPA, respectively. Surprisingly, we found a strictly oxygen-sensitive Clostridia ER capable of functioning efficiently in E. coli even under aerobic conditions. On this basis, reconstitution of the full pathways led to the de novo production of 3PPA and HPPA and the accumulation of the intermediates (cinnamic acid and p-coumaric acid) with cell toxicity. To address this problem, different expression strategies were attempted to optimize individual enzyme׳s expression level and minimize intermediates accumulation. Finally, the titers of 3PPA and HPPA reached 366.77mg/L and 225.10mg/L in shake flasks, respectively. This study not only demonstrated the potential of microbial approach as an alternative to chemical process, but also proved the possibility of using oxygen-sensitive enzymes under aerobic conditions.
authors.affiliation: State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, Beijing 100029, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Sun
authors.affiliation: BiotecEra Inc., Athens, GA 30602, USA.
authors.forename: Yuheng
authors.initials: Y
authors.lastname: Lin
authors.affiliation: State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, Beijing 100029, China.
authors.forename: Xiaolin
authors.initials: X
authors.lastname: Shen
authors.affiliation: College of Engineering, University of Georgia, Athens, GA 30602, USA.
authors.forename: Rachit
authors.initials: R
authors.lastname: Jain
authors.affiliation: State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, Beijing 100029, China.
authors.forename: Xinxiao
authors.initials: X
authors.lastname: Sun
authors.affiliation: State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, Beijing 100029, China. Electronic address: yuanqp@mail.buct.edu.cn.
authors.forename: Qipeng
authors.initials: Q
authors.lastname: Yuan
authors.affiliation: BiotecEra Inc., Athens, GA 30602, USA; BioChemical Engineering Program, College of Engineering, University of Georgia, Athens, GA 30602, USA. Electronic address: yajunyan@uga.edu.
authors.forename: Yajun
authors.initials: Y
authors.lastname: Yan
mesh_terms: D000332:Aerobiosis; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics; D003013:Clostridium / Q000201:enzymology / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D010088:Oxidoreductases / Q000096:biosynthesis / Q000235:genetics; D010100:Oxygen / Q000378:metabolism*; D010666:Phenylpropionates / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D010666:Phenylpropionates; C035253:3-phenylpropionic acid; D010088:Oxidoreductases; D010100:Oxygen
keywords: 3-(4-Hydroxyphenyl) propionic acid; 3-Phenylpropionic acid; Aromatic acids; Enoate reductase; Hydrocinnamic acids