title: Caffeic acid production by simultaneous saccharification and fermentation of kraft pulp using recombinant Escherichia coli.
abstract: Caffeic acid (3,4-dihydroxycinnamic acid) serves as a building block for thermoplastics and a precursor for biologically active compounds and was recently produced from glucose by microbial fermentation. To produce caffeic acid from inedible cellulose, separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) reactions were compared using kraft pulp as lignocellulosic feedstock. Here, a tyrosine-overproducing Escherichia coli strain was metabolically engineered to produce caffeic acid from glucose by introducing the genes encoding a 4-hydroxyphenyllactate 3-hydroxylase (hpaBC) from Pseudomonas aeruginosa and tyrosine ammonia lyase (fevV) from Streptomyces sp. WK-5344. Using the resulting recombinant strain, the maximum yield of caffeic acid in SSF (233 mg/L) far exceeded that by SHF (37.9 mg/L). In the SSF with low cellulase loads (≤2.5 filter paper unit/g glucan), caffeic acid production was markedly increased, while almost no glucose accumulation was detected, indicating that the E. coli cells experienced glucose limitation in this culture condition. Caffeic acid yield was also negatively correlated with the glucose concentration in the fermentation medium. In SHF, the formation of by-product acetate and the accumulation of potential fermentation inhibitors increased significantly with kraft pulp hydrolysate than filter paper hydrolysate. The combination of these inhibitors had synergistic effects on caffeic acid fermentation at low concentrations. With lower loads of cellulase in SSF, less potential fermentation inhibitors (furfural, 5-hydroxymethyfurfural, and 4-hydroxylbenzoic acid) accumulated in the medium. These observations suggest that glucose limitation in SSF is crucial for improving caffeic acid yield, owing to reduced by-product formation and fermentation inhibitor accumulation.
authors.affiliation: Graduate School of Science, Technology and Innovation, Kobe University, 1-1 Rokkodai, Nada, Kobe, 657-8501, Japan.
authors.forename: Hideo
authors.initials: H
authors.lastname: Kawaguchi
authors.affiliation: Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1, Yayoi, Bunkyo, Tokyo, 113-8657, Japan.
authors.forename: Yohei
authors.initials: Y
authors.lastname: Katsuyama
authors.affiliation: Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1, Yayoi, Bunkyo, Tokyo, 113-8657, Japan.
authors.forename: Du
authors.initials: D
authors.lastname: Danyao
authors.affiliation: Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodai, Nada, Kobe, 657-8501, Japan.
authors.forename: Prihardi
authors.initials: P
authors.lastname: Kahar
authors.affiliation: Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodai, Nada, Kobe, 657-8501, Japan.
authors.forename: Sachiko
authors.initials: S
authors.lastname: Nakamura-Tsuruta
authors.affiliation: Graduate School of Science, Technology and Innovation, Kobe University, 1-1 Rokkodai, Nada, Kobe, 657-8501, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Teramura
authors.affiliation: Graduate School of Science, Technology and Innovation, Kobe University, 1-1 Rokkodai, Nada, Kobe, 657-8501, Japan.
authors.forename: Keiko
authors.initials: K
authors.lastname: Wakai
authors.affiliation: Graduate School of Science, Technology and Innovation, Kobe University, 1-1 Rokkodai, Nada, Kobe, 657-8501, Japan.
authors.forename: Kumiko
authors.initials: K
authors.lastname: Yoshihara
authors.affiliation: Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, 1-308, Suematsu, Nonoichi, Ishikawa, 921-8836, Japan.
authors.forename: Hiromichi
authors.initials: H
authors.lastname: Minami
authors.affiliation: Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodai, Nada, Kobe, 657-8501, Japan. ochiaki@port.kobe-u.ac.jp.
authors.forename: Chiaki
authors.initials: C
authors.lastname: Ogino
authors.affiliation: Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1, Yayoi, Bunkyo, Tokyo, 113-8657, Japan.
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Ohnishi
authors.affiliation: Graduate School of Science, Technology and Innovation, Kobe University, 1-1 Rokkodai, Nada, Kobe, 657-8501, Japan.|Biomass Engineering Research Division, RIKEN, 1-7-22 Suehiro, Turumi, Yokohama, Kanagawa, 230-0045, Japan.
authors.forename: Ahikiko
authors.initials: A
authors.lastname: Kondo
mesh_terms: D000085:Acetates / Q000378:metabolism; D000642:Ammonia-Lyases / Q000235:genetics; D018533:Biomass; D002109:Caffeic Acids / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002480:Cellulase / Q000378:metabolism; D003470:Culture Media / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005285:Fermentation; D005662:Furaldehyde / Q000031:analogs & derivatives / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008031:Lignin / Q000378:metabolism*; D060847:Metabolic Engineering / Q000379:methods; D011550:Pseudomonas aeruginosa / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D013302:Streptomyces / Q000235:genetics
chemical_list: D000085:Acetates; D002109:Caffeic Acids; D003470:Culture Media; D011994:Recombinant Proteins; C036909:lignocellulose; C008046:5-hydroxymethylfurfural; D008031:Lignin; D005662:Furaldehyde; D002480:Cellulase; D000642:Ammonia-Lyases; C023208:L-tyrosine ammonia-lyase; D005947:Glucose; C040048:caffeic acid
keywords: Caffeic acid; Escherichia coli; Kraft pulp; Lignocellulosic biomass; Simultaneous saccharification and fermentation