2019 ; Vol.29-4: 625~632
|Author||Ji-Soo Lee, Soon-Kwang Hong, Chang-Ro Lee, Soo-Wan Nam, Sung-Jong Jeon, Yeon-Hee Kim|
|Place of duty||Division of Applied Bioengineering, College of Engineering, Dong-Eui University, Republic of Korea|
|Title||Production of Ethanol from Agarose by Unified Enzymatic Saccharification and Fermentation in Recombinant Yeast|
J. Microbiol. Biotechnol.2019 ;
|Abstract||The unified saccharification and fermentation (USF) system was developed for direct
production of ethanol from agarose. This system contains an enzymatic saccharification
process that uses three types of agarases and a fermentation process by recombinant yeast. The
pGMFα-HGN plasmid harboring AGAH71 and AGAG1 genes encoding β-agarase and the
NABH558 gene encoding neoagarobiose hydrolase was constructed and transformed into the
Saccharomyces cerevisiae 2805 strain. Three secretory agarases were produced by introducing an
S. cerevisiae signal sequence, and they efficiently degraded agarose to galactose, 3,6-anhydro-
L-galactose (AHG), neoagarobiose, and neoagarohexose. To directly produce ethanol from
agarose, the S. cerevisiae 2805/pGMFα-HGN strain was cultivated into YP-containing agarose
medium at 40°C for 48 h (for saccharification) and then 30°C for 72 h (for fermentation).
During the united cultivation process for 120 h, a maximum of 1.97 g/l ethanol from 10 g/l
agarose was produced. This is the first report on a single process containing enzymatic
saccharification and fermentation for direct production of ethanol without chemical
liquefaction (pretreatment) of agarose.|
|Key_word||Unified enzymatic saccharification and fermentation (USF) system, β-agarase, neoagarobiose hydrolase, bioethanol, recombinant yeast|
Yanagisawa M, Kawai S, Murata K. 2013. Strategies for the production of high concentrations of bioethanol from seaweeds: production of high concentrations of bioethanol from seaweeds. Bioengineered 4: 224-235.
John RP, Anisha GS, Nampoothiri KM, Pandey M. 2011. Micro and macroalgal biomass: a renewable source for bioethanol. Bioresour. Technol. 102: 186-193.
Araki CH. 1937. Acetylation of agar like substance of Gelidium amansii. J. Chem. Soc. Japan 58: 1338-1350.
Duckworth M, Yaphe W. 1971. The structure of agar. I. Fractionation of a complex mixture of polysaccharides. Carbohydr. Res. 16: 189-197.
Naik SN, Goud VV, Rout PK, Dalai AK. 2010. Production of first and second generation biofuels: a comprehensive review. Renew. Sust. Energ. Rev. 14: 578-597.
Jung YH, Kim IJ, Kim JJ, Oh KK, Han JI, Choi IG, et al. 2011. Ethanol production from oil palm trunks treated with aqueous ammonia and cellulase. Bioresour. Technol. 102:7307-7312.
Ko JK, Bak JS, Jung MW, Lee HJ, Choi IG, Kim TH, et al. 2009. Ethanol production from rice straw using optimized aqueous-ammonia soaking pretreatment and simultaneous saccharification and fermentation processes. Bioresour. Technol.100: 4374-4380.
Kumar P, Barrett DM, Delwiche MJ, Stroeve P. 2009. Methods for pretreatment of lignocellulosic biomass for efficient hydrolysis and biofuel production. Ind. Eng. Chem. Res. 48: 3713-3729.
Ando S, Arai I, Kiyoto K, Hanai S.1986. Identification of aromatic monomers in steam-exploded poplar and their influences on ethanol fermentation by Saccharomyces cerevisiae J. Ferment. Technol. 64: 567-570.
Olsson L, Hahn-Hägerdal B. 1996. Fermentation of lignocellulosic hydrolysates for ethanol production. Enzyme Microb. Technol. 18: 312-331.
Potin P, Richard C, Rochas C, Kloareg B. 1993. Purification and characterization of the α-agarase from Alteromonas agarlyticus (Cataldi) comb. nov., strain GJ1B. Eur. J. Biochem.214: 599-607.
Kirimura K, Masuda N, Iwasaki Y, Nakagawa H, Kobayashi R, Usami S. 1999. Purification and characterization of a novel β-agarase from an alkalophilic bacterium, Alteromonas sp. E-1. J. Biosci. Bioeng. 87: 436-441.
Ha SC, Lee S, Lee J, Kim HT, Ko HJ, Kim KH, et al. 2011. Crystal structure of a key enzyme in the agarolytic pathway, α-neoagarobiose hydrolase from Saccharophagus degradans 2-40. Biochem. Biophys. Res. Commun. 412: 238-244.
Lee S, Lee JY, Ha SC, Jung J, Shin DH, Kim KH, et al. 2009. Crystallization and preliminary X-ray analysis of neoagarobiose hydrolase from Saccharophagus degradans 2-40. Acta. Crystallogr. F: Struct. Biol. Cryst. Commun. 65: 1299-1301.
Hassairi I, Ben Amar R, Nonus M, Gupta BB. 2001. Production and separation of α -agarase from Altermonas agarlyticus GJ1B. Bioresour. Technol. 79: 47-51.
Seok JH, Kim HS, Hatada Y, Nam SW, Kim YH. 2012. Construction of an expression system for the secretory production of recombinant α-agarase in yeast. Biotechnol. Lett. 34: 1041-1049.
Kim HT, Lee S, Kim KH, Choi IG. 2012. The complete enzymatic saccharification of agarose and its application to simultaneous saccharification and fermentation of agarose for ethanol production. Bioresour. Technol. 107: 301-306.
Winston F, Dollard C, Ricupero-Hovasse SL. 1995. Construction of a set of convenient Saccharomyces cerevisiae strains that are isogenic to S288C. Yeast 11: 53-55.
Park DY, Chi WJ, Park JS, Chang YK, Hong SK. 2015. Cloning, expression, and biochemical characterization of a GH16 β-agarase AgaH71 from Pseudoalteromonas hodoensis H7. Appl. Biochem. Biotechnol. 175: 733-747.
Chi WJ, Park DY, Seo YB, Chang YK, Lee SY, Hong SK. 2014. Cloning, expression, and biochemical characterization of a novel GH16 β-agarase AgaG1 from Alteromonas sp. GNUM-1. Appl. Microbiol. Biotechnol. 98: 4545-4555.
Asghar S, Lee CR, Chi WJ, Kang DK, Hong SK. 2019. Molecular cloning and characterization of a novel coldadapted alkaline 1,3-α-3,6-anhydro-L-galactosidase, Ahg558, from Gayadomonas joobiniege G7. Appl. Biochem. Biotechnol. DOI: 10.1007/s12010-019-02963-w. [Epub ahead of print]
Kim MJ, Kim BH, Nam SW, Choi ES, Shin DH, Cho HY, et al. 2013. Efficient secretory expression of recombinant endoxylanase from Bacillus sp. HY-20 in Saccharomyces cerevisiae. J. Life Sci. 23: 863-868.
Miller GL. 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 31: 426-428.
Kim YH, Heo SY, Kim MJ, Lee JH, Kim YM, Nam SW. 2008. Optimal production of xylooligosaccharide by using recombinant endoxylanase from Bacillus subtilis. Kor. J. Life Sci. 18: 52-57.
Chomczynski P, Sacchi N. 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156-159.
Latchinian-Sadek L, Thomas DY. 1993. Expression, purification, and characterization of the yeast KEX1 gene product, a polypeptide precursor processing carboxypeptidase. J. Biol. Chem. 268: 534-540.
Kim MJ, Kim SH, Lee JH, Seo JH, Lee JH, Kim JH, et al. 2008. High-level secretory expression of human procarboxypeptidase B by Fed-Batch cultivation of Pichia pastoris and its partial characterization. J. Microbiol. Biotechnol. 18: 1938-1944.
Seok JH, Park HG, Lee SH, Nam SW, Jeon SJ, Kim JH, et al. 2010. High-level secretory expression of recombinant β-agarase from Zobellia galactanivorans in Pichia pastoris. Kor. J. Microbiol. Biotechnol. 38: 40-45.
Li RK, Chen Z, Ying XJ, Ng TB, Ye XY.2018. A novel GH16 beta-agarase isolated from a marine bacterium, Microbulbifer sp. BN3 and its characterization and high-level expression in Pichia pastoris. Int. J. Biol. Macromol. 119: 1164-1170.
Syazni, Yanagisawa M, Kasuu M, Ariga O, Nakasaki K. 2016. Direct production of ethanol from neoagarobiose using recombinant yeast that secretes α-neoagarooligosaccharide hydrolase. Enzyme Microb. Technol. 85: 82-89.
Yun EJ, Lee S, Kim HT, Pelton JG, Kim S, Ko HJ. et al. 2015. The novel catabolic pathway of 3,6-anhydro-l-galactose, the main component of red macroalgae, in a marine bacterium. Environ. Microbiol. 17: 1677-1688.