Abstract
Recombinant Corynebacterium glutamicum harboring genes for pyruvate decarboxylase (pdc) and alcohol dehydrogenase (adhB) can produce ethanol under oxygen deprivation. We investigated the effects of elevating the expression levels of glycolytic genes, as well as pdc and adhB, on ethanol production. Overexpression of four glycolytic genes (pgi, pfkA, gapA, and pyk) in C. glutamicum significantly increased the rate of ethanol production. Overexpression of tpi, encoding triosephosphate isomerase, further enhanced productivity. Elevated expression of pdc and adhB increased ethanol yield, but not the rate of production. Fed-batch fermentation using an optimized strain resulted in ethanol production of 119 g/L from 245 g/L glucose with a yield of 95 % of the theoretical maximum. Further metabolic engineering, including integration of the genes for xylose and arabinose metabolism, enabled consumption of glucose, xylose, and arabinose, and ethanol production (83 g/L) at a yield of 90 %. This study demonstrated that C. glutamicum has significant potential for the production of cellulosic ethanol.
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References
Blombach B, Riester T, Wieschalka S, Ziert C, Youn JW, Wendisch VF, Eikmanns BJ (2011) Corynebacterium glutamicum tailored for efficient isobutanol production. Appl Environ Microbiol 77:3300–3310
Cao L, Tang X, Zhang X, Zhang J, Tian X, Wang J, Xiong M, Xiao W (2014) Two-stage transcriptional reprogramming in Saccharomyces cerevisiae for optimizing ethanol production from xylose. Metab Eng 24:150–159
Emmerling M, Bailey JE, Sauer U (1999) Glucose catabolism of Escherichia coli strains with increased activity and altered regulation of key glycolytic enzymes. Metab Eng 1:117–127
Emmerling M, Bailey JE, Sauer U (2000) Altered regulation of pyruvate kinase or co-overexpression of phosphofructokinase increases glycolytic fluxes in resting Escherichia coli. Biotechnol Bioeng 67:623–627
Farwick A, Bruder S, Schadeweg V, Oreb M, Boles E (2014) Engineering of yeast hexose transporters to transport D-xylose without inhibition by D-glucose. Proc Natl Acad Sci U S A 111:5159–5164
Goncalves DL, Matsushika A, de Sales BB, Goshima T, Bon EP, Stambuk BU (2014) Xylose and xylose/glucose co-fermentation by recombinant Saccharomyces cerevisiae strains expressing individual hexose transporters. Enzym Microb Technol 63:13–20
Hasegawa S, Uematsu K, Natsuma Y, Suda M, Hiraga K, Jojima T, Inui M, Yukawa H (2012) Improvement of the redox balance increases L-valine production by Corynebacterium glutamicum under oxygen deprivation. Appl Environ Microbiol 78:865–875
Hasegawa S, Suda M, Uematsu K, Natsuma Y, Hiraga K, Jojima T, Inui M, Yukawa H (2013) Engineering of Corynebacterium glutamicum for high-yield L-valine production under oxygen deprivation conditions. Appl Environ Microbiol 79:1250–1257
Huerta-Beristain G, Utrilla J, Hernandez-Chavez G, Bolivar F, Gosset G, Martinez A (2008) Specific ethanol production rate in ethanologenic Escherichia coli strain KO11 Is limited by pyruvate decarboxylase. J Mol Microbiol Biotechnol 15:55–64
Inui M, Kawaguchi H, Murakami S, Vertès AA, Yukawa H (2004a) Metabolic engineering of Corynebacterium glutamicum for fuel ethanol production under oxygen-deprivation conditions. J Mol Microbiol Biotechnol 8:243–254
Inui M, Murakami S, Okino S, Kawaguchi H, Vertès AA, Yukawa H (2004b) Metabolic analysis of Corynebacterium glutamicum during lactate and succinate productions under oxygen deprivation conditions. J Mol Microbiol Biotechnol 7:182–196
Jojima T, Inui M, Yukawa H (2013) Biorefinery Applications of Corynebacterium glutamicum. In: Yukawa H, Inui M (eds) Corynebacterium glutamicum. Springer, Berlin
Kawaguchi H, Vertès AA, Okino S, Inui M, Yukawa H (2006) Engineering of a xylose metabolic pathway in Corynebacterium glutamicum. Appl Environ Microbiol 72:3418–3428
Kawaguchi H, Sasaki M, Vertès AA, Inui M, Yukawa H (2008) Engineering of an L-arabinose metabolic pathway in Corynebacterium glutamicum. Appl Microbiol Biotechnol 77:1053–1062
Kinoshita S (1985) Glutamic acid bacteria. In: A L Demain and N A Solomon (eds) Biology of Industrial Microorganisms Benjamin Cumings. London pp 115–146
Klinke HB, Thomsen AB, Ahring BK (2004) Inhibition of ethanol-producing yeast and bacteria by degradation products produced during pre-treatment of biomass. Appl Microbiol Biotechnol 66:10–26
Kotrba P, Inui M, Yukawa H (2001) The ptsI gene encoding enzyme I of the phosphotransferase system of Corynebacterium glutamicum. Biochem Biophys Res Commun 289:1307–1313
Lau MW, Gunawan C, Balan V, Dale BE (2010) Comparing the fermentation performance of Escherichia coli KO11, Saccharomyces cerevisiae 424A(LNH-ST) and Zymomonas mobilis AX101 for cellulosic ethanol production. Biotechnol Biofuels 3:11
Lee SM, Jellison T, Alper HS (2014) Systematic and evolutionary engineering of a xylose isomerase-based pathway in Saccharomyces cerevisiae for efficient conversion yields. Biotechnol Biofuels 7:122
Okino S, Inui M, Yukawa H (2005) Production of organic acids by Corynebacterium glutamicum under oxygen deprivation. Appl Microbiol Biotechnol 68:475–480
Oreb M, Dietz H, Farwick A, Boles E (2012) Novel strategies to improve co-fermentation of pentoses with D-glucose by recombinant yeast strains in lignocellulosic hydrolysates. Bioengineering 3:347–351
Peter Smits H, Hauf J, Müller S, Hobley TJ, Zimmermann FK, Hahn-Hägerdal B, Nielsen J, Olsson L (2000) Simultaneous overexpression of enzymes of the lower part of glycolysis can enhance the fermentative capacity of Saccharomyces cerevisiae. Yeast 16:1325–1334
Sakai S, Tsuchida Y, Nakamoto H, Okino S, Ichihashi O, Kawaguchi H, Watanabe T, Inui M, Yukawa H (2007) Effect of lignocellulose-derived inhibitors on growth of and ethanol production by growth-arrested Corynebacterium glutamicum R. Appl Environ Microbiol 73:2349–2353
Sasaki M, Jojima T, Inui M, Yukawa H (2008) Simultaneous utilization of D-cellobiose, D-glucose, and D-xylose by recombinant Corynebacterium glutamicum under oxygen-deprived conditions. Appl Microbiol Biotechnol 81:691–699
Sasaki M, Jojima T, Kawaguchi H, Inui M, Yukawa H (2009) Engineering of pentose transport in Corynebacterium glutamicum to improve simultaneous utilization of mixed sugars. Appl Microbiol Biotechnol 85:105–115
Sasaki M, Jojima T, Inoue T, Yukawa H (2010) Xylitol production by recombinant Corynebacterium glutamicum under oxygen deprivation. Appl Microbiol Biotechnol 86:1057–1066
Smith J, van Rensburg E, Gorgens JF (2014) Simultaneously improving xylose fermentation and tolerance to lignocellulosic inhibitors through evolutionary engineering of recombinant Saccharomyces cerevisiae harbouring xylose isomerase. BMC Biotechnol 14:41
Suzuki N, Nonaka H, Tsuge Y, Okayama S, Inui M, Yukawa H (2005) Multiple large segment deletion method for Corynebacterium glutamicum. Appl Microbiol Biotechnol 69:151–161
Yamamoto S, Gunji W, Suzuki H, Toda H, Suda M, Jojima T, Inui M, Yukawa H (2012) Overexpression of genes encoding glycolytic enzymes in Corynebacterium glutamicum enhances glucose metabolism and alanine production under oxygen deprivation conditions. Appl Environ Microbiol 78:4447–4457
Yamamoto S, Suda M, Niimi S, Inui M, Yukawa H (2013) Strain optimization for efficient isobutanol production using Corynebacterium glutamicum under oxygen deprivation. Biotechnol Bioeng 110:2938–2948
Young EM, Tong A, Bui H, Spofford C, Alper HS (2014) Rewiring yeast sugar transporter preference through modifying a conserved protein motif. Proc Natl Acad Sci U S A 111:131–136
Yukawa H, Omumasaba CA, Nonaka H, Kos P, Okai N, Suzuki N, Suda M, Tsuge Y, Watanabe J, Ikeda Y, Vertès AA, Inui M (2007) Comparative analysis of the Corynebacterium glutamicum group and complete genome sequence of strain R. Microbiology 153:1042–1058
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Jojima, T., Noburyu, R., Sasaki, M. et al. Metabolic engineering for improved production of ethanol by Corynebacterium glutamicum . Appl Microbiol Biotechnol 99, 1165–1172 (2015). https://doi.org/10.1007/s00253-014-6223-4
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DOI: https://doi.org/10.1007/s00253-014-6223-4