Abstract
We engineered efficient 2,3-butanediol (23BD) production from cellobiose using Bacillus subtilis. First, we found that B. subtilis harboring an empty vector could produce 23BD from cellobiose. However, productivity using cellobiose as a carbon source was lower than that when using glucose. This lower productivity was improved by adding purified beta-glucosidase from Thermobifida fusca YX (Tfu_0937) in the fermentation. Encouraged by these findings, we found that hydrolysis of cellobiose to glucose was an important reaction of 23BD biosynthesis in B. subtilis using cellobiose. Hence, we created efficient 23BD production from cellobiose using exogenous Tfu_0937-expressing B. subtilis. Using the engineered strain, 21.2 g L−1 of 23BD was produced after 72 h of cultivation. The productivity and yield were 0.294 g L−1 h−1 and 0.35 g 23BD/g cellobiose, respectively. We successfully demonstrated efficient 23BD production from cellobiose by using BGL-expressing B. subtilis.
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References
Banka AL, Guralp SA, Gulari E (2014) Secretory expression and characterization of two hemicellulases, xylanase, and β-xylosidase, isolated from Bacillus subtilis M015. Appl Biochem Biotechnol 174(8):2702–2710. doi:10.1007/s12010-014-1219-1
Bayer EA, Chanzy H, Lamed R, Shoham Y (1998) Cellulose, cellulases and cellulosomes. Curr Opin Struct Biol 8(5):548–557
Biswas R, Yamaoka M, Nakayama H, Kondo T, Yoshida K, Bisaria VS, Kondo A (2012) Enhanced production of 2,3-butanediol by engineered Bacillus subtilis. Appl Microbiol Biotechnol 94(3):651–658. doi:10.1007/s00253-011-3774-5
Celińska E, Grajek W (2009) Biotechnological production of 2,3-butanediol—current state and prospects. Biotechnol Adv 27(6):715–725. doi:10.1016/j.biotechadv.2009.05.002
Chu H, Xin B, Liu P, Wang Y, Li L, Liu X, Zhang X, Ma C, Xu P, Gao C (2015) Metabolic engineering of Escherichia coli for production of (2S,3S)-butane-2,3-diol from glucose. Biotechnol Biofuels 15(8):143. doi:10.1186/s13068-015-0324-x
Fu J, Wang Z, Chen T, Liu W, Shi T, Wang G, Tang YJ, Zhao X (2014) NADH plays the vital role for chiral pure D-(-)-2,3-butanediol production in Bacillus subtilis under limited oxygen conditions. Biotechnol Bioeng 111(10):2126–2131. doi:10.1002/bit.25265
Gao D, Luan Y, Wang Q, Liang Q, Qi Q (2015) Construction of cellulose-utilizing Escherichia coli based on a secretable cellulase. Microb Cell Fact. 9;14(1):159. doi:10.1186/s12934–015-0349-7.
Jantama K, Polyiam P, Khunnonkwao P, Chan S, Sangproo M, Khor K, Jantama SS, Kanchanatawee S (2015) Efficient reduction of the formation of by-products and improvement of production yield of 2,3-butanediol by a combined deletion of alcohol dehydrogenase, acetate kinase-phosphotransacetylase, and lactate dehydrogenase genes in metabolically engineered Klebsiella oxytoca in mineral salts medium. Metab Eng 30:16–26. doi:10.1016/j.ymben.2015.04.004
John FJS, Rice JD, Preston JF (2006) Characterization of XynC from Bacillus subtilis subsp. subtilis strain 168 and analysis of its role in depolymerization of glucuronoxylan. J Bacteriol 188(24):8617–8626
Jung MY, Mazumdar S, Shin SH, Yang KS, Lee J, Oh MK (2014) Improvement of 2,3-butanediol yield in Klebsiella pneumoniae by deletion of the pyruvate formate-lyase gene. Appl Environ Microbiol 80(19):6195–6203. doi:10.1128/AEM.02069-14
Kay JE, Jewett MC (2015) Lysate of engineered Escherichia coli supports high-level conversion of glucose to 2,3-butanediol. Metab Eng 32:133–142. doi:10.1016/j.ymben.2015.09.015
Kim SJ, Seo SO, Jin YS, Seo JH (2013) Production of 2,3-butanediol by engineered Saccharomyces cerevisiae. Bioresour Technol 146:274–281. doi:10.1016/j.biortech.2013.07.081
Köpke M, Mihalcea C, Liew F, Tizard JH, Ali MS, Conolly JJ, Al-Sinawi B, Simpson SD (2011) 2,3-Butanediol production by acetogenic bacteria, an alternative route to chemical synthesis, using industrial waste gas. Appl Environ Microbiol 77(15):5467–5475. doi:10.1128/AEM.00355-11
Krispin O, Allmansberger R (1998) The Bacillus subtilis AraE protein displays a broad substrate specificity for several different sugars. J Bacteriol 180(12):3250–3252
Li L, Chen C, Li K, Wang Y, Gao C, Ma C, Xu P (2014a) Efficient simultaneous saccharification and fermentation of inulin to 2,3-butanediol by thermophilic Bacillus licheniformis ATCC 14580. Appl Environ Microbiol 80(20):6458–6464. doi:10.1128/AEM.01802-14
Li L, Li K, Wang K, Chen C, Gao C, Ma C, Xu P (2014b) Efficient production of 2,3-butanediol from corn Stover hydrolysate by using a thermophilic Bacillus licheniformis strain. Bioresour Technol 170:256–261. doi:10.1016/j.biortech.2014.07.101
Li L, Li K, Wang Y, Chen C, Xu Y, Zhang L, Han B, Gao C, Tao F, Ma C, Xu P (2015) Metabolic engineering of Enterobacter cloacae for high-yield production of enantiopure (2R,3R)-2,3-butanediol from lignocellulose-derived sugars. Metab Eng 28:19–27. doi:10.1016/j.ymben.2014.11.010
Matsumoto T, Shimada S, Hata Y, Tanaka T, Kondo A (2015) Multi-functional glycoside hydrolase: Blon_0625 from Bifidobacterium longum subsp. infantis ATCC 15697. Enzym Microb Technol 68:10–14. doi:10.1016/j.enzmictec.2014.10.001
Nan H, Seo SO, Oh EJ, Seo JH, Cate JH, Jin YS (2014) 2,3-Butanediol production from cellobiose by engineered Saccharomyces cerevisiae. Appl Microbiol Biotechnol 98(12):5757–5764. doi:10.1007/s00253-014-5683-x
Romero-Garcia S, Hernández-Bustos C, Merino E, Gosset G, Martinez A (2009) Homolactic fermentation from glucose and cellobiose using Bacillus subtilis. Microb Cell Fact 21;8:23. doi:10.1186/1475–2859-8-23
Rutter C, Chen R (2014) Improved cellobiose utilization in E. coli by including both hydrolysis and phosphorolysis mechanisms. Biotechnol Lett 36(2):301–307. doi:10.1007/s10529-013-1355-7
Sekar R, Shin HD, Chen R (2012) Engineering Escherichia coli cells for cellobiose assimilation through a phosphorolytic mechanism. Appl Environ Microbiol 78(5):1611–1614. doi:10.1128/AEM.06693-11
Shin HD, Yoon SH, Wu J, Rutter C, Kim SW, Chen RR (2012) High-yield production of meso-2,3-butanediol from cellodextrin by engineered E. coli biocatalysts. Bioresour Technol 118:367–373. doi:10.1016/j.biortech.2012.04.100
Spiridonov NA, Wilson DB (2001) Cloning and biochemical characterization of BglC, a beta-glucosidase from the cellulolytic actinomycete Thermobifida fusca. Curr Microbiol 42(4):295–301
Tanaka T, Kawabata H, Ogino C, Kondo A (2011) Creation of a cellooligosaccharide-assimilating Escherichia coli strain by displaying active beta-glucosidase on the cell surface via a novel anchor protein. Appl Environ Microbiol 77(17):6265–6270. doi:10.1128/AEM.00459-11
Tobisch S, Glaser P, Krüger S, Hecker M (1997) Identification and characterization of a new beta-glucoside utilization system in Bacillus subtilis. J Bacteriol 179(2):496–506
Wang PZ, Doi RH (1984) Overlapping promoters transcribed by Bacillus subtilis sigma 55 and sigma 37 RNA polymerase holoenzymes during growth and stationary phases. J Biol Chem 10;259(13):8619–25.
Wang Q, Chen T, Zhao X, Chamu J (2012) Metabolic engineering of thermophilic Bacillus licheniformis for chiral pure D-2,3-butanediol production. Biotechnol Bioeng 109(7):1610–1621. doi:10.1002/bit.24427
Westers L, Westers H, Quax WJ (2004) Bacillus subtilis as cell factory for pharmaceutical proteins: a biotechnological approach to optimize the host organism. Biochim Biophys Acta 11;1694(1–3):299–310.
Wang X, Lv M, Zhang L, Li K, Gao C, Ma C, Xu P (2013) Efficient bioconversion of 2,3-butanediol into acetoin using Gluconobacter oxydans DSM 2003. Biotechnol Biofuels. 31;6(1):155. doi:10.1186/1754–6834-6-155.
Yang T, Rao Z, Hu G, Zhang X, Liu M, Dai Y, Xu M, Xu Z, Yang ST (2015) Metabolic engineering of Bacillus subtilis for redistributing the carbon flux to 2,3-butanediol by manipulating NADH levels. Biotechnol Biofuels 27;8:129. doi:10.1186/s13068–015-0320-1
Yang T, Rao Z, Zhang X, Lin Q, Xia H, Xu Z, Yang S (2011) Production of 2,3-butanediol from glucose by GRAS microorganism Bacillus amyloliquefaciens. J Basic Microbiol 51(6):650–658. doi:10.1002/jobm.201100033
Zeng AP, Sabra W (2011) Microbial production of diols as platform chemicals: recent progresses. Curr Opin Biotechnol 22(6):749–757. doi:10.1016/j.copbio.2011.05.005
Zhang B, Li N, Wang Z, Tang YJ, Chen T, Zhao X (2015) Inverse metabolic engineering of Bacillus subtilis for xylose utilization based on adaptive evolution and whole-genome sequencing. Appl Microbiol Biotechnol 99(2):885–896. doi:10.1007/s00253-014-6131-7
Zhang L, Yang Y, Sun J, Shen Y, Wei D, Zhu J, Chu J (2010) Microbial production of 2,3-butanediol by a mutagenized strain of Serratia marcescens H30. Bioresour Technol 101(6):1961–1967. doi:10.1016/j.biortech.2009.10.052
Zhang X, Yang T, Lin Q, Xu M, Xia H, Xu Z, Li H, Rao Z (2011) Isolation and identification of an acetoin high production bacterium that can reverse transform 2,3-butanediol to acetoin at the decline phase of fermentation. World J Microbiol Biothechnol 27:2785–2790
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This work was supported by Special Coordination Funds for Promoting Science and Technology, provided by the Creation of Innovation Centers for Advanced Interdisciplinary Research Areas (Innovative Bioproduction Kobe), MEXT, Japan.
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Tanimura, K., Takashima, S., Matsumoto, T. et al. 2,3-Butanediol production from cellobiose using exogenous beta-glucosidase-expressing Bacillus subtilis . Appl Microbiol Biotechnol 100, 5781–5789 (2016). https://doi.org/10.1007/s00253-016-7326-x
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DOI: https://doi.org/10.1007/s00253-016-7326-x