Abstract
In the present work, Bacillus subtilis was engineered as the cell factory for isobutanol production due to its high tolerance to isobutanol. Initially, an efficient heterologous Ehrlich pathway controlled by the promoter P43 was introduced into B. subtilis for the isobutanol biosynthesis. Further, investigation of acetolactate synthase of B. subtilis, ketol-acid reductoisomerase, and dihydroxy-acid dehydratase of Corynebacterium glutamicum responsible for 2-ketoisovalerate precursor biosynthesis showed that acetolactate synthase played an important role in isobutanol biosynthesis. The overexpression of acetolactate synthase led to a 2.8-fold isobutanol production compared with the control. Apart from isobutanol, alcoholic profile analysis also confirmed the existence of 1.21 g/L ethanol, 1.06 g/L 2-phenylethanol, as well as traces of 2-methyl-1-butanol and 3-methyl-1-butanol in the fermentation broth. Under microaerobic condition, the engineered B. subtilis produced up to 2.62 g/L isobutanol in shake-flask fed-batch fermentation, which was 21.3% higher than that in batch fermentation.
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Anagnostopoulos C, Spizizen J (1961) Requirements for transformation in Bacillus subtilis. J Bacteriol 81:741–746
Atsumi S, Hanai T, Liao JC (2008) Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels. Nature 451:86–89
Atsumi S, Li Z, Liao JC (2009) Acetolactate synthase from Bacillus subtilis serves as a 2-ketoisovalerate decarboxylase for isobutanol biosynthesis in Escherichia coli. Appl Environ Microbiol 75:6306–6311
Atsumi S, Wu T-Y, Eckl E-M, Hawkins SD, Buelter T, Liao JC (2010) Engineering the isobutanol biosynthetic pathway in Escherichia coli by comparison of three aldehyde reductase/alcohol dehydrogenase genes. Appl Microbiol Biotechnol 85:651–657
Bi CH, Zhang XL, Inogram LO, Prestson JF (2009) Genetic engineering of Enterobacter asburiae strain JDR-1 for efficient production of ethanol from hemicellulose hydrolysates. Appl Environ Microbiol 75:5743–5749
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Brochado AR, Matos C, MØller BL, Hansen J, Mortensen UH, Patil KR (2010) Improved vanillin production in baker’s yeast through in silico design. Microb Cell Fact 9:84. doi:10.1186/1475-2859-9-84
Carlini C, Macinai A, Marchionna M, Noviello M, Galletti AMR, Sbrana G (2003) Selective synthesis of isobutanol by means of the Guerbet reaction: part 3: methanol/n-propanol condensation by using bifunctional catalytic systems based on nickel, rhodium and ruthenium species with basic components. J Mol Catal A Chem 206:409–418
Chang A, Scheer M, Grote A, Schomburg I, Schomburg D (2009) BRENDA, AMENDA and FRENDA the enzyme information system: new content and tools in 2009. Nucleic Acid Res 37:588–592
Connor MR, Liao JC (2008) Engineering of an Escherichia coli strain for the production of 3-methyl-1-butanol. Appl Environ Microbiol 74:5769–5775
Cruz-Ramos H, Hoffmann T, Marino M, Nedjari H, Presecan-Siedel E, Dreesen O, Glaser P, Jahn D (2000) Fermentative metabolism of Bacillus subtilis: physiology and regulation of gene expression. J Bacteriol 182:3072–3080
de la Plaza M, Fernández de Palencia P, Peláez C, Requena T (2004) Biochemical and molecular characterization of alpha-ketoisovalerate decarboxylase, an enzyme involved in the formation of aldehydes from amino acid by Lactococcus lactis. FEMS Microbiol Lett 238:367–374
Duan YX, Chen T, Chen X, Zhao XM (2010) Overexpression of glucose-6-phosphate dehydrogenase enhances riboflavin production in Bacillus subtilis. Appl Microbiol Biotechnol 85:1907–1914
Eggeling I, Cordes C, Eggeling L, Sahm H (1987) Regulation of acetohydroxy acid synthase in Corynebacterium glutamicum during fermentation of α-ketobutyrate to L-isoleucine. Appl Microbiol Biotechnol 25:346–351
Etschmann MMW, Schrader J (2006) An aqueous-organic two-phase bioprocess for efficient production of the natural aroma chemicals 2-phenylethanol and 2-phenylethylacetate with yeast. Appl Microbiol Biotehcnol 71:440–443
Feist AM, Zielinski DC, Orth JD, Schellenberger J, Herrgard MJ, Palsson BØ (2010) Model-driven evaluation of the production potential for growth-coupled products of Escherichia coli. Metab Eng 12:173–186
Fischer CR, Klein-Marcuschamer D, Stephanopoulos G (2008) Selection and optimization of microbial hosts for biofuels production. Metab Eng 10:295–304
Garvie EI (1980) Bacterial lactate dehydrogenase. Microbiol Rev 44:106–139
Gollop N, Damri B, Chipman DM, Barak Z (1990) Physiological implications of the substrate specificities of acetohydroxy acid synthases from varied organisms. J Bacteriol 172:3444–3449
Gordillo MA, Sanz A, Sánchez A, Valero F, Montesinos JL, Lafuente J, Solà C (1998) Enhancement of Candida rugosa lipase production by using different control fed-batch operational strategies. Biotechnol Bioeng 60:156–168
Guérout-Fleury AM, Frandsen N, Stragier P (1996) Plasmids for ectopic integration in Bacillus subtilis. Gene 180:57–61
Hädicke O, Klamt S (2010) CASOP: a computational approach for strain optimization aiming at high productivity. J Biotechnol 147:88–101
Haima P, Bron S, Venema G (1987) The effect of restriction on shotgun cloning and plasmid stability in Bacillus subtilis Marburg. Mol Gen Genet 209:335–342
Hayhurst EJ, Kallas L, Hobbs JK, Foster SJ (2008) Cell wall peptidoglycan architecture in Bacillus subtilis. P Natl Acad Sci USA 105:14603–14608
Holtzclaw WD, Chapman LF (1975) Degradative acetolactate synthase of Bacillus subtilis: purification and properties. J Bacteriol 121:917–922
Inui M, Suda M, Kimura S, Yasuda K, Suzuki H, Toda H, Yamamoto S, Okino S, Suzuki N, Yukawa H (2008) Expression of Clostridium acetobutylicum butanol synthetic genes in Escherichia coli. Appl Microbiol Biotechnol 77:1305–1316
Ji XJ, Huang H, Li S, Du J, Lian M (2008) Enhanced 2,3-butanediol production by altering the mixed acid fermentation pathway in Klebsiella oxytoca. Biotechnol Lett 30:731–734
Ji XJ, Huang H, Zhu JG, Ren LJ, Nie ZK, Du J, Li S (2010) Engineering Klebsiella oxytoca for efficient 2,3-butanediol production through insertional inactivation of acetaldehyde dehydrogenase gene. Appl Microbiol Biotechnol 85:1751–1758
Jian J, Zhang SQ, Shi ZY, Wang W, Chen GQ, Wu Q (2010) Production of polyhydroxyalkanoates by Escherichia coli mutants with defected mixed acid fermentation pathways. Appl Microbiol Biotechnol 87:2247–2256
Karabektas M, Hosoz M (2009) Performance and emission characteristics of a diesel engine using isobutanol–diesel fuel blends. Renew Energy 34:1554–1559
Karmazyn-Campelli C, Fluss L, Leighton T, Stragier P (1992) The spoIIN279(ts) mutation affects the FtsA protein of Bacillus subtilis. Biochimie 74:689–694
Kunst F, Ogasawara N, Moszer I, Albertini AM, Alloni G, Azevedo V, Bertero G, Bessières P, Bolotin A, Borchert S, Borriss R, Boursier L, Brans A, Braun M, Brignell SC, Bron S, Brouillet S, Bruschi CV, Caldwell B, Capuano V, Nm C, Choi SK, Codani JJ, Connerton IF, Cummings NJ, Daniel RA, Denizot F, Devine KM, Düsterhöft A, Ehrlich SD, Emmerson PT, Entian KD, Errington J, Fabret C, Ferrari E, Foulger D, Fritz C, Fujita M, Fujita Y, Fuma S, Galizzi A, Galleron N, Ghim SY, Glaser P, Goffeau A, Golightly EJ, Grandi G, Guy BJ, Haga K, Haiech J, Harwood CR, Hénaut A, Hilbert H, Holsappel S, Hosono S, Hullo MF, Itaya M, Jones L, Joris B, Karamata D, Kasahara Y, Klaerr-Blanchard M, Klein C, Kobayashi Y, Koetter P, Koningstein G, Krogh S, Kumano M, Kurita K, Lapidus A, Lardinois S, Lauber J, Lazarevic V, Lee SM, Levine A, Liu H, Masuda S, Mauël C, Médigue C, Medina N, Rp M, Mizuno M, Moestl D, Nakai S, Noback M, Noone D, O’Reilly M, Ogawa K, Ogiwara A, Oudega B, Park SH, Parro V, Pohi TM, Portetelle D, Porwollik S, Prescott AM, Presecan E, Pujic P, Purnelle B, Rapoport G, Rey M, Reynolds S, Rieger M, Rivolta C, Rocha E, Roche B, Rose M, Sadaie Y, Sato T, Scanlan E, Schleich S, Schroeter R, Scoffone F, Sekiguchi J, Sekowska A, Seror SJ, Serror P, Shin BS, Soldo B, Sorokin A, Tacconi E, Takagi T, Takahashi H, Takemaru K, Takeuchi M, Tamakoshi A, Tanaka T, Terpstra P, Tognoni A, Tosato V, Uchiyama S, Vandenbol M, Vannier F, Vassarotti A, Viari A, Wambutt R, Wedler E, Wedler H, Weitzenegger T, Winters P, Wipat A, Yamamoto H, Yamane K, Yasumoto K, Yata K, Yoshida K, Yoshikawa HF, Zumstein E, Yoshikawa H, Danchin A (1997) The complete genome sequence of the Gram-positive bacterium Bacillus subtilis. Nature 390:249–256
Leonard E, Runguphan W, O’Connor S, Prather KJ (2009) Opportunities in metabolic engineering to facilitate scalable alkaloid production. Nat Chem Biol 5:292–300
Leyval D, Uy D, Delaunay S, Goergen JL, Engasser JM (2003) Characterization of the enzyme activities involved in the valine biosynthetic pathway in a valine-producing strain of Corynebacterium glutamicum. J Biotechnol 104:241–252
Liu X, Brune D, Vermaas W, Curtiss R (2010) Production and secretion of fatty acids in genetically engineered cyanobacteria. P Natl Acad Sci USA. doi:10.1073/pnas.1001946107
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method. Methods 25:4020–4408
Marles-Wright J, Grant T, Delumeau O, Delumeau O, van Duinen G, Firbank SJ, Lewis PJ, Murray JW, Newman JA, Quin MB, Race PR, Rohou A, Tichelaar W, van Heel M, Lewis RJ (2008) Molecular architecture of the “Stressosome”, a signal integration and transduction hub. Science 322:92–96
Martin VJJ, Pitera DJ, Withers ST, Newman JD, Keasling JD (2003) Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nat Biotechnol 21:796–802
Menkel E, Thierbach G, Eggeling L, Sahm H (1989) Influence of increased aspartate availability on lysine formation by a recombinant strain of Corynebacterium glutamicum and utilization of fumarate. Appl Environ Microbiol 55:684–688
Mu WM, Liu FL, Jia JH, Chen C, Zhang T, Jiang B (2009) 3-Phenyllactic acid production by substrate feeding and pH-control in fed-batch fermentation of Lactobacillus sp. SK007. Bioresour Technol 100:5226–5229
Mukherji S, van Oudenaarden A (2009) Synthetic biology: understanding biological design from synthetic circuits. Nat Rev Genet 10:859–871
Nakano MM, Dailly YP, Zuber P, Clark DP (1997) Characterization of anaerobic fermentative growth of Bacillus subtilis: identification of fermentation end products and genes required for growth. J Bacteriol 179:6749–6755
Nielsen DR, Leonard E, Yoon SH, Tseng HC, Yuan C, Prather KLJ (2009) Engineering alternative butanol production platforms in heterologous bacteria. Metab Eng 11:262–273
Petersohn A, Brigulla M, Haas S, Hoheisel JD, Völker U, Hecker M (2001) Global analysis of the general stress response of Bacillus subtilis. J Bacteriol 183:5617–5631
Qin X, Taber HW (1996) Transcriptional regulation of the Bacillus subtilis menp1 promoter. J Bacteriol 178:705–713
Renna MC, Najimudin N, Winik LR, Zahler SA (1993) Regulation of the Bacillus subtilis alsS, alsD, and alsR genes involved in post-exponential-phase production of acetoin. J Bacteriol 175:3863–3875
Romer-Garcia S, Hernández-Bustos C, Merino E, Gosset G, Martinez A (2009) Homolactic fermentation from glucose and cellobiose using Bacillus subtilis. Microb Cell Fact 8:23. doi:10.1186/1475-2859-8-23
Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor
Schleifer KH, Kraus J, Dvorak C, Kilpper-Balz R, Collins MD, Fischer W (1985) Transfer of Streptococcus lactis and related streptococci to the genus Lactococcus gen. nov. Syst Appl Microbiol 6:183–195
Smith KM, Cho KM, Liao JC (2010) Engineering Corynebacterium glutamicum for isobutanol production. Appl Microbiol Biotechnol 87:1045–1055
Steen EJ, Chan R, Prasad N, Myers S, Petzold CJ, Redding A, Ouellet M, Keasling JD (2008) Metabolic engineering of Saccharomyces cerevisiae for the production of n-butanol. Microb Cell Fact 7:36. doi:10.1186/1475-2859-7-36
Thykaer J, Nielsen J, Wohlleben W, Weber T, Gutknecht M, Lantz AE, Stegmann E (2010) Increased glycopeptide production after overexpression of shikimate pathway genes being part of the balhimycin biosynthetic gene cluster. Metab Eng 12:455–461
Unrean P, Trinh CT, Srienc F (2010) Rational design and construction of an efficient E. coli for production of diapolycopendioic acid. Metab Eng 12:112–122
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 259:8619–8625
Wong SL (1995) Advances in the use of Bacillus subtilis for the expression and secretion of heterologous proteins. Curr Opin Biotechnol 6:215–223
Zhanga K, Sawaya MR, Eisenberg DS, Liao JC (2008) Expanding metabolism for biosynthesis of nonnatural alcohols. P Natl Acad Sci USA 105:20653–22065
Zhu YB, Chen X, Chen T, Zhao XM (2007) Enhancement of riboflavin production by overexpression of acetolactate synthase in a pta mutant of Bacillus subtilis. FEMS Microbiol Lett 266:224–230
Acknowledgments
The authors appreciate the kind donation of strain B. subtilis 168 and plasmid pHP13, pDG364 and pDG1730 from Dr. Danier R. Zeigler and the Bacillus Genetic Stock Center, The Ohio State University. This research was financially supported by the National 973 Project of China (No. 2007CB714302), the Key Program of National Natural Science Foundation of China (Grant No. 20936002), and National Nature Science Foundation of China (No. 20976124 and No. 20906070).
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Li, S., Wen, J. & Jia, X. Engineering Bacillus subtilis for isobutanol production by heterologous Ehrlich pathway construction and the biosynthetic 2-ketoisovalerate precursor pathway overexpression. Appl Microbiol Biotechnol 91, 577–589 (2011). https://doi.org/10.1007/s00253-011-3280-9
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DOI: https://doi.org/10.1007/s00253-011-3280-9