Abstract
2,3-Butanediol (2,3-BD) is an organic compound, which is widely used as a fuel and fuel additive and applied in chemical, food, and pharmaceutical industries. Contemporary strategies for its economic synthesis include the development of microbial technologies that use starch as cheap and renewable feedstock. The present work encompasses the metabolic engineering of the excellent 2,3-BD producer Klebsiella pneumoniae G31. In order to perform direct starch conversion into 2,3-BD, the amyL gene encoding quite active, liquefying α-amylase in Bacillus licheniformis was cloned under lac promoter control in the recombinant K. pneumoniae G31-A. The enhanced extracellular over-expression of amyL led to the highest extracellular amylase activity (68 U/ml) ever detected in Klebsiella. The recombinant strain was capable of simultaneous saccharification and fermentation (SSF) of potato starch to 2,3-BD. In SSF batch process by the use of 200 g/l starch, the amount of total diols produced was 60.9 g/l (53.8 g/l 2,3-BD and 7.1 g/l acetoin), corresponding to 0.31 g/g conversion rate. The presented results are the first to show successful starch conversion to 2,3-BD by K. pneumoniae in a one-step process.
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References
Al Kazaz M, Desseaux V, Marchis-Mouren G, Prodanov E, Santimone M (1998) The mechanism of porcine pancreatic α-amylase. Inhibition of maltopentaose hydrolysis by acarbose, maltose and maltotriose. Eur J Biochem 252:100–107
Bradford M (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 72:248–254
Cao N, Xia Y, Gong CS, Tsao GT (1997) Production of 2,3-butanediol from pretreated corn cob by Klebsiella pneumoniae in the presence of fungal cellulase. Appl Biochem Biotechnol 63:129–139
Cheng KK, Liu Q, Zhang JA, Li JP, Xu JM, Wang GH (2010) Improved 2,3-butanediol production from corncob acid hydrolysate by fed-batch fermentation using Klebsiella oxytoca. Proc Biochem 45:613–616
Deutch CE (2002) Characterization of a salt-tolerant extracellular α-amylase from Bacillus dipsosauri. Lett Appl Microbiol 35:78–84
Garg SK, Jain A (1995) Fermentative production of 2,3-butanediol: a review. Bioresour Technol 51:103–109
Gawande BN, Patkar AY (2001) Purification and properties of a novel raw starch degrading-cyclodextrin glycosyltransferase from Klebsiella pneumoniae AS-22. Enzym Microb Technol 22:735–743
Hagihara H, Igarashi K, Hayashi Y, Endo K, Ikawa-Kitayama K, Ozaki K, Kawai S, Ito S (2001) Novel α-amylase that is highly resistant to chelating reagents and chemical oxidants from the alkaliphilic Bacillus isolate KSM-K38. Appl Environ Microbiol 67:1744–1750
Hmidet N, Bayoudh A, Berrin JG, Kanoun S, Juge N, Nasri M (2008) Purification and biochemical characterization of a novel α-amylase from Bacillus licheniformis NH1. Cloning, nucleotide sequence and expression of amyN gene in Escherichia coli. Proc Biochem 43:499–510
Ismail NF, Hamdan S, Mahadi NM, Murad AMA, Rabu A, Bakar FDA, Klappa P, Illias R (2011) A mutant l-asparaginase II signal peptide improves the secretion of recombinant cyclodextrin glucanotransferase and the viability of Escherichia coli. Biotechnol Lett 33:999–1005
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
Ji XJ, Huang H, Ouyang PK (2011) Microbial 2,3-butanediol production: a state of-the-art review. Biotechnol Adv 29:351–364
Ji XJ, Xia ZF, Fu NH, Nie ZK, Shen MQ, Tian QQ, Huang H (2013) Cofactor engineering through heterologous expression of an NADH oxidase and its impact on metabolic flux redistribution in Klebsiella pneumoniae. Biotechnol Biofuels 6:7
Jurchescu IM, Hamann J, Xiye Zhou X, Ortmann T, Kuenz A, Ulf Prüße U, Lang S (2013) Enhanced 2,3-butanediol production in fed-batch cultures of free and immobilized Bacillus licheniformis DSM 8785. Appl Microbiol Biotechnol 97:6715–6723
Kim DK, Rathnasingh C, Song H, Lee HJ, Seung D, Chang YK (2013) Metabolic engineering of a novel Klebsiella oxytoca strain for enhanced 2,3-butanediol production. J Biosci Bioeng 116:186–192
Kumar V, Sankaranarayanan M, Jae K, Durgapal M, Ashok S, Ko Y, Sarkar R, Park S (2012) Co-production of 3-hydroxypropionic acid and 1,3-propanediol from glycerol using resting cells of recombinant Klebsiella pneumoniae J2B strain overexpressing aldehyde dehydrogenase. Appl Microbiol Biotechnol 96:373–383
Li Z, Gu Z, Wang M, Du G, Wu J (2009) Delayed supplementation of glycine enhances extracellular secretion of the recombinant α-cyclodextrin glycosyltransferase in Escherichia coli. Appl Microbiol Biotechnol 85:553–561
Li D, Dai JY, Xiu ZL (2010) A novel strategy for integrated utilization of Jerusalem artichoke stalk and tuber for production of 2,3-butanediol by Klebsiella pneumoniae. Bioresour Technol 101:8342–8347
Ma C, Wang A, Qin J, Li L, Ai X, Jiang T, Tang H, Xu P (2009) Enhanced 2,3-butanediol production by Klebsiella pneumoniae SDM. Appl Microbiol Biotechnol 82:49–57
Momma M (2000) Cloning and sequencing of maltohexaose-producing amylase gene of Klebsiella pneumoniae. Biosci Biotechnol Biochem 64:428–431
Nakamura LK (1981) Lactobacillus amylovorus, a new starch-hydrolyzing species from cattle waste-corn fermentations. Int J Syst Bacteriol 31:56–63
Pantschev C, Klenz G, Hafner B (1981) Vergleichende charakterisierung von α-amylasepraparaten. Lebensmittelindustrie 28:71–74
Perego P, Converti A, Del Borghi M (2003) Effects of temperature, inoculum size and starch hydrolysate concentration on butanediol production by Bacillus licheniformis. Bioresour Technol 89:125–131
Petrov K, Petrova P (2009) High production of 2,3-butanediol from glycerol by Klebsiella pneumoniae G31. Appl Microbiol Biotechnol 84:659–665
Petrov K, Petrova P (2010) Enhanced production of 2,3-butanediol from glycerol by forced pH fluctuations. Appl Microbiol Biotechnol 87:943–949
Petrova P, Petrov K, Beschkov V (2009) Production of 1,3-propanediol from glycerol by newly isolated strains of Klebsiella pneumoniae. C R Acad Bulg Sci 62:233–242
Sambrook J, Russell D (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory, New York
Sun LH, Wang XD, Dai JY, Xiu ZL (2009) Microbial production of 2,3-butanediol from Jerusalem artichoke tubers by Klebsiella pneumoniae. Appl Microbiol Biotechnol 82:847–852
Syu MJ (2001) Biological production of 2,3-butanediol. Appl Microbiol Biotechnol 55:10–18
Tonkova A (1991) Effect of glucose and citrate on a-amylase production in Bacillus licheniformis. J Basic Microbiol 31:217–222
Wang AL, Wang Y, Jiang TY, Li LX, Ma CQ, Xu P (2010) Production of 2,3-butanediol from corncob molasses, a waste by-product in xylitol production. Appl Microbiol Biotechnol 87:965–970
Wong CL, Huang CC, Lu WB, Chen WM, Chang JS (2012) Producing 2,3-butanediol from agricultural waste using an indigenous Klebsiella sp. Zmd30 strain. Biochem Eng J 69:32–40
Xiu ZL, Zeng AP (2008) Present state and perspective of downstream processing of biologically produced 1,3-propanediol and 2,3-butanediol. Appl Microbiol Biotechnol 78:917–926
Zeng AP, Sabra W (2011) Microbial production of diols as platform chemicals: recent progresses. Curr Opin Biotechnol 22:749–757
Zheng Y, Zhang H, Zhao L, Wei L, Ma X, Wei D (2008) One-step production of 2,3-butanediol from starch by secretory over-expression of amylase in Klebsiella pneumoniae. J Chem Technol Biotechnol 83:1409–1412
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Tsvetanova, F., Petrova, P. & Petrov, K. 2,3-Butanediol production from starch by engineered Klebsiella pneumoniae G31-A. Appl Microbiol Biotechnol 98, 2441–2451 (2014). https://doi.org/10.1007/s00253-013-5418-4
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DOI: https://doi.org/10.1007/s00253-013-5418-4