Abstract
Thermophilic organisms hold great potential for industry due to their numerous advantages in biotechnological applications such as higher reaction rate, higher substrate loading, decreased susceptibility to reaction contamination, energy savings in industrial fermentations, and ability to express thermostable proteins that can be utilized in many important industrial processes. Bioprospecting for thermophiles will continue to reveal new enzymatic and metabolic paradigms with industrial applicability. In order to translate these paradigms to production scale, routine methods for microbial genetic engineering are needed, yet remain to be developed in many newly isolated thermophiles. Major challenges and recent developments in the establishment of reliable genetic systems in thermophiles are discussed. Here, we use a hyperthermophilic, cellulolytic bacterium, Caldicellulosiruptor bescii, as a case study to demonstrate the development of a genetic system for an industrially useful thermophile, describing in detail methods for transformation, genetic tool utilization, and chromosomal modification using targeted gene deletion and insertion techniques.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Schenk LK, Kelley JH (2010) Mothering an extremely low birth-weight infant: a phenomenological study. Adv Neonat Care 10:88–97
Taylor MP, van Zyl L, Tuffin IM, Leak DJ, Cowan DA (2011) Genetic tool development underpins recent advances in thermophilic whole-cell biocatalysts. Microb Biotechnol 4:438–448
Tamakoshi M, Oshima T (2011) Genetics of thermophiles. In: Horikoshi K (ed) Extremophiles handbook. Springer, Tokyo, pp 547–566
Cava F, Hidalgo A, Berenguer J (2009) Thermus thermophilus as biological model. Extremophiles 13:213–231
Lipscomb GL, Stirrett K, Schut GJ, Yang F, Jenney FE Jr, Scott RA, Adams MW, Westpheling J (2011) Natural competence in the hyperthermophilic archaeon Pyrococcus furiosus facilitates genetic manipulation: construction of markerless deletions of genes encoding the two cytoplasmic hydrogenases. Appl Environ Microbiol 77:2232–2238
Koyama Y, Hoshino T, Tomizuka N, Furukawa K (1986) Genetic transformation of the extreme thermophile Thermus thermophilus and of other Thermus spp. J Bacteriol 166:338–340
Sanders ME, Nicholson MA (1987) A method for genetic transformation of nonprotoplasted Streptococcus lactis. Appl Environ Microbiol 53:1730–1736
Wu LJ, Welker NE (1989) Protoplast transformation of Bacillus stearothermophilus NUB36 by plasmid DNA. J Gen Microbiol 135:1315–1324
Sato T, Fukui T, Atomi H, Imanaka T (2003) Targeted gene disruption by homologous recombination in the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. J Bacteriol 185:210–220
Fukui T, Atomi H, Kanai T, Matsumi R, Fujiwara S, Imanaka T (2005) Complete genome sequence of the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 and comparison with Pyrococcus genomes. Genome Res 15:352–363
Yao S, Mikkelsen MJ (2010) Identification and overexpression of a bifunctional aldehyde/alcohol dehydrogenase responsible for ethanol production in Thermoanaerobacter mathranii. J Mol Microbiol Biotechnol 19:123–133
Chung D, Cha M, Farkas J, Westpheling J (2013) Construction of a stable replicating shuttle vector for Caldicellulosiruptor species: use for extending genetic methodologies to other members of this genus. PLoS One 8:e62881
Olson DG, Lynd LR (2012) Chapter seventeen - transformation of clostridium Thermocellum by electroporation. In: Harry JG (ed) Methods in enzymology. Academic Press, Cambridge, pp 317–330
Maezato Y, Johnson T, McCarthy S, Dana K, Blum P (2012) Metal resistance and lithoautotrophy in the extreme thermoacidophile Metallosphaera sedula. J Bacteriol 194:6856–6863
Guss AM, Olson DG, Caiazza NC, Lynd LR (2012) Dcm methylation is detrimental to plasmid transformation in clostridium thermocellum. Biotechnol Biofuels 5:30
Chung D, Farkas J, Westpheling J (2013) Overcoming restriction as a barrier to DNA transformation in Caldicellulosiruptor species results in efficient marker replacement. Biotechnol Biofuels 6:82
Farkas J, Chung D, Debarry M, Adams MW, Westpheling J (2011) Defining components of the chromosomal origin of replication of the Hyperthermophilic Archaeon Pyrococcus furiosus needed for construction of a stable replicating shuttle vector. Appl Environ Microbiol 77:6343–6349
Tripathi SA, Olson DG, Argyros DA, Miller BB, Barrett TF, Murphy DM, McCool JD, Warner AK, Rajgarhia VB, Lynd LR, Hogsett DA, Caiazza NC (2010) Development of pyrF-based genetic system for targeted gene deletion in clostridium thermocellum and creation of a pta mutant. Appl Environ Microbiol 76:6591–6599
Tsoi TV, Chuvil'skaia NA, Atakishieva I, Dzhavakhishvili T, Akimenko VK (1987) Clostridium thermocellum--a new object of genetic studies. Mol Gen Mikrobiol Virusol 11:18–23
Mai V, Lorenz WW, Wiegel J (1997) Transformation of Thermoanaerobacterium sp. strain JW/SL-YS485 with plasmid pIKM1 conferring kanamycin resistance. FEMS Microbiol Lett 148:163–167
Zeldes BM, Keller MW, Loder AJ, Straub CT, Adams MW, Kelly RM (2015) Extremely thermophilic microorganisms as metabolic engineering platforms for production of fuels and industrial chemicals. Front Microbiol 6:1209
Chung DH, Huddleston JR, Farkas J, Westpheling J (2011) Identification and characterization of CbeI, a novel thermostable restriction enzyme from Caldicellulosiruptor bescii DSM 6725 and a member of a new subfamily of HaeIII-like enzymes. J Ind Microbiol Biotechnol 38:1867–1877
Yang SJ, Kataeva I, Hamilton-Brehm SD, Engle NL, Tschaplinski TJ, Doeppke C, Davis M, Westpheling J, Adams MW (2009) Efficient degradation of lignocellulosic plant biomass, without pretreatment, by the thermophilic anaerobe "Anaerocellum thermophilum" DSM 6725. Appl Environ Microbiol 75:4762–4769
Yang SJ, Kataeva I, Wiegel J, Yin Y, Dam P, Xu Y, Westpheling J, Adams MW (2010) Classification of 'Anaerocellum thermophilum' strain DSM 6725 as Caldicellulosiruptor bescii sp. nov. Int J Syst Evol Microbiol 60:2011–2015
Dam P, Kataeva I, Yang SJ, Zhou F, Yin Y, Chou W, Poole FL 2nd, Westpheling J, Hettich R, Giannone R, Lewis DL, Kelly R, Gilbert HJ, Henrissat B, Xu Y, Adams MW (2011) Insights into plant biomass conversion from the genome of the anaerobic thermophilic bacterium Caldicellulosiruptor bescii DSM 6725. Nucleic Acids Res 39:3240–3254
Brunecky R, Alahuhta M, Xu Q, Donohoe BS, Crowley MF, Kataeva IA, Yang SJ, Resch MG, Adams MW, Lunin VV, Himmel ME, Bomble YJ (2013) Revealing nature's cellulase diversity: the digestion mechanism of Caldicellulosiruptor bescii CelA. Science 342:1513–1516
Clausen A, Mikkelsen MJ, Schroder I, Ahring BK (2004) Cloning, sequencing, and sequence analysis of two novel plasmids from the thermophilic anaerobic bacterium Anaerocellum thermophilum. Plasmid 52:131–138
Svetlichnyi VA, Svetlichnaya TP, Chernykh NA, Zavarzin GA (1990) Anaerocellum Thermophilum Gen. Nov Sp. Nov. an extremely thermophilic cellulolytic eubacterium isolated from hot-springs in the valley of geysers. Microbiology 59:598–604
Farkas J, Chung D, Cha M, Copeland J, Grayeski P, Westpheling J (2013) Improved growth media and culture techniques for genetic analysis and assessment of biomass utilization by Caldicellulosiruptor bescii. J Ind Microbiol Biotechnol 40:41–49
Chung D, Young J, Bomble YJ, Vander Wall TA, Groom J, Himmel ME, Westpheling J (2015) Homologous expression of the Caldicellulosiruptor bescii CelA reveals that the extracellular protein is glycosylated. PLoS One 10:e0119508
Kim SK, Chung D, Himmel ME, Bomble YJ, Westpheling J (2016) Heterologous expression of family 10 xylanases from Acidothermus cellulolyticus enhances the exoproteome of Caldicellulosiruptor bescii and growth on xylan substrates. Biotechnol Biofuels 9:176
Kim SK, Chung D, Himmel ME, Bomble YJ, Westpheling J (2017) In vivo synergistic activity of a CAZyme cassette from Acidothermus cellulolyticus significantly improves the cellulolytic activity of the C. bescii exoproteome. Biotechnol Bioeng 114(11):2474–2480
Kim SK, Chung D, Himmel ME, Bomble YJ, Westpheling J (2017) Engineering the N-terminal end of CelA results in improved performance and growth of Caldicellulosiruptor bescii on crystalline cellulose. Biotechnol Bioeng 114:945–950
Chung D, Pattathil S, Biswal AK, Hahn MG, Mohnen D, Westpheling J (2014) Deletion of a gene cluster encoding pectin degrading enzymes in Caldicellulosiruptor bescii reveals an important role for pectin in plant biomass recalcitrance. Biotechnol Biofuels 7:147
Young J, Chung D, Bomble YJ, Himmel ME, Westpheling J (2014) Deletion of Caldicellulosiruptor bescii CelA reveals its crucial role in the deconstruction of lignocellulosic biomass. Biotechnol Biofuels 7:142
Cha M, Wang H, Chung D, Bennetzen JL, Westpheling J (2013) Isolation and bioinformatic analysis of a novel transposable element, ISCbe4, from the hyperthermophilic bacterium, Caldicellulosiruptor bescii. J Ind Microbiol Biotechnol 40:1443–1448
Widdel F, Pfennig N (1981) Studies on dissimilatory sulfate-reducing bacteria that decompose fatty acids. I. Isolation of new sulfate-reducing bacteria enriched with acetate from saline environments. Description of Desulfobacter postgatei gen. Nov., sp. nov. Arch Microbiol 129:395–400
Wolin EA, Wolin MJ, Wolfe RS (1963) Formation of methane by bacterial extracts. J Biol Chem 238:2882–2886
Adams MW, Holden JF, Menon AL, Schut GJ, Grunden AM, Hou C, Hutchins AM, Jenney FE Jr, Kim C, Ma K, Pan G, Roy R, Sapra R, Story SV, Verhagen MF (2001) Key role for sulfur in peptide metabolism and in regulation of three hydrogenases in the hyperthermophilic archaeon Pyrococcus furiosus. J Bacteriol 183:716–724
Chung D, Farkas J, Huddleston JR, Olivar E, Westpheling J (2012) Methylation by a unique alpha-class N4-cytosine methyltransferase is required for DNA transformation of Caldicellulosiruptor bescii DSM6725. PLoS One 7:e43844
Chung D, Cha M, Guss AM, Westpheling J (2014) Direct conversion of plant biomass to ethanol by engineered Caldicellulosiruptor bescii. Proc Natl Acad Sci U S A 111:8931–8936
Chung D, Cha M, Snyder EN, Elkins JG, Guss AM, Westpheling J (2015) Cellulosic ethanol production via consolidated bioprocessing at 75 degrees C by engineered Caldicellulosiruptor bescii. Biotechnol Biofuels 8:163
Chung D, Cha M, Young J, and Westpheling J (2014) Heterologous expression of extracellular Acidothermus cellulolyticus endoglucanase E1 in Caldicellulosiruptor bescii. In preparation
Cha M, Chung D, Westpheling J (2016) Deletion of a gene cluster for [Ni-Fe] hydrogenase maturation in the anaerobic hyperthermophilic bacterium Caldicellulosiruptor bescii identifies its role in hydrogen metabolism. Appl Microbiol Biotechnol 100:1823–1831
Cha M, Chung D, Elkins J, Guss A, Westpheling J (2013) Metabolic engineering of Caldicellulosiruptor bescii yields increased hydrogen production from lignocellulosic biomass. Biotechnol Biofuels 6:85
Acknowledgments
We thank Gina L. Lipscomb for sharing C. bescii transformation protocols. Funding was provided by the BioEnergy Science Center (BESC) and the Center for Bioenergy Innovation (CBI), from the U.S. Department of Energy Bioenergy Research Centers supported by the Office of Biological and Environmental Research in the DOE Office of Science. This work was authored in part by Alliance for Sustainable Energy, LLC, the Manager and Operator of the National Renewable Energy Laboratory for the U.S. Department of Energy (DOE) under Contract No. DE-AC36-08GO28308. The views expressed in the article do not necessarily represent the views of the DOE or the U.S. Government. The U.S. Government retains and the publisher, by accepting the article for publication, acknowledges that the U.S. Government retains a nonexclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this work, or allow others to do so, for U.S. Government purposes.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2020 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Chung, D., Sarai, N.S., Himmel, M.E., Bomble, Y.J. (2020). Genetics of Unstudied Thermophiles for Industry. In: Himmel, M., Bomble, Y. (eds) Metabolic Pathway Engineering. Methods in Molecular Biology, vol 2096. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0195-2_2
Download citation
DOI: https://doi.org/10.1007/978-1-0716-0195-2_2
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-0194-5
Online ISBN: 978-1-0716-0195-2
eBook Packages: Springer Protocols