Deletion of biofilm synthesis in Eubacterium limosum ATCC 8486 improves handling and transformation efficiency

Sanford, Patrick A.; Miller, Katherine G.; Hoyt, Kathryn O.; Woolston, Benjamin M.

doi:10.1093/femsle/fnad030

Deletion of biofilm synthesis in Eubacterium limosum ATCC 8486 improves handling and transformation efficiency

Journal Article · 01 January 2023 · FEMS Microbiology Letters (Online)

DOI:https://doi.org/10.1093/femsle/fnad030· OSTI ID:2422386

Sanford, Patrick A. ^[1]; Miller, Katherine G.; Hoyt, Kathryn O.;

OSTI

Abstract

Eubacterium limosum is an acetogenic bacterium of potential industrial relevance for its ability to efficiently metabolize a range of single carbon compounds. However, extracellular polymeric substance (EPS) produced by the type strain ATCC 8486 is a serious impediment to bioprocessing and genetic engineering. To remove these barriers, here we bioinformatically identified genes involved in EPS biosynthesis, and targeted several of the most promising candidates for inactivation, using a homologous recombination-based approach. Deletion of a single genomic region encoding homologues for epsABC, ptkA, and tmkA resulted in a strain incapable of producing EPS. This strain is significantly easier to handle by pipetting and centrifugation, and retains important wild-type phenotypes including the ability to grow on methanol and carbon dioxide and limited oxygen tolerance. Additionally, this strain is also more genetically tractable with a 2-fold increase in transformation efficiency compared to the highest previous reports. This work advances a simple, rapid protocol for gene knockouts in E. limosum using only the native homologous recombination machinery. These results will hasten the development of this organism as a workhorse for valorization of single carbon substrates, as well as facilitate exploration of its role in the human gut microbiota.

Research Organization:: Massachusetts Inst. of Technology (MIT), Cambridge, MA (United States)

Sponsoring Organization:: USDOE Advanced Research Projects Agency - Energy (ARPA-E)

DOE Contract Number:: AR0001511

OSTI ID:: 2422386

Journal Information:: FEMS Microbiology Letters (Online), Journal Name: FEMS Microbiology Letters (Online) Vol. 370; ISSN 1574-6968

Publisher:: Federation of European Microbiological Societies

Country of Publication:: United States

Language:: English

References (25)

Genetic engineering system for syngas-utilizing acetogen, Eubacterium limosum KIST612 Jeong, Jiyeong; Kim, Ji-Yeon; Park, Byeonghyeok Bioresource Technology Reports, Vol. 11 https://doi.org/10.1016/j.biteb.2020.100452	journal	September 2020
Carbohydrate analysis by a phenol–sulfuric acid method in microplate format Masuko, Tatsuya; Minami, Akio; Iwasaki, Norimasa Analytical Biochemistry, Vol. 339, Issue 1 https://doi.org/10.1016/j.ab.2004.12.001	journal	April 2005
Determination of the Genome and Primary Transcriptome of Syngas Fermenting Eubacterium limosum ATCC 8486 Song, Yoseb; Shin, Jongoh; Jeong, Yujin Scientific Reports, Vol. 7, Issue 1 https://doi.org/10.1038/s41598-017-14123-3	journal	October 2017
Quantitative Analysis of Polysaccharide Composition in Polyporus umbellatus by HPLC–ESI–TOF–MS Guo, Ning; Bai, Zongli; Jia, Weijuan Molecules, Vol. 24, Issue 14 https://doi.org/10.3390/molecules24142526	journal	July 2019
A Genetic System for Clostridium ljungdahlii: a Chassis for Autotrophic Production of Biocommodities and a Model Homoacetogen Leang, Ching; Ueki, Toshiyuki; Nevin, Kelly P. Applied and Environmental Microbiology, Vol. 79, Issue 4 https://doi.org/10.1128/AEM.02891-12	journal	November 2012
Clostridium acetobutylicum grows vegetatively in a biofilm rich in heteropolysaccharides and cytoplasmic proteins Liu, Dong; Yang, Zhengjiao; Chen, Yong Biotechnology for Biofuels, Vol. 11, Issue 1 https://doi.org/10.1186/s13068-018-1316-4	journal	November 2018
Extracellular polymeric substances are transient media for microbial extracellular electron transfer Xiao, Yong; Zhang, Enhua; Zhang, Jingdong Science Advances, Vol. 3, Issue 7 https://doi.org/10.1126/sciadv.1700623	journal	July 2017
Protocol for high-sensitivity/long linear-range spectrofluorimetric DNA quantification using ethidium bromide Bonasera, Veronica; Alberti, Saverio; Sacchetti, Andrea BioTechniques, Vol. 43, Issue 2 https://doi.org/10.2144/000112500	journal	August 2007
PerR acts as a switch for oxygen tolerance in the strict anaerobe Clostridium acetobutylicum Hillmann, Falk; Fischer, Ralf-Jörg; Saint-Prix, Florence Molecular Microbiology, Vol. 68, Issue 4, p. 848-860 https://doi.org/10.1111/j.1365-2958.2008.06192.x	journal	May 2008
Kinetics and physiological implications of the growth behaviour of Eubacterium limosum on glucose/methanol mixtures Loubiere, P.; Gros, E.; Paquet, V. Journal of General Microbiology, Vol. 138, Issue 5 https://doi.org/10.1099/00221287-138-5-979	journal	May 1992
Features of rumen and sewage sludge strains of Eubacterium limosum, a methanol- and H2-CO2-utilizing species. Genthner, B. R.; Davis, C. L.; Bryant, M. P. Applied and Environmental Microbiology, Vol. 42, Issue 1 https://doi.org/10.1128/aem.42.1.12-19.1981	journal	January 1981
Gut microbes from the phylogenetically diverse genus Eubacterium and their various contributions to gut health Mukherjee, Arghya; Lordan, Cathy; Ross, R. Paul Gut Microbes, Vol. 12, Issue 1 https://doi.org/10.1080/19490976.2020.1802866	journal	August 2020
Gene disruption with PCR products in Saccharomyces cerevisiae Lorenz, Michael C.; Muir, R. Scott; Lim, Eric Gene, Vol. 158, Issue 1 https://doi.org/10.1016/0378-1119(95)00144-U	journal	January 1995
When anaerobes encounter oxygen: mechanisms of oxygen toxicity, tolerance and defence Lu, Zheng; Imlay, James A. Nature Reviews Microbiology, Vol. 19, Issue 12 https://doi.org/10.1038/s41579-021-00583-y	journal	June 2021
The protein tyrosine kinases EpsB and PtkA differentially affect biofilm formation in Bacillus subtilis Gerwig, Jan; Kiley, Taryn B.; Gunka, Katrin Microbiology, Vol. 160, Issue 4 https://doi.org/10.1099/mic.0.074971-0	journal	April 2014
Expanding the genetic engineering toolbox for the metabolically flexible acetogen Eubacterium limosum Sanford, Patrick A.; Woolston, Benjamin M. Journal of Industrial Microbiology and Biotechnology, Vol. 49, Issue 5 https://doi.org/10.1093/jimb/kuac019	journal	July 2022
Cloning, expression, and characterization of a four-component O-demethylase from human intestinal bacterium Eubacterium limosum ZL-II Chen, Jia-Xing; Deng, Chao-Yin; Zhang, Ying-Tao Applied Microbiology and Biotechnology, Vol. 100, Issue 21 https://doi.org/10.1007/s00253-016-7626-1	journal	May 2016
Characterisation of Clostridium difficile Biofilm Formation, a Role for Spo0A Dawson, Lisa F.; Valiente, Esmeralda; Faulds-Pain, Alexandra PLoS ONE, Vol. 7, Issue 12 https://doi.org/10.1371/journal.pone.0050527	journal	December 2012
Development of highly characterized genetic bioparts for efficient gene expression in CO2-fixing Eubacterium limosum Song, Yoseb; Bae, Jiyun; Jin, Sangrak Metabolic Engineering, Vol. 72 https://doi.org/10.1016/j.ymben.2022.03.016	journal	July 2022
Genome Engineering of Eubacterium limosum Using Expanded Genetic Tools and the CRISPR-Cas9 System Shin, Jongoh; Kang, Seulgi; Song, Yoseb ACS Synthetic Biology, Vol. 8, Issue 9 https://doi.org/10.1021/acssynbio.9b00150	journal	August 2019
Production of the biocommodities butanol and acetone from methanol with fluorescent FAST-tagged proteins using metabolically engineered strains of Eubacterium limosum Flaiz, Maximilian; Ludwig, Gideon; Bengelsdorf, Frank R. Biotechnology for Biofuels, Vol. 14, Issue 1 https://doi.org/10.1186/s13068-021-01966-2	journal	May 2021
Rediverting carbon flux in Clostridium ljungdahlii using CRISPR interference (CRISPRi) Woolston, Benjamin M.; Emerson, David F.; Currie, Devin H. Metabolic Engineering, Vol. 48 https://doi.org/10.1016/j.ymben.2018.06.006	journal	July 2018
MtcB, a member of the MttB superfamily from the human gut acetogen Eubacterium limosum, is a cobalamin-dependent carnitine demethylase Kountz, Duncan J.; Behrman, Edward J.; Zhang, Liwen Journal of Biological Chemistry, Vol. 295, Issue 34 https://doi.org/10.1074/jbc.RA120.012934	journal	August 2020
New Horizons in Acetogenic Conversion of One-Carbon Substrates and Biological Hydrogen Storage Müller, Volker Trends in Biotechnology, Vol. 37, Issue 12 https://doi.org/10.1016/j.tibtech.2019.05.008	journal	December 2019
Exopolymeric substances (EPS) from Bacillus subtilis : polymers and genes encoding their synthesis: EPS from Bacillus subtilis Marvasi, Massimiliano; Visscher, Pieter T.; Casillas Martinez, Lilliam FEMS Microbiology Letters, Vol. 313, Issue 1 https://doi.org/10.1111/j.1574-6968.2010.02085.x	journal	November 2010

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