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Inactivation of the Levansucrase Gene in Paenibacillus polymyxa DSM 365 Diminishes Exopolysaccharide Biosynthesis during 2,3-Butanediol Fermentation.
Appl Environ Microbiol. 2020 04 17; 86(9)AE

Abstract

The formation of exopolysaccharides (EPSs) during 2,3-butanediol (2,3-BD) fermentation by Paenibacillus polymyxa increases medium viscosity, which in turn presents considerable technical and economic challenges to 2,3-BD downstream processing. To eliminate EPS production during 2,3-BD fermentation, we used homologous recombination to disable the EPS biosynthetic pathway in P. polymyxa The gene which encodes levansucrase, the major enzyme responsible for EPS biosynthesis in P. polymyxa, was successfully disrupted. The P. polymyxa levansucrase null mutant produced 2.5 ± 0.1 and 1.2 ± 0.2 g/liter EPS on sucrose and glucose, respectively, whereas the wild type produced 21.7 ± 2.5 and 3.1 ± 0.0 g/liter EPS on the same substrates, respectively. These levels of EPS translate to 8.7- and 2.6-fold decreases in EPS formation by the levansucrase null mutant on sucrose and glucose, respectively, relative to that by the wild type, with no significant reduction in 2,3-BD production. Inactivation of EPS biosynthesis led to a considerable increase in growth. On glucose and sucrose, the cell biomass of the levansucrase null mutant (8.1 ± 0.8 and 6.5 ± 0.3 g/liter, respectively) increased 1.4-fold compared to that of the wild type (6.0 ± 0.1 and 4.6 ± 0.3 g/liter, respectively) grown on the same substrates. Evaluation of the genetic stability of the levansucrase null mutant showed that it remained genetically stable over fifty generations, with no observable decrease in growth or 2,3-BD formation, with or without antibiotic supplementation. Hence, the P. polymyxa levansucrase null mutant has potential for use as an industrial biocatalyst for a cost-effective large-scale 2,3-BD fermentation process devoid of EPS-related challenges.IMPORTANCE Given the current barrage of attention and research investments toward the production of next-generation fuels and chemicals, of which 2,3-butanediol (2,3-BD) produced by nonpathogenic Paenibacillus species is perhaps one of the most vigorously pursued, tools for engineering Paenibacillus species are intensely sought after. Exopolysaccharide (EPS) production during 2,3-BD fermentation constitutes a problem during downstream processing. Specifically, EPS negatively impacts 2,3-BD separation from the fermentation broth, thereby increasing the overall cost of 2,3-BD production. The results presented here demonstrate that inactivation of the levansucrase gene in P. polymyxa leads to diminished EPS accumulation. Additionally, a new method for an EPS assay and a simple protocol employing protoplasts for enhanced transformation of P. polymyxa were developed. Overall, although our study shows that levan is not the only EPS produced by P. polymyxa, it represents a significant first step toward developing cost-effective 2,3-BD fermentation devoid of EPS-associated complications during downstream processing.

Authors+Show Affiliations

Department of Animal Sciences, Ohio State Agricultural Research and Development Center (OARDC), The Ohio State University, Wooster, Ohio, USA.Bioenergy and Water Treatment Management Program, Agricultural Technical Institute, The Ohio State University, Wooster, Ohio, USA.Department of Food, Agricultural and Biological Engineering, Ohio State Agricultural Research and Development Center (OARDC), The Ohio State University, Wooster, Ohio, USA. Department of Horticulture and Crop Sciences, Ohio State Agricultural Research and Development Center (OARDC), The Ohio State University, Wooster, Ohio, USA.Department of Animal Sciences, Ohio State Agricultural Research and Development Center (OARDC), The Ohio State University, Wooster, Ohio, USA Ezeji.1@osu.edu.

Pub Type(s)

Journal Article
Research Support, U.S. Gov't, Non-P.H.S.

Language

eng

PubMed ID

32144108

Citation

Okonkwo, Christopher Chukwudi, et al. "Inactivation of the Levansucrase Gene in Paenibacillus Polymyxa DSM 365 Diminishes Exopolysaccharide Biosynthesis During 2,3-Butanediol Fermentation." Applied and Environmental Microbiology, vol. 86, no. 9, 2020.
Okonkwo CC, Ujor V, Cornish K, et al. Inactivation of the Levansucrase Gene in Paenibacillus polymyxa DSM 365 Diminishes Exopolysaccharide Biosynthesis during 2,3-Butanediol Fermentation. Appl Environ Microbiol. 2020;86(9).
Okonkwo, C. C., Ujor, V., Cornish, K., & Ezeji, T. C. (2020). Inactivation of the Levansucrase Gene in Paenibacillus polymyxa DSM 365 Diminishes Exopolysaccharide Biosynthesis during 2,3-Butanediol Fermentation. Applied and Environmental Microbiology, 86(9). https://doi.org/10.1128/AEM.00196-20
Okonkwo CC, et al. Inactivation of the Levansucrase Gene in Paenibacillus Polymyxa DSM 365 Diminishes Exopolysaccharide Biosynthesis During 2,3-Butanediol Fermentation. Appl Environ Microbiol. 2020 04 17;86(9) PubMed PMID: 32144108.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Inactivation of the Levansucrase Gene in Paenibacillus polymyxa DSM 365 Diminishes Exopolysaccharide Biosynthesis during 2,3-Butanediol Fermentation. AU - Okonkwo,Christopher Chukwudi, AU - Ujor,Victor, AU - Cornish,Katrina, AU - Ezeji,Thaddeus Chukwuemeka, Y1 - 2020/04/17/ PY - 2020/01/23/received PY - 2020/02/27/accepted PY - 2020/10/17/pmc-release PY - 2020/3/8/pubmed PY - 2020/3/8/medline PY - 2020/3/8/entrez KW - 2,3-butanediol KW - acetoin KW - cell efficiency KW - exopolysaccharide KW - homologous recombination KW - levan KW - levansucrase KW - polysaccharide polymerase KW - protoplast JF - Applied and environmental microbiology JO - Appl. Environ. Microbiol. VL - 86 IS - 9 N2 - The formation of exopolysaccharides (EPSs) during 2,3-butanediol (2,3-BD) fermentation by Paenibacillus polymyxa increases medium viscosity, which in turn presents considerable technical and economic challenges to 2,3-BD downstream processing. To eliminate EPS production during 2,3-BD fermentation, we used homologous recombination to disable the EPS biosynthetic pathway in P. polymyxa The gene which encodes levansucrase, the major enzyme responsible for EPS biosynthesis in P. polymyxa, was successfully disrupted. The P. polymyxa levansucrase null mutant produced 2.5 ± 0.1 and 1.2 ± 0.2 g/liter EPS on sucrose and glucose, respectively, whereas the wild type produced 21.7 ± 2.5 and 3.1 ± 0.0 g/liter EPS on the same substrates, respectively. These levels of EPS translate to 8.7- and 2.6-fold decreases in EPS formation by the levansucrase null mutant on sucrose and glucose, respectively, relative to that by the wild type, with no significant reduction in 2,3-BD production. Inactivation of EPS biosynthesis led to a considerable increase in growth. On glucose and sucrose, the cell biomass of the levansucrase null mutant (8.1 ± 0.8 and 6.5 ± 0.3 g/liter, respectively) increased 1.4-fold compared to that of the wild type (6.0 ± 0.1 and 4.6 ± 0.3 g/liter, respectively) grown on the same substrates. Evaluation of the genetic stability of the levansucrase null mutant showed that it remained genetically stable over fifty generations, with no observable decrease in growth or 2,3-BD formation, with or without antibiotic supplementation. Hence, the P. polymyxa levansucrase null mutant has potential for use as an industrial biocatalyst for a cost-effective large-scale 2,3-BD fermentation process devoid of EPS-related challenges.IMPORTANCE Given the current barrage of attention and research investments toward the production of next-generation fuels and chemicals, of which 2,3-butanediol (2,3-BD) produced by nonpathogenic Paenibacillus species is perhaps one of the most vigorously pursued, tools for engineering Paenibacillus species are intensely sought after. Exopolysaccharide (EPS) production during 2,3-BD fermentation constitutes a problem during downstream processing. Specifically, EPS negatively impacts 2,3-BD separation from the fermentation broth, thereby increasing the overall cost of 2,3-BD production. The results presented here demonstrate that inactivation of the levansucrase gene in P. polymyxa leads to diminished EPS accumulation. Additionally, a new method for an EPS assay and a simple protocol employing protoplasts for enhanced transformation of P. polymyxa were developed. Overall, although our study shows that levan is not the only EPS produced by P. polymyxa, it represents a significant first step toward developing cost-effective 2,3-BD fermentation devoid of EPS-associated complications during downstream processing. SN - 1098-5336 UR - https://www.unboundmedicine.com/medline/citation/32144108/Inactivation_of_the_Levansucrase_Gene_in_Paenibacillus_polymyxa_DSM_365_Diminishes_Exopolysaccharide_Biosynthesis_during_2,3-Butanediol_Fermentation L2 - http://aem.asm.org/cgi/pmidlookup?view=long&pmid=32144108 DB - PRIME DP - Unbound Medicine ER -
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