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Development of both type I-B and type II CRISPR/Cas genome editing systems in the cellulolytic bacterium Clostridium thermocellum.

ABSTRACT: The robust lignocellulose-solubilizing activity of C. thermocellum makes it a top candidate for consolidated bioprocessing for biofuel production. Genetic techniques for C. thermocellum have lagged behind model organisms thus limiting attempts to improve biofuel production. To improve our ability to engineer C. thermocellum, we characterized a native Type I-B and heterologous Type II Clustered Regularly-Interspaced Short Palindromic Repeat (CRISPR)/cas (CRISPR associated) systems. We repurposed the native Type I-B system for genome editing. We tested three thermophilic Cas9 variants (Type II) and found that GeoCas9, isolated from Geobacillus stearothermophilus, is active in C. thermocellum. We employed CRISPR-mediated homology directed repair to introduce a nonsense mutation into pyrF. For both editing systems, homologous recombination between the repair template and the genome appeared to be the limiting step. To overcome this limitation, we tested three novel thermophilic recombinases and demonstrated that exo/beta homologs, isolated from Acidithiobacillus caldus, are functional in C. thermocellum. For the Type I-B system an engineered strain, termed LL1586, yielded 40% genome editing efficiency at the pyrF locus and when recombineering machinery was expressed this increased to 71%. For the Type II GeoCas9 system, 12.5% genome editing efficiency was observed and when recombineering machinery was expressed, this increased to 94%. By combining the thermophilic CRISPR system (either Type I-B or Type II) with the recombinases, we developed a new tool that allows for efficient CRISPR editing. We are now poised to enable CRISPR technologies to better engineer C. thermocellum for both increased lignocellulose degradation and biofuel production.

SUBMITTER: Walker JE

PROVIDER: S-EPMC6926293 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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Development of both type I-B and type II CRISPR/Cas genome editing systems in the cellulolytic bacterium Clostridium thermocellum.

Walker Julie E JE Lanahan Anthony A AA Zheng Tianyong T Toruno Camilo C Lynd Lee R LR Cameron Jeffrey C JC Olson Daniel G DG Eckert Carrie A CA

Metabolic engineering communications 20191128

The robust lignocellulose-solubilizing activity of C. thermocellum makes it a top candidate for consolidated bioprocessing for biofuel production. Genetic techniques for C. thermocellum have lagged behind model organisms thus limiting attempts to improve biofuel production. To improve our ability to engineer C. thermocellum, we characterized a native Type I-B and heterologous Type II Clustered Regularly-Interspaced Short Palindromic Repeat (CRISPR)/cas (CRISPR associated) sy ...[more]

PMID: 31890588

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Project description:BackgroundMicrobial cellulose conversion by Clostridium thermocellum 27405 occurs predominantly through the activity of substrate-adherent bacteria organized in thin, primarily single cell-layered biofilms. The importance of cellulosic surface exposure to microbial hydrolysis has received little attention despite its implied impact on conversion kinetics.ResultsWe showed the spatial heterogeneity of fiber distribution in pure cellulosic sheets, which made direct measurements of biofilm colonization and surface penetration impossible. Therefore, we utilized on-line measurements of carbon dioxide (CO2) production in continuous-flow reactors, in conjunction with confocal imaging, to observe patterns of biofilm invasion and to indirectly estimate microbial accessibility to the substrate's surface and the resulting limitations on conversion kinetics. A strong positive correlation was found between cellulose consumption and CO2 production (R2?=?0.996) and between surface area and maximum biofilm activity (R2?=?0.981). We observed an initial biofilm development rate (0.46 h-1, 0.34 h-1 and 0.33 h-1) on Whatman sheets (#1, #598 and #3, respectively) that stabilized when the accessible surface was maximally colonized. The results suggest that cellulose conversion kinetics is initially subject to a microbial limitation period where the substrate is in excess, followed by a substrate limitation period where cellular mass, in the form of biofilms, is not limiting. Accessible surface area acts as an important determinant of the respective lengths of these two distinct periods. At end-point fermentation, all sheets were digested predominantly under substrate accessibility limitations (e.g., up to 81% of total CO2 production for Whatman #1). Integration of CO2 production rates over time showed Whatman #3 underwent the fastest conversion efficiency under microbial limitation, suggestive of best biofilm penetration, while Whatman #1 exhibited the least recalcitrance and the faster degradation during the substrate limitation period.ConclusionThe results showed that the specific biofilm development rate of cellulolytic bacteria such as C. thermocellum has a notable effect on overall reactor kinetics during the period of microbial limitation, when ca. 20% of cellulose conversion occurs. The study further demonstrated the utility of on-line CO2 measurements as a method to assess biofilm development and substrate digestibility pertaining to microbial solubilization of cellulose, which is relevant when considering feedstock pre-treatment options.

Dataset Information

Development of both type I-B and type II CRISPR/Cas genome editing systems in the cellulolytic bacterium Clostridium thermocellum.

Publications

Development of both type I-B and type II CRISPR/Cas genome editing systems in the cellulolytic bacterium <i>Clostridium thermocellum</i>.

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