Breaking the Restriction Barriers and Applying CRISPRi as a Gene Silencing Tool in Pseudoclostridium thermosuccinogenes.

Pseudoclostridium thermosuccinogenes is a thermophilic bacterium capable of producing succinate from lignocellulosic-derived sugars and has the potential to be exploited as a platform organism. However, exploitation of P. thermosuccinogenes has been limited partly due to the genetic inaccessibility and lack of genome engineering tools. In this study, we established the genetic accessibility for P. thermosuccinogenes DSM 5809. By overcoming restriction barriers, transformation efficiencies of 102 CFU/µg plasmid DNA were achieved. To this end, the plasmid DNA was methylated in vivo when transformed into an engineered E. coli HST04 strain expressing three native methylation systems of the thermophile. This protocol was used to introduce a ThermodCas9-based CRISPRi tool targeting the gene encoding malic enzyme in P. thermosuccinogenes, demonstrating the principle of gene silencing. This resulted in 75% downregulation of its expression and had an impact on the strain's fermentation profile. Although the details of the functioning of the restriction modification systems require further study, in vivo methylation can already be applied to improve transformation efficiency of P. thermosuccinogenes. Making use of the ThermodCas9-based CRISPRi, this is the first example demonstrating that genetic engineering in P. thermosuccinogenes is feasible and establishing the way for metabolic engineering of this bacterium.

Characterization of sporulation dynamics of Pseudoclostridium thermosuccinogenes using flow cytometry.

Single-cell analysis of microbial population heterogeneity is a fast growing research area in microbiology due to its potential to identify and quantify the impact of subpopulations on microbial performance in, for example, industrial biotechnology, environmental biology, and pathogenesis. Although several tools have been developed, determination of population heterogenity in anaerobic bacteria, especially spore-forming clostridia species has been amply studied. In this study we applied single cell analysis techniques such as flow cytometry (FCM) and fluorescence-assisted cell sorting (FACS) on the spore-forming succinate producer Pseudoclostridium thermosuccinogenes. By combining FCM and FACS with fluorescent staining, we differentiated and enriched all sporulation-related morphologies of P. thermosuccinogenes. To evaluate the presence of metabolically active vegetative cells, a blend of the dyes propidium iodide (PI) and carboxy fluorescein diacetate (cFDA) tested best. Side scatter (SSC-H) in combination with metabolic indicator cFDA dye provided the best separation of sporulation populations. Based on this protocol, we successfully determined culture heterogeneity of P. thermosuccinogenes by discriminating between mature spores, forespores, dark and bright phase endospores, and vegetative cells populations. Henceforth, this methodology can be applied to further study sporulation dynamics and its impact on fermentation performance and product formation by P. thermosuccinogenes.

CRISPR interference (CRISPRi) as transcriptional repression tool for Hungateiclostridium thermocellum DSM 1313.

Hungateiclostridium thermocellum DSM 1313 has biotechnological potential as a whole-cell biocatalyst for ethanol production using lignocellulosic renewable sources. The full exploitation of H. thermocellum has been hampered due to the lack of simple and high-throughput genome engineering tools. Recently in our research group, a thermophilic bacterial CRISPR-Cas9-based system has been developed as a transcriptional suppression tool for regulation of gene expression. We applied ThermoCas9-based CRISPR interference (CRISPRi) to repress the H. thermocellum central metabolic lactate dehydrogenase (ldh) and phosphotransacetylase (pta) genes. The effects of repression on target genes were studied based on transcriptional expression and product formation. Single-guide RNA (sgRNA) under the control of native intergenic 16S/23S rRNA promoter from H. thermocellum directing the ThermodCas9 to the promoter region of both pta and ldh silencing transformants reduced expression up to 67% and 62% respectively. This resulted in 24% and 17% decrease in lactate and acetate production, correspondingly. Hence, the CRISPRi approach for H. thermocellum to downregulate metabolic genes can be used for remodelling of metabolic pathways without the requisite for genome engineering. These data established for the first time the feasibility of employing CRISPRi-mediated gene repression of metabolic genes in H. thermocellum DSM 1313.

Hijacking CRISPR-Cas for high-throughput bacterial metabolic engineering: advances and prospects.

High engineering efficiencies are required for industrial strain development. Due to its user-friendliness and its stringency, CRISPR-Cas-based technologies have strongly increased genome engineering efficiencies in bacteria. This has enabled more rapid metabolic engineering of both the model host Escherichia coli and non-model organisms like Clostridia, Bacilli, Streptomycetes and cyanobacteria, opening new possibilities to use these organisms as improved cell factories. The discovery of novel Cas9-like systems from diverse microbial environments will extend the repertoire of applications and broaden the range of organisms in which it can be used to create novel production hosts. This review analyses the current status of prokaryotic metabolic engineering towards the production of biotechnologically relevant products, based on the exploitation of different CRISPR-related DNA/RNA endonuclease variants.