RRNPP-type quorum-sensing systems regulate solvent formation, sporulation and cell motility in Clostridium saccharoperbutylacetonicum.

BACKGROUND: Clostridium saccharoperbutylacetonicum N1-4 (HMT) is a strictly anaerobic, spore-forming Gram-positive bacterium capable of hyper-butanol production through the well-known acetone-butanol-ethanol fermentation process. Recently, five putative RRNPP-type QSSs (here designated as QSS1 to QSS5) were predicted in this bacterial strain, each of which comprises a putative RRNPP-type regulator (QssR1 to QssR5) and a cognate signaling peptide precursor (QssP1 to QssP5). In addition, both proteins are encoded by the same operon. The functions of these multiple RRNPP-type QSSs are unknown. RESULTS: To elucidate the function of multiple RRNPP-type QSSs as related to cell metabolism and solvent production in N1-4 (HMT), we constructed qssR-deficient mutants ΔR1, ΔR2, ΔR3 and ΔR5 through gene deletion using CRISPR-Cas9 and N1-4-dcas9-R4 (with the QssR4 expression suppressed using CRISPR-dCas9). We also constructed complementation strains by overexpressing the corresponding regulator gene. Based on systematic characterization, results indicate that QSS1, QSS2, QSS3, and QSS5 positively regulate the sol operon expression and thus solvent production, but they likely negatively regulate cell motility. Consequently, QSS4 might not directly regulate solvent production, but positively affect cell migration. In addition, QSS3 and QSS5 appear to positively regulate sporulation efficiency. CONCLUSIONS: Our study provides the first insights into the roles of multiple RRNPP-type QSSs of C. saccharoperbutylacetonicum for the regulation of solvent production, cell motility, and sporulation. Results of this study expand our knowledge of how multiple paralogous QSSs are involved in the regulation of essential bacterial metabolism pathways.

Markerless genome editing in Clostridium beijerinckii using the CRISPR-Cpf1 system.

CRISPR-Cpf1 is a type V CRISPR system that has recently been exploited for genome engineering purposes. Compared to the well-known Streptococcus pyogenes CRISPR-Cas9 system, the effector protein Cpf1 recognizes T-rich protospacer-adjacent motif (PAM) instead of G-rich PAM (used by CRISPR-Cas9), which could offer a substantial expansion of the existing genetic toolbox for genome editing. In this study, we report the implementation of the Acidaminococcus sp. Cpf1 (AsCpf1) for markerless genome engineering in Clostridium beijerinckii, a prominent species for biosolvent production through the well-known Acetone-Butanol-Ethanol (ABE) pathway. A lactose inducible promoter was used to control the expression of AsCpf1 to decrease its toxicity, while a constitutive small RNA promoter was employed to drive the expression of pre-crRNA. A One-Step-Assembly (OSA) approach was employed to construct the CRISPR-Cpf1-based vector in one single step, which simplified and streamlined the plasmid construction process. Using the customized CRISPR-Cpf1 system, we successfully deleted spo0A and pta genes in C. beijerinckii, with an editing efficiency of up to 100%. Altogether, our results demonstrated the easy programmability and high efficiency of the CRISPR-Cpf1 system for versatile genome engineering purposes. This study provides valuable guidance and essential references for repurposing the CRISPR-Cpf1 system for genome engineering in other microorganisms.

Exploiting endogenous CRISPR-Cas system for multiplex genome editing in Clostridium tyrobutyricum and engineer the strain for high-level butanol production.

Although CRISPR-Cas9/Cpf1 have been employed as powerful genome engineering tools, heterologous CRISPR-Cas9/Cpf1 are often difficult to introduce into bacteria and archaea due to their severe toxicity. Since most prokaryotes harbor native CRISPR-Cas systems, genome engineering can be achieved by harnessing these endogenous immune systems. Here, we report the exploitation of Type I-B CRISPR-Cas of Clostridium tyrobutyricum for genome engineering. In silico CRISPR array analysis and plasmid interference assay revealed that TCA or TCG at the 5'-end of the protospacer was the functional protospacer adjacent motif (PAM) for CRISPR targeting. With a lactose inducible promoter for CRISPR array expression, we significantly decreased the toxicity of CRISPR-Cas and enhanced the transformation efficiency, and successfully deleted spo0A with an editing efficiency of 100%. We further evaluated effects of the spacer length on genome editing efficiency. Interestingly, spacers ≤ 20 nt led to unsuccessful transformation consistently, likely due to severe off-target effects; while a spacer of 30-38 nt is most appropriate to ensure successful transformation and high genome editing efficiency. Moreover, multiplex genome editing for the deletion of spo0A and pyrF was achieved in a single transformation, with an editing efficiency of up to 100%. Finally, with the integration of the alcohol dehydrogenase gene (adhE1 or adhE2) to replace cat1 (the key gene responsible for butyrate production and previously could not be deleted), two mutants were created for n-butanol production, with the butanol titer reached historically record high of 26.2 g/L in a batch fermentation. Altogether, our results demonstrated the easy programmability and high efficiency of endogenous CRISPR-Cas. The developed protocol herein has a broader applicability to other prokaryotes containing endogenous CRISPR-Cas systems. C. tyrobutyricum could be employed as an excellent platform to be engineered for biofuel and biochemical production using the CRISPR-Cas based genome engineering toolkit.

[Engineering of Escherichia coli for convenient expression of [FeFe]-hydrogenase].

OBJECTIVE: A new method used to heterologously express [FeFe]-hydrogenase in Escherichia coli was investigated in our present study. METHODS: By homologous recombination, three assistant genes (hydE, hydF and hydG) for hydrogenase were integrated into the chromosome of E. coli BW 25113-10, in which all hydrogenase genes were inactivated. A hydrogenase structural gene hydA from Clostridium butyricum was used to test the hydrogenase maturation ability of the recombined E. coli. BW 25113-13. RESULTS: The corrected integration of the three assistant genes was confirmed by PCR, and RT-PCR results indicated that the three accessory genes were transcripted in the recombinant. The active expression of hydA indicated that the constitutively expressed accessory proteins could assist the maturation of the [FeFe]-hydrogenases. CONCLUSIONS: A simplified [FeFe]-hydrogenase expression recombinant E. coli BW25113-13 was constructed. It would lay foundations for the functional screening of [FeFe]-hydrogenases and the construction of novel hydrogen producing pathways in E. coli.

Succession of the bacterial community and dynamics of hydrogen producers in a hydrogen-producing bioreactor.

Variation in the hydrogen production rate was consistent with the succession of dominant bacteria during the batch fermentation process. Denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes and quantitative analysis of the hydA genes at both the DNA and mRNA levels confirmed that Clostridium perfringens was the most dominant hydrogen producer in the bioreactor.