Dam and Dcm methylations prevent gene transfer into Clostridium pasteurianum NRRL B-598: development of methods for electrotransformation, conjugation, and sonoporation

Kolek, Jan; Sedlář, Karel; Provazník, Ivo; Patáková, Petra

doi:10.1186/s13068-016-0436-y

Cited by 36 publications

(24 citation statements)

References 47 publications

(77 reference statements)

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“…C. pasteurianum genome sequences have been reported by at least five additional groups, covering the type strain (ATCC 6013/DSM 525) (16,(21)(22)(23) and two nontype strains (BC1 and NRRL B-598) (24). A method for electroporation of a unique acetone-producing aerotolerant strain was also described recently (25), demonstrating widespread interest in C. pasteurianum as a biocatalyst for crude glycerol valorization. Despite these advancements, only two targeted mutant strains possessing improved n-butanol-producing or glycerol-utilizing properties have been described.…”

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confidence: 99%

Disruption of the Reductive 1,3-Propanediol Pathway Triggers Production of 1,2-Propanediol for Sustained Glycerol Fermentation by Clostridium pasteurianum

Pyne

Sokolenko

Liu

et al. 2016

Appl Environ Microbiol

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Crude glycerol, the major by-product of biodiesel production, is an attractive bioprocessing feedstock owing to its abundance, low cost, and high degree of reduction. In line with the advent of the biodiesel industry, Clostridium pasteurianum has gained prominence as a result of its unique capacity to convert waste glycerol into n-butanol, a high-energy biofuel. However, no efforts have been directed at abolishing the production of 1,3-propanediol (1,3-PDO), the chief competing product of C. pasteurianum glycerol fermentation. Here, we report rational metabolic engineering of C. pasteurianum for enhanced n-butanol production through inactivation of the gene encoding 1,3-PDO dehydrogenase (dhaT). In spite of current models of anaerobic glycerol dissimilation, culture growth and glycerol utilization were unaffected in the dhaT disruption mutant (dhaT::Ll.LtrB). Metabolite characterization of the dhaT::Ll.LtrB mutant revealed an 83% decrease in 1,3-PDO production, encompassing the lowest C. pasteurianum 1,3-PDO titer reported to date (0.58 g liter ؊1 ). With 1,3-PDO formation nearly abolished, glycerol was converted almost exclusively to n-butanol (8.6 g liter ؊1 ), yielding a high n-butanol selectivity of 0.83 g n-butanol g ؊1 of solvents compared to 0.51 g n-butanol g ؊1 of solvents for the wild-type strain. Unexpectedly, high-performance liquid chromatography (HPLC) analysis of dhaT::Ll.LtrB mutant culture supernatants identified a metabolite peak consistent with 1,2-propanediol (1,2-PDO), which was confirmed by nuclear magnetic resonance (NMR). Based on these findings, we propose a new model for glycerol dissimilation by C. pasteurianum, whereby the production of 1,3-PDO by the wild-type strain and low levels of both 1,3-PDO and 1,2-PDO by the engineered mutant balance the reducing equivalents generated during cell mass synthesis from glycerol. IMPORTANCEOrganisms from the genus Clostridium are perhaps the most notable native cellular factories, owing to their vast substrate utilization range and equally diverse variety of metabolites produced. The ability of C. pasteurianum to sustain redox balance and glycerol fermentation despite inactivation of the 1,3-PDO pathway is a testament to the exceptional metabolic flexibility exhibited by clostridia. Moreover, identification of a previously unknown 1,2-PDO-formation pathway, as detailed herein, provides a deeper understanding of fermentative glycerol utilization in clostridia and will inform future metabolic engineering endeavors involving C. pasteurianum. To our knowledge, the C. pasteurianum dhaT disruption mutant derived in this study is the only organism that produces both 1,2-and 1,3-PDOs. Most importantly, the engineered strain provides an excellent platform for highly selective production of n-butanol from waste glycerol.A s a result of a 6-fold increase in global production between 2005 and 2012, biodiesel is presently the second most common biofuel in the world next to ethanol (1). This dramatic increase has generated an exceptional surplus of cru...

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confidence: 99%

Disruption of the Reductive 1,3-Propanediol Pathway Triggers Production of 1,2-Propanediol for Sustained Glycerol Fermentation by Clostridium pasteurianum

Pyne

Sokolenko

Liu

et al. 2016

Appl Environ Microbiol

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“…However, the two species have divergent RM systems, with R. cellulolyticum shown to contain an isoschizomer of Msp I that recognizes the sequence CCGG. Dcm methylation was also reported to decrease the transformation efficiency of some plasmids in R. thermocellum and Clostridium pasteurianum . Thus, all of the plasmids used for transformation of R. josui in the current study were prepared from E. coli C2925 ( dam − / dcm − ).…”

Section: Discussionmentioning

confidence: 99%

Development of an efficient host‐vector system of Ruminiclostridium josui

et al. 2018

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Although Ruminiclostridium josui (formerly Clostridium josui), a strictly anaerobic mesophilic cellulolytic bacterium, is a promising candidate for biomass utilization via consolidated bioprocessing, its host-vector system has not yet been established. The existence of a restriction and modification system is a significant barrier to the transformation of R. josui. Here, we partially purified restriction endonuclease RjoI from R. josui cell extract using column chromatography. Further characterization showed that RjoI is an isoschizomer of DpnI, recognizing the sequence 5'-G ATC-3', where the A nucleotide is Dam-methylated. RjoI cleaved the recognition sequence between the A and T nucleotides, producing blunt ends. We then successfully introduced plasmids prepared from Escherichia coli C2925 (dam /dcm ) into R. josui by electroporation. The highest transformation efficiency of 6.6 × 10 transformants/μg of DNA was obtained using a square-wave pulse (750 V, 1 ms). When the R. josui cel48A gene, devoid of the dockerin-encoding region, cloned into newly developed plasmid pKKM801 was introduced into R. josui, a truncated form of RjCel48A, RjCel48AΔdoc, was detected in the culture supernatant but not in the intracellular fraction. This is the first report on the establishment of fundamental technology for molecular breeding of R. josui.

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“…The plasmids were then isolated and electroporated into C. beijerinckii as previously described (62, 63). dam + dcm + DNA and dam - dcm - DNA were used for C. beijerinckii NCIMB 8052 and DSM 6423, respectively.…”

Section: Methodsmentioning

confidence: 99%

σ⁵⁴ (σ^L) plays a central role in carbon metabolism in the industrially relevant Clostridium beijerinckii

Hocq

Bouilloux-Lafont

Ferreira

et al. 2018

Preprint

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9Microbial production of butanol and isopropanol, two high value-added chemicals, is 10 naturally occurring in the solventogenic Clostridium beijerinckii DSM 6423. Despite its 11 ancient discovery, the precise mechanisms controlling alcohol synthesis in this 12 microorganism are poorly understood. In this work, an allyl alcohol tolerant strain 13 obtained by random mutagenesis was characterized. This strain, designated as the AA 14 mutant, shows a dominant production of acids, a severely diminished butanol synthesis 15 capacity, and produces acetone instead of isopropanol. Interestingly, this solvent-16 deficient strain was also found to have a limited consumption of two carbohydrates and 17 to be still able to form spores, highlighting its particular phenotype. Sequencing of the 18 AA mutant revealed point mutations in several genes including CIBE_0767 (sigL), which 19 encodes the σ 54 sigma factor. Complementation with the wild-type sigL gene fully 20 restored solvent production and sugar assimilation, demonstrating that σ 54 plays a 21 central role in regulating these pathways in C. beijerinckii DSM 6423. Genomic 22 comparison with other strains further revealed that these functions are probably 23 conserved among the C. beijerinckii strains. The importance of σ 54 in C. beijerinckii was 24 further assessed by the characterization of a sigL deletion mutant of the model strain 25 NCIMB 8052 obtained with a CRISPR/Cas9 tool. The resulting mutant exhibited 26 phenotypic traits similar to the AA strain, and was subsequently complemented with the 27 sigL gene from either the wild type or the AA strains. The results of this experiment 28 confirmed the crucial role of σ 54 in the regulation of both solventogenesis and sugar 29 consumption pathways in C. beijerinckii. 30 3 Importance 31Clostridium beijerinckii shows a significant potential for producing valuable biochemicals 32 and biofuels. One of the major hurdles impeding its widespread usage is its low 33 endogenous production of alcohols, which could be alleviated by metabolic engineering 34 approaches. Despite its former long-time use in the industrial acetone-butanol-ethanol 35 process, the molecular mechanisms controlling solventogenesis in the Clostridium 36 genus still remain elusive, preventing genetic engineering approaches for strain 37 enhancement. In this context, our study provides novel insights into the crucial role of 38 the σ 54 transcriptional factor in solvent synthesis regulation in two C. beijerinckii strains. 39Furthermore, we show that this sigma factor also controls sugar consumption and is 40 therefore a key controller of carbon metabolism in this species. 41 42 80 highly conserved DNA-binding region of the encoded protein. We hypothesize that this 81 mutation results in the loss of nucleic acid binding capacity and thus impedes 82 transcription of the σ 54 regulon. According to in silico analyses, this regulon is apparently 83 conserved in other C. beijerinckii strains. To confirm this hypothesis, sigL (Cbei_0595) 84was deleted in the acet...

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Dam and Dcm methylations prevent gene transfer into Clostridium pasteurianum NRRL B-598: development of methods for electrotransformation, conjugation, and sonoporation

Cited by 36 publications

References 47 publications

Disruption of the Reductive 1,3-Propanediol Pathway Triggers Production of 1,2-Propanediol for Sustained Glycerol Fermentation by Clostridium pasteurianum

Disruption of the Reductive 1,3-Propanediol Pathway Triggers Production of 1,2-Propanediol for Sustained Glycerol Fermentation by Clostridium pasteurianum

Development of an efficient host‐vector system of Ruminiclostridium josui

σ⁵⁴ (σ^L) plays a central role in carbon metabolism in the industrially relevant Clostridium beijerinckii

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Dam and Dcm methylations prevent gene transfer into Clostridium pasteurianum NRRL B-598: development of methods for electrotransformation, conjugation, and sonoporation

Cited by 36 publications

References 47 publications

Disruption of the Reductive 1,3-Propanediol Pathway Triggers Production of 1,2-Propanediol for Sustained Glycerol Fermentation by Clostridium pasteurianum

Disruption of the Reductive 1,3-Propanediol Pathway Triggers Production of 1,2-Propanediol for Sustained Glycerol Fermentation by Clostridium pasteurianum

Development of an efficient host‐vector system of Ruminiclostridium josui

σ54 (σL) plays a central role in carbon metabolism in the industrially relevant Clostridium beijerinckii

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σ⁵⁴ (σ^L) plays a central role in carbon metabolism in the industrially relevant Clostridium beijerinckii