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18 pages, 3465 KiB

Open AccessArticle

Improvement of the Genome Editing Tools Based on 5FC/5FU Counter Selection in Clostridium acetobutylicum

by Eglantine Boudignon, Céline Foulquier and Philippe Soucaille

Microorganisms 2023, 11(11), 2696; https://doi.org/10.3390/microorganisms11112696 - 03 Nov 2023

Cited by 1 | Viewed by 592

Several genetic tools have been developed for genome engineering in Clostridium acetobutylicum utilizing 5-fluorouracil (5FU) or 5-fluorocytosine (5FC) resistance as a selection method. In our group, a method based on the integration, by single crossing over, of a suicide plasmid (pCat-upp) [...] Read more.

Several genetic tools have been developed for genome engineering in Clostridium acetobutylicum utilizing 5-fluorouracil (5FU) or 5-fluorocytosine (5FC) resistance as a selection method. In our group, a method based on the integration, by single crossing over, of a suicide plasmid (pCat-upp) followed by selection for the second crossing over using a counter-selectable marker (the upp gene and 5FU resistance) was recently developed for genome editing in C. acetobutylicum. This method allows genome modification without leaving any marker or scar in a strain of C. acetobutylicum that is ∆upp. Unfortunately, 5FU has strong mutagenic properties, inducing mutations in the strain’s genome. After numerous applications of the pCat-upp/5FU system for genome modification in C. acetobutylicum, the CAB1060 mutant strain became entirely resistant to 5FU in the presence of the upp gene, resulting in failure when selecting on 5FU for the second crossing over. It was found that the potential repressor of the pyrimidine operon, PyrR, was mutated at position A115, leading to the 5FU resistance of the strain. To fix this problem, we created a corrective replicative plasmid expressing the pyrR gene, which was shown to restore the 5FU sensitivity of the strain. Furthermore, in order to avoid the occurrence of the problem observed with the CAB1060 strain, a preventive suicide plasmid, pCat-upp-pyrR*, was also developed, featuring the introduction of a synthetic codon-optimized pyrR gene, which was referred to as pyrR* with low nucleotide sequence homology to pyrR. Finally, to minimize the mutagenic effect of 5FU, we also improved the pCat-upp/5FU system by reducing the concentration of 5FU from 1 mM to 5 µM using a defined synthetic medium. The optimized system/conditions were used to successfully replace the ldh gene by the sadh-hydG operon to convert acetone into isopropanol. Full article

(This article belongs to the Special Issue Physiology, Genetic and Industrial Applications of Clostridia)

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19 pages, 3176 KiB

Open AccessArticle

Pleiotropic Regulator GssR Positively Regulates Autotrophic Growth of Gas-Fermenting Clostridium ljungdahlii

by Huan Zhang, Can Zhang, Xiaoqun Nie, Yuwei Wu, Chen Yang, Weihong Jiang and Yang Gu

Microorganisms 2023, 11(8), 1968; https://doi.org/10.3390/microorganisms11081968 - 31 Jul 2023

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Abstract

Clostridium ljungdahlii is a representative autotrophic acetogen capable of producing multiple chemicals from one-carbon gases (CO₂/CO). The metabolic and regulatory networks of this carbon-fixing bacterium are interesting, but still remain minimally explored. Here, based on bioinformatics analysis followed by functional screening, [...] Read more.

Clostridium ljungdahlii is a representative autotrophic acetogen capable of producing multiple chemicals from one-carbon gases (CO₂/CO). The metabolic and regulatory networks of this carbon-fixing bacterium are interesting, but still remain minimally explored. Here, based on bioinformatics analysis followed by functional screening, we identified a RpiR family transcription factor (TF) that can regulate the autotrophic growth and carbon fixation of C. ljungdahlii. After deletion of the corresponding gene, the resulting mutant strain exhibited significantly impaired growth in gas fermentation, thus reducing the production of acetic acid and ethanol. In contrast, the overexpression of this TF gene could promote cell growth, indicating a positive regulatory effect of this TF in C. ljungdahlii. Thus, we named the TF as GssR (growth and solvent synthesis regulator). Through the following comparative transcriptomic analysis and biochemical verification, we discovered three important genes (encoding pyruvate carboxylase, carbon hunger protein CstA, and a BlaI family transcription factor) that were directly regulated by GssR. Furthermore, an upstream regulator, BirA, that could directly bind to gssR was found; thus, these two regulators may form a cascade regulation and jointly affect the physiology and metabolism of C. ljungdahlii. These findings substantively expand our understanding on the metabolic regulation of carbon fixation in gas-fermenting Clostridium species. Full article

(This article belongs to the Special Issue Physiology, Genetic and Industrial Applications of Clostridia)

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13 pages, 9414 KiB

Open AccessArticle

Role of the Solute-Binding Protein CuaD in the Signaling and Regulating Pathway of Cellobiose and Cellulose Utilization in Ruminiclostridium cellulolyticum

by Aurélie Fosses, Nathalie Franche, Goetz Parsiegla, Yann Denis, Maria Maté, Pascale de Philip, Henri-Pierre Fierobe and Stéphanie Perret

Microorganisms 2023, 11(7), 1732; https://doi.org/10.3390/microorganisms11071732 - 01 Jul 2023

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Abstract

In Ruminiclostridium cellulolyticum, cellobiose is imported by the CuaABC ATP-binding cassette transporter containing the solute-binding protein (SBP) CuaA and is further degraded in the cytosol by the cellobiose phosphorylase CbpA. The genes encoding these proteins have been shown to be essential for [...] Read more.

In Ruminiclostridium cellulolyticum, cellobiose is imported by the CuaABC ATP-binding cassette transporter containing the solute-binding protein (SBP) CuaA and is further degraded in the cytosol by the cellobiose phosphorylase CbpA. The genes encoding these proteins have been shown to be essential for cellobiose and cellulose utilization. Here, we show that a second SBP (CuaD), whose gene is adjacent to two genes encoding a putative two-component regulation system (CuaSR), forms a three-component system with CuaS and CuaR. Studies of mutant and recombinant strains of R. cellulolyticum have indicated that cuaD is important for the growth of strains on cellobiose and cellulose. Furthermore, the results of our RT-qPCR experiments suggest that both the three (CuaDSR)- and the two (CuaSR)-component systems are able to perceive the cellobiose signal. However, the strain producing the three-component system is more efficient in its cellobiose and cellulose utilization. As CuaD binds to CuaS, we propose an in-silico model of the complex made up of two extracellular domains of CuaS and two of CuaD. CuaD allows microorganisms to detect very low concentrations of cellobiose due to its high affinity and specificity for this disaccharide, and together with CuaSR, it triggers the expression of the cuaABC-cbpA genes involved in cellodextrins uptake. Full article

(This article belongs to the Special Issue Physiology, Genetic and Industrial Applications of Clostridia)

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19 pages, 3069 KiB

Open AccessArticle

The Lanthipeptide Synthetase-like Protein CA_C0082 Is an Effector of Agr Quorum Sensing in Clostridium acetobutylicum

by Jonathan R. Humphreys, Zak Bean, Jamie Twycross and Klaus Winzer

Microorganisms 2023, 11(6), 1460; https://doi.org/10.3390/microorganisms11061460 - 31 May 2023

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Abstract

Lanthipeptide synthetases are present in all domains of life. They catalyze a crucial step during lanthipeptide biosynthesis by introducing thioether linkages during posttranslational peptide modification. Lanthipeptides have a wide range of functions, including antimicrobial and morphogenetic activities. Intriguingly, several Clostridium species contain lanthipeptide [...] Read more.

Lanthipeptide synthetases are present in all domains of life. They catalyze a crucial step during lanthipeptide biosynthesis by introducing thioether linkages during posttranslational peptide modification. Lanthipeptides have a wide range of functions, including antimicrobial and morphogenetic activities. Intriguingly, several Clostridium species contain lanthipeptide synthetase-like genes of the class II (lanM) family but lack other components of the lanthipeptide biosynthetic machinery. In all instances, these genes are located immediately downstream of putative agr quorum sensing operons. The physiological role and mode of action of the encoded LanM-like proteins remain uncertain as they lack conserved catalytic residues. Here we show for the industrial organism Clostridium acetobutylicum that the LanM-like protein CA_C0082 is not required for the production of active AgrD-derived signaling peptide but nevertheless acts as an effector of Agr quorum sensing. Expression of CA_C0082 was shown to be controlled by the Agr system and is a prerequisite for granulose (storage polymer) formation. The accumulation of granulose, in turn, was shown to be required for maximal spore formation but also to reduce early solvent formation. CA_C0082 and its putative homologs appear to be closely associated with Agr systems predicted to employ signaling peptides with six-membered ring structures and may represent a new subfamily of LanM-like proteins. This is the first time their contribution to bacterial Agr signaling has been described. Full article

(This article belongs to the Special Issue Physiology, Genetic and Industrial Applications of Clostridia)

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13 pages, 2888 KiB

Open AccessArticle

Exploitation of a Type 1 Toxin–Antitoxin System as an Inducible Counter-Selective Marker for Genome Editing in the Acetogen Eubacterium limosum

by James Millard, Alexander Agius, Ying Zhang, Philippe Soucaille and Nigel Peter Minton

Microorganisms 2023, 11(5), 1256; https://doi.org/10.3390/microorganisms11051256 - 10 May 2023

Cited by 1 | Viewed by 1420

Abstract

Targeted mutations in the anaerobic methylotroph Eubacterium limosum have previously been obtained using CRISPR-based mutagenesis methods. In this study, a RelB-family toxin from Eubacterium callanderi was placed under the control of an anhydrotetracycline-sensitive promoter, forming an inducible counter-selective system. This inducible system was [...] Read more.

Targeted mutations in the anaerobic methylotroph Eubacterium limosum have previously been obtained using CRISPR-based mutagenesis methods. In this study, a RelB-family toxin from Eubacterium callanderi was placed under the control of an anhydrotetracycline-sensitive promoter, forming an inducible counter-selective system. This inducible system was coupled with a non-replicative integrating mutagenesis vector to create precise gene deletions in Eubacterium limosum B2. The genes targeted in this study were those encoding the histidine biosynthesis gene hisI, the methanol methyltransferase and corrinoid protein mtaA and mtaC, and mtcB, encoding an Mttb-family methyltransferase which has previously been shown to demethylate L-carnitine. A targeted deletion within hisI brought about the expected histidine auxotrophy, and deletions of mtaA and mtaC both abolished autotrophic growth on methanol. Deletion of mtcB was shown to abolish the growth of E. limosum on L-carnitine. After an initial selection step to isolate transformant colonies, only a single induction step was required to obtain mutant colonies for the desired targets. The combination of an inducible counter-selective marker and a non-replicating integrative plasmid allows for quick gene editing of E. limosum. Full article

(This article belongs to the Special Issue Physiology, Genetic and Industrial Applications of Clostridia)

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16 pages, 2424 KiB

Open AccessArticle

Heterologous 1,3-Propanediol Production Using Different Recombinant Clostridium beijerinckii DSM 6423 Strains

by Teresa Schoch, Tina Baur, Johanna Kunz, Sophia Stöferle and Peter Dürre

Microorganisms 2023, 11(3), 784; https://doi.org/10.3390/microorganisms11030784 - 18 Mar 2023

Cited by 3 | Viewed by 1510

Abstract

1,3-propanediol (1,3-PDO) is a valuable basic chemical, especially in the polymer industry to produce polytrimethylene terephthalate. Unfortunately, the production of 1,3-PDO mainly depends on petroleum products as precursors. Furthermore, the chemical routes have significant disadvantages, such as environmental issues. An alternative is the [...] Read more.

1,3-propanediol (1,3-PDO) is a valuable basic chemical, especially in the polymer industry to produce polytrimethylene terephthalate. Unfortunately, the production of 1,3-PDO mainly depends on petroleum products as precursors. Furthermore, the chemical routes have significant disadvantages, such as environmental issues. An alternative is the biobased fermentation of 1,3-PDO from cheap glycerol. Clostridium beijerinckii DSM 6423 was originally reported to produce 1,3-PDO. However, this could not be confirmed, and a genome analysis revealed the loss of an essential gene. Thus, 1,3-PDO production was genetically reinstalled. Genes for 1,3-PDO production from Clostridium pasteurianum DSM 525 and Clostridium beijerinckii DSM 15410 (formerly Clostridium diolis) were introduced into C. beijerinckii DSM 6423 to enable 1,3-PDO production from glycerol. 1,3-PDO production by recombinant C. beijerinckii strains were investigated under different growth conditions. 1,3-PDO production was only observed for C. beijerinckii [pMTL83251_P_pta-ack_1,3-PDO.diolis], which harbors the genes of C. beijerinckii DSM 15410. By buffering the growth medium, production could be increased by 74%. Furthermore, the effect of four different promoters was analyzed. The use of the constitutive thlA promoter from Clostridium acetobutylicum led to a 167% increase in 1,3-PDO production compared to the initial recombinant approach. Full article

(This article belongs to the Special Issue Physiology, Genetic and Industrial Applications of Clostridia)

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16 pages, 2308 KiB

Open AccessArticle

New Insights into the Physiology of the Propionate Producers Anaerotignum propionicum and Anaerotignum neopropionicum (Formerly Clostridium propionicum and Clostridium neopropionicum)

by Tina Baur and Peter Dürre

Microorganisms 2023, 11(3), 685; https://doi.org/10.3390/microorganisms11030685 - 07 Mar 2023

Cited by 2 | Viewed by 1738

Abstract

Propionate is an important platform chemical that is available through petrochemical synthesis. Bacterial propionate formation is considered an alternative, as bacteria can convert waste substrates into valuable products. In this regard, research primarily focused on propionibacteria due to high propionate titers achieved from [...] Read more.

Propionate is an important platform chemical that is available through petrochemical synthesis. Bacterial propionate formation is considered an alternative, as bacteria can convert waste substrates into valuable products. In this regard, research primarily focused on propionibacteria due to high propionate titers achieved from different substrates. Whether other bacteria could also be attractive producers is unclear, mostly because little is known about these strains. Therefore, two thus far less researched strains, Anaerotignum propionicum and Anaerotignum neopropionicum, were investigated with regard to their morphologic and metabolic features. Microscopic analyses revealed a negative Gram reaction despite a Gram-positive cell wall as well as surface layers for both strains. Furthermore, growth, product profiles, and the potential for propionate formation from sustainable substrates, i.e., ethanol or lignocellulosic sugars, were assessed. Results showed that both strains can oxidize ethanol to different extents. While A. propionicum only partially used ethanol, A. neopropionicum converted 28.3 mM ethanol to 16.4 mM propionate. Additionally, the ability of A. neopropionicum to produce propionate from lignocellulose-derived substrates was analyzed, leading to propionate concentrations of up to 14.5 mM. Overall, this work provides new insights into the physiology of the Anaerotignum strains, which can be used to develop effective propionate producer strains. Full article

(This article belongs to the Special Issue Physiology, Genetic and Industrial Applications of Clostridia)

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Review

Jump to: Research

55 pages, 5789 KiB

Open AccessReview

Solvent-Producing Clostridia Revisited

by David T. Jones, Frederik Schulz, Simon Roux and Steven D. Brown

Microorganisms 2023, 11(9), 2253; https://doi.org/10.3390/microorganisms11092253 - 07 Sep 2023

Cited by 1 | Viewed by 1961

Abstract

The review provides an overview of the current status of the solvent-producing clostridia. The origin and development of industrial clostridial species, as well as the history of the industrial Acetone Butanol Ethanol fermentation process, is reexamined, and the recent resurgence of interest in [...] Read more.

The review provides an overview of the current status of the solvent-producing clostridia. The origin and development of industrial clostridial species, as well as the history of the industrial Acetone Butanol Ethanol fermentation process, is reexamined, and the recent resurgence of interest in the production of biobutanol is reviewed. Over 300 fully sequenced genomes for solvent-producing and closely related clostridial species are currently available in public databases. These include 270 genomes sourced from the David Jones culture collection. These genomes were allocated arbitrary DJ codes, and a conversion table to identify the species and strains has now been provided. The expanded genomic database facilitated new comparative genomic and phylogenetic analysis. A synopsis of the common features, molecular taxonomy, and phylogeny of solvent-producing clostridia and the application of comparative phylogenomics are evaluated. A survey and analysis of resident prophages in solvent-producing clostridia are discussed, and the discovery, occurrence, and role of novel R-type tailocins are reported. Prophage genomes with R-type tailocin-like features were detected in all 12 species investigated. The widespread occurrence of tailocins in Gram-negative species is well documented; this survey has indicated that they may also be widespread in clostridia. Full article

(This article belongs to the Special Issue Physiology, Genetic and Industrial Applications of Clostridia)

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