Forward and reverse genetics in industrially important Clostridia

The bacterial genus Clostridium is composed of Gram-positive, spore-forming rods with widespread biotechnological applications. This study focused mainly on Clostridium pasteurianum DSM 525, a saccharolytic species which is able to convert glycerol, the by-product of the biodiesel industry, into the valuable chemical commodities n-butanol, ethanol and 1,3-propanediol. The aim was to formulate reproducible methods for the creation of mutants, both directed and random, and use the tools developed to investigate genes, and their products, important in solvent production. A prerequisite for the deployment of the envisaged genetic tools was a reproducible means for their introduction into the cell. Following the observation of low frequencies of plasmid transfer by electroporation it was hypothesised that the low level of transformants observed were a consequence of the presence of rare hypertransformable variants within the population. Accordingly, successfully transformed clonal populations were cured of their acquired plasmid and retransformed. In a number of instances the cured cell lines proved hypertransformable, with plasmid transformation frequencies obtained that were 5 orders of magnitude higher than those obtained with the progenitor strain. All of the hypertransformable strains isolated were shown by whole genome sequence to contain single nucleotide polymorphisms (SNPs) in one or more genes. In one instance, the single SNP present was shown to be directly responsible for the increased transformation frequency by its deliberate restoration to wild type using the allelic exchange procedures subsequently developed. Having established reproducible, high frequencies of plasmid transformation reverse genetics was employed to establish gene function. Accordingly, allelic exchange gene knock-out procedures were used to target genes coding for enzymes of the central energy metabolism in C. pasteurianum and the phenotypes of the mutants obtained were analysed in laboratory scale fermentations. Strains in which the genes encoding the redox response regulator (rex) and a hydrogenase (hyd) were deleted showed increased n-butanol titres, representing first steps towards utilisation of C. pasteurianum as a chassis for this important chemical. With the inactivation of the dhaBCE gene, encoding glycerol dehydratase, production of 1,3-propanediol was entirely eliminated, demonstrating the importance of the reductive pathway for growth and redox homeostasis of this organism when grown on glycerol. In order to allow forward genetic approaches, a mariner-transposon system previously exemplified in Clostridium difficile was adapted for use in alternative clostridial hosts. In the absence of an efficient transformation system for C. pasteurianum, the initial exemplification of the system was undertaken in Clostridium acetobutylicum and Clostridium sporogenes. Successful transposon delivery was demonstrated through the use of a plasmid conditional for replication and through the insertion of a gene encoding an alternate sigma-factor, TcdR, into their genomes. Transposition was shown to be entirely random and the libraries obtained of sufficient size to allow the isolation of both auxotrophic and sporulation/germination deficient mutants. Steps were taken to develop the same system in C. pasteurianum which was successful by using a suicide delivery plasmid, which was only possible with the high transformation efficiency achieved as part of this study. This study presents an essential forward genetics procedure for industrially important Clostridium species and a comprehensive genetic engineering approach for the important biofuel producer C. pasteurianum

Forward and reverse genetics in industrially important Clostridia

The bacterial genus Clostridium is composed of Gram-positive, spore-forming rods with widespread biotechnological applications. This study focused mainly on Clostridium pasteurianum DSM 525, a saccharolytic species which is able to convert glycerol, the by-product of the biodiesel industry, into the valuable chemical commodities n-butanol, ethanol and 1,3-propanediol. The aim was to formulate reproducible methods for the creation of mutants, both directed and random, and use the tools developed to investigate genes, and their products, important in solvent production. A prerequisite for the deployment of the envisaged genetic tools was a reproducible means for their introduction into the cell. Following the observation of low frequencies of plasmid transfer by electroporation it was hypothesised that the low level of transformants observed were a consequence of the presence of rare hypertransformable variants within the population. Accordingly, successfully transformed clonal populations were cured of their acquired plasmid and retransformed. In a number of instances the cured cell lines proved hypertransformable, with plasmid transformation frequencies obtained that were 5 orders of magnitude higher than those obtained with the progenitor strain. All of the hypertransformable strains isolated were shown by whole genome sequence to contain single nucleotide polymorphisms (SNPs) in one or more genes. In one instance, the single SNP present was shown to be directly responsible for the increased transformation frequency by its deliberate restoration to wild type using the allelic exchange procedures subsequently developed. Having established reproducible, high frequencies of plasmid transformation reverse genetics was employed to establish gene function. Accordingly, allelic exchange gene knock-out procedures were used to target genes coding for enzymes of the central energy metabolism in C. pasteurianum and the phenotypes of the mutants obtained were analysed in laboratory scale fermentations. Strains in which the genes encoding the redox response regulator (rex) and a hydrogenase (hyd) were deleted showed increased n-butanol titres, representing first steps towards utilisation of C. pasteurianum as a chassis for this important chemical. With the inactivation of the dhaBCE gene, encoding glycerol dehydratase, production of 1,3-propanediol was entirely eliminated, demonstrating the importance of the reductive pathway for growth and redox homeostasis of this organism when grown on glycerol. In order to allow forward genetic approaches, a mariner-transposon system previously exemplified in Clostridium difficile was adapted for use in alternative clostridial hosts. In the absence of an efficient transformation system for C. pasteurianum, the initial exemplification of the system was undertaken in Clostridium acetobutylicum and Clostridium sporogenes. Successful transposon delivery was demonstrated through the use of a plasmid conditional for replication and through the insertion of a gene encoding an alternate sigma-factor, TcdR, into their genomes. Transposition was shown to be entirely random and the libraries obtained of sufficient size to allow the isolation of both auxotrophic and sporulation/germination deficient mutants. Steps were taken to develop the same system in C. pasteurianum which was successful by using a suicide delivery plasmid, which was only possible with the high transformation efficiency achieved as part of this study. This study presents an essential forward genetics procedure for industrially important Clostridium species and a comprehensive genetic engineering approach for the important biofuel producer C. pasteurianum

Improving gene transfer in Clostridium pasteurianum through the isolation of rare hypertransformable variants

Effective microbial metabolic engineering is reliant on efficient gene transfer. Here we present a simple screening strategy that may be deployed to isolate rare, hypertransformable variants. The procedure was used to increase the frequency of transformation of the solvent producing organism Clostridium pasteurianum by three to four orders of magnitude

Development of Clostridium saccharoperbutylacetonicum as a Whole Cell Biocatalyst for Production of Chirally Pure (R)-1,3-Butanediol

Chirally pure (R)-1,3-butanediol ((R)-1,3-BDO) is a valuable intermediate for the production of fragrances, pheromones, insecticides and antibiotics. Biotechnological production results in superior enantiomeric excess over chemical production and is therefore the preferred production route. In this study (R)-1,3-BDO was produced in the industrially important whole cell biocatalyst Clostridium saccharoperbutylacetonicum through expression of the enantio-specific phaB gene from Cupriavidus necator. The heterologous pathway was optimised in three ways: at the transcriptional level choosing strongly expressed promoters and comparing plasmid borne with chromosomal gene expression, at the translational level by optimising the codon usage of the gene to fit the inherent codon adaptation index of C. saccharoperbutylacetonicum, and at the enzyme level by introducing point mutations which led to increased enzymatic activity. The resulting whole cell catalyst produced up to 20 mM (1.8 g/l) (R)-1,3-BDO in non-optimised batch fermentation which is a promising starting position for economical production of this chiral chemical

Quantitative Bioreactor Monitoring of Intracellular Bacterial Metabolites in Clostridium autoethanogenum Using Liquid Chromatography–Isotope Dilution Mass Spectrometry

We report a liquid chromatography–isotope dilution mass spectrometry method for the simultaneous quantification of 131 intracellular bacterial metabolites of Clostridium autoethanogenum. A comprehensive mixture of uniformly 13C-labeled internal standards (U-13C IS) was biosynthesized from the closely related bacterium Clostridium pasteurianum using 4% 13C–glucose as a carbon source. The U-13C IS mixture combined with 12C authentic standards was used to validate the linearity, precision, accuracy, repeatability, limits of detection, and quantification for each metabolite. A robust-fitting algorithm was employed to reduce the weight of the outliers on the quantification data. The metabolite calibration curves were linear with R2 ≥ 0.99, limits of detection were ≤1.0 μM, limits of quantification were ≤10 μM, and precision/accuracy was within RSDs of 15% for all metabolites. The method was subsequently applied for the daily monitoring of the intracellular metabolites of C. autoethanogenum during a CO gas fermentation over 40 days as part of a study to optimize biofuel production. The concentrations of the metabolites were estimated at steady states of different pH levels using the robust-fitting mathematical approach, and we demonstrate improved accuracy of results compared to conventional regression. Metabolic pathway analysis showed that reactions of the incomplete (branched) tricarboxylic acid “cycle” were the most affected pathways associated with the pH shift in the bioreactor fermentation of C. autoethanogenum and the concomitant changes in ethanol production

A universal mariner transposon system for forward genetic studies in the genus Clostridium.

DNA transposons represent an essential tool in the armoury of the molecular microbiologist. We previously developed a catP-based mini transposon system for Clostridium difficile in which the expression of the transposase gene was dependent on a sigma factor unique to C. difficile, TcdR. Here we have shown that the host range of the transposon is easily extended through the rapid chromosomal insertion of the tcdR gene at the pyrE locus of the intended clostridial target using Allele-Coupled Exchange (ACE). To increase the effectiveness of the system, a novel replicon conditional for plasmid maintenance was developed, which no longer supports the effective retention of the transposon delivery vehicle in the presence of the inducer isopropyl β-D-1-thiogalactopyranoside (IPTG). As a consequence, those thiamphenicol resistant colonies that arise in clostridial recipients, following plating on agar medium supplemented with IPTG, are almost exclusively due to insertion of the mini transposon into the genome. The system has been exemplified in both Clostridium acetobutylicum and Clostridium sporogenes, where transposon insertion has been shown to be entirely random. Moreover, appropriate screening of both libraries resulted in the isolation of auxotrophic mutants as well as cells deficient in spore formation/germination. This strategy is capable of being implemented in any Clostridium species

Vector map of plasmid pMTL-YZ14.

<p>Expression of the hyperactive <i>mariner</i> transposase gene <i>Himar1 C9</i> was driven by the <i>C</i>. <i>difficile</i> toxin B promoter, P_tcdB. The plasmid backbone consisted of the conditional replicon between restriction sites AscI and FseI, the macrolide-lincosamide-streptogramin B antibiotic resistance gene <i>ermB</i>, and the Gram-negative replicon, ColE1. The whole mariner element (i.e., transposase gene and <i>catP</i> mini-transposon) can be excised as a SbfI fragment. The control plasmid pMTL-YZ13 was identical, except that the Gram-positive replicon is the pCB102 replicon from <i>C</i>. <i>butyricum</i>. This plasmid conforms to the pMTL80000 modular system for <i>Clostridium</i> shuttle plasmids [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122411#pone.0122411.ref032" target="_blank">32</a>].</p

Schematic diagram of the <i>lac</i>-based, IPTG inducible expression cassette in pMTL-YZ2 (A), and the demonstration of its function in <i>C</i>.

<p><b><i>acetobutylicum</i> (B) and <i>C</i>. <i>sporogenes</i> (C). A</b>: Schematic diagram of the <i>lac</i>-based, IPTG inducible expression cassette. Key: LacI is the LacI repressor protein gene. LacI binds to the indicated <i>lacO</i> region, blocking transcription from the P_<b>fac</b> promoter. The P_<b>ptb</b> promoter (derived from the <i>C</i>. <i>beijerinckii</i> gene encoding phosphotransbutyrylase) directs the transcription of the <i>lacI</i> gene. <b>B</b> and <b>C</b>: IPTG induction of CAT production in of <i>C</i>. <i>acetobutylicum</i> (<b>B</b>) and <i>C</i>. <i>sporogenes</i> (<b>C</b>) harbouring pMTL-YZ2. Circles equate to cells which received no IPTG, squares represents samples from cells that were induced with IPTG. The arrow indicates the time of adding IPTG. Activity is expressed as units of CAT activity per mg of soluble protein.</p

Analysis of the functionality of the exogenous TcdR in two clostridial strains (CRG3011 and CRG3817).

<p>Plasmid maps of the pMTL82254-P_<b>tcdB</b> (<b>A</b>) and pMTL82254-P_<b>fdx</b> (<b>B</b>). Key: CD0164 terminator, a transcriptional terminator isolated from downstream of the <i>C</i>. <i>difficile</i> strain 630 CD0164 gene; <i>catP</i>, a <i>C</i>. <i>perfringens</i>-derived gene encoding chloramphenicol acetyltransferase; Cpa fdx terminator, transcriptional terminator of the ferredoxin gene of <i>C</i>. <i>pasteurianum</i>; <i>repA</i> and <i>orf2</i>, replication region of the <i>C</i>. <i>botulinum</i> plasmid pBP1; <i>ermB</i>, the macrolide-lincosamide-streptogramin B antibiotic resistance gene of plasmid pAMß1; ColE1, the replication origin of plasmid ColE1, and; <i>traJ</i>, transfer function of the RP4 <i>oriT</i> region. CD <i>tcdB</i> promoter, the promoter region of the <i>C</i>. <i>difficile tcdB</i> gene; Csp fdx promoter: the promoter region of the <i>C</i>. <i>sporogenes fdx</i> gene. (<b>C)</b>: CAT activity of either <i>C</i>. <i>acetobutylicum</i> ATCC 824 wild type or CRG3011 (<i>tcdR</i> containing <i>C</i>. <i>acetobutylicum</i> ATCC 824) carrying plasmids pMTL82254-P_<b>tcdB</b> and pMTL82254-P_<b>fdx</b>. (<b>D)</b>: CAT activity of either <i>C</i>. <i>sporogenes</i> NCIMB 10969 wild type or CRG3817 (<i>tcdR</i> containing <i>C</i>. <i>sporogenes</i> NCIMB 10969) carrying plasmids pMTL82254-P_<b>tcdB</b> and pMTL82254-P_<b>fdx</b>. Black circles ●, wild type with pMTL82254-P_<b>tcdB</b>; black squares ■, wild type with pMTL82254-P_<b>fdx</b>; black triangles ▲, CRG3011/CRG3817 with pMTL82254-P_<b>tcdB</b>; black triangles ▼, CRG3011/CRG3817 with pMTL82254-P_<b>fdx</b>.</p

Characterization of the found auxotroph mutant with different additives in P2 minimal medium.

<p>No growth(˗˗), slight growth(+) and normal growth(++).</p><p>Characterization of the found auxotroph mutant with different additives in P2 minimal medium.</p