Endogenous CRISPR/Cas systems for genome engineering in the acetogens Acetobacterium woodii and Clostridium autoethanogenum.

Acetogenic bacteria can play a major role in achieving Net Zero through their ability to convert CO2 into industrially relevant chemicals and fuels. Full exploitation of this potential will be reliant on effective metabolic engineering tools, such as those based on the Streptococcus pyogenes CRISPR/Cas9 system. However, attempts to introduce cas9-containing vectors into Acetobacterium woodii were unsuccessful, most likely as a consequence of Cas9 nuclease toxicity and the presence of a recognition site for an endogenous A. woodii restriction-modification (R-M) system in the cas9 gene. As an alternative, this study aims to facilitate the exploitation of CRISPR/Cas endogenous systems as genome engineering tools. Accordingly, a Python script was developed to automate the prediction of protospacer adjacent motif (PAM) sequences and used to identify PAM candidates of the A. woodii Type I-B CRISPR/Cas system. The identified PAMs and the native leader sequence were characterized in vivo by interference assay and RT-qPCR, respectively. Expression of synthetic CRISPR arrays, consisting of the native leader sequence, direct repeats, and adequate spacer, along with an editing template for homologous recombination, successfully led to the creation of 300 bp and 354 bp in-frame deletions of pyrE and pheA, respectively. To further validate the method, a 3.2 kb deletion of hsdR1 was also generated, as well as the knock-in of the fluorescence-activating and absorption-shifting tag (FAST) reporter gene at the pheA locus. Homology arm length, cell density, and the amount of DNA used for transformation were found to significantly impact editing efficiencies. The devised workflow was subsequently applied to the Type I-B CRISPR/Cas system of Clostridium autoethanogenum, enabling the generation of a 561 bp in-frame deletion of pyrE with 100% editing efficiency. This is the first report of genome engineering of both A. woodii and C. autoethanogenum using their endogenous CRISPR/Cas systems.

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

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.

Improving carbon monoxide tolerance of Cupriavidus necator H16 through adaptive laboratory evolution.

Background: The toxic gas carbon monoxide (CO) is abundantly present in synthesis gas (syngas) and certain industrial waste gases that can serve as feedstocks for the biological production of industrially significant chemicals and fuels. For efficient bacterial growth to occur, and to increase productivity and titres, a high resistance to the gas is required. The aerobic bacterium Cupriavidus necator H16 can grow on CO2 + H2, although it cannot utilise CO as a source of carbon and energy. This study aimed to increase its CO resistance through adaptive laboratory evolution. Results: To increase the tolerance of C. necator to CO, the organism was continually subcultured in the presence of CO both heterotrophically and autotrophically. Ten individual cultures were evolved heterotrophically with fructose in this manner and eventually displayed a clear growth advantage over the wild type strain. Next-generation sequencing revealed several mutations, including a single point mutation upstream of a cytochrome bd ubiquinol oxidase operon (cydA2B2), which was present in all evolved isolates. When a subset of these mutations was engineered into the parental H16 strain, only the cydA2B2 upstream mutation enabled faster growth in the presence of CO. Expression analysis, mutation, overexpression and complementation suggested that cydA2B2 transcription is upregulated in the evolved isolates, resulting in increased CO tolerance under heterotrophic but not autotrophic conditions. However, through subculturing on a syngas-like mixture with increasing CO concentrations, C. necator could also be evolved to tolerate high CO concentrations under autotrophic conditions. A mutation in the gene for the soluble [NiFe]-hydrogenase subunit hoxH was identified in the evolved isolates. When the resulting amino acid change was engineered into the parental strain, autotrophic CO resistance was conferred. A strain constitutively expressing cydA2B2 and the mutated hoxH gene exhibited high CO tolerance under both heterotrophic and autotrophic conditions. Conclusion: C. necator was evolved to tolerate high concentrations of CO, a phenomenon which was dependent on the terminal respiratory cytochrome bd ubiquinol oxidase when grown heterotrophically and the soluble [NiFe]-hydrogenase when grown autotrophically. A strain exhibiting high tolerance under both conditions was created and presents a promising chassis for syngas-based bioproduction processes.

Growth rate and nutrient limitation as key drivers of extracellular quorum sensing signal molecule accumulation in Pseudomonas aeruginosa.

In Pseudomonas aeruginosa, quorum sensing (QS) depends on an interconnected regulatory hierarchy involving the Las, Rhl and Pqs systems, which are collectively responsible for the co-ordinated synthesis of a diverse repertoire of N-acylhomoserine lactones (AHLs) and 2-alkyl-4-quinolones (AQs). Apparent population density-dependent phenomena such as QS may, however, be due to growth rate and/or nutrient exhaustion in batch culture. Using continuous culture, we show that growth rate and population density independently modulate the accumulation of AHLs and AQs such that the highest concentrations are observed at a slow growth rate and high population density. Carbon source (notably succinate), nutrient limitation (C, N, Fe, Mg) or growth at 25 °C generally reduces AHL and AQ levels, except for P and S limitation, which result in substantially higher concentrations of AQs, particularly AQ N-oxides, despite the lower population densities achieved. Principal component analysis indicates that ~26 % variation is due to nutrient limitation and a further 30 % is due to growth rate. The formation of N-(3-oxododecanoyl)-l-homoserine lactone (3OC12-HSL) turnover products such as the ring opened form and tetramic acid varies with the limiting nutrient limitation and anaerobiosis. Differential ratios of N-butanoyl-homoserine lactone (C4-HSL), 3OC12-HSL and the AQs as a function of growth environment are clearly apparent. Inactivation of QS by mutation of three key genes required for QS signal synthesis (lasI, rhlI and pqsA) substantially increases the concentrations of key substrates from the activated methyl cycle and aromatic amino acid biosynthesis, as well as ATP levels, highlighting the energetic drain that AHL and AQ synthesis and hence QS impose on P. aeruginosa.

Metabolic engineering of Cupriavidus necator H16 for heterotrophic and autotrophic production of 3-hydroxypropionic acid.

3-Hydroxypropionate (3-HP) is a versatile compound for chemical synthesis and a potential building block for biodegradable polymers. Cupriavidus necator H16, a facultative chemolithoautotroph, is an attractive production chassis and has been extensively studied as a model organism for biopolymer production. Here, we engineered C. necator H16 for 3-HP biosynthesis from its central metabolism. Wild type C. necator H16 can use 3-HP as a carbon source, a highly undesirable trait for a 3-HP production chassis. However, deletion of its three (methyl-)malonate semialdehyde dehydrogenases (mmsA1, mmsA2 and mmsA3) resulted in a strain that cannot grow on 3-HP as the sole carbon source, and this strain was selected as our production host. A stepwise approach was used to construct pathways for 3-HP production via ß-alanine. Two additional gene deletion targets were identified during the pathway construction process. Deletion of the 3-hydroxypropionate dehydrogenase, encoded by hpdH, prevented the re-consumption of the 3-HP produced by our engineered strains, while deletion of gdhA1, annotated as a glutamate dehydrogenase, prevented the utilization of aspartate as a carbon source, one of the key pathway intermediates. The final strain carrying these deletions was able to produce up to 8 mM 3-HP heterotrophically. Furthermore, an engineered strain was able to produce 0.5 mM 3-HP under autotrophic conditions, using CO2 as sole carbon source. These results form the basis for establishing C. necator H16 as an efficient platform for the production of 3-HP and 3-HP-containing polymers.

Metabolic modeling-based drug repurposing in Glioblastoma.

The manifestation of intra- and inter-tumor heterogeneity hinders the development of ubiquitous cancer treatments, thus requiring a tailored therapy for each cancer type. Specifically, the reprogramming of cellular metabolism has been identified as a source of potential drug targets. Drug discovery is a long and resource-demanding process aiming at identifying and testing compounds early in the drug development pipeline. While drug repurposing efforts (i.e., inspecting readily available approved drugs) can be supported by a mechanistic rationale, strategies to further reduce and prioritize the list of potential candidates are still needed to facilitate feasible studies. Although a variety of 'omics' data are widely gathered, a standard integration method with modeling approaches is lacking. For instance, flux balance analysis is a metabolic modeling technique that mainly relies on the stoichiometry of the metabolic network. However, exploring the network's topology typically neglects biologically relevant information. Here we introduce Transcriptomics-Informed Stoichiometric Modelling And Network analysis (TISMAN) in a recombinant innovation manner, allowing identification and validation of genes as targets for drug repurposing using glioblastoma as an exemplar.

A genome-scale metabolic model of Cupriavidus necator H16 integrated with TraDIS and transcriptomic data reveals metabolic insights for biotechnological applications.

Exploiting biological processes to recycle renewable carbon into high value platform chemicals provides a sustainable and greener alternative to current reliance on petrochemicals. In this regard Cupriavidus necator H16 represents a particularly promising microbial chassis due to its ability to grow on a wide range of low-cost feedstocks, including the waste gas carbon dioxide, whilst also naturally producing large quantities of polyhydroxybutyrate (PHB) during nutrient-limited conditions. Understanding the complex metabolic behaviour of this bacterium is a prerequisite for the design of successful engineering strategies for optimising product yields. We present a genome-scale metabolic model (GSM) of C. necator H16 (denoted iCN1361), which is directly constructed from the BioCyc database to improve the readability and reusability of the model. After the initial automated construction, we have performed extensive curation and both theoretical and experimental validation. By carrying out a genome-wide essentiality screening using a Transposon-directed Insertion site Sequencing (TraDIS) approach, we showed that the model could predict gene knockout phenotypes with a high level of accuracy. Importantly, we indicate how experimental and computational predictions can be used to improve model structure and, thus, model accuracy as well as to evaluate potential false positives identified in the experiments. Finally, by integrating transcriptomics data with iCN1361 we create a condition-specific model, which, importantly, better reflects PHB production in C. necator H16. Observed changes in the omics data and in-silico-estimated alterations in fluxes were then used to predict the regulatory control of key cellular processes. The results presented demonstrate that iCN1361 is a valuable tool for unravelling the system-level metabolic behaviour of C. necator H16 and can provide useful insights for designing metabolic engineering strategies.

Required Gene Set for Autotrophic Growth of Clostridium autoethanogenum.

The majority of the genes present in bacterial genomes remain poorly characterized, with up to one-third of those that are protein encoding having no definitive function. Transposon insertion sequencing represents a high-throughput technique that can help rectify this deficiency. The technology, however, can only be realistically applied to those species in which high rates of DNA transfer can be achieved. Here, we have developed a number of approaches that overcome this barrier in the autotrophic species Clostridium autoethanogenum by using a mariner-based transposon system. The inherent instability of such systems in the Escherichia coli conjugation donor due to transposition events was counteracted through the incorporation of a conditionally lethal codA marker on the plasmid backbone. Relatively low frequencies of transformation of the plasmid into C. autoethanogenum were circumvented through the use of a plasmid that is conditional for replication coupled with the routine implementation of an Illumina library preparation protocol that eliminates plasmid-based reads. A transposon library was then used to determine the essential genes needed for growth using carbon monoxide as the sole carbon and energy source. IMPORTANCE Although microbial genome sequences are relatively easily determined, assigning gene function remains a bottleneck. Consequently, relatively few genes are well characterized, leaving the function of many as either hypothetical or entirely unknown. High-throughput transposon sequencing can help remedy this deficiency, but is generally only applicable to microbes with efficient DNA transfer procedures. These exclude many microorganisms of importance to humankind either as agents of disease or as industrial process organisms. Here, we developed approaches to facilitate transposon insertion sequencing in the acetogen Clostridium autoethanogenum, a chassis being exploited to convert single-carbon waste gases CO and CO2 into chemicals and fuels at an industrial scale. This allowed the determination of gene essentiality under heterotrophic and autotrophic growth, providing insights into the utilization of CO as a sole carbon and energy source. The strategies implemented are translatable and will allow others to apply transposon insertion sequencing to other microbes where DNA transfer has until now represented a barrier to progress.

Agr Quorum Sensing influences the Wood-Ljungdahl pathway in Clostridium autoethanogenum.

Acetogenic bacteria are capable of fermenting CO2 and carbon monoxide containing waste-gases into a range of platform chemicals and fuels. Despite major advances in genetic engineering and improving these biocatalysts, several important physiological functions remain elusive. Among these is quorum sensing, a bacterial communication mechanism known to coordinate gene expression in response to cell population density. Two putative agr systems have been identified in the genome of Clostridium autoethanogenum suggesting bacterial communication via autoinducing signal molecules. Signal molecule-encoding agrD1 and agrD2 genes were targeted for in-frame deletion. During heterotrophic growth on fructose as a carbon and energy source, single deletions of either gene did not produce an observable phenotype. However, when both genes were simultaneously inactivated, final product concentrations in the double mutant shifted to a 1.5:1 ratio of ethanol:acetate, compared to a 0.2:1 ratio observed in the wild type control, making ethanol the dominant fermentation product. Moreover, CO2 re-assimilation was also notably reduced in both hetero- and autotrophic growth conditions. These findings were supported through comparative proteomics, which showed lower expression of carbon monoxide dehydrogenase, formate dehydrogenase A and hydrogenases in the ∆agrD1∆agrD2 double mutant, but higher levels of putative alcohol and aldehyde dehydrogenases and bacterial micro-compartment proteins. These findings suggest that Agr quorum sensing, and by inference, cell density play a role in carbon resource management and use of the Wood-Ljungdahl pathway as an electron sink.

Clostridium beijerinckii strain degeneration is driven by the loss of Spo0A activity.

Solventogenic clostridia represent a diverse group of anaerobic, spore-forming bacteria capable of producing acetone, butanol and ethanol through their unique biphasic metabolism. An intrinsic problem with these organisms however is their tendency to degenerate when repeatedly subcultured or when grown continuously. This phenomenon sees cells lose their ability to produce solvents and spores, posing a significant problem for industrial applications. To investigate the mechanistic and evolutionary basis of degeneration we combined comparative genomics, ultra-deep sequencing, and concepts of sociomicrobiology using Clostridium beijerinckii NCIMB 8052 as our model organism. These approaches revealed spo0A, the master regulator gene involved in spore and solvent formation, to be key to the degeneration process in this strain. Comparative genomics of 71 degenerate variants revealed four distinct hotspot regions that contained considerably more mutations than the rest of the genome. These included spo0A as well as genes suspected to regulate its expression and activity. Ultra-deep sequencing of populations during the subculturing process showed transient increases in mutations we believe linked to the spo0A network, however, these were ultimately dominated by mutations in the master regulator itself. Through frequency-dependent fitness assays, we found that spo0A mutants gained a fitness advantage, relative to the wild type, presumably allowing for propagation throughout the culture. Combined, our data provides new insights into the phenomenon of clostridial strain degeneration and the C. beijerinckii NCIMB 8052 solvent and spore regulation network.

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 R 2 ≥ 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.

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.

Physicochemical and metabolic constraints for thermodynamics-based stoichiometric modelling under mesophilic growth conditions.

Metabolic engineering in the post-genomic era is characterised by the development of new methods for metabolomics and fluxomics, supported by the integration of genetic engineering tools and mathematical modelling. Particularly, constraint-based stoichiometric models have been widely studied: (i) flux balance analysis (FBA) (in silico), and (ii) metabolic flux analysis (MFA) (in vivo). Recent studies have enabled the incorporation of thermodynamics and metabolomics data to improve the predictive capabilities of these approaches. However, an in-depth comparison and evaluation of these methods is lacking. This study presents a thorough analysis of two different in silico methods tested against experimental data (metabolomics and 13C-MFA) for the mesophile Escherichia coli. In particular, a modified version of the recently published matTFA toolbox was created, providing a broader range of physicochemical parameters. Validating against experimental data allowed the determination of the best physicochemical parameters to perform the TFA (Thermodynamics-based Flux Analysis). An analysis of flux pattern changes in the central carbon metabolism between 13C-MFA and TFA highlighted the limited capabilities of both approaches for elucidating the anaplerotic fluxes. In addition, a method based on centrality measures was suggested to identify important metabolites that (if quantified) would allow to further constrain the TFA. Finally, this study emphasised the need for standardisation in the fluxomics community: novel approaches are frequently released but a thorough comparison with currently accepted methods is not always performed.

RRNPP-type quorum sensing affects solvent formation and sporulation in Clostridium acetobutylicum.

The strictly anaerobic bacterium Clostridium acetobutylicum is well known for its ability to convert sugars into organic acids and solvents, most notably the potential biofuel butanol. However, the regulation of its fermentation metabolism, in particular the shift from acid to solvent production, remains poorly understood. The aim of this study was to investigate whether cell-cell communication plays a role in controlling the timing of this shift or the extent of solvent formation. Analysis of the available C. acetobutylicum genome sequences revealed the presence of eight putative RRNPP-type quorum-sensing systems, here designated qssA to qssH, each consisting of an RRNPP-type regulator gene followed by a small open reading frame encoding a putative signalling peptide precursor. The identified regulator and signal peptide precursor genes were designated qsrA to qsrH and qspA to qspH, respectively. Triplicate regulator mutants were generated in strain ATCC 824 for each of the eight systems and screened for phenotypic changes. The qsrB mutants showed increased solvent formation during early solventogenesis and hence the QssB system was selected for further characterization. Overexpression of qsrB severely reduced solvent and endospore formation and this effect could be overcome by adding short synthetic peptides to the culture medium representing a specific region of the QspB signalling peptide precursor. In addition, overexpression of qspB increased the production of acetone and butanol and the initial (48 h) titre of heat-resistant endospores. Together, these findings establish a role for QssB quorum sensing in the regulation of early solventogenesis and sporulation in C. acetobutylicum.

Complete Genome Sequence of Cupriavidus necator H16 (DSM 428).

The hydrogen-utilizing strain Cupriavidus necator H16 (DSM 428) was sequenced using a combination of PacBio and Illumina sequencing. Annotation of this strain reveals 6,543 protein-coding genes, 263 pseudogenes, 64 tRNA genes, and 15 rRNA genes.

A novel conjugal donor strain for improved DNA transfer into Clostridium spp.

Clostridium encompasses species which are relevant to human and animal disease as well as species which have industrial potential, for instance, as producers of chemicals and fuels or as tumour delivery vehicles. Genetic manipulation of these target organisms is critical for advances in these fields. DNA transfer efficiencies, however, vary between species. Low efficiencies can impede the progress of research efforts. A novel conjugal donor strain of Escherichia coli has been created which exhibits a greater than 10-fold increases in conjugation efficiency compared to the traditionally used CA434 strain in the three species tested; C. autoethanogenum DSM 10061, C. sporogenes NCIMB 10696 and C. difficile R20291. The novel strain, designated 'sExpress', does not methylate DNA at Dcm sites (CCWGG) which allows circumvention of cytosine-specific Type IV restriction systems. A robust protocol for conjugation is presented which routinely produces in the order of 105 transconjugants per millilitre of donor cells for C. autoethanogenum, 106 for C. sporogenes and 102 for C. difficile R20291. The novel strain created is predicted to be a superior conjugal donor in a wide range of species which possess Type IV restriction systems.

The carbonic anhydrase of Clostridium autoethanogenum represents a new subclass of ß-carbonic anhydrases.

Carbonic anhydrase catalyses the interconversion of carbon dioxide and water to bicarbonate and protons. It was unknown if the industrial-relevant acetogen Clostridium autoethanogenum possesses these enzymes. We identified two putative carbonic anhydrase genes in its genome, one of the ß class and one of the γ class. Carbonic anhydrase activity was found for the purified ß class enzyme, but not the γ class candidate. Functional complementation of an Escherichia coli carbonic anhydrase knock-out mutant showed that the ß class carbonic anhydrase could complement this activity, but not the γ class candidate gene. Phylogenetic analysis showed that the ß class carbonic anhydrase of Clostridium autoethanogenum represents a novel sub-class of ß class carbonic anhydrases that form the F-clade. The members of this clade have the shortest primary structure of any known carbonic anhydrase.

The genetic basis of 3-hydroxypropanoate metabolism in Cupriavidus necator H16.

BACKGROUND: 3-Hydroxypropionic acid (3-HP) is a promising platform chemical with various industrial applications. Several metabolic routes to produce 3-HP from organic substrates such as sugars or glycerol have been implemented in yeast, enterobacterial species and other microorganisms. In this study, the native 3-HP metabolism of Cupriavidus necator was investigated and manipulated as it represents a promising chassis for the production of 3-HP and other fatty acid derivatives from CO2 and H2. RESULTS: When testing C. necator for its tolerance towards 3-HP, it was noted that it could utilise the compound as the sole source of carbon and energy, a highly undesirable trait in the context of biological 3-HP production which required elimination. Inactivation of the methylcitrate pathway needed for propionate utilisation did not affect the organism's ability to grow on 3-HP. Putative genes involved in 3-HP degradation were identified by bioinformatics means and confirmed by transcriptomic analyses, the latter revealing considerably increased expression in the presence of 3-HP. Genes identified in this manner encoded three putative (methyl)malonate semialdehyde dehydrogenases (mmsA1, mmsA2 and mmsA3) and two putative dehydrogenases (hpdH and hbdH). These genes, which are part of three separate mmsA operons, were inactivated through deletion of the entire coding region, either singly or in various combinations, to engineer strains unable to grow on 3-HP. Whilst inactivation of single genes or double deletions could only delay but not abolish growth, a triple ∆mmsA1∆mmsA2∆mmsA3 knock-out strain was unable utilise 3-HP as the sole source of carbon and energy. Under the used conditions this strain was also unable to co-metabolise 3-HP alongside other carbon and energy sources such as fructose and CO2/H2. Further analysis suggested primary roles for the different mmsA operons in the utilisation of ß-alanine generating substrates (mmsA1), degradation of 3-HP (mmsA2), and breakdown of valine (mmsA3). CONCLUSIONS: Three different (methyl)malonate semialdehyde dehydrogenases contribute to 3-HP breakdown in C. necator H16. The created triple ∆mmsA1∆mmsA2∆mmsA3 knock-out strain represents an ideal chassis for autotrophic 3-HP production.

Engineering of vitamin prototrophy in Clostridium ljungdahlii and Clostridium autoethanogenum.

Clostridium autoethanogenum and Clostridium ljungdahlii are physiologically and genetically very similar strict anaerobic acetogens capable of growth on carbon monoxide as sole carbon source. While exact nutritional requirements have not been reported, we observed that for growth, the addition of vitamins to media already containing yeast extract was required, an indication that these are fastidious microorganisms. Elimination of complex components and individual vitamins from the medium revealed that the only organic compounds required for growth were pantothenate, biotin and thiamine. Analysis of the genome sequences revealed that three genes were missing from pantothenate and thiamine biosynthetic pathways, and five genes were absent from the pathway for biotin biosynthesis. Prototrophy in C. autoethanogenum and C. ljungdahlii for pantothenate was obtained by the introduction of plasmids carrying the heterologous gene clusters panBCD from Clostridium acetobutylicum, and for thiamine by the introduction of the thiC-purF operon from Clostridium ragsdalei. Integration of panBCD into the chromosome through allele-coupled exchange also conveyed prototrophy. C. autoethanogenum was converted to biotin prototrophy with gene sets bioBDF and bioHCA from Desulfotomaculum nigrificans strain CO-1-SRB, on plasmid and integrated in the chromosome. The genes could be used as auxotrophic selection markers in recombinant DNA technology. Additionally, transformation with a subset of the genes for pantothenate biosynthesis extended selection options with the pantothenate precursors pantolactone and/or beta-alanine. Similarly, growth was obtained with the biotin precursor pimelate combined with genes bioYDA from C. acetobutylicum. The work raises questions whether alternative steps exist in biotin and thiamine biosynthesis pathways in these acetogens.

Gsmodutils: a python based framework for test-driven genome scale metabolic model development.

MOTIVATION: Genome scale metabolic models (GSMMs) are increasingly important for systems biology and metabolic engineering research as they are capable of simulating complex steady-state behaviour. Constraints based models of this form can include thousands of reactions and metabolites, with many crucial pathways that only become activated in specific simulation settings. However, despite their widespread use, power and the availability of tools to aid with the construction and analysis of large scale models, little methodology is suggested for their continued management. For example, when genome annotations are updated or new understanding regarding behaviour is discovered, models often need to be altered to reflect this. This is quickly becoming an issue for industrial systems and synthetic biotechnology applications, which require good quality reusable models integral to the design, build, test and learn cycle. RESULTS: As part of an ongoing effort to improve genome scale metabolic analysis, we have developed a test-driven development methodology for the continuous integration of validation data from different sources. Contributing to the open source technology based around COBRApy, we have developed the gsmodutils modelling framework placing an emphasis on test-driven design of models through defined test cases. Crucially, different conditions are configurable allowing users to examine how different designs or curation impact a wide range of system behaviours, minimizing error between model versions. AVAILABILITY AND IMPLEMENTATION: The software framework described within this paper is open source and freely available from http://github.com/SBRCNottingham/gsmodutils. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.