Endogenous CRISPR/Cas systems for genome engineering in the acetogens and .

Acetogenic bacteria can play a major role in achieving Net Zero through their ability to convert CO 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 CRISPR/Cas9 system. However, attempts to introduce -containing vectors into were unsuccessful, most likely as a consequence of Cas9 nuclease toxicity and the presence of a recognition site for an endogenous restriction-modification (R-M) system in the gene.

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

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.

Improving carbon monoxide tolerance of H16 through adaptive laboratory evolution.

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 H16 can grow on CO + H, although it cannot utilise CO as a source of carbon and energy.

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

In , quorum sensing (QS) depends on an interconnected regulatory hierarchy involving the Las, Rhl and Pq systems, which are collectively responsible for the co-ordinated synthesis of a diverse repertoire of -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.

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.

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.

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.

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.

Required Gene Set for Autotrophic Growth of .

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.

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

Acetogenic bacteria are capable of fermenting CO 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.

Quantitative Bioreactor Monitoring of Intracellular Bacterial Metabolites in 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 . A comprehensive mixture of uniformly C-labeled internal standards (U-C IS) was biosynthesized from the closely related bacterium using 4% C-glucose as a carbon source. The U-C IS mixture combined with C authentic standards was used to validate the linearity, precision, accuracy, repeatability, limits of detection, and quantification for each metabolite.

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

Chirally pure ()-1,3-butanediol (()-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 ()-1,3-BDO was produced in the industrially important whole cell biocatalyst through expression of the enantio-specific gene from .

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.

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

The strictly anaerobic bacterium 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.

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

The hydrogen-utilizing strain 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.

The carbonic anhydrase of Clostridium autoethanogenum represents a new subclass of β-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 β class and one of the γ class.

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.

The genetic basis of 3-hydroxypropanoate metabolism in 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 was investigated and manipulated as it represents a promising chassis for the production of 3-HP and other fatty acid derivatives from CO and H.

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.

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.

Functional Genetic Elements for Controlling Gene Expression in Cupriavidus necator H16.

A robust and predictable control of gene expression plays an important role in synthetic biology and biotechnology applications. Development and quantitative evaluation of functional genetic elements, such as constitutive and inducible promoters as well as ribosome binding sites (RBSs), are required. In this study, we designed, built, and tested promoters and RBSs for controlling gene expression in the model lithoautotroph H16.

Progress towards platform chemical production using .

Since 2013, there has been an explosion in the number of research articles published on , an acetogen capable of producing platform chemicals such as ethanol and 2,3-butanediol from greenhouse gases. However, no review focusing solely on has appeared in the literature. This review outlines the research conducted into this organism in three broad categories (Enzymology, Genetics, and Systems Biology) and suggestions for future research are offered.

The Butanol Producing Microbe Clostridium beijerinckii NCIMB 14988 Manipulated Using Forward and Reverse Genetic Tools.

The solventogenic anaerobe Clostridium beijerinckii has potential for use in the sustainable bioconversion of plant-derived carbohydrates into solvents, such as butanol or acetone. However, relatively few strains have been extensively characterised either at the genomic level or through exemplification of a complete genetic toolkit. To remedy this situation, a new strain of C.

Quantitative Isotope-Dilution High-Resolution-Mass-Spectrometry Analysis of Multiple Intracellular Metabolites in Clostridium autoethanogenum with Uniformly C-Labeled Standards Derived from Spirulina.

We have investigated the applicability of commercially available lyophilized spirulina ( Arthrospira platensis), a microorganism uniformly labeled with C, as a readily accessible source of multiple C-labeled metabolites suitable as internal standards for the quantitative determination of intracellular bacterial metabolites. Metabolites of interest were analyzed by hydrophilic-interaction liquid chromatography coupled with high-resolution mass spectrometry. Multiple internal standards obtained from uniformly (U)-C-labeled extracts from spirulina were used to enable isotope-dilution mass spectrometry (IDMS) in the identification and quantification of intracellular metabolites.