The potential of clostridial spores as therapeutic delivery vehicles in tumour therapy

AbstractDespite substantial investment in prevention, treatment and aftercare, cancer remains a leading cause of death worldwide. More effective and accessible therapies are required. A potential solution is the use of endospore forming Clostridium species, either on their own, or as a tumour delivery vehicle for anti-cancer drugs. This is because intravenously injected spores of these obligate anaerobes can exclusively germinate in the hypoxic/necrotic regions present in solid tumours and nowhere else in the body. Research aimed at exploiting this unique phenomenon in anti-tumour strategies has been ongoing since the early part of the 20th century. Only in the last decade, however, has there been significant progress in the development and refinement of strategies based on spore-mediated tumour colonisation using a range of clostridial species. Much of this progress has been due to advances in genomics and our ability to modify strains using more sophisticated gene tools

Upscaling diffusion through first-order volumetric sinks: a homogenization of bacterial nutrient uptake

In mathematical models that include nutrient delivery to bacteria, it is prohibitively expensive to include a pointwise nutrient uptake within small bacterial regions over bioreactor length-scales, and so such models often impose an effective uptake instead. In this paper, we systematically investigate how the effective uptake should scale with bacterial size and other microscale properties under first-order uptake kinetics. We homogenize the unsteady problem of nutrient diffusing through a locally periodic array of spherical bacteria, within which it is absorbed. We introduce a general model that could also be applied to other single-cell microorganisms, such as cyanobacteria, microalgae, protozoa, and yeast and we consider generalizations to arbitrary bacterial shapes, including some analytic results for ellipsoidal bacteria. We explore in detail the three distinguished limits of the system on the timescale of diffusion over the macroscale. When the bacterial size is of the same order as the distance between them, the effective uptake has two limiting behaviours, scaling with the bacterial volume for weak uptake and with the bacterial surface area for strong uptake. We derive the function that smoothly transitions between these two behaviours as the system parameters vary. Additionally, we explore the distinguished limit in which bacteria are much smaller than the distance between them and have a very strong uptake. In this limit, we find that the effective uptake is bounded above as the uptake rate grows without bound; we are able to quantify this and characterise the transition to the other limits we consider

Applying asymptotic methods to synthetic biology: modelling the reaction kinetics of the mevalonate pathway

The mevalonate pathway is normally found in eukaryotes, and allows for the production of isoprenoids, a useful class of organic compounds. This pathway has been successfully introduced to Escherichia coli, enabling a biosynthetic production route for many isoprenoids. In this paper, we develop and solve a mathematical model for the concentration of metabolites in the mevalonate pathway over time, accounting for the loss of acetyl-CoA to other metabolic pathways. Additionally, we successfully test our theoretical predictions experimentally by introducing part of the pathway into Cupriavidus necator. In our model, we exploit the natural separation of time scales as well as of metabolite concentrations to make significant asymptotic progress in understanding the system. We confirm that our asymptotic results agree well with numerical simulations, the former enabling us to predict the most important reactions to increase isopentenyl diphosphate production whilst minimizing the levels of HMG-CoA, which inhibits cell growth. Thus, our mathematical model allows us to recommend the upregulation of certain combinations of enzymes to improve production through the mevalonate pathway

Applying asymptotic methods to synthetic biology: modelling the reaction kinetics of the mevalonate pathway

The mevalonate pathway is normally found in eukaryotes, and allows for the production of isoprenoids, a useful class of organic compounds. This pathway has been successfully introduced to Escherichia coli, enabling a biosynthetic production route for many isoprenoids. In this paper, we develop and solve a mathematical model for the concentration of metabolites in the mevalonate pathway over time, accounting for the loss of acetyl-CoA to other metabolic pathways. Additionally, we successfully test our theoretical predictions experimentally by introducing part of the pathway into Cupriavidus necator. In our model, we exploit the natural separation of time scales as well as of metabolite concentrations to make significant asymptotic progress in understanding the system. We confirm that our asymptotic results agree well with numerical simulations, the former enabling us to predict the most important reactions to increase isopentenyl diphosphate production whilst minimizing the levels of HMG-CoA, which inhibits cell growth. Thus, our mathematical model allows us to recommend the upregulation of certain combinations of enzymes to improve production through the mevalonate pathway

Biosynthesis of Poly(3HB- co-3HP) with Variable Monomer Composition in Recombinant Cupriavidus necator H16

Polyhydroxyalkanoates are attractive alternatives to traditional plastics. However, although polyhydroxybutyrate (PHB) is produced in large quantities by Cupriavidus necator H16, its properties are far from ideal for the manufacture of plastic products. These properties may be improved through its coproduction with 3-hydroxypropionate (3HP), which leads to the formation of the copolymer poly(3-hydroxybutyrate-co-3-hydroxypropionate) (poly(3HB-co-3HP). To achieve this, a pathway was designed to enable C. necator H16 to convert β-alanine to 3HP. The initial low levels of incorporation of 3HP into the copolymer were overcome by the overproduction of the native propionyl-CoA transferase together with PHA synthase from Chromobacterium sp. USM2. Following optimization of 3HP incorporation into the copolymer, the molar fraction of 3HP could be controlled by cultivation in medium containing different concentrations of β-alanine. Between 0 and 80 mol % 3HP could be achieved. Further supplementation with 2 mM cysteine increased the maximum 3HP molar fraction to 89%. Additionally, the effect of deletions of the phaA and phaB1 genes of the phaCAB operon on 3HP molar fraction were investigated. A phaAB1 double knockout resulted in a copolymer containing 91 mol % 3HP without the need for cysteine supplementation

Homologous overexpression of hydrogenase and glycerol dehydrogenase in Clostridium pasteurianum to enhance hydrogen production from crude glycerol

This study reports engineering of a hypertransformable variant of C. pasteurianum for bioconversion of glycerol into hydrogen (H2). A functional glycerol-triggered hydrogen pathway was engineered based on two approaches: (1) increasing product yield by overexpression of immediate enzyme catalyzing H2 production, (2) increasing substrate uptake by overexpression of enzymes involved in glycerol utilization. The first strategy aimed at overexpression of hydA gene encoding hydrogenase, and the second one, through combination of overexpression of dhaD1 and dhaK genes encoding glycerol dehydrogenase and dihydroxyacetone kinase. These genetic manipulations resulted in two recombinant strains (hydA ++ /dhaD1K ++) capable of producing 97% H2 (v/v), with yields of 1.1 mol H2/mol glycerol in hydA overexpressed strain, and 0.93 mol H2/mol glycerol in dhaD1K overexpressed strain, which was 1.5 fold higher than wild type. Among two strains, dhaD1K ++ consumed more glycerol than hydA ++ which proves that overexpression of glycerol enzymes has enhanced glycerol intake rate

Using singular perturbation theory to determine kinetic parameters in a non-standard coupled enzyme assay

We investigate how to characterize the kinetic parameters of an aminotransaminase using a non-standard coupled (or auxiliary) enzyme assay, where the peculiarity arises for two reasons. First, one of the products of the auxiliary enzyme is a substrate for the primary enzyme and, second, we explicitly account for the reversibility of the auxiliary enzyme reaction. Using singular perturbation theory, we characterize the two distinguished asymptotic limits in terms of the strength of the reverse reaction, which allows us to determine how to deduce the kinetic parameters of the primary enzyme for a characterized auxiliary enzyme. This establishes a parameter-estimation algorithm that is applicable more generally to similar reaction networks. We demonstrate the applicability of our theory by performing enzyme assays to characterize a novel putative aminotransaminase enzyme, CnAptA (UniProtKB Q0KEZ8) from Cupriavidus necator H16, for two different omega-amino acid substrates

RiboCas: A Universal CRISPR-Based Editing Tool for Clostridium

Members of the genus Clostridium represent a diverse assemblage of species exhibiting both medical and industrial importance. Deriving both a greater understanding of their biology, while at the same time enhancing their exploitable properties, requires effective genome editing tools. Here, we demonstrate the first implementation in the genus of theophylline-dependent, synthetic riboswitches exhibiting a full set of dynamic ranges, also suitable for applications where tight control of gene expression is required. Their utility was highlighted by generating a novel riboswitch-based editing tool—RiboCas—that overcomes the main obstacles associated with CRISPR/Cas9 systems, including low transformation efficiencies and excessive Cas9 toxicity. The universal nature of the tool was established by obtaining chromosomal modifications in C. pasteurianum, C. difficile, and C. sporogenes, as well as by carrying out the first reported example of CRISPR-targeted gene disruption in C. botulinum. The high efficiency (100% mutant generation) and ease of application of RiboCas make it suitable for use in a diverse range of microorganisms

Heterologous gene expression in the human gut bacteria Eubacterium rectale and Roseburia inulinivorans by means of conjugative plasmids

Acknowledgements The Rowett Institute (University of Aberdeen) receives financial support from the Scottish Government Rural and Environmental Sciences and Analytical Services (RESAS). POS was a PhD student supported by the Scottish Government (RESAS) and the Science Foundation Ireland, through a centre award (12/RC/2273) to APC Microbiome Ireland, Cork, Ireland.Peer reviewedPostprin