Biology subjects

Swartz, J.

Publications and source records attributed to Swartz, J..

5 recordsLinked to original sources

bioRxiv2026

Hydrogen-Driven Cell-Free Cofactor Regeneration Enables Stoichiometric Bioconversion of Pyruvate to Lactate

The declining cost of green hydrogen--projected below 1.5 USD/kg by 2030--opens new avenues for its use beyond fuel cells and industrial heating. Here we demonstrate that H2 can serve as a stoichiometric electron donor for cell-free enzymatic cofactor regeneration, coupling H2 oxidation to NADPH production and driving the complete bioconversion of pyruvate to lactate. A partially purified enzyme ensemble from Escherichia coli overexpressing Clostridium pasteurianum ferredoxin, augmented with [FeFe]-hydrogenase CpII, delivers NADP+ reduction rates of 103 M min-1 (27-fold enhancement) with superlinear dependence on H2 partial pressure. Reconstitution from purified components (CpI or CpII, CpFd, AnFNR, LDH) uncovers a redox-potential-dependent lag phase: the NADPH/NADP+ ratio must exceed 0.85 before pyruvate reduction becomes thermodynamically spontaneous, after which the rate accelerates exponentially. These results position hydrogen-driven cofactor regeneration as a scalable, byproduct-free platform for reductive biotransformations powered by renewable H2.

bioengineering↗

bioRxiv2026

Genome mining reveals a sporulation associated protein with ferredoxin NADP+ reductase activity in Clostridium pasteurianum: structural and kinetic characterization

Ferredoxin-NADP+ reductases (FNRs) are ubiquitous flavoenzymes that catalyse the reversible transfer of electrons between iron-sulfur ferredoxins and the pyridine nucleotide pool, thereby occupying a central position in diverse redox metabolic pathways including photosynthesis, nitrogen fixation, and detoxification of reactive oxygen species. Although FNR activity was demonstrated in cell extracts of Clostridium pasteurianum more than five decades ago, the gene encoding this activity has remained unidentified. In the present study, a systematic bioinformatic screen of all 3,797 predicted proteins from the C. pasteurianum genome was conducted using conserved FAD- and NAD(P)+-interacting residues from structurally characterised reductases as search templates. This analysis identified a single candidate, AQ984_05830, which is annotated as a sporulation protein but possesses all six predicted cofactor-interacting residues. Heterologous expression and cytochrome c reduction assays confirmed ferredoxin-dependent reductase activity, with a wild-type kcat of 0.007 min-1--a value orders of magnitude lower than those reported for canonical FNRs. A parallel genome-wide screen further revealed a repertoire of ferredoxin-like carriers, suggesting that C. pasteurianum distributes hydrogen-derived electrons among multiple ferredoxins to serve diverse metabolic fates, of which NADP reduction by CpFNR is one. Alanine scanning mutagenesis of five predicted cofactor-interacting residues revealed that K68A and K73A mutations abolished activity, whereas T64A, T185A and S202A mutations improved catalytic efficiency (kcat/Km) for NADH by 14 to 18 folds. AlphaFold structure prediction combined with SwissDock and ClusPro molecular docking simulations placed the FAD binding site centrally between the NAD(P)H and ferredoxin binding domains, consistent with the expected electron relay architecture. Structural analysis of the beneficial mutations suggests that disruption of hydrogen bonds flanking a flexible coil (residues 186-199) propagates conformational effects to the NAD(P)H binding loops, rationalising the improved substrate affinities. These findings expand the known functional diversity of the FNR superfamily and suggest an unrecognised role for redox regulation during endospore formation in C. pasteurianum.

biochemistry↗

bioRxiv2025

On-demand insulin manufacturing using cell-free systems with an "on-column" conversion approach

Recent studies project that the prevalence of diabetes is expected to increase significantly and lead to escalating demand on the insulin supply chain. Despite being the first recombinant therapeutic approved by the FDA, insulin remains challenging to access for many around the globe. Here we report on advancements in manufacturing insulin using cell-free protein synthesis (CFPS) systems to rapidly produce mature desB30-insulin in less than a day. This is a major advance towards decentralizing insulin manufacturing and bringing production closer to the point-of-care, thereby improving diabetic patient accessibility. To this end, a purified cell-free extract, PUREfrex(R) 2.1, was utilized to synthesize a tagged proinsulin construct that can be captured and converted into mature insulin using an on-column affinity chromatography process. Notably, two chaperones, peptidyl prolyl isomerase (FkpA), and seven kilodalton protein (Skp) were observed to play a critical role during cell-free expression of proinsulin. The proinsulin was then immobilized on a Ni-NTA column where the purification and conversion of cell-free products were performed sequentially to yield desB30-insulin. Following further optimization, this method serves as a time and resource-efficient production process as compared to current methods. When applied simultaneously, the cell-free expression and on-column conversion methods reported here can be adopted to enable rapid on-demand insulin manufacturing in order to improve the accessibility of insulin and prevent future shortages.

synthetic biology↗

bioRxiv2017

Toward a Genome Scale Sequence Specific Dynamic Model of Cell-Free Protein Synthesis in Escherichia coli

Cell-free protein expression systems have become widely used in systems and synthetic biology. In this study, we developed an ensemble of dynamic E. coli cell-free protein synthesis (CFPS) models. Model parameters were estimated from a training dataset for the cell-free production of a protein product, chloramphenicol acetyltransferase (CAT). The dataset consisted of measurements of glucose, organic acids, energy species, amino acids, and CAT. The ensemble accurately predicted these measurements, especially those of the central carbon metabolism. We then used the trained model to evaluate the optimality of protein production. CAT was produced with an energy efficiency of 12%, suggesting that the process could be further optimized. Reaction group knockouts showed that protein productivity and the metabolism as a whole depend most on oxidative phosphorylation and glycolysis and gluco-neogenesis. Amino acid biosynthesis is also important for productivity, while the overflow metabolism and TCA cycle affect the overall system state. In addition, the translation rate is shown to be more important to productivity than the transcription rate. Finally, CAT production was robust to allosteric control, as was most of the network, with the exception of the organic acids in central carbon metabolism. This study is the first to use kinetic modeling to predict dynamic protein production in a cell-free E. coli system, and should provide a foundation for genome scale, dynamic modeling of cell-free E. coli protein synthesis.

synthetic biology↗

bioRxiv2017

Sequence Specific Modeling of E. coli Cell-Free Protein Synthesis

Cell-free protein synthesis (CFPS) is a widely used research tool in systems and synthetic biology. However, if CFPS is to become a mainstream technology for applications such as point of care manufacturing, we must understand the performance limits and costs of these systems. Toward this question, we used sequence specific constraint based modeling to evaluate the performance of E. coli cell-free protein synthesis. A core E. coli metabolic network, describing glycolysis, the pentose phosphate pathway, energy metabolism, amino acid biosynthesis and degradation was augmented with sequence specific descriptions of transcription and translation and effective models of promoter function. Model parameters were largely taken from literature, thus the constraint based approach coupled the transcription and translation of the protein product, and the regulation of gene expression, with the availability of metabolic resources using only a limited number of adjustable model parameters. We tested this approach by simulating the expression of two model proteins: chloramphenicol acetyltransferase and dual emission green fluorescent protein, for which we have training data sets; we then expanded the simulations to a range of additional proteins. Protein expression simulations were consistent with measurements for a variety of cases. The constraint based simulations confirmed that oxidative phosphorylation was active in the CAT cell-free extract, as without it there was no feasible solution within the experimental constraints of the system. We then compared the metabolism of theoretically optimal and experimentally constrained CFPS reactions, and developed parameter free correlations which could be used to estimate productivity as a function of protein length and promoter type. Lastly, global sensitivity analysis identified the key metabolic processes that controlled CFPS productivity and energy efficiency. In summary, sequence specific constraint based modeling of CFPS offered a novel means to a priori estimate the performance of a cell-free system, using only a limited number of adjustable parameters. While we modeled the production of a single protein in this study, the approach could easily be extended to multi-protein synthetic circuits, RNA circuits or the cell free production of small molecule products.

synthetic biology↗