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There is an increasing interest in the upgrading of inexpensive and abundant C

There are several natural and anthropomorphic environments where iron- and/or sulfur-oxidizing bacteria thrive in extremely acidic conditions. These acidophilic chemolithautotrophs play important roles in biogeochemical iron and sulfur cycles, are critical catalysts for industrial metal bioleaching operations, and have underexplored potential in future biotechnological applications. However, their unique growth conditions complicate the development of genetic techniques. Over the past few decades genetic tools have been successfully developed for Acidithiobacillus ferrooxidans, which serves as a model organism that exhibits both iron- and sulfur-oxidizing capabilities. Conjugal transfer of plasmids has enabled gene overexpression, gene knockouts, and some preliminary metabolic engineering. We highlight the development of genetic systems and recent genetic engineering of A. ferrooxidans, and discuss future perspectives.

A hydrometallurgical process is developed to lower the costs of copper production and thereby sustain the use of copper throughout the global transition to renewable energy technologies. The unique feature of the hydrometallurgical process is the reductive treatment of chalcopyrite, which is in contrast to the oxidative treatment more commonly pursued in the literature. Chalcopyrite reduction by chromium(II) ion is described for the first time and superior kinetics are shown. At high concentrate loadings of 39, 78, and 117 g L

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Acidithiobacillus ferrooxidans is an acidophilic chemolithoautotroph that is commonly reported to exhibit diauxic population growth behavior where ferrous iron is oxidized before elemental sulfur when both are available, despite the higher energy content of sulfur. We have discovered sulfur dispersion formulations that enables sulfur oxidation before ferrous iron oxidation. The oxidation of dispersed sulfur can lower the culture pH within days below the range where aerobic ferrous iron oxidation can occur. Thus, ferric iron reduction can be observed quickly which had previously been reported over extended incubation periods with untreated sulfur. Therefore, we demonstrate that this substrate utilization pattern is strongly dependent on the cell loading in relation to sulfur concentration, sulfur surface hydrophobicity, and the pH of the culture. Our dispersed sulfur formulation, lig-sulfur, can be used to support the rapid antibiotic selection of plasmid-transformed cells, which is not possible in liquid cultures where ferrous iron is the main source of energy for these acidophiles. Furthermore, we find that media containing lig-sulfur supports higher production of green fluorescent protein compared to media containing ferrous iron. The use of dispersed sulfur is a valuable new tool for the development of engineered A. ferrooxidans strains and it provides a new method to control iron and sulfur oxidation behaviors.

Acidithiobacillus ferrooxidans cells can oxidize iron and sulfur and are key members of the microbial biomining communities that are exploited in the large-scale bioleaching of metal sulfide ores. Some minerals are recalcitrant to bioleaching due to the presence of other inhibitory materials in the ore bodies. Additives are intentionally included in processed metals to reduce environmental impacts and microbially influenced corrosion. We have previously reported a new aerobic corrosion mechanism where A. ferrooxidans cells combined with pyrite and chloride can oxidize low-grade stainless steel (SS304) with a thiosulfate-mediated mechanism. Here we explore process conditions and genetic engineering of the cells that enable corrosion of a higher grade steel (SS316). The addition of elemental sulfur and an increase in the cell loading resulted in a 74% increase in the corrosion of SS316 as compared to the initial sulfur- and cell-free control experiments containing only pyrite. The overexpression of the endogenous rus gene, which is involved in the cellular iron oxidation pathway, led to a further 85% increase in the corrosion of the steel in addition to the improvements made by changes to the process conditions. Thus, the modification of the culturing conditions and the use of rus-overexpressing cells led to a more than threefold increase in the corrosion of SS316 stainless steel, such that 15% of the metal coupons was dissolved in just 2 weeks. This study demonstrates how the engineering of cells and the optimization of their cultivation conditions can be used to discover conditions that lead to the corrosion of a complex metal target.

© 2020 Wiley Periodicals LLC Today's Biochemical Engineer may contribute to advances in a wide range of technical areas. The recent Biochemical and Molecular Engineering XXI conference focused on “The Next Generation of Biochemical and Molecular Engineering: The role of emerging technologies in tomorrow's products and processes”. On the basis of topical discussions at this conference, this perspective synthesizes one vision on where investment in research areas is needed for biotechnology to continue contributing to some of the world's grand challenges.

A hydrometallurgical process has been demonstrated to electrochemically convert chalcopyrite (CuFeS2) to less refractory mineral phases for subsequent chemical oxidation. The electrochemical reaction mechanisms are not well understood; consequently, researchers have been unable to improve the process. In this study, the bulk and surface phases of the chalcopyrite mineral during the progression of the electrochemical reactions are monitored using x-ray diffraction and x-ray photoelectron spectroscopy, respectively. The results suggest that chalcopyrite reacts at the cathode of the electrochemical reactor to release iron and form an intermediate chalcocite (Cu2S) mineral phase. Allowing Cu2S to contact the anode leads to the formation of covellite (CuS), whereas preventing the mineral from anode contact leads to the formation of cuprite (Cu2O). It was shown that copper ions are more easily extracted from Cu2O than CuS; therefore, it may be desirable to isolate the anode from mineral contact during the electrochemical process.

The kcat/KM ratio holds substantial significance as a comparative figure of merit in the evaluation of enzymes with uni-molecular mechanisms. However, its applicability in the evaluation of multi-reactant systems is frequently not justified. Here, we derive figures of merit for multi-reactant enzyme mechanisms based on the stabilization of the transition states and the kinetic Haldane equilibrium relationships. For most bi-molecular reactions, the ratio of kcat/KiaKb (where Kia is the dissociation constant of A and Kb is the Michaelis constant of B) is best suited for comparing enzyme performance, especially at non-saturating reactant concentrations. The value of this parameter for assessing the performance of a series of oxidoreductase mutants is demonstrated. Figures of merit for other common enzymatic mechanisms are derived, which enable a theory-based approach for comparing steady-state catalytic performances of enzymes with multiple reactants.

Protein engineering has been used to enhance the activities, selectivities, and stabilities of enzymes. Frequently tradeoffs are observed, where improvements in some features can come at the expense of others. Nature uses modular assembly of active sites for complex, multi-step reactions, and natural "swing arm" mechanisms have evolved to transfer intermediates between active sites. Biomimetic polyethylene glycol (PEG) swing arms modified with NAD(H) have been explored to introduce synthetic swing arms into fused oxidoreductases. Here we report that increasing NAD(H)-PEG swing arms can improve the activity of synthetic formate:malate oxidoreductases as well as the thermal and operational stabilities of the biocatalysts. The modular assembly approach enables the K M values of new enzymes to be predictable, based on the parental enzymes. We describe four unique synthetic transhydrogenases that have no native homologs, and this platform could be easily extended for the predictive design of additional synthetic cofactor-independent transhydrogenases.

Acidithiobacillus ferrooxidans are well-studied iron- and sulfur-oxidizing acidophilic chemolithoautotrophs that are exploited for their ability to participate in the bioleaching of metal sulfides. Here, we overexpressed the endogenous glutamate--cysteine ligase and glutathione synthetase genes in separate strains and found that glutathione synthetase overexpression increased intracellular glutathione levels. We explored the impact of pH on the halotolerance of iron oxidation in wild type and engineered cultures. The increase in glutathione allowed the modified cells to grow under salt concentrations and pH conditions that are fully inhibitory to wild type cells. These results indicate that glutathione overexpression can be used to increase halotolerance in A. ferrooxidans and would likely be a useful strategy on other acidophilic bacteria.ImportanceThe use of acidophilic bacteria in the hydrometallurgical processing of sulfide ores can enable many benefits including the potential reduction of environmental impacts. The cells involved in bioleaching tend to have limited halotolerance, and increased halotolerance could enable several benefits, including a reduction in the need for fresh water resources. We show that the genetic modification of A. ferrooxidans for the overproduction of glutathione is a promising strategy to enable cells to resist the oxidative stress that can occur during growth in the presence of salt.

In this study, we use a modeling approach to evaluate the potential impact of microbial metabolism on the organic composition of cloud droplets and atmospheric aerosols. Microbial consumption rates...

The enzymatic microenvironment can impact biocatalytic activity; however, these effects can be difficult to investigate as mutations and fusions can introduce multiple variables and overlapping effects. The fusion of a supercharged protein is a potentially facile means to alter the enzymatic microenvironment. We have investigated complexes made between a thermostable alcohol dehydrogenase (AdhD) and superfolding green fluorescent protein (sfGFP) mutants with extreme surface charges. Three charged sfGFP variants, -30, 0, and +36 were covalently attached to AdhD through the SpyCatcher/SpyTag system. Specific rates for the NAD+ -dependent oxidation of butane-2,3-diol were significantly increased in the -30 sfGFP complex, a mixed effect was seen for the 0 sfGFP complexes, and the rates were unaffected by +36 sfGFP complexation. Reactions performed at various pH values (7.8-9.8) and salt concentrations (7.75-500 mm) showed that there was a complex interplay between these effects that was consistent with fusion proteins affecting the local ionic strength, as opposed to the local pH. Steady-state kinetic analyses were performed with the -30 and 0 AdhD-sfGFP complexes. The overall catalytic efficiency was dependent on the charge of the fused sfGFP variant; the -30 sfGFP fusions exhibited the largest beneficial effects at pH 8.8. The impact of the fusions on the apparent ionic strength provides further insight into the effects of charged patches observed on metabolon-forming enzyme complexes.

Copper is expected to be in high demand in the coming decades due to the emergence of wind and solar technologies, which require about five times as much copper as traditional energy sources. Copper, however, is expected to be in short supply in the coming decades due to the high costs associated with the mining, concentrating, and processing of chalcopyrite (CuFeS2), which accounts for about 70% of all copper reserves. This work introduces a potentially transformative hydrometallurgical process for domestic production of copper from CuFeS2. Commercialization of such a process could sustain a high rate of copper production throughout the 21st century. Chalcopyrite is reacted with a redox couple to enable the rapid, clean, and complete recovery of copper. The reductant may be regenerated by an electrolysis unit. Reactions 1 and 2 show the direct electrochemical reduction of CuFeS2 to Cu2S and Cu0, respectively 2 CuFeS2 + 6H+ + 2e- → Cu2S + 2 Fe2+ + 3 H2 S [1] Cu2S + 2H+ + 2e- → 2 Cu0 + H2 S [2] The cathodic reduction of CuFeS2 competes with the hydrogen evolution reaction and therefore becomes inefficient at current densities exceeding 40 mA/cm2. Conversely, the cathodic reduction of an electron mediator circumvents the hydrogen evolution reaction and enables current densities exceeding 100 mA/cm2. Figure 1 shows a result that highlights the use of an electron mediator to facilitate the rapid and complete reduction of chalcopyrite, followed by the dissolution of the resultant solid product into sulfuric acid for the complete recovery of copper. In figure 1a, the release of Fe2+ ions to solution during the progression of the reaction with electron mediator is shown. In all cases, the solution contains 4M H2SO4 and various loadings of CuFeS2 concentrate. Error bars show standard deviations of replicates in triplicate. The reaction nearly goes to completion in 10 minutes. In figure 1b, the subsequent extraction of Cu2+ from mineral products dissolution into 1M H2SO4. The resulting concentration of copper in the sulfuric acid is amenable to electrowinning. Results are shown for high loadings of chalcopyrite, within a practical range for economic viability. In this work, we report successful results from two redox couples that can be effectively regenerated via electrolysis. A preliminary technoeconomic analysis is discussed, identifying potential opportunities as well as technical challenges. Figure 1

Cyc2 is the key protein in the outer membrane of Acidithiobacillus ferrooxidans that mediates electron transfer between extracellular inorganic iron and the intracellular central metabolism. This cytochrome c is specific for iron and interacts with periplasmic proteins to complete a reversible electron transport chain. A structure of Cyc2 has not yet been characterized experimentally. Here we describe a structural model of Cyc2, and associated proteins, to highlight a plausible mechanism for the ferrous iron electron transfer chain. A comparative modeling protocol specific for trans membrane beta barrel (TMBB) proteins in acidophilic conditions (pH ~2) was applied to the primary sequence of Cyc2. The proposed structure has three main regimes: extracellular loops exposed to low-pH conditions, a TMBB, and a N-terminal cytochrome-like region within the periplasmic space. The Cyc2 model was further refined by identifying likely iron and heme docking sites. This represents the first computational model of Cyc2 that accounts for the membrane microenvironment and the acidity in the extracellular matrix. This approach can be used to model other TMBBs which can be critical for chemolithotrophic microbial growth.Importance of workAcidithiobacillus ferrooxidans can oxidize both iron and reduced sulfur compounds and plays a key role in metal sulfide ore bioleaching used for the industrial recovery of metals. A. ferrooxidans has also been explored as a potential organism for emerging technologies such as e-waste recycling and biofuel production. Synthetic biology efforts are hampered by lack of knowledge about the mechanisms of iron oxidation and reduction, which is mediated by the Cyc2 transmembrane beta barrel (TMBB) protein.

Acidithiobacillus ferrooxidans are acidophilic chemolithoautotrophs that are commonly reported to exhibit diauxic population growth behavior where ferrous iron is oxidized before elemental sulfur when both are available, despite the higher energy content of sulfur. We have discovered sulfur dispersion formulations that enables sulfur oxidation before ferrous iron oxidation. The oxidation of dispersed sulfur can lower the culture pH within days below the range where aerobic ferrous iron oxidation can occur so that ferric iron reduction occurs which had previously been reported over extended incubation periods with untreated sulfur. Therefore, we demonstrate that this substrate utilization pattern is strongly dependent on the cell loading in relation to sulfur concentration, sulfur surface hydrophobicity, and the pH of the culture. Our dispersed sulfur formulation, lig-sulfur, can be used to support the rapid antibiotic selection of plasmid-transformed cells, which is not possible in liquid cultures where ferrous iron is the main source of energy for these acidophiles. Furthermore, we find that media containing lig-sulfur supports higher production of green fluorescent protein (GFP) compared to media containing ferrous iron. The use of dispersed sulfur is a valuable new tool for the development of engineered A. ferrooxidans strains and it provides a new method to control iron and sulfur oxidation behaviors.

Enzymatic biocatalysis can be limited by the necessity of soluble cofactors. Here, we introduced PEGylated nicotinamide adenine dinucleotide (NAD(H)) swing arms to two covalently fused dehydrogenase enzymes to eliminate their nicotinamide cofactor requirements. A formate dehydrogenase and cytosolic malate dehydrogenase were connected via SpyCatcher-SpyTag fusions. Bifunctionalized polyethylene glycol chains tethered NAD(H) to the fusion protein. This produced a formate:malate oxidoreductase that exhibited cofactor-independent ping-pong kinetics with predictable Michaelis constants. Kinetic modeling was used to explore the effective cofactor concentrations available for electron transfer in the complexes. This approach could be used to create additional cofactor-independent transhydrogenase biocatalysts by swapping fused dehydrogenases.

The RTX domains found in some secreted proteins fold into the β-roll secondary structure motif upon calcium binding, which enables folding to be localized extracellularly. We inserted an RTX domain from the adenylate cyclase of Bordetella pertussis into a loop near the catalytic active site of the thermostable alcohol dehydrogenase D (AdhD) from Pyrococcus furiosus. The resultant chimera, β-AdhD, gained the calcium-binding ability of the β-roll, retained the thermostable activity of AdhD, and exhibited reduced overall alcohol dehydrogenase activity. However, the addition of calcium to β-AdhD preferentially inhibited NAD+-dependent activity in comparison to NADP+-dependent activity. Calcium was found to be a competitive inhibitor of AdhD, and the addition of the RTX domain introduced calcium-dependent noncompetitive inhibition to β-AdhD affecting NAD+-dependent activity. Thus, the insertion of an intrinsically disordered calcium-binding domain into a key loop in a cofactor-dependent enzyme results in an en...

There are many biotechnology applications that would benefit from simple, stable proteins with engineered biomolecular recognition. Here, we explored the hypothesis that a thermostable alcohol dehydrogenase (AdhD from Pyrococcus furiosus) could be engineered to bind a small molecule instead of a cofactor or molecules involved in the catalytic transition state. We chose the explosive molecule 1,3,5-trinitro-1,3,5-triazine (royal demolition explosive, RDX) as a proof-of-concept. Its low solubility in water was exploited for immobilization for biopanning by using ribosome display. Docking simulations were used to identify two potential binding sites in AdhD, and a randomized library focused on tyrosine or serine mutations was used to determine that RDX was binding in the substrate binding pocket of the enzyme. A fully randomized binding pocket library was selected, and affinity maturation by error-prone PCR led to the identification of a mutant (EP-16) that gained the ability to bind RDX with an affinity of (73±11) μm. These results underscore the way in which thermostable enzymes can be useful scaffolds for expanding the biomolecular recognition toolbox.

Protein engineering provides a means to alter protein structure leading to new functions. Much work has focused on the engineering of enzyme active sites to enhance catalytic activity, however there is an increasing trend towards engineering other aspects of biocatalysts as these efforts can also lead to useful improvements. This tutorial discusses recent advances in engineering an enzyme's local chemical and physical environment, with the goal of enhancing enzyme reaction kinetics, substrate selectivity, and activity in harsh conditions (e.g., low or high pH). By introducing stimuli-responsiveness to these enzyme modifications, dynamic control of activity also becomes possible. These new biomolecular and protein engineering techniques are separate and independent from traditional active site engineering and can therefore be applied synergistically to create new biocatalyst technologies with novel functions.

Alternative scaffolds for biomolecular recognition are being developed to overcome some of the limitations associated with immunoglobulin domains. The repeat-in-toxin (RTX) domain is a repeat protein sequence that reversibly adopts the β-roll secondary structure motif specifically upon calcium binding. This conformational change was exploited for controlled biomolecular recognition. Using ribosome display, an RTX peptide library was selected to identify binders to a model protein, lysozyme, exclusively in the folded state of the peptide. Several mutants were identified with low micromolar dissociation constants. After concatenation of the mutants, a 500-fold increase in the overall affinity for lysozyme was achieved leading to a peptide with an apparent dissociation constant of 65 nM. This mutant was immobilized for affinity chromatography experiments, and the on/off nature of the molecular recognition was demonstrated as the target is captured from a mixture in the presence of calcium and is released in the absence of calcium as the RTX peptides lose their β-roll structure. This work presents the design of a new stimulus-responsive scaffold that can be used for environmentally responsive specific molecular recognition and self-assembly.

The utilization of cofactor-dependent redox enzymes in bioprocess technologies requires low cost cofactor regeneration methods. PEGylated NAD(H) (PEG-NAD(H)) has been utilized in enzyme membrane reactors as a means to recover the cofactor; however, there is a lack of understanding of the effect of PEGylation on enzymatic activity, especially on the relationship between biocatalysis and transport phenomena. To explore this further, two redox enzymes (formate dehydrogenase (FDH) from Saccharomyces cerevisiae and NAD(H)-dependent d-lactate dehydrogenase (nLDH) from Escherichia coli) have been chosen and the kinetic effects caused by cofactor modifications (with PEG of three different chain lengths) have been investigated. The PEGylation did not impact the cofactor dissociation constants and mass transfer was not the rate-limiting step in biocatalysis for either enzyme. However, the PEG chain length had different impacts on the formation of enzyme/cofactor and/or enzyme/cofactor/substrate ternary complexes for the enzymes. © 2017 American Institute of Chemical Engineers AIChE J, 2017

A. ferrooxidans is an important iron- and sulfur-oxidizing acidophilic chemolithoautotroph that is used extensively in metal extraction and refining, and more recently in the bioproduction of chemicals. However, a lack of genetic tools has limited the further development of this organism for industrial bioprocesses. Using prior microarray studies that identified genes which may express differentially in response to the availability of iron and sulfur, the cycA1 and tusA promoter sequences have been characterized for their ability to drive green fluorescent protein (GFP) expression. The promoters exhibited opposite control behavior, where the cycA1 sequence was repressed and the tusA promoter was induced by the presence of sulfur in the growth medium. Sulfur was found to be the dominant signal. The sulfur IC50 for cycA1 was 0.56 mM (18 mg/L) while the sulfur EC50 of tusA was 2.5 mM (80 mg/L). Together these sequences provide two new tools to selectively induce or repress gene expression in A. ferrooxidans. A. ferrooxidans is an important industrial organism; however, genetic tools for control of gene expression do not exist. Here we report the identification of promoter sequences that allow for the development of control of gene expression for engineering this organism. This article is protected by copyright. All rights reserved

Electrofuels processes are potentially promising platforms for biochemical production from CO2 using renewable energy. When coupled to solar panels, this approach could avoid the inefficiencies of photosynthesis and there is no competition with food agriculture. In addition, these systems could potentially be used to store intermittent or stranded electricity generated from other renewable sources. Here we develop reactor configurations for continuous electrofuels processes to convert electricity and CO2 to isobutyric acid (IBA) using genetically modified (GM) chemolithoautotrophic Acidithiobacillus ferrooxidans. These cells oxidize ferrous iron which can be electrochemically reduced. During two weeks of cultivation on ferrous iron, stable cell growth and continuous IBA production from CO2 were achieved in a process where media was circulated between electrochemical and biochemical rectors. An alternative process with an additional electrochemical cell for accelerated ferrous production was developed, and this system achieved an almost three-fold increase in steady state cell densities, and an almost 4-fold increase in the ferrous iron oxidation rate. Combined, this led to an almost 8-fold increase in the steady state volumetric productivity of IBA up to 0.063±0.012mg/L/h, without a decline in energy efficiency from previous work. Continued development of reactor configurations which can increase the delivery of energy to the genetically modified cells will be required to increase product titers and volumetric productivities.

Microbially influenced corrosion (MIC) results in significant damage to metallic materials in many industries. Anaerobic sulfate-reducing bacteria (SRB) have been well studied for their involvement in these processes. Highly corrosive environments are also found in pulp and paper processing, where chloride and thiosulfate lead to the corrosion of stainless steels. Acidithiobacillus ferrooxidans is a critically important chemolithotrophic acidophile exploited in metal biomining operations, and there is interest in using A. ferrooxidans cells for emerging processes such as electronic waste recycling. We explored conditions under which A. ferrooxidans could enable the corrosion of stainless steel. Acidic medium with iron, chloride, low sulfate, and pyrite supplementation created an environment where unstable thiosulfate was continuously generated. When combined with the chloride, acid, and iron, the thiosulfate enabled substantial corrosion of stainless steel (SS304) coupons (mass loss, 5.4 ± 1.1 mg/cm2 over 13 days), which is an order of magnitude higher than what has been reported for SRB. There results were verified in an abiotic flow reactor, and the importance of mixing was also demonstrated. Overall, these results indicate that A. ferrooxidans and related pyrite-oxidizing bacteria could produce aggressive MIC conditions in certain environmental milieus. IMPORTANCE MIC of industrial equipment, gas pipelines, and military material leads to billions of dollars in damage annually. Thus, there is a clear need to better understand MIC processes and chemistries as efforts are made to ameliorate these effects. Additionally, A. ferrooxidans is a valuable acidophile with high metal tolerance which can continuously generate ferric iron, making it critical to copper and other biomining operations as well as a potential biocatalyst for electronic waste recycling. New MIC mechanisms may expand the utility of these cells in future metal resource recovery operations.

Proteins designed for supramolecular assembly provide a simple means to immobilize and organize enzymes for biotechnology applications. We have genetically fused the thermostable alcohol dehydrogenase D (AdhD) from Pyrococcus furiosus to a computationally designed cage-forming protein (O3-33). The trimeric form of the O3-33-AdhD fusion protein was most active in solution. The immobilization of the fusion protein on bioelectrodes leads to a doubling of the electrochemical operational stability as compared to the unfused control proteins. Thus, the fusion of enzymes to the designed self-assembling domains offers a simple strategy to increase the stability in biocatalytic systems.

The RTX domains found in some pathogenic proteins encode repetitive peptide sequences that reversibly bind calcium and fold into the unique the β-roll secondary structure. Several of these domains have been studied in isolation, yielding key insights into their structure/function relationships. These domains are increasingly being used in protein engineering applications, where the calcium-induced control over structure can be exploited to gain new functions. Here we review recent advances in the use of RTX domains in the creation of calcium responsive biomaterials.

The development of biomaterials with embedded enzymatic activities has been driven by a range of applications including tissue engineering, biosensors, and bioenergy applications. Advances in the design and production of peptide-based biomaterials have inspired protein engineers to begin creating enzymes with self-assembling, biomaterial forming capabilities. Outfitting enzymes with cross-link forming domains allows biomaterials to be created with a range of benefits including simple low-cost production, homogenous dispersion of activity in the hydrogels, and the ability to colocalize enzymes to create multistep cascades in the hydrogels. Just as natural hydrogels have evolved to exhibit important material and catalytic properties, designed bifunctional proteins that enable colocalization of activity within biomaterials are poised to further advance a range of biocatalytic, biomedical, and biotechnological applications.

The development of Acidithiobacillus ferrooxidans as a non-model host organism for synthetic biology is hampered by a lack of genetic tools and techniques. New plating and liquid-based selection methods were developed to improve the identification of transformed cell lines. Enabled by these methods, a hyperactive transposase was used to generate mutants with integrated genes for the expression of the superfolder green fluorescent protein (sfGFP) gene or a 2-keto decarboxylase (KDC) gene, which enabled the production and secretion of isobutyric acid (IBA). An inverse PCR method was used to identify the insertion sites of the KDC gene in several mutants, leading to the identification of a region on the chromosome that may be suitable for future genetic insertions. These results demonstrate that functional exogenous metabolic genes have been chromosomally integrated into A. ferrooxidans, and this advance will facilitate the future development of these cells for new biotechnology applications. IMPORTANCEAcidithiobacillus ferrooxidans is an iron- and sulfur-oxidizing chemolithoautotroph and is a key member of the microbial consortia used in industrial biomining applications. There is interest in exploiting these cells for other metal recovery applications as well as in developing them as unique nonmodel microbial cell factories. Plasmid-driven expression of exogenous genes has been reported, and homologous recombination has been used to knock out some gene expression. Here, new selection protocols facilitated the development of a transposition method for chromosomal integration of exogenous genes into A. ferrooxidans. This greatly expands the available genetic toolbox, which will open the door to greater metabolic engineering efforts for these cells.

Supramolecular assembly of enzymes into metabolon structures is thought to enable efficient transport of reactants between active sites via substrate channeling. Recombinant versions of porcine citrate synthase (CS), mitochondrial malate dehydrogenase (mMDH), and aconitase (Aco) were found to adopt a homogeneous native-like metabolon structure in vitro. Site-directed mutagenesis performed on highly conserved arginine residues located in the positively charged channel connecting mMDH and CS active sites led to the identification of CS(R65A) which retained high catalytic efficiency. Substrate channeling between the CS mutant and mMDH was severely impaired and the overall channeling probability decreased from 0.99 to 0.023. This work provides direct mechanistic evidence for the channeling of reaction intermediates, and disruption of this interaction would have important implications on the control of flux in central carbon metabolism.

The development of 3D structures exploring the properties of nano-materials and biological molecules has been shown through the years as an effective path forward for the design of advanced bio-nano architectures for enzymatic fuel cells, photo-bio energy harvesting devices, nano-biosensors and bio-actuators and other bio-nano-interfacial architectures. In this study we demonstrate a scaffold design utilizing carbon nanotubes, deoxyribose nucleic acid (DNA) and a specific DNA binding transcription factor that allows for directed immobilization of a single enzyme. Functionalized carbon nanotubes were covalently bonded to a diazonium salt modified gold surface through carbodiimide chemistry creating a brush-type nanotube alignment. The aligned nanotubes created a highly ordered structure with high surface area that allowed for the attachment of a protein assembly through a designed DNA scaffold. The enzyme immobilization was controlled by a zinc finger (ZNF) protein domain that binds to a specific dsDNA sequence. ZNF 268 was genetically fused to the small laccase (SLAC) from Streptomyces coelicolor, an enzyme belonging to the family of multi-copper oxidases, and used to demonstrate the applicability of the developed approach. Analytical techniques such as X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), and enzymatic activity analysis, allowed characterization at each stage of development of the bio-nano architecture. This article is part of a Special Issue entitled Biodesign for Bioenergetics--the design and engineering of electronic transfer cofactors, proteins and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson.

Millions of years of evolution have produced biological systems capable of efficient one-pot multi-step catalysis. The underlying mechanisms that facilitate these reaction processes are increasingly providing inspiration in synthetic chemistry. Substrate channelling, where intermediates between enzymatic steps are not in equilibrium with the bulk solution, enables increased efficiencies and yields in reaction and diffusion processes. Here, we review different mechanisms of substrate channelling found in nature and provide an overview of the analytical methods used to quantify these effects. The incorporation of substrate channelling into synthetic cascades is a rapidly developing concept, and recent examples of the fabrication of cascades with controlled diffusion and flux of intermediates are presented.

Here we developed a flow-based system resulting in improved performance of enzyme cascade-based biofuel cells. A paper-based biofuel cell with passive laminar flow was build to show the impact of flow on the performance of two different enzyme cascades – methanol and ethanol cascade. Both cascades demonstrated enhanced electrochemical output as a consequence of the decreased diffusion path of reaction intermediates identifying the intermediates diffusion in between enzymatic active sites as the rate limiting step in cascade operating systems.