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Acidithiobacillus ferrooxidans is an acidophilic chemolithoautotroph that plays an important role in biogeochemical iron and sulfur cycling and is a member of the consortia used in industrial hydrometallurgical processing of copper. Metal sulfide bioleaching is catalyzed by the regeneration of ferric iron; however, bioleaching of chalcopyrite, the dominant unmined form of copper on Earth, is inhibited by surface passivation. Here, we report the implementation of CRISPR interference (CRISPRi) using the catalytically inactive Cas12a (dCas12a) in A. ferrooxidans to knock down the expression of genes in the petI and petII operons. These operons encode bc 1 complex proteins and knockdown of these genes enabled the manipulation (enhancement or repression) of iron oxidation. The petB2 gene knockdown strain enhanced iron oxidation, leading to enhanced pyrite and chalcopyrite oxidation, which correlated with reduced biofilm formation and decreased surface passivation of the minerals. These findings highlight the utility of CRISPRi/dCas12a technology for engineering A. ferrooxidans while unveiling a new strategy to manipulate and improve bioleaching efficiency., Competing Interests: Conflict of interest The authors declare that they have no conflict of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The engineering of enzymatic activity generally involves alteration of the protein primary sequences, which introduce structural changes that give rise to functional improvements. Mechanical forces have been used to interrogate protein biophysics, leading to deep mechanistic insights in single-molecule studies. Here, we use simple DNA springs to apply small pulling forces to perturb the active site of a thermostable alcohol dehydrogenase. Methods were developed to enable the study of different spring lengths and spring orientations under bulk catalysis conditions. Tension applied across the active site expanded the binding pocket volume and shifted the preference of the enzyme for longer chain-length substrates, which could be tuned by altering the spring length and the resultant applied force. The substrate specificity changes did not occur when the DNA spring was either severed or rotated by ∼90°. These findings demonstrate an alternative approach in protein engineering, where active site architectures can be dynamically and reversibly remodeled using applied mechanical forces.

The Block V of the RTX domain of the adenylate cyclase protein from Bordetella pertussis is disordered, and upon binding eight calcium ions, it folds into a beta roll domain with a C-terminal capping group. Due to their similar ionic radii and coordination geometries, trivalent lanthanide ions have been used to probe and identify calcium-binding sites in many proteins. Here, we report using a FRET-based assay that the RTX domain can bind rare earth elements (REEs) with higher affinities than calcium. The apparent disassociation constants for lanthanide ions ranged from 20 to 75 μM, which are an order of magnitude higher than the affinity for calcium, with a higher selectivity toward heavy REEs over light REEs. Most proteins release bound ions at mildly acidic conditions (pH 5-6), and the high affinity REE-binding lanmodulin protein can bind 3-4 REE ions at pH as low as ∼2.5. Circular dichroism (CD) spectra of the RTX domain demonstrate pH-induced folding of the beta roll domain in the absence of ions, indicating that protonation of key amino acids enables structure formation in low pH solutions. The beta roll domain coordinates up to four ions in extreme pH conditions (pH < 1), as determined by equilibrium ultrafiltration experiments. Finally, to demonstrate a potential application of the RTX domain, REE ions (Nd 3+ and Dy 3+ ) were recovered from other non-REEs (Fe 2+ and Co 2+ ) in a NdFeB magnet simulant solution (at pH 6).

Molybdenum and rhenium are critically important metals for a number of emerging technologies. We identified and characterized a molybdenum/tungsten transport protein (ModA/WtpA) of Acidithiobacillus ferrooxidans and demonstrated the binding of tungstate, molybdate, and chromate. We used computational design to expand the binding capabilities of the protein to include perrhenate. A disulfide bond was engineered into the binding pocket of ModA/WtpA to introduce a more favorable geometric coordination and surface charge distribution for oxyanion binding. The mutant protein experimentally demonstrated a 2-fold higher binding affinity for molybdate and 6-fold higher affinity for perrhenate. The overexpression of the wild-type and mutant ModA/WtpA proteins in A. ferrooxidans cells enhanced the innate tungstate, molybdate, and chromate binding capacities of the cells to up to 2-fold higher. In addition, the engineered cells expressing the mutant protein exhibited enhanced perrhenate binding, showing 5-fold and 2-fold higher binding capacities compared to the wild-type and ModA/WtpA-overexpressing cells, respectively. Furthermore, the engineered cell lines enhanced biocorrosion of stainless steel as well as the recovered valuable metals from an acidic wastewater generated from molybdenite processing. The improved binding efficiency for the oxyanion metals, along with the high selectivity over nontargeted metals under mixed metal environments, highlights the potential value of the engineered strains for practical microbial metal reclamation under low pH conditions., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

As global demands for rare-earth elements (REEs) continue to grow, the biological recovery of REEs has been explored as a promising strategy, driven by potential economic and environmental benefits. It is known that calcium-binding domains, including helix-loop-helix EF hands and repeats-in-toxin (RTX) domains, can bind lanthanide ions due to their similar ionic radii and coordination preference to calcium. Recently, the lanmodulin protein from Methylorubrum extorquens was reported, which has evolved a high affinity for lanthanide ions over calcium. Acidithiobacillus ferrooxidans is a chemolithoautotrophic acidophile, which has been explored for use in bioleaching for metal recovery. In this report, A. ferrooxidans was engineered for the recombinant intracellular expression of lanmodulin. In addition, an RTX domain from the adenylate cyclase protein of Bordetella pertussis , which has previously been shown to bind Tb 3+ , was expressed periplasmically via fusion with the endogenous rusticyanin protein. The binding of lanthanides (Tb 3+ , Pr 3+ , Nd 3+ , and La 3+ ) was improved by up to 4-fold for cells expressing lanmodulin and 13-fold for cells expressing the RTX domains in both pure and mixed metal solutions. Interestingly, the presence of lanthanides in the growth media enhanced protein expression, likely by influencing protein stability. Both engineered cell lines exhibited higher recoveries and selectivities for four tested lanthanides (Tb 3+ , Pr 3+ , Nd 3+ , and La 3+ ) over non-REEs (Fe 2+ and Co 2+ ) in a synthetic magnet leachate, demonstrating the potential of these new strains for future REE reclamation and recycling applications.

ATP is the energy currency of the cell and new methods for ATP regeneration will benefit a range of emerging biotechnology applications including synthetic cells. We designed and assembled a membraneless ATP-regenerating enzymatic cascade by exploiting the substrate specificities of selected NAD(P)(H)-dependent oxidoreductases combined with substrate-specific kinases. The enzymes in the NAD(P)(H) cycle were selected to avoid cross-reactions, and the cascade was driven by irreversible fuel oxidation. As a proof-of-concept, formate oxidation was chosen as the fueling reaction. ATP regeneration was accomplished via the phosphorylation of NADH to NADPH and the subsequent transfer of the phosphate to ADP by a reversible NAD + kinase. The cascade was able to regenerate ATP at a high rate (up to 0.74 mmol/L/h) for hours, and >90% conversion of ADP to ATP using monophosphate was also demonstrated. The cascade was used to regenerate ATP for use in cell free protein synthesis reactions, and the ATP production rate was further enhanced when powered by the multistep oxidation of methanol. The NAD(P)(H) cycle provides a simple cascade for the in vitro regeneration of ATP without the need for a pH-gradient or costly phosphate donors.

The functionalization of proteins with DNA through the formation of covalent bonds enables a wide range of biotechnology advancements. For example, single-molecule analytical methods rely on bioconjugated DNA as elastic biolinkers for protein immobilization. Labeling proteins with DNA enables facile protein identification, as well as spatial and temporal organization and control of protein within DNA-protein networks. Bioconjugation reactions can target native, engineered, and non-canonical amino acids (NCAAs) within proteins. In addition, further protein engineering via the incorporation of peptide tags and self-labeling proteins can also be used for conjugation reactions. The selection of techniques will depend on application requirements such as yield, selectivity, conjugation position, potential for steric hindrance, cost, commercial availability, and potential impact on protein function., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Cell adhesion is generally a prerequisite to the microbial bioleaching of sulfide minerals, and surface biofilm formation is modulated via quorum sensing (QS) communication. We explored the impact of the overexpression of endogenous QS machinery on the covellite bioleaching capabilities of Acidithiobacillus ferrooxidans , a representative acidophilic chemolithoautotrophic bacterium. Cells were engineered to overexpress the endogenous qs-I operon or just the afeI gene under control of the tac promoter. Both strains exhibited increased transcriptional gene expression of afeI and improved cell adhesion to covellite, including increased production of extracellular polymeric substances and increased biofilm formation. Under low iron conditions, the improved bioleaching of covellite was more evident when afeI was overexpressed alone as compared to the native operon. These observations demonstrate the potential for the genetic modulation of QS as a mechanism for increasing the bioleaching efficiency of covellite, and potentially other copper sulfide minerals., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors. Published by Elsevier B.V.)

Oxidoreductases catalyze essential redox reactions, and many require a diffusible cofactor for electron transport, such as NAD(H). Non-canonical cofactor analogs have been explored as a means to create enzymatic reactions that operate orthogonally to existing metabolism. Here, we aimed to engineer the formate dehydrogenase from Candid boidinii (CbFDH) for activity with the non-canonical cofactor nicotinamide adenine dinucleotide 3'-phosphate (3'-NADP(H)). We used PyRosetta, the Cofactor Specificity Reversal Structural Analysis and Library Design (CSR-SALAD), and structure-guided saturation mutagenesis to identify mutations that enable CbFDH to use 3'-NADP+. Two single mutants, D195A and D195G, had the highest activities with 3'-NADP+, while the double mutant D195G/Y196S exhibited the highest cofactor selectivity reversal behavior. Steady state kinetic analyses were performed; the D195A mutant exhibited the highest KTS value with 3'-NADP+. This work compares the utility of computational approaches for cofactor specificity engineering while demonstrating the engineering of an important enzyme for novel non-canonical cofactor selectivity., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

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 -1 , chalcopyrite reacted completely within minutes at room temperature and pressure. The XRD, SEM-EDS, and XPS measurements indicate that chalcopyrite reacts to form copper(I) chloride (CuCl). After the reductive treatment, the mineral products are leached by iron(III) sulfate to demonstrate the complete extraction of copper. The chromium(II) ion may be regenerated by an electrolysis unit inspired by an iron chromium flow battery in a practical industrial process., (© 2023 The Authors. Published by Wiley-VCH GmbH.)

There is an increasing interest in the upgrading of inexpensive and abundant C 1 feedstocks to higher carbon products. Linear carbon ligation routes are of particular interest due to their simplicity and potential for high carbon efficiencies. The formolase (FLS) enzyme was computationally designed to catalyze the formose reaction, where formaldehyde molecules are coupled to produce a mixture of C 2 (glycolaldehyde) and C 3 (dihydroxyacetone) molecules. Recent protein engineering efforts have resulted in the introduction of several FLS variants with altered catalytic properties. As is often the case with enzymes catalyzing reactions with complex and/or nonnatural trajectories, there are no mechanistic kinetic models that fully describe the activity of the FLS enzyme. FLS variants are typically evaluated by fitting rate data to empirical rate laws, with some variation of the k cat /K M ratio used to report and rank performances. The apparent parameters estimated in this manner are unlikely to capture the full catalytic performance of these enzymes. In this study, we derive a mechanistic rate law describing FLS activity as well as theory-based figures of merit to rank FLS performance under relevant conditions. We proceed to fit the rate equation to initial rate data obtained from several FLS mutants, and use the figures of merit to compare the mutations. This study provides a theoretical framework for comparing FLS enzymes which will be essential as novel carbon ligation pathways are devised and implemented., (© 2022 Wiley Periodicals LLC.)

The NAD + kinase (NADK) is the only known enzyme capable of phosphorylating NAD(H) to NADP(H) and therefore it plays a crucial role in maintaining NAD(P)(H) homeostasis. All domains of life contain at least one NADK gene, and the commonly investigated isoforms have been measured, or assumed, to be functionally irreversible. In 1977, the kinetics of native pigeon liver NADK were thoroughly investigated, and it was reported to exhibit reversible activity, such that ATP and NAD + can be formed from ADP and NADP + . We hypothesized that the reverse activity of the pigeon enzyme may enable compensation of the high picolinic acid carboxylase (PC) activity present in pigeon livers, which inhibits NAD + biosynthesis from dietary tryptophan. Here, we report the characterization of four recombinantly expressed NADKs and explore their reversible activities. Duck and cat livers have higher PC activity than pigeon livers, and the recombinant duck and cat NADKs exhibit high activity in the reverse direction. The human NADK has an affinity for NAD + that is ∼600 times higher than the pigeon, duck, and cat isoforms, and we conclude that NAD + serves as a potent product inhibitor for the reverse activity of the human NADK, which accounts for the observed irreversible behavior. These results demonstrate that while all four NADKs are reversible, the reverse activity of the human enzyme alone is impeded via product inhibition. This mechanism─the conversion of a reversible to a unidirectional reaction by product inhibition─may be valuable in future metabolic engineering applications.

The high efficiency of cascade reactions in supramolecular enzyme nanoassemblies, known as metabolons, has attracted substantial attention in various fields ranging from fundamental biochemistry and molecular biology to recent applications in biofuel cells, biosensors, and chemical synthesis. One reason for the high efficiency of metabolons is the structures formed by sequential enzymes that allow the direct transport of intermediates between consecutive active sites. The supercomplex of malate dehydrogenase (MDH) and citrate synthase (CS) is an ideal example of the controlled transport of intermediates via electrostatic channeling. Here, using a combination of molecular dynamics (MD) simulations and a Markov state model (MSM), we examined the transport process of the intermediate oxaloacetate (OAA) from MDH to CS. The MSM enables the identification of the dominant transport pathways of OAA from MDH to CS. Analysis of all pathways using a hub score approach reveals a small set of residues that control OAA transport. This set includes an arginine residue previously identified experimentally. MSM analysis of a mutated complex, where the identified arginine is replaced by alanine, led to a 2-fold decrease in transfer efficiency, also consistent with experimental results. This work provides a molecular-level understanding of the electrostatic channeling mechanism and will enable the further design of catalytic nanostructures utilizing electrostatic channeling., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

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., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Metal processing using microorganisms has many advantages including the potential for reduced environmental impacts as compared to conventional technologies. Acidithiobacillus ferrooxidans is an iron- and sulfur-oxidizing chemolithoautotroph that is known to participate in metal bioleaching, and its metabolic capabilities have been exploited for industrial-scale copper and gold biomining. In addition to bioleaching, microorganisms could also be engineered for selective metal binding, enabling new opportunities for metal bioseparation and recovery. Here, we explored the ability of polyhistidine (polyHis) tags appended to two recombinantly expressed endogenous proteins to enhance the metal binding capacity of A. ferrooxidans . The genetically engineered cells achieved enhanced cobalt and copper binding capacities, and the Langmuir isotherm captures their interaction behavior with these divalent metals. Additionally, the cellular localization of the recombinant proteins correlated with kinetic modeling of the binding interactions, where the outer membrane-associated polyHis-tagged licanantase peptide bound the metals faster than the periplasmically expressed polyHis-tagged rusticyanin protein. The selectivity of the polyHis sequences for cobalt over copper from mixed metal solutions suggests potential utility in practical applications, and further engineering could be used to create metal-selective bioleaching microorganisms.

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., (© 2021 Wiley-VCH GmbH.)

Natural cascades frequently use spatial organization to introduce beneficial substrate channeling mechanisms, a strategy that has been widely mimicked in many engineered multienzyme cascades with enhanced catalysis. Enabled by new molecular scaffolds it is now possible to test the effects of spatial organization on cascade kinetics; however, these scaffolds can also alter the microenvironment experienced by the assembled enzymes. We know from decades of enzyme immobilization research that the microenvironment affects enzymatic activity, thus complicating kinetic analysis. Here, we review these effects and discuss examples that exploit the microenvironment to improve single enzyme and cascade catalysis. In doing so, we highlight the challenges in ascribing kinetic enhancements directly to substrate channeling without first determining the effects of the microenvironment., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Acidithiobacillus ferrooxidans is a well-studied iron- and sulfur-oxidizing acidophilic chemolithoautotroph that is exploited for its 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. Furthermore, we found that improved iron oxidation ability in the presence of chloride also resulted in higher levels of intracellular reactive oxygen species (ROS) in the strain. 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. IMPORTANCE The 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 the use of freshwater 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.

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 an 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., (© 2021 The Protein Society.)

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., (© 2021 Wiley Periodicals LLC.)

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., (© 2020 Wiley Periodicals LLC.)

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/cm 2 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., (Copyright © 2019 American Society for Microbiology.)

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., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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., (© 2019 Elsevier Inc. All rights reserved.)

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. IMPORTANCE Acidithiobacillus 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., (Copyright © 2018 American Society for Microbiology.)

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.

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.

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., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Acidithiobacillus 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 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 IC 50 for cycA1 was 0.56 mM (18 mg/L), whereas the sulfur EC 50 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. Acidithiobacillus 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., (© 2016 International Union of Biochemistry and Molecular Biology, Inc.)

The isolated Block V repeats-in-toxin (RTX) peptide domain of adenylate cyclase (CyaA) from Bordetella pertussis reversibly folds into a β-roll secondary structure upon calcium binding. In this review, we discuss how the conformationally dynamic nature of the peptide is being engineered and employed as a switching mechanism to mediate different protein functions and protein-protein interactions. The peptide has been used as a scaffold for diverse applications including: a precipitation tag for bioseparations, a cross-linking domain for protein hydrogel formation and as an alternative scaffold for biomolecular recognition applications. Proteins and peptides such as the RTX domains that exhibit natural stimulus-responsive behavior are valuable building blocks for emerging synthetic biology applications., Competing Interests: The authors declare no conflict of interest.

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.

Stimulus-responsive biomaterials have applications in many areas of biotechnology, such as tissue engineering, drug delivery, and bioelectrocatalysis. The intrinsically disordered repeat-in-toxin (RTX) domain is a conformationally dynamic peptide that gains β-roll secondary structure when bound to calcium ions. A smart hydrogel platform was constructed by genetically fusing two rationally designed mutant RTX domains: first, a mutant peptide with hydrophobic interfaces capable of calcium-dependent network assembly, and second, another mutant that conditionally binds the model target protein lysozyme. In this way, the calcium-induced control over the secondary structure of the β-roll peptide was exploited to regulate both the cross-linking and lysozyme-binding functionalities. The constructed biomaterial exhibited calcium-dependent gelation and target molecule retention, and erosion experiments showed that β-roll peptides with a higher affinity for lysozyme produced more robust hydrogel networks. This work demonstrates the use of RTX domains for introducing two useful features simultaneously, network cross-linking and target protein binding, and that the calcium-dependent regulation of these systems can be useful for controlling bulk self-assembly and controlled release capabilities.

Developing new feedstocks for the efficient production of biochemicals and biofuels will be a critical challenge as we diversify away from petrochemicals. One possible opportunity is the utilization of sulfide-based minerals in the Earth's crust. Non-photosynthetic chemolithoautotrophic bacteria are starting to be developed to produce biochemicals from CO 2 using energy obtained from the oxidation of inorganic feedstocks. Biomining of metals like gold and copper already exploit the native metabolism of these bacteria and these represent perhaps the largest-scale bioprocesses ever developed. The metabolic engineering of these bacteria could be a desirable alternative to classical heterotrophic bioproduction. In this review, we discuss biomining operations and the challenges and advances in the engineering of associated chemolithoautotrophic bacteria for biofuel production. The co-generation of biofuels integrated with mining operations is a largely unexplored opportunity that will require advances in fundamental microbiology and the development of new genetic tools and techniques for these organisms. Although this approach is presently in its infancy, the production of biochemicals using energy from non-petroleum mineral resources is an exciting new biotechnology opportunity., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

There have been many reports exploring the engineering of the cofactor specificity of aldo-keto reductases (AKRs), as this class of proteins is ubiquitous and exhibits many useful activities. A common approach is the mutagenesis of amino acids involved in interactions with the 2'-phosphate group of NADP(H) in the cofactor binding pocket. We recently performed a 'loop-grafting' approach to engineer the substrate specificity of the thermostable alcohol dehydrogenase D (AdhD) from Pyrococcus furiosus and we found that a loop insertion after residue 211, which is on the back side of the cofactor binding pocket, could also alter cofactor specificity. Here, we further explore this approach by introducing single point mutations and single amino acid insertions at the loop insertion site. Six different mutants of AdhD were created by either converting glycine 211 to cysteine or serine or by inserting alanine, serine, glycine or cysteine between the 211 and 212 residues. Several mutants gained activity with NADP+ above the wild-type enzyme. And remarkably, it was found that all of the mutants investigated resulted in some degree of reversal of cofactor specificity in the oxidative direction. These changes were generally a result of changes in conformations of the ternary enzyme/cofactor/substrate complexes as opposed to changes in affinities or binding energies of the cofactors. This study highlights the role that amino acids which are distal to the cofactor binding pocket but are involved in substrate interactions can influence cofactor specificity in AdhD, and this strategy should translate to other AKR family members., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Electrofuels processes are potentially promising platforms for biochemical production from CO 2 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 CO 2 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 CO 2 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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Alcohol dehydrogenase D (AdhD) is a monomeric thermostable alcohol dehydrogenase from the aldo-keto reductase (AKR) superfamily of proteins. We have been exploring various strategies of engineering the activity of AdhD so that it could be employed in future biotechnology applications. Driven by insights made in other AKRs, we have made mutations in the cofactor-binding pocket of the enzyme and broadened its cofactor specificity. A pre-steady state kinetic analysis yielded new insights into the conformational behavior of this enzyme. The most active mutant enzyme concomitantly gained activity with a non-native cofactor, nicotinamide mononucleotide, NMN(H), and an enzymatic biofuel cell was demonstrated with this enzyme/cofactor pair. Substrate specificity was altered by grafting loop regions near the active site pocket from a mesostable human aldose reductase (hAR) onto the thermostable AdhD. These moves not only transferred the substrate specificity of hAR but also the cofactor specificity of hAR. We have added alpha-helical appendages to AdhD to enable it to self-assemble into a thermostable catalytic proteinaceous hydrogel. As our understanding of the structure/function relationship in AdhD and other AKRs advances, this ubiquitous protein scaffold could be engineered for a variety of catalytic activities that will be useful for many future applications., (Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)