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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.)