RESUMO
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 knockdown the expression of genes in the petI and petII operons. These operons encode bc1 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.
RESUMO
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 â¼2.5. 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 (Nd3+ and Dy3+) were recovered from other non-REEs (Fe2+ and Co2+) in a NdFeB magnet simulant solution (at pH 6).
Assuntos
Metais Terras Raras , Metais Terras Raras/química , Concentração de Íons de Hidrogênio , Elementos da Série dos Lantanídeos/química , Bordetella pertussis/enzimologia , Bordetella pertussis/química , Sítios de Ligação , Ligação Proteica , Domínios Proteicos , Cálcio/química , Cálcio/metabolismoRESUMO
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 Tb3+, was expressed periplasmically via fusion with the endogenous rusticyanin protein. The binding of lanthanides (Tb3+, Pr3+, Nd3+, and La3+) 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 (Tb3+, Pr3+, Nd3+, and La3+) over non-REEs (Fe2+ and Co2+) in a synthetic magnet leachate, demonstrating the potential of these new strains for future REE reclamation and recycling applications.
Assuntos
Acidithiobacillus , Elementos da Série dos Lantanídeos , Metais Terras Raras , Cálcio/metabolismo , Acidithiobacillus/genética , Acidithiobacillus/química , Acidithiobacillus/metabolismo , Elementos da Série dos Lantanídeos/metabolismo , Íons/metabolismoRESUMO
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 â¼600 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 mechanismâthe conversion of a reversible to a unidirectional reaction by product inhibitionâmay be valuable in future metabolic engineering applications.
Assuntos
NAD , Fosfotransferases (Aceptor do Grupo Álcool) , Humanos , NADP/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismoRESUMO
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 KM 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.
Assuntos
NADP Trans-Hidrogenases/metabolismo , NAD/metabolismo , Polietilenoglicóis/metabolismo , Estabilidade Enzimática , Modelos Moleculares , NAD/química , NADP Trans-Hidrogenases/química , Polietilenoglicóis/química , Engenharia de ProteínasRESUMO
There is an increasing interest in the upgrading of inexpensive and abundant C1 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 C2 (glycolaldehyde) and C3 (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 kcat /KM 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.
Assuntos
Di-Hidroxiacetona , Formaldeído , Carbono , Catálise , Enzimas/metabolismo , CinéticaRESUMO
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.
Assuntos
Acidithiobacillus/genética , Acidithiobacillus/metabolismo , Glutationa Sintase/genética , Ferro/metabolismo , Tolerância ao Sal/genética , Acidithiobacillus/efeitos dos fármacos , Escherichia coli/genética , Glutationa/biossíntese , Concentração de Íons de Hidrogênio , Oxirredução , Espécies Reativas de Oxigênio/metabolismo , Cloreto de Sódio/farmacologiaRESUMO
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.
Assuntos
Acidithiobacillus/crescimento & desenvolvimento , Meios de Cultura/química , Ferro/metabolismo , Enxofre/metabolismo , OxirreduçãoRESUMO
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.
Assuntos
Acidithiobacillus , Azurina , Aço Inoxidável , Acidithiobacillus/genética , Acidithiobacillus/metabolismo , Azurina/genética , Azurina/metabolismo , Corrosão , Ferro/metabolismo , Oxirredução , Sulfetos/metabolismoRESUMO
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.
Assuntos
Bioquímica , Bioengenharia , Biotecnologia , HumanosRESUMO
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 (7.75-500â 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 pHâ 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.
Assuntos
Álcool Desidrogenase/química , Biocatálise , Proteínas de Fluorescência Verde/química , Animais , Proteínas Arqueais/química , Butileno Glicóis/química , Hidrozoários/química , Cinética , Oxirredução , Peptídeos/química , Pyrococcus furiosus/enzimologia , TermodinâmicaRESUMO
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.
Assuntos
Acidithiobacillus/metabolismo , Ferro/química , Aço Inoxidável/química , Sulfatos/química , Tiossulfatos/química , Ligas , Crescimento Quimioautotrófico , Cobre , Corrosão , Elétrons , Microbiologia Industrial , Mineração , Oxidantes , Oxirredução , Sulfetos , Propriedades de SuperfícieRESUMO
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.
Assuntos
Enzimas/metabolismo , Engenharia de Proteínas , Álcool Desidrogenase/metabolismo , Biocatálise , Cálcio/metabolismo , Citocromos c/química , Citocromos c/metabolismo , DNA/química , DNA/metabolismo , Enzimas/genética , Peroxidase do Rábano Silvestre/química , Peroxidase do Rábano Silvestre/metabolismo , Cinética , Especificidade por SubstratoRESUMO
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)â µm. These results underscore the way in which thermostable enzymes can be useful scaffolds for expanding the biomolecular recognition toolbox.
Assuntos
Álcool Desidrogenase/química , Engenharia de Proteínas , Pyrococcus furiosus/enzimologia , Temperatura , Triazinas/química , Álcool Desidrogenase/metabolismo , Estabilidade Enzimática , Simulação de Acoplamento Molecular , Solubilidade , Água/químicaRESUMO
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.
Assuntos
Acidithiobacillus/genética , Cromossomos Bacterianos/genética , Engenharia Genética/métodos , Transposases/metabolismo , Acidithiobacillus/metabolismo , Regulação Bacteriana da Expressão Gênica , Mutagênese Insercional , Transposases/genéticaRESUMO
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.
Assuntos
Materiais Biomiméticos , DNA/química , Metabolismo Energético , Enzimas Imobilizadas/química , Nanotubos de Carbono/química , Bioengenharia/métodos , Materiais Biomiméticos/síntese química , Materiais Biomiméticos/química , Técnicas Biossensoriais/métodos , DNA/metabolismo , Enzimas Imobilizadas/metabolismo , Ouro/química , Humanos , Lacase/química , Lacase/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Multimerização Proteica , Estrutura Quaternária de Proteína , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Streptomyces coelicolor , Propriedades de SuperfícieRESUMO
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 ß-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 ß-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 ß-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.
Assuntos
Cálcio/química , Hidrogéis , Modelos Químicos , Peptídeos , Engenharia de Proteínas , Hidrogéis/síntese química , Hidrogéis/química , Peptídeos/síntese química , Peptídeos/química , Estrutura Secundária de ProteínaRESUMO
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 IC50 for cycA1 was 0.56 mM (18 mg/L), whereas 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. 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.
Assuntos
Acidithiobacillus/genética , Regulação Bacteriana da Expressão Gênica/genética , Regiões Promotoras Genéticas/genética , Acidithiobacillus/citologia , Acidithiobacillus/crescimento & desenvolvimento , Células Cultivadas , Perfilação da Expressão Gênica , Engenharia GenéticaRESUMO
The chemolithoautotrophic bacterium Acidithiobacillus ferrooxidans has previously been genetically modified to produce isobutyric acid (IBA) from carbon dioxide while obtaining energy from the oxidation of ferrous iron. Here, a combinatorial approach was used to explore the influence of medium composition in both batch and chemostat cultures in order to improve IBA yields (g IBA/mol Fe(2+)) and productivities (g IBA/L/d). Medium pH, ferrous concentration (Fe(2+)), and inclusion of iron chelators all had positive impact on the IBA yield. In batch experiments, gluconate was found to be a superior iron chelator because its use resulted in smaller excursions in pH. In batch cultures, IBA yields decreased linearly with increases in the final effective Fe(3+) concentrations. Chemostat cultures followed similar trends as observed in batch cultures. Specific cellular productivities were found to be a function of the steady state ORP (Oxidation-reduction potential) of the growth medium, which is primarily determined by the Fe(3+) to Fe(2+) ratio. By operating at low ORP, chemostat cultures were able to achieve volumetric productivities as high as 3.8 ± 0.2 mg IBA/L/d which is a 14-fold increase over the previously reported value.