Improved Solubility and Stability of a Thermostable Carbonic Anhydrase via Fusion with Marine-Derived Intrinsically Disordered Solubility Enhancers.

Carbonic anhydrase (CA), an enzyme catalyzing the reversible hydration reaction of carbon dioxide (CO2), is considered a promising biocatalyst for CO2 reduction. The α-CA of Thermovibrio ammonificans (taCA) has emerged as a compelling candidate due to its high thermostability, a critical factor for industrial applications. However, the low-level expression and poor in vitro solubility have hampered further utilization of taCA. Recently, these limitations have been addressed through the fusion of the NEXT tag, a marine-derived, intrinsically disordered small peptide that enhances protein expression and solubility. In this study, the solubility and stability of NEXT-taCA were further investigated. When the linker length between the NEXT tag and the taCA was shortened, the expression level decreased without compromising solubility-enhancing performance. A comparison between the NEXT tag and the NT11 tag demonstrated the NEXT tag's superiority in improving both the expression and solubility of taCA. While the thermostability of taCA was lower than that of the extensively engineered DvCA10, the NEXT-tagged taCA exhibited a 30% improvement in long-term thermostability compared to the untagged taCA, suggesting that enhanced solubility can contribute to enzyme thermostability. Furthermore, the bioprospecting of two intrinsically disordered peptides (Hcr and Hku tags) as novel solubility-enhancing fusion tags was explored, demonstrating their performance in improving the expression and solubility of taCA. These efforts will advance the practical application of taCA and provide tools and insights for enzyme biochemistry and bioengineering.

An Intrinsically Disordered Peptide Tag that Confers an Unusual Solubility to Aggregation-Prone Proteins.

There is a high demand for the production of recombinant proteins in Escherichia coli for biotechnological applications, but their production is still limited by their insolubility. Fusion tags have been successfully used to enhance the solubility of aggregation-prone proteins; however, smaller and more powerful tags are desired for increasing the yield and quality of target proteins. Here, the NEXT tag, a 53-amino-acid-long solubility enhancer, is described. The NEXT tag showed outstanding ability to improve both in vivo and in vitro solubilities, with minimal effect on passenger proteins. The C-terminal region of the tag was mostly responsible for in vitro solubility, while the N-terminal region was essential for in vivo soluble expression. The NEXT tag appeared to be intrinsically disordered and seemed to exclude neighboring molecules and prevent protein aggregation by acting as an entropic bristle. This novel peptide tag should have general use as a fusion partner to increase the yield and quality of difficult-to-express proteins. IMPORTANCE Production of recombinant proteins in Escherichia coli still suffers from the insolubility problem. Conventional solubility enhancers with large sizes, represented by maltose-binding protein (MBP), have remained the first-choice tags; however, the success of the soluble expression of tagged proteins is largely unpredictable. In addition, the large tags can negatively affect the function of target proteins. In this work, the NEXT tag, an intrinsically disordered peptide, was introduced as a small but powerful alternative to MBP. The NEXT tag could significantly improve both the expression level and the solubility of target proteins, including a thermostable carbonic anhydrase and a polyethylene terephthalate (PET)-degrading enzyme that are remarkable enzymes for environmental bioremediation.

Carbonic anhydrase for CO2 capture, conversion and utilization.

Carbonic anhydrase (CA) enzymes, catalyzing the CO2 hydration at a high turnover number, can be employed in expediting CO2 capture, conversion and utilization to aid in carbon neutrality. Despite extensive research over the last decade, there remain challenges in CA-related technologies due to poor stability and suboptimal use of CAs. Herein, we discuss recent advances in CA stabilization by protein engineering and enzyme immobilization, and shed light on state-of-the-art of in vitro and in vivo CA-mediated CO2 conversion for improved production of value-added chemicals using CO2 as a feedstock.

Enhanced Production of a Thermostable Carbonic Anhydrase in Escherichia coli by Using a Modified NEXT Tag.

Carbonic anhydrase (CA) is an ultrafast enzyme that catalyzes the reversible conversion of carbon dioxide (CO2) to bicarbonate. CA is considered to be a green catalyst for enzyme-based CO2 capture and utilization. In particular, the CA of Thermovibrio ammonificans (taCA) has attracted increasing attention as a highly stable enzyme. However, the poor solubility and the low expression level in Escherichia coli have hampered further utilization of taCA. In a recent study, these limitations were partly resolved by using a small solubility-enhancing fusion tag named NEXT, which originates from the N-terminal extension of Hydrogenovibrio marinus CA. In this study, the NEXT tag was engineered by adding small peptides to the N terminus to further increase the production yield of NEXT-tagged taCA. The addition of ng3 peptide (His-Gly-Asn) originating from the N-terminal sequence of Neisseria gonorrhoeae CA improved the expression of NEXT-taCA, while the previously developed translation-enhancing element (TEE) and Ser-Lys-Ile-Lys (SKIK) tag were not effective. The expression test with all 16 codon combinations for the ng3 sequence revealed that the change in translation initiation rate brought about by the change in nucleotide sequence was not the primary determinant for the change in expression level. The modified ng3-NEXT tag may be applied to increase the production yields of various recombinant proteins.

Engineering a Plant Viral Coat Protein for In Vitro Hybrid Self-Assembly of CO2-Capturing Catalytic Nanofilaments.

Plant virus-based nanoparticles are used as self-assembled protein scaffolds for the construction of enzyme nanocarriers. To date, one-pot production and coupling of both enzymes and scaffolds by genetic conjugation have been demonstrated only in plants. Herein, we report bacterial production and in vitro self-assembly of nanofilaments for CO2 capture. Filamentous virus-like particles (VLPs) were successfully formed by genetically fusing carbonic anhydrase from Hydrogenovibrio marinus (hmCA) to the N terminus of the coat protein (CPPVY) of potato virus Y with a flexible linker. The instability of VLPs against proteolytic degradation was circumvented by the periplasmic export of the fusion protein. The truncated form of CPPVY coexpressed by internal translation was crucial for the successful formation of long filamentous VLPs by alleviating steric hindrance via hybrid assembly. The fast and economic bottom-up fabrication of highly active nanobiocatalyst allows the nanofilaments to be efficiently used and recovered in potential biocatalytic and biosensor systems.

Stability-Controllable Self-Immobilization of Carbonic Anhydrase Fused with a Silica-Binding Tag onto Diatom Biosilica for Enzymatic CO2 Capture and Utilization.

Exploiting carbonic anhydrase (CA), an enzyme that catalyzes the hydration of CO2, is a powerful route for eco-friendly and cost-effective carbon capture and utilization. For successful industrial applications, the stability and reusability of CA should be improved, which necessitates enzyme immobilization. Herein, the ribosomal protein L2 (Si-tag) from Escherichia coli was utilized for the immobilization of CA onto diatom biosilica, a promising renewable support material. The Si-tag was redesigned (L2NC) and genetically fused to CA from the marine bacterium Hydrogenovibrio marinus (hmCA). One-step self-immobilization of hmCA-L2NC onto diatom biosilica by simple mixing was successfully achieved via Si-tag-mediated strong binding, showing multilayer adsorption with a maximal loading of 1.4 wt %. The immobilized enzyme showed high reusability and no enzyme leakage even under high temperature conditions. The activity of hmCA-L2NC was inversely proportional to the enzyme loading, while the stability was directly proportional to the enzyme loading. This discovered activity-stability trade-off phenomenon could be attributed to macromolecular crowding on the highly dense surface of the enzyme-immobilized biosilica. Collectively, our system not only facilitates the stability-controllable self-immobilization of enzyme via Si-tag on a diatom biosilica support for the robust, facile, and green construction of stable biocatalysts, but is also a unique model for studying the macromolecular crowding effect on surface-immobilized enzymes.

Engineering the genetic components of a whole-cell catalyst for improved enzymatic CO2 capture and utilization.

Carbonic anhydrase (CA) is a diffusion-limited enzyme that rapidly catalyzes the hydration of carbon dioxide (CO2 ). CA has been proposed as an eco-friendly yet powerful catalyst for CO2 capture and utilization. A bacterial whole-cell biocatalyst equipped with periplasmic CA provides an option for a cost-effective CO2 -capturing system. However, further utilization of the previously constructed periplasmic system has been limited by its relatively low activity and stability. Herein, we engineered three genetic components of the periplasmic system for the construction of a highly efficient whole-cell catalyst: a CA-coding gene, a signal sequence, and a ribosome-binding site (RBS). A stable and halotolerant CA (hmCA) from the marine bacterium Hydrogenovibrio marinus was employed to improve both the activity and stability of the system. The improved secretion and folding of hmCA and increased membrane permeability were achieved by translocation via the Sec-dependent pathway. The engineering of RBS strength further enhanced whole-cell activity by improving both the secretion and folding of hmCA. The newly engineered biocatalyst displayed 5.7-fold higher activity and 780-fold higher stability at 60°C compared with those of the previously constructed periplasmic system, providing new opportunities for applications in CO2 capture and utilization.

Characterization and High-Level Periplasmic Expression of Thermostable α-Carbonic Anhydrase from Thermosulfurimonas Dismutans in Escherichia Coli for CO2 Capture and Utilization.

Carbonic anhydrase (CA) is a diffusion-controlled enzyme that rapidly catalyzes carbon dioxide (CO2) hydration. CA has been considered as a powerful and green catalyst for bioinspired CO2 capture and utilization (CCU). For successful industrial applications, it is necessary to expand the pool of thermostable CAs to meet the stability requirement under various operational conditions. In addition, high-level expression of thermostable CA is desirable for the economical production of the enzyme. In this study, a thermostable CA (tdCA) of Thermosulfurimonas dismutans isolated from a deep-sea hydrothermal vent was expressed in Escherichia coli and characterized in terms of expression level, solubility, activity and stability. tdCA showed higher solubility, activity, and stability compared to those of CA from Thermovibrio ammonificans, one of the most thermostable CAs, under low-salt aqueous conditions. tdCA was engineered for high-level expression by the introduction of a point mutation and periplasmic expression via the Sec-dependent pathway. The combined strategy resulted in a variant showing at least an 8.3-fold higher expression level compared to that of wild-type tdCA. The E. coli cells with the periplasmic tdCA variant were also investigated as an ultra-efficient whole-cell biocatalyst. The engineered bacterium displayed an 11.9-fold higher activity compared to that of the recently reported system with a halophilic CA. Collectively these results demonstrate that the highly expressed periplasmic tdCA variant, either in an isolated form or within a whole-cell platform, is a promising biocatalyst with high activity and stability for CCU applications.

Control of nacre biomineralization by Pif80 in pearl oyster.

Molluscan nacre is a fascinating biomineral consisting of a highly organized calcium carbonate composite that provides unique fracture toughness and an iridescent color. Organisms elaborately control biomineralization using organic macromolecules. We propose the involvement of the matrix protein Pif80 from the pearl oyster Pinctada fucata in the development of the inorganic phase during nacre biomineralization, based on experiments using the recombinant form of Pif80. Through interactions with calcium ions, Pif80 participates in the formation of polymer-induced liquid precursor-like amorphous calcium carbonate granules and stabilizes these granules by forming calcium ion-induced coacervates. At the calcification site, the disruption of Pif80 coacervates destabilizes the amorphous mineral precursors, resulting in the growth of a crystalline structure. The redissolved Pif80 controls the growth of aragonite on the polysaccharide substrate, which contributes to the formation of polygonal tablet structure of nacre. Our findings provide insight into the use of organic macromolecules by living organisms in biomineralization.

Engineering de novo disulfide bond in bacterial α-type carbonic anhydrase for thermostable carbon sequestration.

Exploiting carbonic anhydrase (CA), an enzyme that rapidly catalyzes carbon dioxide hydration, is an attractive biomimetic route for carbon sequestration due to its environmental compatibility and potential economic viability. However, the industrial applications of CA are strongly hampered by the unstable nature of enzymes. In this work, we introduced in silico designed, de novo disulfide bond in a bacterial α-type CA to enhance thermostability. Three variants were selected and expressed in Escherichia coli with an additional disulfide bridge. One of the variants showed great enhancement in terms of both kinetic and thermodynamic stabilities. This improvement could be attributed to the loss of conformational entropy of the unfolded state, showing increased rigidity. The variant showed an upward-shifted optimal temperature and appeared to be thermoactivated, which compensated for the lowered activity at 25 °C. Collectively, the variant constructed by the rapid and effective de novo disulfide engineering can be used as an efficient biocatalyst for carbon sequestration under high temperature conditions.

Versatile signal peptide of Flavobacterium-originated organophosphorus hydrolase for efficient periplasmic translocation of heterologous proteins in Escherichia coli.

Organophosphorus hydrolase (OPH) from Flavobacterium species is a membrane-associated homodimeric metalloenzyme and has its own signal peptide in its N-terminus. We found that OPH was translocated into the periplasmic space when the original signal peptide-containing OPH was expressed in recombinant Escherichia coli even though its translocation efficiency was relatively low. To investigate the usability of this OPH signal peptide for periplasmic expression of heterologous proteins in an E. coli system, we employed green fluorescent protein (GFP) as a cytoplasmic folding reporter and alkaline phosphatase (ALP) as a periplasmic folding reporter. We found that the OPH signal peptide was able to use both twin-arginine translocation (Tat) and general secretory (Sec) machineries by switching translocation pathways according to the nature of target proteins in E. coli. These results might be due to the lack of Sec-avoidance sequence in the c-region and a moderate hydrophobicity of the OPH signal peptide. Interestingly, the OPH signal peptide considerably enhanced the translocation efficiencies for both GFP and ALP compared with commonly used TorA and PelB signal peptides that have Tat and Sec pathway dependences, respectively. Therefore, this OPH signal peptide could be successfully used in recombinant E. coli system for efficient periplasmic production of target protein regardless of the subcellular localization where functional folding of the protein occurs. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:848-854, 2016.

Activation of formate hydrogen-lyase via expression of uptake [NiFe]-hydrogenase in Escherichia coli BL21(DE3).

BACKGROUND: Several recent studies have reported successful hydrogen (H2) production achieved via recombinant expression of uptake [NiFe]-hydrogenases from Hydrogenovibrio marinus, Rhodobacter sphaeroides, and Escherichia coli (hydrogenase-1) in E. coli BL21(DE3), a strain that lacks H2-evolving activity. However, there are some unclear points that do not support the conclusion that the recombinant hydrogenases are responsible for the in vivo H2 production. RESULTS: Unlike wild-type BL21(DE3), the recombinant BL21(DE3) strains possessed formate hydrogen-lyase (FHL) activities. Through experiments using fdhF (formate dehydrogenase-H) or hycE (hydrogenase-3) mutants, it was shown that H2 production was almost exclusively dependent on FHL. Upon expression of hydrogenase, extracellular formate concentration was changed even in the mutant strains lacking FHL, indicating that formate metabolism other than FHL was also affected. The two subunits of H. marinus uptake [NiFe]-hydrogenase could activate FHL independently of each other, implying the presence of more than two different mechanisms for FHL activation in BL21(DE3). It was also revealed that the signal peptide in the small subunit was essential for activation of FHL via the small subunit. CONCLUSIONS: Herein, we demonstrated that the production of H2 was indeed induced via native FHL activated by the expression of recombinant hydrogenases. The recombinant strains with [NiFe]-hydrogenase appear to be unsuitable for practical in vivo H2 production due to their relatively low H2 yields and productivities. We suggest that an improved H2-producing cell factory could be designed by constructing a well characterized and overproduced synthetic H2 pathway and fully activating the native FHL in BL21(DE3).

Draft genome sequence of Hydrogenovibrio marinus MH-110, a model organism for aerobic H2 metabolism.

Hydrogenovibrio marinus, an obligate chemolithoautotroph isolated from oceanic surface water, is a Knallgas bacterium that conserves energy by oxidizing H2 in the presence of O2. The strain possesses a periplasmic membrane-bound respiratory [NiFe]-hydrogenase with high O2 tolerance, hence is of great biotechnological importance in the development of H2-based technologies for a promising alternative energy. Here, we report the draft genome of H. marinus MH-110, providing genomic information on the biosynthesis of the hydrogenase, aerobic H2 metabolism, and autotrophic carbon assimilation.

Engineered Escherichia coli with periplasmic carbonic anhydrase as a biocatalyst for CO2 sequestration.

Carbonic anhydrase is an enzyme that reversibly catalyzes the hydration of carbon dioxide (CO2). It has been suggested recently that this remarkably fast enzyme can be used for sequestration of CO2, a major greenhouse gas, making this a promising alternative for chemical CO2 mitigation. To promote the economical use of enzymes, we engineered the carbonic anhydrase from Neisseria gonorrhoeae (ngCA) in the periplasm of Escherichia coli, thereby creating a bacterial whole-cell catalyst. We then investigated the application of this system to CO2 sequestration by mineral carbonation, a process with the potential to store large quantities of CO2. ngCA was highly expressed in the periplasm of E. coli in a soluble form, and the recombinant bacterial cell displayed the distinct ability to hydrate CO2 compared with its cytoplasmic ngCA counterpart and previously reported whole-cell CA systems. The expression of ngCA in the periplasm of E. coli greatly accelerated the rate of calcium carbonate (CaCO3) formation and exerted a striking impact on the maximal amount of CaCO3 produced under conditions of relatively low pH. It was also shown that the thermal stability of the periplasmic enzyme was significantly improved. These results demonstrate that the engineered bacterial cell with periplasmic ngCA can successfully serve as an efficient biocatalyst for CO2 sequestration.

Biomineralization-based conversion of carbon dioxide to calcium carbonate using recombinant carbonic anhydrase.

Recently, as a mimic of the natural biomineralization process, the use of carbonic anhydrase (CA), which is an enzyme catalyzing fast reversible hydration of carbon dioxide to bicarbonate, has been suggested for biological conversion of CO(2) to valuable chemicals. While purified bovine CA (BCA) has been used in previous studies, its practical utilization in CO(2) conversion has been limited due to the expense of BCA preparation. In the present work, we investigated conversion of CO(2) into calcium carbonate as a target carbonate mineral by using a more economical, recombinant CA. To our knowledge, this is the first report of the usage of recombinant CA for biological CO(2) conversion. Recombinant α-type CA originating in Neisseria gonorrhoeae (NCA) was highly expressed as a soluble form in Escherichia coli. We found that purified recombinant NCA which showed comparable CO(2) hydration activity to commercial BCA significantly promoted formation of solid CaCO(3) through the acceleration of CO(2) hydration rate, which is naturally slow. In addition, the rate of calcite crystal formation was also accelerated using recombinant NCA. Moreover, non-purified crude recombinant NCA also showed relatively significant ability. Therefore, recombinant CA could be an effective, economical biocatalyst in practical CO(2) conversion system.

Improved production of biohydrogen in light-powered Escherichia coli by co-expression of proteorhodopsin and heterologous hydrogenase.

BACKGROUND: Solar energy is the ultimate energy source on the Earth. The conversion of solar energy into fuels and energy sources can be an ideal solution to address energy problems. The recent discovery of proteorhodopsin in uncultured marine γ-proteobacteria has made it possible to construct recombinant Escherichia coli with the function of light-driven proton pumps. Protons that translocate across membranes by proteorhodopsin generate a proton motive force for ATP synthesis by ATPase. Excess protons can also be substrates for hydrogen (H(2)) production by hydrogenase in the periplasmic space. In the present work, we investigated the effect of the co-expression of proteorhodopsin and hydrogenase on H(2) production yield under light conditions. RESULTS: Recombinant E. coli BL21(DE3) co-expressing proteorhodopsin and [NiFe]-hydrogenase from Hydrogenovibrio marinus produced ~1.3-fold more H(2) in the presence of exogenous retinal than in the absence of retinal under light conditions (70 µmole photon/(m2·s)). We also observed the synergistic effect of proteorhodopsin with endogenous retinal on H(2) production (~1.3-fold more) with a dual plasmid system compared to the strain with a single plasmid for the sole expression of hydrogenase. The increase of light intensity from 70 to 130 µmole photon/(m(2)·s) led to an increase (~1.8-fold) in H(2) production from 287.3 to 525.7 mL H(2)/L-culture in the culture of recombinant E. coli co-expressing hydrogenase and proteorhodopsin in conjunction with endogenous retinal. The conversion efficiency of light energy to H(2) achieved in this study was ~3.4%. CONCLUSION: Here, we report for the first time the potential application of proteorhodopsin for the production of biohydrogen, a promising alternative fuel. We showed that H(2) production was enhanced by the co-expression of proteorhodopsin and [NiFe]-hydrogenase in recombinant E. coli BL21(DE3) in a light intensity-dependent manner. These results demonstrate that E. coli can be applied as light-powered cell factories for biohydrogen production by introducing proteorhodopsin.

Production of biohydrogen by heterologous expression of oxygen-tolerant Hydrogenovibrio marinus [NiFe]-hydrogenase in Escherichia coli.

Oxygen sensitivity of hydrogenase is a critical issue in efficient biological hydrogen production. In the present study, oxygen-tolerant [NiFe]-hydrogenase from the marine bacterium, Hydrogenovibrio marinus, was heterologously expressed in Escherichia coli, for the first time. Recombinant E. coli BL21 expressing H. marinus [NiFe]-hydrogenase actively produced hydrogen, but the parent strain did not. Recombinant H. marinus hydrogenase required both nickel and iron for biological activity. Compared to the recombinant E. coli [NiFe]-hydrogenase 1 described in our previous report, recombinant H. marinus [NiFe]-hydrogenase displayed 1.6- to 1.7-fold higher hydrogen production activity in vitro. Importantly, H. marinus [NiFe]-hydrogenase exhibited relatively good oxygen tolerance in analyses involving changes of surface aeration and oxygen proportion within a gas mixture. Specifically, recombinant H. marinus [NiFe]-hydrogenase produced ∼7- to 9-fold more hydrogen than did E. coli [NiFe]-hydrogenase 1 in a gaseous environment containing 5-10% (v/v) oxygen. In addition, purified H. marinus [NiFe]-hydrogenase displayed a hydrogen evolution activity of ∼28.8 nmol H2/(minmg protein) under normal aerobic purification conditions. Based on these results, we suggest that oxygen-tolerant H. marinus [NiFe]-hydrogenase can be employed for in vivo and in vitro biohydrogen production without requirement for strictly anaerobic facilities.

Production of biohydrogen by recombinant expression of [NiFe]-hydrogenase 1 in Escherichia coli.

BACKGROUND: Hydrogenases catalyze reversible reaction between hydrogen (H2) and proton. Inactivation of hydrogenase by exposure to oxygen is a critical limitation in biohydrogen production since strict anaerobic conditions are required. While [FeFe]-hydrogenases are irreversibly inactivated by oxygen, it was known that [NiFe]-hydrogenases are generally more tolerant to oxygen. The physiological function of [NiFe]-hydrogenase 1 is still ambiguous. We herein investigated the H2 production potential of [NiFe]-hydrogenase 1 of Escherichia coli in vivo and in vitro. The hyaA and hyaB genes corresponding to the small and large subunits of [NiFe]-hydrogenase 1 core enzyme, respectively, were expressed in BL21, an E. coli strain without H2 producing ability. RESULTS: Recombinant BL21 expressing [NiFe]-hydrogenase 1 actively produced H2 (12.5 mL H2/(h.L) in 400 mL glucose minimal medium under micro-aerobic condition, whereas the wild type BL21 did not produce H2 even when formate was added as substrate for formate hydrogenlyase (FHL) pathway. The majority of recombinant protein was produced as an insoluble form, with translocation of a small fraction to the membrane. However, the membrane fraction displayed high activity (approximately 65% of total cell fraction), based on unit protein mass. Supplement of nickel and iron to media showed these metals contribute essentially to the function of [NiFe]-hydrogenase 1 as components of catalytic site. In addition, purified E. coli [NiFe]-hydrogenase 1 using his6-tag displayed oxygen-tolerant activity of approximately 12 nmol H2/(min.mg protein) under a normal aeration environment, compared to [FeFe]-hydrogenase, which remains inactive under this condition. CONCLUSIONS: This is the first report on physiological function of E. coli [NiFe]-hydrogenase 1 for H2 production. We found that [NiFe]-hydrogenase 1 has H2 production ability even under the existence of oxygen. This oxygen-tolerant property is a significant advantage because it is not necessary to protect the H2 production process from oxygen. Therefore, we propose that [NiFe]-hydrogenase can be successfully applied as an efficient biohydrogen production tool under micro-aerobic conditions.