Byung Hoon Jo

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.

Engineered Escherichia coli with Periplasmic Carbonic Anhydrase as a Biocatalyst for CO2 Sequestration

ABSTRACT 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.

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

BackgroundHydrogenases 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 hya A and hya B 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.ResultsRecombinant 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 (~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 ~12 nmol H2/(min·mg protein) under a normal aeration environment, compared to [FeFe]-hydrogenase, which remains inactive under this condition.ConclusionsThis 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.

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

BackgroundSolar 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 (H2) production by hydrogenase in the periplasmic space. In the present work, we investigated the effect of the co-expression of proteorhodopsin and hydrogenase on H2 production yield under light conditions.ResultsRecombinant E. coli BL21(DE3) co-expressing proteorhodopsin and [NiFe]-hydrogenase from Hydrogenovibrio marinus produced ~1.3-fold more H2 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 H2 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/(m2·s) led to an increase (~1.8-fold) in H2 production from 287.3 to 525.7 mL H2/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 H2 achieved in this study was ~3.4%.ConclusionHere, we report for the first time the potential application of proteorhodopsin for the production of biohydrogen, a promising alternative fuel. We showed that H2 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.

Recent developments and applications of bioinspired silicification

Bioinspired synthesis of silica has attracted attention from a wide range of researchers as novel route for fabrication of various nanomaterials. Proteins including silaffins and silicateins as well as polyamines from marine diatoms and sponges are key biomolecules in these biomimetic silicification processes. These methods allow silica mineralization from various silica precursors under mild, biologically compatible conditions in an unprecedentedly fast and facile manner. Notably, the silica polycondensation entails the concomitant encapsulation of other molecules in the reaction solutions. Due to the efficient encapsulation and synergetic effects brought by the encapsulated molecules and the characteristics of biomimetic silica synthesis as well as the mechanical and chemical properties of silica itself, the silica- biomolecule nanocomposites have broad applications in biocatalysis, biosensor, and biomedical areas. Introduction and combination of novel template, precursors, inorganics, or enzymes with the previously used strategies will allow construction of more efficient, purpose-optimized silica nanomaterials with controlled size, composition, and morphology.

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.

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.

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

BackgroundSeveral 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.ResultsUnlike 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.ConclusionsHerein, 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).