-
[show abstract]
[hide abstract]
ABSTRACT: BACKGROUND: Biologically active inclusion bodies (IBs) have gained much attention in recent years. Fusion with IB-inducing partner has been shown to be an efficient strategy for generating active IBs. To make full use of the advantages of active IBs, one of the key issues will be to improve the activity yield of IBs when expressed in cells, which would need more choices on IB-inducing fusion partners and approaches for engineering IBs. Green fluorescent protein (GFP) has been reported to aggregate when overexpressed, but GFP fusion has not been considered as an IB-inducing approach for these fusion proteins so far. In addition, the role of linker in fusion proteins has been shown to be important for protein characteristics, yet impact of linker on active IBs has never been reported. RESULTS: Here we report that by fusing GFP and acid phosphatase PhoC via a linker region, the resultant PhoC-GFPs were expressed largely as IBs. These IBs show high levels of specific fluorescence and specific PhoC activities (phosphatase and phosphotransferase), and can account for up to over 80% of the total PhoC activities in the cells. We further demonstrated that the aggregation of GFP moiety in the fusion protein plays an essential role in the formation of PhoC-GFP IBs. In addition, PhoC-GFP IBs with linkers of different flexibility were found to exhibit different levels of activities and ratios in the cells, suggesting that the linker region can be utilized to manipulate the characteristics of active IBs. CONCLUSIONS: Our results show that active IBs of PhoC can be generated by GFP fusion, demonstrating for the first time the potential of GFP fusion to induce active IB formation of another soluble protein. We also show that the linker sequence in PhoC-GFP fusion proteins plays an important role on the regulation of IB characteristics, providing an alternative and important approach for engineering of active IBs with the goal of obtaining high activity yield of IBs.
Microbial Cell Factories 03/2013; 12(1):25. · 3.55 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: A novel thermostable alcohol dehydrogenase (ADH) showing activity toward aromatic secondary alcohols was identified from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1 (TkADH). The gene, tk0845, which encodes an aldo-keto reductase, was heterogeneously expressed in Escherichia coli. The enzyme was found to be a monomer with a molecular mass of 31 kDa. It was highly thermostable with an optimal temperature of 90°C and a half-life of 4.5 h at 95°C. The apparent K(m) values for the cofactor NAD(+), NADP(+), NADH and NADPH were similar within a range of 66-127 μM. TkADH preferred secondary alcohols and accepted various ketones and aldehydes as substrates. Interestingly, the enzyme could oxidize 1-phenylethanol and its derivatives having substituents at meta- and para-positions with high enantioselectivity (E-values from >290 to >1000), yielding the corresponding (R)-alcohols with optical purities of greater than 99.8% enantiomeric excess (ee). TkADH could also reduce 2,2,2-trifluoroacetophenone to (R)-2,2,2-trifluoro-1-phenylethanol with high enantioselectivity (>99.6% ee). Furthermore, the enzyme showed high resistance to organic solvents, and was particularly highly active in the presence of H(2)O/20% 2-propanol and H(2)O/50% n-hexane or n-octane. This ADH is expected to be a useful tool for the production of aromatic chiral alcohols.
Applied and environmental microbiology 01/2013; · 3.69 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Hythane (H(2)+CH(4)) has attracted growing attention due to its versatile advantages as, for instance vehicle fuel. Biohythane consisting of biohydrogen and biomethane via two-stage fermentation is a potential high-value solution for the valorization of waste biomass resources and probably an alternative to the fossil based hythane. However, the significance and application potential of biohythane have not yet been fully recognized. This review focuses on the progress of biohydrogen and subsequent biomethane fermentation in terms of substrate, microbial consortium, reactor configuration, as well as the H(2)/CH(4) ratio from the perspective of the feasibility of biohythane production in the past ten years. The current paper also covers how controls of the microbial consortium and bioprocess, system integration influence the biohythane productivity. Challenges and perspectives on biohythane technology will finally be addressed. This review provides a state-of-the-art technological insight into biohythane production by two-stage dark fermentation from biomass.
Bioresource technology 10/2012; · 4.25 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Enzymatic degradation of heparin has great potential as an ecological and specific way to produce low molecular weight heparin. However, the commercial use of heparinase I (HepA), one of the most important heparin lyases, has been hampered by low productivity and poor thermostability. Fusion with green fluorescent protein (GFP) or maltose-binding protein (MBP) has shown potential in facilitating the industrial use of HepA. Thus, tripartite fusion of GFP, MBP and HepA would be a promising approach. Therefore, in the present study, the tripartite fusion strategy was systematically studied, mainly focusing on the fusion order and the linker sequence, to obtain a fusion protein offering one-step purification and real-time detection of HepA activity by fluorescence as well as high HepA activity and thermostability. Our results show that fusion order is important for MBP binding affinity and HepA activity, while the linker sequences at domain junctions have significant effects on protein expression level, HepA activity and thermostability as well as GFP fluorescence. The best tripartite fusion was identified as MBP-(EAAAK)(3)-GFP-(GGGGS)(3)-HepA, which shows potential to facilitate the production of HepA and its application in industrial preparation of low molecular weight heparin.
Journal of biotechnology 10/2012; · 2.88 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Heparinase I (HepI), which specifically cleaves heparin and heparan sulfate, is one of the most extensively studied glycosaminoglycan lyases. Low productivity of HepI has largely hindered its industrial and pharmaceutical applications. Loss of bacterial HepI enzyme activity through poor thermostability during its expression and purification process in production can be an important issue. In this study, using a thermostabilization strategy combining site-directed mutagenesis and calcium ion addition during its production markedly improved the yield of maltose-binding protein-fused HepI (MBP-HepI) from recombinant Escherichia coli. Substitution of Cys297 to serine in MBP-HepI offered a 30.6 % increase in the recovered total enzyme activity due to a mutation-induced thermostabilizing effect. Furthermore, upon addition of Ca(2+) as a stabilizer at optimized concentrations throughout its expression, extraction, and purification process, purified mutant MBP-HepI showed a specific activity of 56.3 IU/mg, 206 % higher than that of the wild type obtained without Ca(2+) addition, along with a 177 % increase in the recovered total enzyme activity. The enzyme obtained through this novel approach also exhibited significantly enhanced thermostability, as indicated by both experimental data and the kinetic modeling. High-yield production of thermostable MBP-HepI using the present system will facilitate its applications in laboratory-scale heparin analysis as well as industrial-scale production of low molecular weight heparin as an improved anticoagulant substitute.
Applied Microbiology and Biotechnology 05/2012; · 3.42 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Engineering and redesign of enzymes are important to industrial biocatalysis. Fusion enzyme technology, based on fusion protein design, is frequently used in multifunctional enzyme construction and enzyme proximity control. Here, we reviewed the recent progress in molecular design strategy and application studies of fusion enzymes. The concept and features of fusion enzymes were introduced, followed by a systematical summary of the design strategy of fusion enzymes. In particular, the effects of different linker properties on fusion enzymes and their possible mechanisms were discussed. In addition, recent studies on fusion enzyme applications were also discussed. Finally, based on our own studies on fusion enzymes and the current research progress, the key problems in fusion enzyme technology and perspectives of this field were discussed.
Sheng wu gong cheng xue bao = Chinese journal of biotechnology 04/2012; 28(4):393-409.
-
[show abstract]
[hide abstract]
ABSTRACT: BACKGROUND: Bacterial hydrogen evolution releases excess electrons or energy formed during the metabolic oxidations. Until now, few studies have investigated the global regulation of cellular energy flows, pertaining to bacterial hydrogen production for the improvement of hydrogen production. In this study, the cellular energy metabolism of Enterobacter aerogenes was regulated by the addition of pyrophosphate (PPi) and the overexpression of polyphosphate kinase (PPK), to improve hydrogen production. RESULTS: By overexpressing PPK at 10 mmol L−1 PPi, total hydrogen yields were increased by 32.7%. Metabolic flux analysis demonstrated that overexpression of PPK in E. aerogenes resulted in a higher cellular ATP level and a higher NADH consumption rate, which changed the cellular redox state and allowed more electrons to flow into the hydrogen production pathway. CONCLUSION: Alteration of the energy metabolism in E. aerogenes can affect hydrogen production. This implies that if one could accurately control the energy flow or the electron flow in the cells, hydrogen productivity would be further greatly improved. Copyright © 2012 Society of Chemical Industry
Journal of Chemical Technology & Biotechnology 01/2012; 87(7):996-1003. · 2.17 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Metabolic engineering is recognized as one of the most important technologies for improving fermentative hydrogen yield. A vector with hydrogenase genes (hoxEFUYH) from Synechocystis sp. PCC 6803 under an alkB promoter was constructed, and introduced into Escherichia coli DH5α to alter the hydrogen metabolism with glucose as the sole carbon source. The recombinant strain reached a highest hydrogen yield of 1.89 mol/mol glucose, which was 95% of the theoretical hydrogen yield of E. coli. Hydrogenase activities for hydrogen evolution were increased and formic acid assimilation was accelerated with the expression of hoxEFUYH. The expression of hoxEFUYH suppressed the transcription of native hydrogenase 1 and hydrogenase 2, which were responsible for hydrogen uptake activity, while it had no influence on the transcription of the hydrogenase 3. Moreover, as the electron donor of HoxEFUYH is NADH, the expressed HoxEFUYH expanded the substrate specificity of the hydrogen-evolving hydrogenase in E. coli.
Biochemical Engineering Journal. 01/2012; 60:81-86.
-
[show abstract]
[hide abstract]
ABSTRACT: An NADH dehydrogenase encoded by the nuo cluster was isolated and impaired by knocking out the nuoB gene in Enterobacter aerogenes to examine its effect on hydrogen production. Three nuoB-deleted mutant strains were constructed from the wild-type strain E. aerogenes IAM1183 and two recombinant strains, IAM1183-A (ΔhycA) and IAM1183-O (ΔhybO), from which the hycA and hybO genes had already been deleted previously, respectively. Compared with the performance of the wild-type strain, the overall hydrogen production of the mutants IAM1183-B (ΔnuoB), IAM1183-AB (ΔhycA/ΔnuoB) and IAM1183-BO (ΔhybO/ΔnuoB) was increased by 49.2%, 54.0%, and 52.4% in batch culture, respectively. The hydrogen yields from glucose by the three mutants IAM1183-B, IAM1183-AB, IAM1183-BO were 1.38, 1.49, and 1.39 mol H2/mol glucose, respectively, while it was 1.16 mol H2/mol glucose in the wild-type strain. Metabolic flux analysis indicated that all three mutants exhibited reduced fluxes to lactate production, and enhanced fluxes toward the generation of hydrogen, acetate, ethanol, succinate and 2,3-butanediol. Both the formate pathway and the NADH pathway contributed to increased hydrogen production in the mutant strains. The assay of 4 NADH-mediated enzyme activities (H2ase, LDH, ADH and BDDH) was in accordance with the redistributions of the metabolic fluxes in the mutant strains.
International Journal of Hydrogen Energy 01/2012; 37(21):15875-15885. · 4.05 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: With the growing interest in using surfactants to improve microbial cell performance for whole-cell biocatalysis and bioremediation, understanding the interactions between surfactants and bacteria is of great importance. By using cyanine fluorescent protein (CFP) and bacterial luciferase (LUX) as dual bioreporters, the effects of n-dodecyltrimethylammonium bromide (DTAB) on the whole cells and intracellular proteins in Pseudomonas putida cultures were quantitatively and systematically studied. The dual reporter system was shown to be a useful indicator to assess the effect of DTAB treatment on whole-cell metabolic activity, membrane permeability, and cellular enzyme activity. CFP was useful to assess the leakage of intracellular enzymes and the lysis of cells and was able to reflect the activities of most cellular enzymes, while LUX reflected the permeability of cell membranes and cellular metabolic activity. The validity of CFP-LUX dual bioreporters was further confirmed by detecting changes in extracellular proteins, membrane potential, oxygen consumption rate (OUR), and intracellular catechol 2,3-dioxygenase (C23O) activity with the addition of DTAB. The dual LUX-CFP bioreporter is a useful tool for analyzing the surfactant-bacterium interactions for biotechnological applications.
Applied Microbiology and Biotechnology 11/2011; 93(1):393-400. · 3.42 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Enterobacter aerogenes is one of the most widely-studied model strains for fermentative hydrogen production. To improve the hydrogen yield of E. aerogenes, the bioengineering on a biomolecular level and metabolic network level is of importance. In this review, the fermentative technology of E. aerogenes for hydrogen production will be first briefly summarized. And then the bioengineering of E. aerogenes for the improvement of hydrogen yield will be thoroughly reviewed, including the anaerobic metabolic networks for hydrogen evolution in E. aerogenes, metabolic engineering for improving hydrogen production in E. aerogenes and mixed culture of E. aerogenes with other hydrogen-producing bacteria to enhance the overall yield in anaerobic cultivation. Finally, a perspective on E. aerogenes as a hydrogen producer including systems bioengineering approach for improving the hydrogen yield and application of the engineered E. aerogenes in mixed culture will be presented.
Bioresource technology 09/2011; 102(18):8344-9. · 4.25 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: A novel thermostable NAD(P)H oxidase from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1 (TkNOX) catalyzes oxidation of NADH and NADPH with oxygen from atmospheric air as an electron acceptor. Although the optimal temperature of TkNOX is >90°C, it also shows activity at 30°C. This enzyme was used for the regeneration of both NADP(+) and NAD(+) in alcohol dehydrogenase (ADH)-catalyzed enantioselective oxidation of racemic 1-phenylethanol. NADP(+) regeneration at 30°C was performed by TkNOX coupled with (R)-specific ADH from Lactobacillus kefir, resulting in successful acquisition of optically pure (S)-1-phenylethanol. The use of TkNOX with moderately thermostable (S)-specific ADH from Rhodococcus erythropolis enabled us to operate the enantioselective bioconversion accompanying NAD(+) regeneration at high temperatures. Optically pure (R)-1-phenylethanol was successfully obtained by this system after a shorter reaction time at 45-60°C than that at 30°C, demonstrating an advantage of the combination of thermostable enzymes. The ability of TkNOX to oxidize both NADH and NADPH with remarkable thermostability renders this enzyme a versatile tool for regeneration of the oxidized nicotinamide cofactors without the need for extra substrates other than dissolved oxygen from air.
Biotechnology and Bioengineering 08/2011; 109(1):53-62. · 3.95 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: To obtain oleaginous yeast mutants with improved lipid production and growth rates, an atmospheric and room temperature plasma (ARTP) jet was used with a 96-well plate for high throughput screening. Mutants with changes in growth rates and lipid contents were obtained. At a lethality rate of 99%, the positive mutation rate of the yeast cells was 27.2% evaluated by the growth rates of the mutants and the comparison with the wild strain. The fermentation in a medium composed of yeast extract (10 g/L), peptone (10 g/L) and D-glucose (20 g/L) resulted in the lipid yield of the mutant (C4) with 4.07% (W/W) compared with that of the wild strain (1.87%).
Sheng wu gong cheng xue bao = Chinese journal of biotechnology 03/2011; 27(3):461-7.
-
[show abstract]
[hide abstract]
ABSTRACT: Effects of different microaerophilic conditions on cell growth, glucose consumption, hydrogen production and cellular metabolism of wild Enterobacter aerogenes strain and polyphosphate kinase (PPK) overexpressing strain were systematically studied in this paper, using NaH(2)PO(4) as the phosphate sources. Under different microaerophilic conditions, PPK-overexpressing strain showed better cell growth, glucose consumption and hydrogen production than the wild strain. In the presence of limited oxygen (2.1%) and by PPK overexpression, the hydrogen production per liter of culture, the hydrogen production per cell and the hydrogen yield per mol of glucose increased by 20.1%, 12.3% and 10.8%, respectively, compared with the wild strain under strict anaerobic conditions. Metabolic analysis showed that the increase of the total hydrogen yield was attributed to the improvement of NADH pathway. The result of more reductive cellular oxidation state balance also further demonstrated that, under proper initial microaerophilic conditions and by PPK overexpression, the cell could adjust the cellular redox states and make more energy flow into hydrogen production pathways.
Enzyme and microbial technology. 02/2011; 48(2):187-92.
-
[show abstract]
[hide abstract]
ABSTRACT: A novel atmospheric and room temperature plasma (ARTP) which used helium as the working gas was employed to generate mutants of Enterobacter aerogenes for improving the hydrogen production. For the mutation, 50 μl of the E. aerogenes culture (OD600 = 2.0) was dipped onto a sterilized stainless steel plate (5 mm in diameter). The plate was then treated for 3 min by ARTP at the gas flow rate of 15.0 slpm (standard liters per minute) and 100 W of radio-frequency power input. The positive mutation rate defined by the ratio of the mutants with the increase of hydrogen productivity to the total mutants reached about 10%, and a mutant with improved cell growth and hydrogen productivity was then selected. The total hydrogen yield per mole glucose was increased by 26.4%, mainly dependent on the increase of hydrogen production by the NADH pathway. Two important parameters of the ATP yield and oxidation state balance indicated that the cellular metabolism was changed in the mutant. The genetic stability of the mutant for hydrogen production was maintained after more than 25 subcultures.
Biochemical Engineering Journal. 01/2011; 55(1):17-22.
-
[show abstract]
[hide abstract]
ABSTRACT: Violacein is a bacteria-originated indolocarbazole pigment with potential applications due to its various bioactivities such as anti-tumor, antiviral, and antifungal activities. However, stable mass production of this pigment is difficult due to its low productivities and the instability of wild-type violacein-producing strains. In order to establish a stable and efficient production system for violacein, the violacein synthesis pathway from a new species of Duganella sp. B2 was reconstructed in different bacterial strains including Escherichia coli, Citrobacter freundii, and Enterobacter aerogenes by using different vectors. The gene cluster that encodes five enzymes involved in the violacein biosynthetic pathway was first isolated from Duganella sp. B2, and three recombinant expression vectors were constructed using the T7 promoter or the alkane-responsive promoter PalkB. Our results showed that violacein could be stably synthesized in E. coli, C. freundii, and E. aerogenes. Interestingly, we found that there were great differences between the different recombinant strains, not only in the protein expression profiles pertaining to violacein biosynthesis but also in the productivity and composition of crude violacein. Among the host strains tested, the crude violacein production by the recombinant C. freundii strain reached 1.68 g L(-1) in shake flask cultures, which was 4-fold higher than the highest production previously reported in flask culture by other groups. To the best of our knowledge, this is the first report on the efficient production of violacein by genetically engineered strains.
Applied Microbiology and Biotechnology 12/2009; 86(4):1077-88. · 3.42 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The NAD+-dependent formate dehydrogenase FDH1 gene (fdh1), cloned from Candida boidinii, was expressed in the ldh-deleted mutant of Enterobacter aerogenes IAM1183 strain. The plasmid of pCom10 driven by the PalkB promoter was used to construct the fdh1 expression system and thus introduce a new dihydronicotinamide adenine dinucleotide (NADH) regeneration pathway from formate in the ldh-deleted mutant. The knockout of NADH-consuming lactate pathway affected the whole cellular metabolism, and the hydrogen yield increased by 11.4% compared with the wild strain. Expression of fdh1 in the ldh-deleted mutant caused lower final cell concentration and final pH after 16 h cultivation, and finally resulted in 86.8% of increase in hydrogen yield per mole consumed glucose. The analysis of cellular metabolites and estimated redox state balance in the fdhl-expressed strain showed that more excess of reducing power was formed by the rewired NADH regeneration pathway, changing the metabolic distribution and promoting the hydrogen production.
Applied Microbiology and Biotechnology 10/2009; 86(1):255-62. · 3.42 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Methanotrophs use methane as the sole carbon and energy source, which cause slow growth, low cell density and hinder its industrial applications. One promising solution is to heterologously express methane monooxygenase (MMO) in other host cells that can be easily cultivated at high cell density. We systematically exploited the possibility of functional expression of pMMO by choosing different promoters and different host cells. The results showed that the recombinants could oxidize methane to methanol. In particular, ethanol could also be detected in the oxidized products, but the enzyme activity was instable, implying that some changes of pMMO expressed in the host cells might have occurred. In addition, SDS-PAGE analysis showed that many recombinants could express the subunits of pMMO, but the enzyme activity could not be detected. In conclusion, correct fold of pMMO in the host cells is important for its functional expression.
Sheng wu gong cheng xue bao = Chinese journal of biotechnology 08/2009; 25(8):1151-9.
-
[show abstract]
[hide abstract]
ABSTRACT: An expression system for NAD(+)-dependent formate dehydrogenase gene (fdh1), from Candida boidinii, was constructed and cloned into Enterobacter aerogenes IAM1183. With the fdh1 expression, the total H(2) yield was attributed to a decrease in activity of the lactate pathway and an increase of the formate pathway flux due to the NADH regeneration. Analysis of the redox state balance and ethanol-to-acetate ratio in the fdhl-expressed strain showed that increased reducing power arose from the reconstruction of NADH regeneration pathway from formate thereby contributing to the improved H(2) production.
Biotechnology Letters 07/2009; 31(10):1525-30. · 1.68 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: By constructing the expression system for fusion protein of GFPmut1 (a green fluorescent protein mutant) with the hyperthermophilic xylanase obtained from Dictyoglomus thermophilum Rt46B.1, the effects of temperature on the fluorescence of GFP and its relationship with the activities of GFP-fused xylanase have been studied. The fluorescence intensities of both GFP and GFP-xylanase have proved to be thermally sensitive, with the thermal sensitivity of the fluorescence intensity of GFP-xylanase being 15% higher than that of GFP. The lost fluorescence intensity of GFP inactivated at high temperature of below 60 degrees C in either single or fusion form can be completely recovered by treatment at 0 degrees C. By the fluorescence recovery of GFP domain at low temperature, the ratios of fluorescence intensity to xylanase activity (Rgfp/Axyl) at 15 degrees C and 37 degrees C have been compared. Even though the numbers of molecules of GFP and xylanase are equivalent, the Rgfp/Axyl ratio at 15 degrees C is ten times of that at 37 degrees C. This is mainly due to the fact that lower temperature is more conducive to the correct folding of GFP than the hyperthermophilic xylanase during the expression. This study has indicated that the ratio of GFP fluorescence to the thermophilic enzyme activity for the fusion proteins expressed at different temperatures could be helpful in understanding the folding properties of the two fusion partners and in design of the fusion proteins.
Applied Microbiology and Biotechnology 05/2009; 84(3):511-7. · 3.42 Impact Factor
