Efficiently utilizing all available carbon from lignocellulosic feedstock presents a major barrier to the production of economically feasible biofuel. Previously, to enable xylose utilization, we introduced a cofactor-dependent xylose reductase (XR) and xylitol dehydrogenase (XDH) pathway, or a cofactor-independent xylose isomerase (XI) pathway, into Saccharomyces cerevisiae. The resulting strains metabolized xylose with high efficiency. However, in both pathway recombinant strains, the cofactor imbalance caused accumulation of the byproducts glycerol and/or xylitol and reduced the ethanol production efficiency.
In this study, we introduced NADH oxidase from Lactococcus lactis into both XI and XR-XDH pathway recombinant strains. To reduce byproduct accumulation while maintaining xylose metabolism, we optimized the expression level of NADH oxidase by comparing its expression under the control of different promoters and plasmids. In recombinant XI strains, NADH oxidase was expressed at different levels, regulated by the GPD2 promoter or TEF1 promoter in the 2 μ plasmid. The expression under the control of GPD2 promoter decreased glycerol production by 84% and increased the ethanol yield and specific growth rate by 8% and 12%, respectively. In contrast, in the recombinant XR-XDH strains, such expression level was not efficient enough to decrease the byproduct accumulation. Therefore, higher NADH oxidase expression levels were tested. In the strain expressing NADH oxidase under the control of the TEF1 promoter in the centromeric plasmids, xylitol and glycerol production were reduced by 60% and 83%, respectively, without significantly affecting xylose consumption.
By fine-tuning NADH oxidase expression, we decreased the glycerol or/and xylitol production in both recombinant XI and XR-XDH xylose-metabolizing yeast strains. The optimal NADH oxidase expression levels depend on metabolic pathways. Similar cofactor engineering strategies could maximize the production of other redox dependent metabolites.
NADH oxidase; Xylose metabolism pathways; Cofactor; Glycerol; Xylitol
Carbon sources for biofuel production are wide-ranging and their availability depends on the climate and soil conditions of the land where the production chain is located. Henequen (Agave fourcroydes Lem.) is cultivated in Yucatán, Mexico to produce natural fibers from the leaves, and a juice containing fructans is produced during this process. Fructans can be hydrolyzed to fructose and glucose and metabolized into ethanol by appropriate yeasts. In Mexico, different Agave species provide the carbon source for (distilled and non-distilled) alcoholic beverage production using the stem of the plant, whilst the leaves are discarded. In this work, we investigated the effect of thermal acid and enzymatic hydrolysis of the juice on the amount of reducing sugars released. Growth curves were generated with the yeasts Saccharomyces cerevisiae and Kluyveromyces marxianus and fermentations were then carried out with Kluyveromyces marxianus to determine alcohol yields.
With thermal acid hydrolysis, the greatest increase in reducing sugars (82.6%) was obtained using 5% H2SO4 at 100°C with a 30 min reaction time. Statistically similar results can be obtained using the same acid concentration at a lower temperature and with a shorter reaction time (60°C, 15 min), or by using 1% H2SO4 at 100°C with a 30 min reaction time. In the case of enzymatic hydrolysis, the use of 5.75, 11.47 and 22.82 U of enzyme did not produce significant differences in the increase in reducing sugars. Although both hydrolysis processes obtained similar results, the difference was observed after fermentation. Ethanol yields were 50.3 ± 4 and 80.04 ± 5.29% of the theoretical yield respectively.
Final reducing sugars concentrations obtained with both thermal acid and enzymatic hydrolysis were similar. Saccharomyces cerevisiae, a good ethanol producer, did not grow in the hydrolysates. Only Kluyveromyces marxianus was able to grow in them, giving a higher ethanol yield with the enzymatic hydrolysate. The leaves account for a non-negligible weight of the total agave plant biomass, so this work complements the knowledge already developed on agave fermentations by making it possible to produce ethanol from almost the entire plant (stem and leaves).
Biofuel; Sugars; Oligofructans; Hydrolysis; Pretreatments
Metal borides are a class of inorganic solids that is much less known and investigated than for example metal oxides or intermetallics. At the same time it is a highly versatile and interesting class of compounds in terms of physical and chemical properties, like semiconductivity, ferromagnetism, or catalytic activity. This makes these substances attractive for the generation of new materials. Very little is known about the interaction between organic materials and borides. To generate nanostructured and composite materials which consist of metal borides and organic modifiers it is necessary to develop new synthetic strategies. Phage peptide display libraries are commonly used to select peptides that bind specifically to metals, metal oxides, and semiconductors. Further, these binding peptides can serve as templates to control the nucleation and growth of inorganic nanoparticles. Additionally, the combination of two different binding motifs into a single bifunctional phage could be useful for the generation of new composite materials.
In this study, we have identified a unique set of sequences that bind to amorphous and crystalline nickel boride (Ni3B) nanoparticles, from a random peptide library using the phage display technique. Using this technique, strong binders were identified that are selective for nickel boride. Sequence analysis of the peptides revealed that the sequences exhibit similar, yet subtle different patterns of amino acid usage. Although a predominant binding motif was not observed, certain charged amino acids emerged as essential in specific binding to both substrates. The 7-mer peptide sequence LGFREKE, isolated on amorphous Ni3B emerged as the best binder for both substrates. Fluorescence microscopy and atomic force microscopy confirmed the specific binding affinity of LGFREKE expressing phage to amorphous and crystalline Ni3B nanoparticles.
This study is, to our knowledge, the first to identify peptides that bind specifically to amorphous and to crystalline Ni3B nanoparticles. We think that the identified strong binding sequences described here could potentially serve for the utilisation of M13 phage as a viable alternative to other methods to create tailor-made boride composite materials or new catalytic surfaces by a biologically driven nano-assembly synthesis and structuring.
The editors of BMC Biotechnology would like to thank all our reviewers who have contributed to the journal in Volume 13 (2013).
Methicillin-resistant Staphylococcus aureus (MRSA) has become one of the most prevalent pathogens responsible for nosocomial infections throughout the world. As clinical MRSA diagnosis is concerned, current diagnostic methodologies are restricted by significant drawbacks and novel methods are required for MRSA detection. This study aimed at developing a simple loop-mediated isothermal amplification (LAMP) assay targeting on orfX for the rapid detection of methicillin-resistance Staphylococcus aureus (MRSA).
The protocol was designed by targeting orfX, a highly conserved open reading frame in S. aureus. One hundred and sixteen reference strains, including 52 Gram-positive and 64 Gram-negative isolates, were included for evaluation and optimization of the orfX-LAMP assay. This assay had been further performed on 667 Staphylococcus (566 MRSA, 25 MSSA, 53 MRCNS and 23 MSCNS) strains and were comparatively validated by PCR assay using primers F3 and B3, with rapid template DNA processing, simple equipments (water bath) and direct result determination (both naked eye and under UV light) applied. The indispensability of each primer had been confirmed, and the optimal amplification was obtained under 65°C for 45 min. The 25 μl reactant was found to be the most cost-efficient volume, and the detection limit was determined to be 10 DNA copies and 10 CFU/reaction. High specificity was observed when orfX-LAMP assay was subjected to 116 reference strains. For application, 557 (98.4%, 557/566) and 519 (91.7%, 519/566) tested strains had been detected positive by LAMP and PCR assays. The detection rate, positive predictive value (PPV) and negative predictive value (NPV) of orfX-LAMP were 98.4%, 100% and 92.7% respectively.
The established orfX-LAMP assay had been demonstrated to be a valid and rapid detection method on MRSA.
Loop-mediated isothermal amplification (LAMP); MRSA; OrfX
Eukaryotic ubiquitin and SUMO are frequently used as tags to enhance the fusion protein expression in microbial host. They increase the solubility and stability, and protect the peptides from proteolytic degradation due to their stable and highly conserved structures. Few of prokaryotic ubiquitin-like proteins was used as fusion tags except ThiS, which enhances the fusion expression, however, reduces the solubility and stability of the expressed peptides in E. coli. Hence, we investigated if MoaD, a conserved small sulfur carrier in prokaryotes with the similar structure of ubiquitin, could also be used as fusion tag in heterologous expression in E. coli.
Fusion of MoaD to either end of EGFP enhanced the expression yield of EGFP with a similar efficacy of ThiS. However, the major parts of the fusion proteins were expressed in the aggregated form, which was associated with the retarded folding of EGFP, similar to ThiS fusions. Fusion of MoaD to insulin chain A or B did not boost their expression as efficiently as ThiS tag did, probably due to a less efficient aggregation of products. Interestingly, fusion of MoaD to the murine ribonuclease inhibitor enhanced protein expression by completely protecting the protein from intracellular degradation in contrast to ThiS fusion, which enhanced degradation of this unstable protein when expressed in E. coli.
Prokaryotic ubiquitin-like protein MoaD can act as a fusion tag to promote the fusion expression with varying mechanisms, which enriches the arsenal of fusion tags in the category of insoluble expression.
MoaD; Prokaryote; Ubiquitin; Expression; Degradation; Protein folding
Penicillin G acylase (PGA) is used industrially to catalyze the hydrolysis of penicillin G to obtain 6-aminopenicillanic acid. In Escherichia coli, the most-studied microorganism for PGA production, this enzyme accumulates in the periplasmic cell space, and temperature plays an important role in the correct synthesis of its subunits.
This work investigates the influence of medium composition, cultivation strategy, and temperature on PGA production by recombinant E. coli cells. Shake flask cultures carried out using induction temperatures ranging from 18 to 28°C revealed that the specific enzyme activity achieved at 20°C (3000 IU gDCW-1) was 6-fold higher than the value obtained at 28°C. Auto-induction and high cell density fed-batch bioreactor cultures were performed using the selected induction temperature, with both defined and complex media, and IPTG and lactose as inducers. Final biomass concentrations of 100 and 120 gDCW L-1, and maximum enzyme productivities of 7800 and 5556 IU L-1 h-1, were achieved for high cell density cultures using complex and defined media, respectively.
To the best of our knowledge, the volumetric enzyme activity and productivity values achieved using the complex medium are the highest ever reported for PGA production using E. coli. Overall PGA recovery yields of 64 and 72% after purification were achieved for crude extracts obtained from cells cultivated in defined and complex media, respectively. The complex medium was the most cost-effective for PGA production, and could be used in both high cell density and straightforward auto-induction protocols.
Penicillin G acylase; Recombinant E. coli; Auto-induction medium; Fed-batch culture
Protein Kinases are key regulators of cell function and play essential roles in the occurrence and development of many human diseases. Many kinase inhibitors have been used for molecular targeted treatment of those diseases such as cancer and inflammation. However, those highly hydrophobic kinase inhibitors shared the common features of poor bioavailability and limited in vivo half-life, which strongly impeded their practical applications. Our previous study demonstrated that microbial synthesized biodegradable polyester poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx), a member of polyhydroxyalkanoates (PHAs) family, could serve as a promising delivery nanocarrier for those hydrophobic kinase inhibitors. Recently, a novel natural synthesized hybrid copolymer, PEG200 end-capped PHBHHx (PHBHHxPEG) was produced by Aeromonas hydrophila fermentation. In this study, the novel PHBHHxPEG NPs were prepared and investigated to serve as intracellular delivery nanocarriers for sustained release of hydrophobic kinase inhibitors.
PHBHHxPEG nanoparticles (NPs) prepared by an emulsification–solvent evaporation method were spherical with a diameter around 200 nm. The entrapment efficiency on rapamycin in PHBHHxPEG NPs was 91.9% and the sustained release of rapamycin from PHBHHxPEG NPs could be achieved for almost 10 days. The cellular uptake of PHBHHxPEG NPs was significant higher than that of PHBHHx NPs. The anti-proliferation effect and mTOR inhibition ability of rapamycin-loaded PHBHHxPEG NPs was stronger than that of drug-loaded PHBHHx NPs and free rapamycin.
PHBHHxPEG NPs could achieve the efficient entrapment and sustained release of rapamycin. The novel biodegradable PHBHHxPEG appeared a promising nanocarrier for sustained delivery of hydrophobic kinase inhibitors with improved cellular uptake and kinase inhibition efficiency.
Polyhydroxyalkanoate; PEG; Rapamycin; Nanoparticle; Drug delivery
Signalling proteins often contain several well defined and conserved protein domains. Structural analyses of such domains by nuclear magnetic spectroscopy or X-ray crystallography may greatly inform the function of proteins. A limiting step is often the production of sufficient amounts of the recombinant protein. However, there is no particular way to predict whether a protein will be soluble when expressed in E.coli. Here we report our experience with expression of a Src homology 2 (SH2) domain.
The SH2 domain of the SH2D2A protein (or T cell specific adapter protein, TSAd) forms insoluble aggregates when expressed as various GST-fusion proteins in Escherichia coli (E. coli). Alteration of the flanking sequences, or growth temperature influenced expression and solubility of TSAd-SH2, however overall yield of soluble protein remained low. The algorithm TANGO, which predicts amyloid fibril formation in eukaryotic cells, identified a hydrophobic sequence within the TSAd-SH2 domain with high propensity for beta-aggregation. Mutation to the corresponding amino acids of the related HSH2- (or ALX) SH2 domain increased the yield of soluble TSAd-SH2 domains. High beta-aggregation values predicted by TANGO correlated with low solubility of recombinant SH2 domains as reported in the literature.
Solubility of recombinant proteins expressed in E.coli can be predicted by TANGO, an algorithm developed to determine the aggregation propensity of peptides. Targeted mutations representing corresponding amino acids in similar protein domains may increase solubility of recombinant proteins.
Bacterial inclusion bodies; Protein aggregation; Recombinant protein expression; SH2 domain; SH2D2A; Protein solubility
Streptococcus pneumoniae (S. pneumoniae) causes several serious diseases including pneumonia, septicemia and meningitis. The World Health Organization estimates that streptococcal pneumonia is the cause of approximately 1.9 million deaths of children under five years of age each year. The large number of serotypes underlying the disease spectrum, which would be reflected in the high production cost of a commercial vaccine effective to protect against all of them and the higher level of amino acid sequence conservation as compared to polysaccharide structure, has prompted us to attempt to use conserved proteins for the development of a simpler vaccine. One of the most prominent proteins is pneumolysin (Ply), present in almost all the serotypes known at the moment, which shows an effective protection against S. pneumoniae infections.
We have cloned the pneumolysin gene from S. pneumoniae serotype 14 and studied the effects of eight variables related to medium composition and induction conditions on the soluble expression of rPly in Escherichia coli (E. coli) and a 28-4 factorial design was applied. Statistical analysis was carried out to compare the conditions used to evaluate the expression of soluble pneumolysin; rPly activity was evaluated by hemolytic activity assay and served as the main response to evaluate the proper protein expression and folding. The optimized conditions, validated by the use of triplicates, include growth until an absorbance of 0.8 (measured at 600 nm) with 0.1 mM IPTG during 4 h at 25°C in a 5 g/L yeast extract, 5 g/L tryptone, 10 g/L NaCl, 1 g/L glucose medium, with addition of 30 μg/mL kanamycin.
This experimental design methodology allowed the development of an adequate process condition to attain high levels (250 mg/L) of soluble expression of functional rPly in E. coli, which should contribute to reduce operational costs. It was possible to recover the protein in its active form with 75% homogeneity.
Soluble expression; Experimental design; Design of experiment (DoE); rPly; Recombinant E. coli; Hemolytic activity
The Rho-associated coiled-coil kinase-2 (ROCK2) is an important signaling transducer in the transmission of extracellular signals effecting organization of the actin cytoskeleton. ROCK2 has been implicated in numerous pathologies and the current focus is on understanding the molecular events that couple ROCK2 activity to biological function. To aid in the search for new ROCK2 substrates, we have developed an analog-sensitive (AS) ROCK2 protein that allows the use of selective ATP analogs that are not efficiently utilized by other protein kinases.
The analog sensitive protein, M160A ROCK2, was highly active and could phosphorylate proteins from a cellular homogenate with
32P-N6 (benzyl)ATP. We show the utility of this approach by identifying a putative ROCK2 substrate, elongation initiation factor-1-α1. We further show that the major site of ROCK2 phosphorylation of EIF1α1 is Thr432.
Our work demonstrates that AS-ROCK2 could be useful in a systematic proteomic approach for identifying novel ROCK2 substrates.
ROCK2; Protein kinase; Chemical engineering; Cell signaling
Gas vesicles are hollow, buoyant organelles bounded by a thin and extremely stable protein membrane. They are coded by a cluster of gvp genes in the halophilic archaeon, Halobacterium sp. NRC-1. Using an expression vector containing the entire gvp gene cluster, gas vesicle nanoparticles (GVNPs) have been successfully bioengineered for antigen display by constructing gene fusions between the gvpC gene and coding sequences from bacterial and viral pathogens.
To improve and streamline the genetic system for bioengineering of GVNPs, we first constructed a strain of Halobacterium sp. NRC-1 deleted solely for the gvpC gene. The deleted strain contained smaller, more spindle-shaped nanoparticles observable by transmission electron microscopy, confirming a shape-determining role for GvpC in gas vesicle biogenesis. Next, we constructed expression plasmids containing N-terminal coding portions or the complete gvpC gene. After introducing the expression plasmids into the Halobacterium sp. NRC-1 ΔgvpC strain, GvpC protein and variants were localized to the GVNPs by Western blotting analysis and their effects on increasing the size and shape of nanoparticles established by electron microscopy. Finally, a synthetic gene coding for Gaussia princeps luciferase was fused to the gvpC gene fragments on expression plasmids, resulting in an enzymatically active GvpC-luciferase fusion protein bound to the buoyant nanoparticles from Halobacterium.
GvpC protein and its N-terminal fragments expressed from plasmid constructs complemented a Halobacterium sp. NRC-1 ΔgvpC strain and bound to buoyant GVNPs. Fusion of the luciferase reporter gene from Gaussia princeps to the gvpC gene derivatives in expression plasmids produced GVNPs with enzymatically active luciferase bound. These results establish a significantly improved genetic system for displaying foreign proteins on Halobacterium gas vesicles and extend the bioengineering potential of these novel nanoparticles to catalytically active enzymes.
Vaccine; Halophiles; Archaea; Luciferase
Nanoparticles appear to be promising devices for application in the agriculture and food industries, but information regarding the response of plants to contact with nano-devices is scarce. Toxic effects may be imposed depending on the type and concentration of nanoparticle as well as time of exposure. A number of mechanisms may underlie the ability of nanoparticles to cause genotoxicity, besides the activation of ROS scavenging mechanisms. In a previous study, we showed that plant cells accumulate 3-Mercaptopropanoic acid-CdSe/ZnS quantum dots (MPA-CdSe/ZnS QD) in their cytosol and nucleus and increased production of ROS in a dose dependent manner when exposed to QD and that a concentration of 10 nM should be cyto-compatible.
When Medicago sativa cells were exposed to 10, 50 and 100 nM MPA-CdSe/ZnS QD a correspondent increase in the activity of Superoxide dismutase, Catalase and Glutathione reductase was registered. Different versions of the COMET assay were used to assess the genotoxicity of MPA-CdSe/ZnS QD. The number of DNA single and double strand breaks increased with increasing concentrations of MPA-CdSe/ZnS QD. At the highest concentrations, tested purine bases were more oxidized than the pyrimidine ones. The transcription of the DNA repair enzymes Formamidopyrimidine DNA glycosylase, Tyrosyl-DNA phosphodiesterase I and DNA Topoisomerase I was up-regulated in the presence of increasing concentrations of MPA-CdSe/ZnS QD.
Concentrations as low as 10 nM MPA-CdSe/ZnS Quantum Dots are cytotoxic and genotoxic to plant cells, although not lethal. This sets a limit for the concentrations to be used when practical applications using nanodevices of this type on plants are being considered. This work describes for the first time the genotoxic effect of Quantum Dots in plant cells and demonstrates that both the DNA repair genes (Tdp1β, Top1β and Fpg) and the ROS scavenging mechanisms are activated when MPA-CdSe/ZnS QD contact M. sativa cells.
CdSe/ZnS quantum dots; 3-Mercaptopropanoic acid; Plant cells; Medicago sativa; Cytotoxicity; Genotoxicity
It remains a challenge for recombinant S. cerevisiae to convert xylose in lignocellulosic biomass hydrolysates to ethanol. Although industrial diploid strains are more robust compared to laboratory haploid strains, however, industrial diploid S. cerevisiae strains have been less pursued in previous studies. This work aims to construct fast xylose-fermenting yeast using an industrial ethanol-producing diploid S. cerevisiae strain as a host.
Fast xylose-fermenting yeast was constructed by genome integration of xylose-utilizing genes and adaptive evolution, including 1) Piromyces XYLA was introduced to enable the host strain to convert xylose to xylulose; 2) endogenous genes (XKS1, RKI1, RPE1, TKL1, and TAL1) were overexpressed to accelerate conversion of xylulose to ethanol; 3) Candida intermedia GXF1, which encodes a xylose transporter, was introduced at the GRE3 locus to improve xylose uptake; 4) aerobic evolution in rich xylose media was carried out to increase growth and xylose consumption rates. The best evolved strain CIBTS0735 consumed 80 g/l glucose and 40 g/l xylose in rich media within 24 hours at an initial OD600 of 1.0 (0.63 g DCW/l) and produced 53 g/l ethanol.
Based on the above fermentation performance, we conclude that CIBTS0735 shows great potential for ethanol production from lignocellulosic biomass.
Saccharomyces cerevisiae; Xylose isomerase; Adaptive evolution; Xylose fermentation
Affinity precipitation has been reported as a potential technology for the purification of proteins at the early stage of downstream processing. The technology could be achieved using reversible soluble-insoluble polymers coupled with an affinity ligand to purify proteins from large volumes of dilute solution material such as fermentation broths or plasma. In this study, a thermo-response polymer was synthesized using N-methylol acrylamide, N-isopropyl acrylamide and butyl acrylate as monomers. The molecular weight of the polymer measured by the viscosity method was 3.06 × 104 Da and the lower critical solution temperature (LCST) was 28.0°C.The recovery of the polymer above the LCST was over 95.0%. Human serum albumin (HSA) is the most abundant protein in the human serum system, and it has important functions in the human body. High purity HSA is required in pharmaceuticals. Safe and efficient purification is a crucial process during HSA production.
A thermo-response polymer was synthesized and L-thyroxin immobilized on the polymer as an affinity ligand to enable affinity precipitation of HSA. The LCST of the affinity polymer was 31.0°C and the recovery was 99.6% of its original amount after recycling three times. The optimal adsorption condition was 0.02 M Tris–HCl buffer (pH 7.0) and the HSA adsorption capacity was 14.9 mg/g polymer during affinity precipitation. Circular dichroism spectra and a ForteBio Octet system were used to analyze the interactions between the affinity polymer and HSA during adsorption and desorption. The recovery of total HSA by elution with 1.0 mol/L NaSCN was 93.6%. When the affinity polymer was applied to purification of HSA from human serum, HSA could be purified to single-band purity according to SDS-PAGE.
A thermo-response polymer was synthesized and L-thyroxin was attached to the polymer. Affinity precipitation was used to purify HSA from human serum.
Affinity precipitation; L-thyroxin; Thermo-response polymer; Human serum albumin
Staphylococcus aureus produces several toxins, including Panton-Valentine leukocidin (PVL). The involvement of PVL in primary skin infections, necrotizing pneumonia, musculoskeletal disorders, brain abscess, and other diseases, some of which are life-threatening, has been reported. Following expert opinion, we aimed to provide the tools for establishment of sequence-based diagnostics and therapeutics for those conditions. We engineered the synergistic S and F (LukS-PV and LukF-PV respectively) pro-toxin subunits from Staphylococcus aureus USA400 into separate expression E. coli BL21(DE3)-pLysS hosts.
Following Nickel affinity chromatography (NAC), the F subunit came out without bands of impurity. The S sub-unit did not come off very pure after NAC thus necessitating further purification by size exclusion and ion-exchange chromatography. The purification plots showed that the BioLogic-LP and AKTA systems are reliable for following the progress of the chromatographic purification in real-time. Computer predicted Mw for the 6His-LukF-PV and 6His-LukS-PV were 35645.41 Da and 33530.04 Da respectively, while the mass spectrometry results were 35643.57 Da and 33528.34 Da respectively.
The BioLogic-LP and AKTA systems are commendable for reliability and user-friendliness. As a recent work elsewhere also reported that a second round of chromatography was necessary to purify the S subunit after the first attempt, we speculate that the S subunit might contain yet unidentified motif(s) requiring further treatment. The purified S and F sub-units of PVL were supplied to the Nottingham Cancer Immunotherapy group who used them to establish sequence-based monoclonal antibodies for diagnostic and therapeutic uses targeting PVL.
Staphylococcus aureus; Panton-Valentine leukocidin; Leukocytolytic exotoxin; Chromatography; Mass spectrometry; Immuno-therapy
The complete degradation of the cellulose requires the synergistic action of endo-β-glucanase, exo-β-glucanase, and β-glucosidase. But endo-β-glucanase and exo-β-glucanase can be recovered by solid–liquid separation in cellulose hydrolysis by their cellulose binding domain (CBD), however, the β-glucosidases cannot be recovered because of most β-glucosidases without the CBD, so additional β-glucosidases are necessary for the next cellulose degradation. This will increase the cost of cellulose degradation.
The glucose-tolerant β-glucosidase (BGL) from Thermoanaerobacterium thermosaccharolyticum DSM 571 was fused with cellulose binding domain (CBD) of Clostridium cellulovorans cellulosome anchoring protein by a peptide linker. The fusion enzyme (BGL-CBD) gene was overexpressed in Escherichia coli with the maximum β-glucosidase activity of 17 U/mL. Recombinant BGL-CBD was purified by heat treatment and following by Ni-NTA affinity. The enzymatic characteristics of the BGL-CBD showed optimal activities at pH 6.0 and 65°C. The fusion of CBD structure enhanced the hydrolytic efficiency of the BGL-CBD against cellobiose, which displayed a 6-fold increase in V
in comparison with the BGL. A gram of cellulose was found to absorb 643 U of the fusion enzyme (BGL-CBD) in pH 6.0 at 50°C for 25 min with a high immobilization efficiency of 90%. Using the BGL-CBD as the catalyst, the yield of glucose reached a maximum of 90% from 100 g/L cellobiose and the BGL-CBD could retain over 85% activity after five batches with the yield of glucose all above 70%. The performance of the BGL-CBD on microcrystalline cellulose was also studied. The yield of the glucose was increased from 47% to 58% by adding the BGL-CBD to the cellulase, instead of adding the Novozyme 188.
The hydrolytic activity of BGL-CBD is greater than that of the Novozyme 188 in cellulose degradation. The article provides a prospect to decrease significantly the operational cost of the hydrolysis process.
Glucose-tolerant β-glucosidase; Cellulose binding domain (CBD); Fusion protein; Immobilization
β-Fructofuranosidases (or invertases) catalyse the commercially-important biotransformation of sucrose into short-chain fructooligosaccharides with wide-scale application as a prebiotic in the functional foods and pharmaceutical industries.
We identified a β-fructofuranosidase gene (CmINV) from a Ceratocystis moniliformis genome sequence using protein homology and phylogenetic analysis. The predicted 615 amino acid protein, CmINV, grouped with an existing clade within the glycoside hydrolase (GH) family 32 and showed typical conserved motifs of this enzyme family. Heterologous expression of the CmINV gene in Saccharomyces cerevisiae BY4742∆suc2 provided further evidence that CmINV indeed functions as a β-fructofuranosidase. Firstly, expression of the CmINV gene complemented the inability of the ∆suc2 deletion mutant strain of S. cerevisiae to grow on sucrose as sole carbohydrate source. Secondly, the recombinant protein was capable of producing short-chain fructooligosaccharides (scFOS) when incubated in the presence of 10% sucrose. Purified deglycosylated CmINV protein showed a molecular weight of ca. 66 kDa and a Km and Vmax on sucrose of 7.50 mM and 986 μmol/min/mg protein, respectively. Its optimal pH and temperature conditions were determined to be 6.0 and 62.5°C, respectively. The addition of 50 mM LiCl led to a 186% increase in CmINV activity. Another striking feature was the relatively high volumetric production of this protein in S. cerevisiae as one mL of supernatant was calculated to contain 197 ± 6 International Units of enzyme.
The properties of the CmINV enzyme make it an attractive alternative to other invertases being used in industry.
β-fructofuranosidase; Short-chain fructooligosaccharides; Ceratocystis moniliformis; Saccharomyces cerevisiae; Heterologous expression
The starting material for RNA sequencing (RNA-seq) studies is usually total RNA or polyA+ RNA. Both forms of RNA represent heterogeneous pools of RNA molecules at different levels of maturation and processing. Such heterogeneity, in addition to the biases associated with polyA+ purification steps, may influence the analysis, sensitivity and the interpretation of RNA-seq data. We hypothesize that subcellular fractions of RNA may provide a more accurate picture of gene expression.
We present results for sequencing of cytoplasmic and nuclear RNA after cellular fractionation of tissue samples. In comparison with conventional polyA+ RNA, the cytoplasmic RNA contains a significantly higher fraction of exonic sequence, providing increased sensitivity in expression analysis and splice junction detection, and in improved de novo assembly of RNA-seq data. Conversely, the nuclear fraction shows an enrichment of unprocessed RNA compared with total RNA-seq, making it suitable for analysis of nascent transcripts and RNA processing dynamics.
Our results show that cellular fractionation is a more rapid and cost effective approach than conventional polyA+ enrichment when studying mature RNAs. Thus, RNA-seq of separated cytosolic and nuclear RNA can significantly improve the analysis of complex transcriptomes from mammalian tissues.
RNA sequencing; Transcriptomics; RNA splicing; RNA purification; PolyA+ selection; Cytoplasmic RNA; Nuclear RNA; Nascent transcripts; De novo assembly; Transcription profiling
Lentiviral vectors have emerged as efficient vehicles for transgene delivery in both dividing and non-dividing cells. A number of different modifications in vector design have increased biosafety and transgene expression. However, despite these advances, the transduction of primary human T cells is still challenging and methods to achieve efficient gene transfer are often expensive and time-consuming.
Here we present a simple optimised protocol for the generation and transduction of lentivirus in primary human CD45RA+ T cells. We show that generation of high-titre lentivirus with improved primary T cell transduction is dependent upon optimised ultracentrifuge speed during viral concentration. Moreover, we demonstrate that transduction efficiency can be increased with simple modifications to the culturing conditions. Overall, a transduction efficiency of up to 89% in primary human CD45RA+ cells is achievable when these modifications are used in conjunction.
The optimised protocol described here is easy to implement and should facilitate the production of high-titre lentivirus with superior transduction efficiency in primary human T cells without the need for further purification methods.
Titre; Primary lymphocytes; CD45RA+; Ultracentrifugation
Cyclic AMP (cAMP) and cyclic GMP (cGMP) have roles in relaying external signals and modifying gene expression within cells in all phyla. Currently there are no reporter systems suitable for bacteria and plant cells that measure alterations in downstream gene expression following changes in intracellular levels of cyclic nucleotides. As the plant protein OLIGOPEPTIDE TRANSPORTER X (OPTX) is upregulated by cGMP, we fused the OPTX promoter to a luciferase reporter gene (OPTX:LUC) to develop a plant cell reporter of cGMP-induced gene expression. We prepared a second construct augmented with three mammalian cGMP response elements (OPTXcGMPRE:LUC) and a third construct containing five gibberellic acid response elements (OPTXGARE:LUC). All three constructs were tested in bacteria and isolated plant protoplasts.
Membrane permeable cGMP enhanced luciferase activity of OPTX:LUC and OPTXGARE:LUC in protoplasts. Treatment with the plant hormone gibberellic acid which acts via cGMP also generated downstream luciferase activity. However, membrane permeable cAMP induced similar responses to cGMP in protoplasts. Significantly increased luciferase activity occurred in bacteria transformed with either OPTXcGMPRE:LUC or OPTXGARE:LUC in response to membrane permeable cAMP and cGMP. Bacteria co-transformed with OPTXcGMPRE:LUC or OPTXGARE:LUC and the soluble cytoplasmic domain of phytosulfokine receptor1 (PSKR1; a novel guanylate cyclase) had enhanced luciferase activity following induction of PSKR1 expression.
We have developed promoter reporter systems based on the plant OPTX promoter that can be employed in bacteria and isolated plant cells. We have shown that it can be used in bacteria to screen recombinant proteins for guanylate cyclase activity as increases in intracellular cGMP levels result in altered gene transcription and luciferase activity.
Cyclic GMP; Cyclic AMP; Luciferase reporter; OPTX promoter
Clostridial co-culture containing cellulolytic and solventogenic species is a potential consolidated bioprocessing (CBP) approach for producing biochemicals and biofuels from cellulosic biomass. It has been demonstrated that the rate of cellulose utilization in the co-culture of Clostridium acetobutylicum and Clostridium cellulolyticum is improved compared to the mono-culture of C. cellulolyticum (BL 5:119-124, 1983). However, the metabolic interactions in this co-culture are not well understood. To investigate the metabolic interactions in this co-culture we dynamically characterized the physiology and microbial composition using qPCR.
The qPCR data suggested a higher growth rate of C. cellulolyticum in the co-culture compared to its mono-culture. Our results also showed that in contrast to the mono-culture of C. cellulolyticum, which did not show any cellulolytic activity under conditions similar to those of co-culture, the co-culture did show cellulolytic activity even superior to the C. cellulolyticum mono-culture at its optimal pH of 7.2. Moreover, experiments indicated that the co-culture cellulolytic activity depends on the concentration of C. acetobutylicum in the co-culture, as no cellulolytic activity was observed at low concentration of C. acetobutylicum, and thus confirming the essential role of C. acetobutylicum in improving C. cellulolyticum growth in the co-culture. Furthermore, butanol concentration of 350 mg/L was detected in the co-culture batch experiments.
These results suggest the presence of synergism between these two species, while C. acetobutylicum metabolic activity significantly improves the cellulolytic activity in the co-culture, and allows C. cellulolyticum to survive under harsh co-culture conditions, which do not allow C. cellulolyticum to grow and metabolize cellulose independently. It is likely that C. acetobutylicum improves the cellulolytic activity of C. cellulolyticum in the co-culture through exchange of metabolites such as pyruvate, enabling it to grow and metabolize cellulose under harsh co-culture conditions.
Consolidated bioprocessing; Clostridial co-culture; qPCR analysis; Clostridium acetobutylicum; Clostridium cellulolyticum
There is an imperative necessity for alternative sources of energy able to reduce the world dependence of fossil oil. One of the most successful options is ethanol obtained mainly from sugarcane and corn fermentation. The foremost residue from sugarcane industry is the bagasse, a rich lignocellulosic raw material uses for the production of ethanol second generation (2G). New cellulolytic and hemicellulytic enzymes are needed, in order to optimize the degradation of bagasse and production of ethanol 2G.
The ability to produce hemicellulases and related enzymes, suitable for lignocellulosic biomass deconstruction, was explored using 110 endophytic fungi and 9 fungi isolated from spoiled books in Brazil. Two initial selections were performed, one employing the esculin gel diffusion assay, and the other by culturing on agar plate media with beechwood xylan and liquor from the hydrothermal pretreatment of sugar cane bagasse. A total of 56 isolates were then grown at 29°C on steam-exploded delignified sugar cane bagasse (DEB) plus soybean bran (SB) (3:1), with measurement of the xylanase, pectinase, β-glucosidase, CMCase, and FPase activities. Twelve strains were selected, and their enzyme extracts were assessed using different substrates. Finally, the best six strains were grown under xylan and pectin, and several glycohydrolases activities were also assessed. These strains were identified morphologically and by sequencing the internal transcribed spacer (ITS) regions and the partial β-tubulin gene (BT2). The best six strains were identified as Aspergillus niger DR02, Trichoderma atroviride DR17 and DR19, Alternaria sp. DR45, Annulohypoxylon stigyum DR47 and Talaromyces wortmannii DR49. These strains produced glycohydrolases with different profiles, and production was highly influenced by the carbon sources in the media.
The selected endophytic fungi Aspergillus niger DR02, Trichoderma atroviride DR17 and DR19, Alternaria sp. DR45, Annulohypoxylon stigyum DR47 and Talaromyces wortmannii DR49 are excellent producers of hydrolytic enzymes to be used as part of blends to decompose sugarcane biomass at industrial level.
Endophytic fungi; Xylanase; Hemicellulases; Accessory enzymes
Genome scale metabolic reconstructions are developed to efficiently engineer biocatalysts and bioprocesses based on a rational approach. However, in most reconstructions, due to the lack of appropriate measurements, experimentally determined growth parameters are simply taken from literature including other organisms, which reduces the usefulness and suitability of these models. Pseudomonas putida KT2440 is an outstanding biocatalyst given its versatile metabolism, its ability to generate sufficient energy and turnover of NADH and NAD. To apply this strain optimally in industrial production, a previously developed genome-scale metabolic model (iJP815) was experimentally assessed and streamlined to enable accurate predictions of the outcome of metabolic engineering approaches.
To substantially improve the accuracy of the genome scale model (iJP815), continuous bioreactor cultures on a mineral medium with glucose as a sole carbon source were carried out at different dilution rates, which covered pulling analysis of the macromolecular composition of the biomass. Besides, the maximum biomass yield (on substrate) of 0.397 gDCW · gglc-1, the maintenance coefficient of 0.037 gglc · gDCW-1 · h-1 and the maximum specific growth rate of 0.59 h-1 were determined. Only the DNA fraction increased with the specific growth rate. This resulted in reliable estimation for the Growth-Associated Maintenance (GAM) of 85 mmolATP · gDCW-1 and the Non Growth-Associated Maintenance (NGAM) of 3.96 mmolATP · gDCW-1 · h-1. Both values were found significantly different from previous assignment as a consequence of a lower yield and higher maintenance coefficient than originally assumed. Contrasting already published 13C flux measurements and the improved model allowed for constraining the solution space, by eliminating futile cycles. Furthermore, the model predictions were compared with transcriptomic data at overall good consistency, which helped to identify missing links.
By careful interpretation of growth stoichiometry and kinetics when grown in the presence of glucose, this work reports on an accurate genome scale metabolic model of Pseudomonas putida, providing a solid basis for its use in designing superior strains for biocatalysis. By consideration of substrate specific variation in stoichiometry and kinetics, it can be extended to other substrates and new mutants.
Continuous cultivation; P. putida KT2440; Glucose; Metabolic modeling; Biomass composition; Transcriptomics
Various DNA manipulation methods have been developed to prepare mutant genes for protein engineering. However, development of more efficient and convenient method is still demanded. Homologous DNA assembly methods, which do not depend on restriction enzymes, have been used as convenient tools for cloning and have been applied to site-directed mutagenesis recently. This study describes an optimized homologous DNA assembly method, termed as multiple patch cloning (MUPAC), for multiple site-directed and saturation mutagenesis.
To demonstrate MUPAC, we introduced five back mutations to a mutant green fluorescent protein (GFPuv) with five deleterious mutations at specific sites and transformed Escherichia coli (E. coli) with the plasmids obtained. We observed that the over 90% of resulting colonies possessed the plasmids containing the reverted GFPuv gene and exhibited fluorescence. We extended the test to introduce up to nine mutations in Moloney Murine Leukemia Virus reverse transcriptase (M-MLV RT) by assembling 11 DNA fragments using MUPAC. Analysis of the cloned plasmid by electrophoresis and DNA sequencing revealed that approximately 30% of colonies had the objective mutant M-MLV RT gene. Furthermore, we also utilized this method to prepare a library of mutant GFPuv genes containing saturation mutations at five specific sites, and we found that MUPAC successfully introduced NNK codons at all five sites, whereas other site remained intact.
MUPAC could efficiently introduce various mutations at multiple specific sites within a gene. Furthermore, it could facilitate the preparation of experimental gene materials important to molecular and synthetic biology research.