Metabolically engineered Saccharomyces cerevisiae for enhanced isoamyl alcohol production.

Higher chain alcohols have gained much attention as next generation transport fuels because of their higher energy density and low moisture absorption capacity compared to ethanol. In the present study, we attempted to engineer Saccharomyces cerevisiae for the synthesis of isoamyl alcohol via de novo leucine biosynthetic pathway coupled with Ehrlich degradation pathway. To achieve high-level production of isoamyl alcohol, two strategies are used in the current study: (1) reconstruction of a chromosome-based leucine biosynthetic pathway under the control of galactose-inducible promoters; (2) overexpression of the mitochondrial 2-isopropylmalate (α-IPM) transporter to boost the transportation of α-IPM from mitochondria to the cytosol. We found engineered yeast cells with a combinatorially assembled leucine biosynthetic pathway coupled with the Ehrlich degradation pathway resulted in high-level production of isoamyl alcohol; however, there was still a significant amount of isobutanol co-formed during the fermentation process. Further introducing an α-IPM transporter not only boosted the isoamyl alcohol biosynthetic pathway activity but also reduced isobutanol to a much lower level. Taken together, our work represents the first study to construct a chromosome-based leucine biosynthetic pathway for isoamyl alcohol production. Furthermore, the utilization of the mitochondrial compartment coupled with the transporter engineering serves as an effective approach to minimize the by-product formation and to improve the isoamyl alcohol production.

Engineering the leucine biosynthetic pathway for isoamyl alcohol overproduction in Saccharomyces cerevisiae.

Isoamyl alcohol can be used not only as a biofuel, but also as a precursor for various chemicals. Saccharomyces cerevisiae inherently produces a small amount of isoamyl alcohol via the leucine degradation pathway, but the yield is very low. In the current study, several strategies were devised to overproduce isoamyl alcohol in budding yeast. The engineered yeast cells with the cytosolic isoamyl alcohol biosynthetic pathway produced significantly higher amounts of isobutanol over isoamyl alcohol, suggesting that the majority of the metabolic flux was diverted to the isobutanol biosynthesis due to the broad substrate specificity of Ehrlich pathway enzymes. To channel the key intermediate 2-ketosiovalerate (KIV) towards α-IPM biosynthesis, we introduced an artificial protein scaffold to pull dihydroxyacid dehydratase and α-IPM synthase into the close proximity, and the resulting strain yielded more than twofold improvement of isoamyl alcohol. The best isoamyl alcohol producer yielded 522.76 ± 38.88 mg/L isoamyl alcohol, together with 540.30 ± 48.26 mg/L isobutanol and 82.56 ± 8.22 mg/L 2-methyl-1-butanol. To our best knowledge, our work represents the first study to bypass the native compartmentalized α-IPM biosynthesis pathway for the isoamyl alcohol overproduction in budding yeast. More importantly, artificial protein scaffold based on the feature of quaternary structure of enzymes would be useful in improving the catalytic efficiency and the product specificity of other enzymatic reactions.

Metabolically engineered Saccharomyces cerevisiae for branched-chain ester productions.

Medium branched-chain esters can be used not only as a biofuel but are also useful chemicals with various industrial applications. The development of economically feasible and environment friendly bio-based fuels requires efficient cell factories capable of producing desired products in high yield. Herein, we sought to use a number of strategies to engineer Saccharomyces cerevisiae for high-level production of branched-chain esters. Mitochondrion-based expression of ATF1 gene in a base strain with an overexpressed valine biosynthetic pathway together with expression of mitochondrion-relocalized α-ketoacid decarboxylase (encoded by ARO10) and alcohol dehydrogenase (encoded by ADH7) not only produced isobutyl acetate, but also 3-methyl-1-butyl acetate and 2-methyl-1-butyl acetate. Further segmentation of the downstream esterification step into the cytosol to utilize the cytosolic acetyl-CoA pool for acetyltransferase (ATF)-mediated condensation enabled an additional fold improvement of ester productions. The best titre attained in the present study is 260.2mg/L isobutyl acetate, 296.1mg/L 3-methyl-1-butyl acetate and 289.6mg/L 2-methyl-1-butyl acetate.

Programming Saposin-Mediated Compensatory Metabolic Sinks for Enhanced Ubiquinone Production.

Microbial synthesis of ubiquinone by fermentation processes has been emerging in recent years. However, as ubiquinone is a primary metabolite that is tightly regulated by the host central metabolism, tweaking the individual pathway components could only result in a marginal improvement on the ubiquinone production. Given that ubiquinone is stored in the lipid bilayer, we hypothesized that introducing additional metabolic sink for storing ubiquinone might improve the CoQ10 production. As human lipid binding/transfer protein saposin B (hSapB) was reported to extract ubiquinone from the lipid bilayer and form the water-soluble complex, hSapB was chosen to build a compensatory metabolic sink for the ubiquinone storage. As a proof-of-concept, hSapB-mediated metabolic sink systems were devised and systematically investigated in the model organism of Escherichia coli. The hSapB-mediated periplasmic sink resulted in more than 200% improvement of CoQ8 over the wild type strain. Further investigation revealed that hSapB-mediated sink systems could also improve the CoQ10 production in a CoQ10-hyperproducing E. coli strain obtained by a modular pathway rewiring approach. As the design principles and the engineering strategies reported here are generalizable to other microbes, compensatory sink systems will be a method of significant interest to the synthetic biology community.

Mitochondrial acetyl-CoA utilization pathway for terpenoid productions.

Acetyl-CoA is a central molecule in the metabolism of the cell, which is also a precursor molecule to a variety of value-added products such as terpenoids and fatty acid derived molecules. Considering subcellular compartmentalization of metabolic pathways allows higher concentrations of enzymes, substrates and intermediates, and bypasses competing pathways, mitochondrion-compartmentalized acetyl-CoA utilization pathways might offer better pathway activities with improved product yields. As a proof-of-concept, we sought to explore a mitochondrial farnesyl pyrophosphate (FPP) biosynthetic pathway for the biosynthesis of amorpha-4,11-diene in budding yeast. In the present study, the eight-gene FPP biosynthetic pathway was successfully expressed inside yeast mitochondria to enable high-level amorpha-4,11-diene production. In addition, we also found the mitochondrial compartment serves as a partial barrier for the translocation of FPP from mitochondria into the cytosol, which would potentially allow minimized loss of FPP to cytosolic competing pathways. To our best knowledge, this is the first report to harness yeast mitochondria for terpenoid productions from the mitochondrial acetyl-CoA pool. We envision subcellular metabolic engineering might also be employed for an efficient production of other bio-products from the mitochondrial acetyl-CoA in other eukaryotic organisms.

Development of Colorimetric-Based Whole-Cell Biosensor for Organophosphorus Compounds by Engineering Transcription Regulator DmpR.

It is useful for whole-cell biosensors to be based on colorimetric detection because the output signal can be easily visualized. However, colorimetric-based whole-cell biosensors suffer higher detection limits as compared to bioluminescence- or fluorescence-based biosensors. In this work, we attempt to reduce the detection limit for a colorimetric-based whole-cell biosensor by applying directed evolution techniques on a transcription regulator, DmpR, to alter the expression level of its cognate promoter, which was fused to mRFP1 to output red coloration in the presence of organophosphate pesticides containing a phenolic group. We selected the two best-performing mutants, DM01 and DM12, which were able to develop red coloration in the presence of parathion as low as 10 µM after just 6 h of induction at 30 °C. This suggests that engineering of the transcription regulator in the sensing domain is useful for improving various properties of whole-cell biosensors, such as reducing the detection limit for simple colorimetric detection of organophosphate pesticides.

Genome-scale metabolic modeling and in silico analysis of lipid accumulating yeast Candida tropicalis for dicarboxylic acid production.

Recently, the bio-production of α,ω-dicarboxylic acids (DCAs) has gained significant attention, which potentially leads to the replacement of the conventional petroleum-based products. In this regard, the lipid accumulating yeast Candida tropicalis, has been recognized as a promising microbial host for DCA biosynthesis: it possess the unique ω-oxidation pathway where the terminal carbon of α-fatty acids is oxidized to form DCAs with varying chain lengths. However, despite such industrial importance, its cellular physiology and lipid accumulation capability remain largely uncharacterized. Thus, it is imperative to better understand the metabolic behavior of this lipogenic yeast, which could be achieved by a systems biological approach. To this end, herein, we reconstructed the genome-scale metabolic model of C. tropicalis, iCT646, accounting for 646 unique genes, 945 metabolic reactions, and 712 metabolites. Initially, the comparative network analysis of iCT646 with other yeasts revealed several distinctive metabolic reactions, mainly within the amino acid and lipid metabolism including the ω-oxidation pathway. Constraints-based flux analysis was, then, employed to predict the in silico growth rates of C. tropicalis which are highly consistent with the cellular phenotype observed in glucose and xylose minimal media chemostat cultures. Subsequently, the lipid accumulation capability of C. tropicalis was explored in comparison with Saccharomyces cerevisiae, indicating that the formation of "citrate pyruvate cycle" is essential to the lipid accumulation in oleaginous yeasts. The in silico flux analysis also highlighted the enhanced ability of pentose phosphate pathway as NADPH source rather than malic enzyme during lipogenesis. Finally, iCT646 was successfully utilized to highlight the key directions of C. tropicalis strain design for the whole cell biotransformation application to produce long-chain DCAs from alkanes. Biotechnol. Bioeng. 2016;113: 1993-2004. © 2016 Wiley Periodicals, Inc.

Dynamic control of ERG9 expression for improved amorpha-4,11-diene production in Saccharomyces cerevisiae.

BACKGROUND: To achieve high-level production of non-native isoprenoid products, it requires the metabolic flux to be diverted from the production of sterols to the heterologous metabolic reactions. However, there are limited tools for restricting metabolic flux towards ergosterol synthesis. In the present study, we explored dynamic control of ERG9 expression using different ergosterol-responsive promoters to improve the production of non-native isoprenoids. RESULTS: Several ergosterol-responsive promoters were identified using quantitative real-time PCR (qRT-PCR) analysis in an engineered strain with relatively high mevalonate pathway activity. We found mRNA levels for ERG11, ERG2 and ERG3 expression were significantly lower in the engineered strain over the reference strain BY4742, indicating these genes are transcriptionally down-regulated when ergosterol is in excess. Further replacement of the native ERG9 promoter with these ergosterol-responsive promoters revealed that all engineered strains improved amorpha-4,11-diene by 2~5-fold over the reference strain with ERG9 under its native promoter. The best engineered strain with ERG9 under the control of P ERG1 produced amorpha-4,11-diene to a titer around 350 mg/L after 96 h cultivation in shake-flasks. CONCLUSIONS: We envision dynamic control at the branching step using feedback regulation at transcriptional level could serve as a generalized approach for redirecting the metabolic flux towards product-of-interest.

Combinatorial assembly of large biochemical pathways into yeast chromosomes for improved production of value-added compounds.

Saccharomyces cerevisiae as a eukaryotic organism is particularly suitable as microbial cell factory because it has interesting features such as membrane environments for supporting membrane-associated enzymes and its capability for post-translational modifications of enzymes from plants. However, S. cerevisiae does not readily express polycistronic transcriptional units, which represents a significant challenge for constructing large biochemical pathways in budding yeast. In the present study, we developed a novel approach for rapid construction of large biochemical pathways into yeast chromosomes. Our approach takes advantage of antibiotic selection for combinatorial assembly of large pathways into the δ-sites of retrotransposon elements of yeast chromosomes. As proof-of-principle, a five-gene isobutanol pathway and an eight-gene mevalonate pathway were successfully assembled into yeast chromosomes in one-step fashion. To our knowledge, this is the first report to exploit δ-integration coupled with antibiotic selection for rapid assembly of large biochemical pathways in budding yeast. We envision our new approach could serve as a generalized technique for large pathway construction in yeast-a method that would be of significant interest to the synthetic biology community.

Combinatorial engineering of mevalonate pathway for improved amorpha-4,11-diene production in budding yeast.

Combinatorial genome integration of mevalonate pathway genes was performed with the aim of optimizing the metabolic flux for improved production of terpenoids in budding yeast. In the present study, we developed a novel δ-integration platform to achieve multiple genome integrations through modulating the concentration of antibiotics. By exploiting carotenoid biosynthesis as screening module, we successfully created a library of yeast colonies appeared with various intensities of orange color. As proof-of-concept that carotenoid overproducers could serve to boost the titer of other terpenoids, we further tested engineered strains for the production of amorpha-4,11-diene, an important precursor for antimalarial drug. However, we experienced some limitations of the carotenoid-based screening approach as it was only effective in detecting a small range of pathway activity improvement and further increasing mevalonate pathway activity led to a decreased orange color. By far, we were only able to obtain one mutant strain yielded more than 13-fold amorpha-4,11-diene over parental strains, which was approximately 64 mg/L of caryophyllene equivalents. Further qPCR studies confirmed that erg10, erg13, thmg1 and erg12 involved in mevalonate pathway were overexpressed in this mutant strain. We envision the current δ-integration platform would form the basis of a generalized technique for multiple gene integrations in yeast-a method that would be of significant interest to the metabolic engineering community.

Metabolic reconstruction and flux analysis of industrial Pichia yeasts.

Pichia yeasts have been recognized as important microbial cell factories in the biotechnological industry. Notably, the Pichia pastoris and Pichia stipitis species have attracted much research interest due to their unique cellular physiology and metabolic capability: P. pastoris has the ability to utilize methanol for cell growth and recombinant protein production, while P. stipitis is capable of assimilating xylose to produce ethanol under oxygen-limited conditions. To harness these characteristics for biotechnological applications, it is highly required to characterize their metabolic behavior. Recently, following the genome sequencing of these two Pichia species, genome-scale metabolic networks have been reconstructed to model the yeasts' metabolism from a systems perspective. To date, there are three genome-scale models available for each of P. pastoris and P. stipitis. In this mini-review, we provide an overview of the models, discuss certain limitations of previous studies, and propose potential future works that can be conducted to better understand and engineer Pichia yeasts for industrial applications.

Flexible all-solid-state asymmetric supercapacitors based on free-standing carbon nanotube/graphene and Mn3O4 nanoparticle/graphene paper electrodes.

We report the design of all-solid-state asymmetric supercapacitors based on free-standing carbon nanotube/graphene (CNTG) and Mn(3)O(4) nanoparticles/graphene (MG) paper electrodes with a polymer gel electrolyte of potassium polyacrylate/KCl. The composite paper electrodes with carbon nanotubes or Mn(3)O(4) nanoparticles uniformly intercalated between the graphene nanosheets exhibited excellent mechanical stability, greatly improved active surface areas, and enhanced ion transportation, in comparison with the pristine graphene paper. The combination of the two paper electrodes with the polymer gel electrolyte endowed our asymmetric supercapacitor of CNTG//MG an increased cell voltage of 1.8 V, a stable cycling performance (capacitance retention of 86.0% after 10,000 continuous charge/discharge cycles), more than 2-fold increase of energy density (32.7 Wh/kg) compared with the symmetric supercapacitors, and importantly a distinguished mechanical flexibility.

High-performance asymmetric supercapacitor based on graphene hydrogel and nanostructured MnO2.

We have successfully fabricated an asymmetric supercapacitor with high energy and power densities using graphene hydrogel (GH) with 3D interconnected pores as the negative electrode and vertically aligned MnO(2) nanoplates on nickel foam (MnO(2)-NF) as the positive electrode in a neutral aqueous Na(2)SO(4) electrolyte. Because of the desirable porous structure, high specific capacitance and rate capability of GH and MnO(2)-NF, complementary potential window of the two electrodes, and the elimination of polymer binders and conducting additives, the asymmetric supercapacitor can be cycled reversibly in a wide potential window of 0-2.0 V and exhibits an energy density of 23.2 Wh kg(-1) with a power density of 1.0 kW kg(-1). Energy density of the asymmetric supercapacitor is significantly improved in comparison with those of symmetric supercapacitors based on GH (5.5 Wh kg(-1)) and MnO(2)-NF (6.7 Wh kg(-1)). Even at a high power density of 10.0 kW kg(-1), the asymmetric supercapacitor can deliver a high energy density of 14.9 Wh kg(-1). The asymmetric supercapacitor also presents stable cycling performance with 83.4% capacitance retention after 5000 cycles.

EBV up-regulates cytochrome c through VDAC1 regulations and decreases the release of cytoplasmic Ca2+ in the NPC cell line.

EBV (Epstein-Barr virus) is considered to be a major factor that causes NPC (nasopharyngeal carcinoma), which is one of the sneakiest cancers frequently occurring in Southeast Asia and Southern China. Apoptosis and pro-apoptotic signals have been studied for decades; however, few have extended the prevailing view of EBV to its impact on NPC in perspective of apoptosis. One of the important proteins named VDAC1 (voltage-dependent anion protein 1) on the mitochondrial outer membrane controls the pro-apoptotic signals in mammalian cells. The impact of EBV infection on VDAC1 and related apoptotic signals remains unclear. In order to study the VDAC1's role in EBV-infected NPC cells, we employ siRNA (small interfering RNA) inhibition to analyse the release of Ca2+ and Cyto c (cytochrome c) signals in the cytoplasm, as they are important pro-apoptotic signals. The results show a decrease of Ca2+ release and up-regulation of Cyto c with EBV infection. After siRNA transfection, the dysregulation of Cyto c is neutralized, which is evidence that the level of Cyto c release in virus-infected NPC cells is the as same as that of non-infected NPC cells. This result indicates that EBV infection changes the cytoplasmic level of Cyto c through regulating VDAC1. In summary, this study reports that EBV changes the release of Ca2+ and Cyto c in the cytoplasm of NPC cells, and that Cyto c changes are mediated by VDAC1 regulation.

Engineering global transcription factor cyclic AMP receptor protein of Escherichia coli for improved 1-butanol tolerance.

One major challenge in biofuel production, including biobutanol production, is the low tolerance of the microbial host towards increasing biofuel concentration during fermentation. Here, we have demonstrated that Escherichia coli 1-butanol tolerance can be greatly enhanced through random mutagenesis of global transcription factor cyclic AMP receptor protein (CRP). Four mutants (MT1-MT4) with elevated 1-butanol tolerance were isolated from error-prone PCR libraries through an enrichment screening. A DNA shuffling library was then constructed using MT1-MT4 as templates and one mutant (MT5) that exhibited the best tolerance ability among all variants was selected. In the presence of 0.8 % (v/v, 6.5 g/l) 1-butanol, the growth rate of MT5 was found to be 0.28 h(-1) while that of wild type was 0.20 h(-1). When 1-butanol concentration increased to 1.2 % (9.7 g/l), the growth rate of MT5 (0.18 h(-1)) became twice that of the wild type (0.09 h(-1)). Microbial adhesion to hydrocarbon test showed that cell surface of MT5 was less hydrophobic and its cell length became significantly longer in the presence of 1-butanol, as observed by scanning electron microscopy. Quantitative real-time reverse transcription PCR analysis revealed that several CRP regulated, 1-butanol stress response related genes (rpoH, ompF, sodA, manX, male, and marA) demonstrated differential expression in MT5 in the presence or absence of 1-butanol. In conclusion, direct manipulation of the transcript profile through engineering global transcription factor CRP can provide a useful tool in strain engineering.

Improvement of biomass properties by pretreatment with ionic liquids for bioconversion process.

Cassava pulp residue and rice straw were used as a precursor for pretreatment with ionic liquids to study the effects of pretreatment conditions on product yield and properties. Cassava pulp residue is a potential biomass in the bioconversion process due to it requiring mild pretreatment conditions while providing a high sugar conversion. The maximum sugar conversion and lignin extraction are attained from pretreatment of biomasses with particle size of <38 µm and ionic liquid of 1-Ethyl-3-methylimidazolium acetate at 120°C for 24h. The effectiveness of ionic liquid for biomass pretreatment process follows the sequence: 1-Ethyl-3-methylimidazolium acetate>1-Ethyl-3-methylimidazolium diethyl phosphate>1,3-Dimethylimidazolium methyl sulfate. The increase of pretreatment temperature from 25 to 120°C and decrease of biomass particle size renders higher sugar conversion, lignin extraction and lower crystallinity index. However, pretreatment at temperatures higher than 120°C shows a sharp decline of regenerated biomass yield, sugar conversion and lignin extraction and giving higher crystallinity index at pretreatment temperature of 180°C.

The imminent role of protein engineering in synthetic biology.

Protein engineering has for decades been a powerful tool in biotechnology for generating vast numbers of useful enzymes for industrial applications. Today, protein engineering has a crucial role in advancing the emerging field of synthetic biology, where metabolic engineering efforts alone are insufficient to maximize the full potential of synthetic biology. This article reviews the advancements in protein engineering techniques for improving biocatalytic properties to optimize engineered pathways in host systems, which are instrumental to achieve high titer production of target molecules. We also discuss the specific means by which protein engineering has improved metabolic engineering efforts and provide our assessment on its potential to continue to advance biology engineering as a whole.

Random mutagenesis of global transcription factor cAMP receptor protein for improved osmotolerance.

The naturally existing microbial hosts can rarely satisfy industrial requirements, thus there has always been an intense effort in strain engineering to meet the needs of these bioprocesses. Here, in this work, we want to prove the concept that engineering global transcription factor cAMP receptor protein (CRP) of Escherichia coli can improve cell phenotypes. CRP is one of the global regulatory proteins that can regulate the transcription of over 400 genes in E. coli. The target phenotype in this study is strain osmotolerance. Amino acid mutations were introduced to CRP by either error-prone PCR or DNA shuffling, and the random mutagenesis libraries were subjected to enrichment selection under NaCl stress. Five CRP mutants (MT1-MT5) were selected from the error-prone PCR libraries with enhanced osmotolerance. DNA shuffling technique was employed to generate mutant MT6 with MT1-MT5 as templates. All of these variants showed much better growth in the presence of NaCl compared to the wild type, and MT6 presented the best tolerance towards NaCl. In the presence of 0.9 M NaCl, the growth rate of MT6 is 0.113 h(-1), while that of WT is 0.077 h(-1). MT6 also exhibited resistance to other osmotic stressors, such as KCl, glucose, and sucrose. DNA microarray analysis showed that genes involved in colanic acid biosynthesis are up-regulated in the absence of salt stress, whereas carbohydrate metabolic genes are differentially expressed under NaCl stress when comparing MT6 to WT. Scanning electron microscopy images confirmed the elongation of both WT and MT6 when exposed to NaCl but the cell surface of MT6 was relatively smooth.

Nanotube-supported bioproduction of 4-hydroxy-2-butanone via in situ cofactor regeneration.

Nicotinamide cofactor-dependent oxidoreductases have been widely employed during the bioproduction of varieties of useful compounds. Efficient cofactor regeneration is often required for these biotransformation reactions. Herein, we report the synthesis of an important pharmaceutical intermediate 4-hydroxy-2-butanone (4H2B) via an immobilized in situ cofactor regeneration system composed of NAD(+)-dependent glycerol dehydrogenase (GlyDH) and NAD(+)-regenerating NADH oxidase (nox). Both enzymes were immobilized on functionalized single-walled carbon nanotubes (SWCNTs) through the specific interaction between the His-tagged enzymes and the modified SWCNTs. GlyDH demonstrated ca. 100% native enzyme activity after immobilization. The GlyDH/nox ratio, pH, and amount of nicotinamide cofactor were examined to establish the optimum reaction conditions for 4H2B production. The nanoparticle-supported cofactor regeneration system become more stable and the yield of 4H2B turned out to be almost twice (37%) that of the free enzyme system after a 12-h reaction. Thus, we believe that this non-covalent specific immobilization procedure can be applied to cofactor regeneration system for bioconversions.

Comparative protein profile of human hepatoma HepG2 cells treated with graphene and single-walled carbon nanotubes: an iTRAQ-coupled 2D LC-MS/MS proteome analysis.

Graphitic nanomaterials are promising candidates for applications in electronics, energy, materials and biomedical areas. Nevertheless, few detailed studies related to the mechanistic understanding of these nanomaterials with the living systems have been performed to date. In the present study, our group applied the iTRAQ-coupled 2D LC-MS/MS approach to analyze the protein profile change of human hepatoma HepG2 cells treated with graphene and single-walled carbon nanotubes (SWCNTs), with the purpose of characterizing the interactions between living system and these nanomaterials at molecular level. Overall 37 differentially expressed proteins involved in metabolic pathway, redox regulation, cytoskeleton formation and cell growth were identified. Based on the protein profile, we found SWCNTs severely interfered the intracellular metabolic routes, protein synthesis and cytoskeletal systems. Moreover, our data suggested that SWCNTs might induce oxidative stress, thereby activating p53-mediated DNA damage checkpoint signals and leading to apoptosis. However, only moderate variation of protein levels for the cells treated with graphene was observed, which indicated graphene was less toxic and might be promising candidate for biomedical applications. We envision that this systematic characterization of cellular response at protein expression level will be of great importance to evaluate biocompatibility of nanomaterials.