Novel Bacterial Surface Display System Based on the Escherichia coli Protein MipA.

Bacterial surface display systems have been developed for various applications in biotechnology and industry. Particularly, the discovery and design of anchoring motifs is highly important for the successful display of a target protein or peptide on the surface of bacteria. In this study, an efficient display system on Escherichia coli was developed using novel anchoring motifs designed from the E. coli mipA gene. Using the C-terminal fusion system of an industrial enzyme, Pseudomonas fluorescens lipase, six possible fusion sites, V140, V176, K179, V226, V232, and K234, which were truncated from the C-terminal end of the mipA gene (MV140, MV176, MV179, MV226, MV232, and MV234) were examined. The whole-cell lipase activities showed that MV140 was the best among the six anchoring motifs. Furthermore, the lipase activity obtained using MV140 as the anchoring motif was approximately 20-fold higher than that of the previous anchoring motifs FadL and OprF but slightly higher than that of YiaTR232. Western blotting and confocal microscopy further confirmed the localization of the fusion lipase displayed on the E. coli surface using the truncated MV140. Additionally the MV140 motif could be used for successfully displaying another industrial enzyme, α-amylase from Bacillus subtilis. These results showed that the fusion proteins using the MV140 motif had notably high enzyme activities and did not exert any adverse effects on either cell growth or outer membrane integrity. Thus, this study shows that MipA can be used as a novel anchoring motif for more efficient bacterial surface display in the biotechnological and industrial fields.

Comprehensive analysis of the cardiac proteome in a rat model of myocardial ischemia-reperfusion using a TMT-based quantitative proteomic strategy.

BACKGROUND: Traditional studies of the cardiac proteome have mainly investigated in an animal model by two-dimensional gel electrophoresis (2-DE). However, the results have not been of satisfactory quality for an understanding of the underlying mechanism. Recent quantitative proteomic methods have been improved to overcome these limitations. To comprehensively study the cardiac proteome in a rat model of ischemia-reperfusion (IR), we developed a tandem mass tag (TMT)-based quantitative proteomic strategy. Furthermore, using this strategy, we examined the molecular mechanisms underlying the prevention of myocardial infarction by the intake of Triticum aestivum L. extract (TALE), a representative dietary fiber grain. METHODS: Cardiac proteomes were analyzed by 2-DE as a gel-based approach, and TMT labeling coupled with two-dimensional liquid chromatography (2D-LC) and tandem mass spectrometry (MS/MS) as a non-gel-based quantitative approach. Additionally, gene ontology annotation was conducted by PANTHER database. Several proteins of interest were verified by a Western blot analysis. RESULTS: Total 641 proteins were identified commonly from two independent MS datasets using 2D-LC MS/MS. Among these, we identified 151 IR-related proteins that were differentially expressed between the sham-operation group and IR group, comprising 62 up-regulated proteins and 89 down-regulated proteins. Most of the reduced proteins were involved in metabolic processes. In addition, 57 of the IR-related proteins were affected by TALE intake, representing 25 up-regulated proteins and 32 down-regulated proteins. In particular, TALE intake leads to a switch in metabolism to reduce the loss of high-energy phosphates and the accumulation of harmful catabolites (especially reactive oxygen species (ROS)) and to maintain cytoskeleton balance, leading to a reduction in cardiac IR injury. CONCLUSIONS: Our study provides a comprehensive proteome map of IR-related proteins and potential target proteins and identifies mechanisms implicated in the prevention of myocardial infarction by TALE intake in a rat IR model.

Impact of Endoscopists' Personality Traits on Adenoma and Polyp Detection Rates in Colonoscopy: A KASID Multicenter Study.

BACKGROUND: The personality traits of endoscopists have been suggested to affect the adenoma detection rate (ADR). We thus evaluated the relationship between endoscopists' personality traits and the ADR during colonoscopy using the Minnesota Multiphasic Personality Inventory-2 (MMPI-2). METHODS: In total, 1230 patients (asymptomatic and aged 50-80 years) who underwent screening or surveillance (≥ 5 years) colonoscopy were recruited from 13 university hospitals by 20 endoscopists between September 2015 and December 2017. We retrospectively measured the ADR, polyp detection rate (PDR), and number of adenomas per colonoscopy (APC). All 20 endoscopists completed all 567 true/false MMPI-2 items. RESULTS: The overall mean colonoscopy withdrawal time, PDR, ADR, and APC were 7.3 ± 2.8 min, 55%, 45.3%, and 0.97 ± 1.58, respectively. No significant difference was observed in the MMPI-2 clinical scales (e.g., hypochondriasis and psychasthenia), content scales (e.g., obsessiveness and type A character), or supplementary scales (e.g., dominance and social responsibility) between the high ADR group (ADR ≥45%, n = 10) and the low ADR group (ADR < 45%, n = 10). In multivariate logistic regression analysis, the ADR was associated significantly with patient age and sex. The ADR was related significantly to endoscopists' colonoscopy experience and the per-minute increase in the colonoscopy withdrawal time (OR 1.21, 95% CI 1.06-1.38, p = 0.005). In a logistic regression analysis adjusted for patient factors, the ADR was associated significantly with ego strength (OR 1.04, 95% CI 1.00-1.09, p = 0.044), as measured by the MMPI-2. CONCLUSIONS: With the exception of ego strength, the endoscopists' personality traits were not associated with adenoma or polyp detection.

Protective Effects of Arabinogalactan-Peptide Isolated from Wheat Flour against Myocardial Injury in an Ischemia/Reperfusion Rat Model.

We have previously shown that supplementation of wheat with hot-water extract reduces myocardial injury by inhibiting apoptosis in a rat model of myocardial infarction (MI). Arabinogalactan-peptide (AGP), a cell wall polysaccharide of wheat, was also responsible for the protection. However, the underlying mechanisms were not elucidated. In this study, we investigated the underlying mechanisms for how AGP supplementation reduces myocardial injury. First, we isolated highly pure AGP from all-purpose wheat flour. We supplemented rats with AGP at a dose of 100 mg/kg/d for 3 days, and subjected the rats to ischemia (30 min) through ligation of the left anterior descending coronary artery followed by reperfusion (3 h) through a release of the ligation. Supplementation with AGP significantly reduced the infarct size in the heart. In addition, AGP intake inhibited the apoptotic cascade, determined through decreased mitogen-activated protein kinases (p38 and c-Jun N-terminal kinase) phosphorylation, decreased Bcl-2-associated X protein/B-cell lymphoma ratios, and decreased generation of nicked DNA, which was confirmed through western blotting and terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling staining. These findings indicate that AGP intake can protect against myocardial injury. Traditionally, consumption of dietary fiber such as AGP has been shown to reduce MI risk by inhibiting preocclusion steps through reducing risk factors. Our findings suggest that AGP intake can also reduce MI risk by inhibiting postocclusion steps. This study describes a better dietary recommendation and new prevention strategy for reducing MI risk through regular consumption of wheat rich in AGP.

Comprehensive Analysis of Proteomic Differences between Escherichia coli K-12 and B Strains Using Multiplexed Isobaric Tandem Mass Tag (TMT) Labeling.

The Escherichia coli K-12 and B strains are among the most frequently used bacterial hosts for scientific research and biotechnological applications. However, omics analyses have revealed that E. coli K-12 and B exhibit notably different genotypic and phenotypic attributes, even though they were derived from the same ancestor. In a previous study, we identified a limited number of proteins from the two strains using two-dimensional gel electrophoresis and tandem mass spectrometry (MS/MS). In this study, an in-depth analysis of the physiological behavior of the E. coli K-12 and B strains at the proteomic level was performed using six-plex isobaric tandem mass tag-based quantitative MS. Additionally, the best lysis buffer for increasing the efficiency of protein extraction was selected from three tested buffers prior to the quantitative proteomic analysis. This study identifies the largest number of proteins in the two E. coli strains reported to date and is the first to show the dynamics of these proteins. Notable differences in proteins associated with key cellular properties, including some metabolic pathways, the biosynthesis and degradation of amino acids, membrane integrity, cellular tolerance, and motility, were found between the two representative strains. Compared with previous studies, these proteomic results provide a more holistic view of the overall state of E. coli cells based on a single proteomic study and reveal significant insights into why the two strains show distinct phenotypes. Additionally, the resulting data provide in-depth information that will help fine-tune processes in the future.

Plant-based foods containing cell wall polysaccharides rich in specific active monosaccharides protect against myocardial injury in rat myocardial infarction models.

Many cohort studies have shown that consumption of diets containing a higher composition of foods derived from plants reduces mortality from coronary heart disease (CHD). Here, we examined the active components of a plant-based diet and the underlying mechanisms that reduce the risk of CHD using three rat models and a quantitative proteomics approach. In a short-term myocardial infarction (MI) model, intake of wheat extract (WE), the representative cardioprotectant identified by screening approximately 4,000 samples, reduced myocardial injury by inhibiting apoptosis, enhancing ATP production, and maintaining protein homeostasis. In long-term post-MI models, this myocardial protection resulted in ameliorating adverse left-ventricular remodelling, which is a predictor of heart failure. Among the wheat components, arabinose and xylose were identified as active components responsible for the observed efficacy of WE, which was administered via ingestion and tail-vein injections. Finally, the food components of plant-based diets that contained cell wall polysaccharides rich in arabinose, xylose, and possibly fucose were found to confer protection against myocardial injury. These results show for the first time that specific monosaccharides found in the cell wall polysaccharides in plant-based diets can act as active ingredients that reduce CHD by inhibiting postocclusion steps, including MI and heart failure.

Deciphering Clostridium tyrobutyricum Metabolism Based on the Whole-Genome Sequence and Proteome Analyses.

UNLABELLED: Clostridium tyrobutyricum is a Gram-positive anaerobic bacterium that efficiently produces butyric acid and is considered a promising host for anaerobic production of bulk chemicals. Due to limited knowledge on the genetic and metabolic characteristics of this strain, however, little progress has been made in metabolic engineering of this strain. Here we report the complete genome sequence of C. tyrobutyricum KCTC 5387 (ATCC 25755), which consists of a 3.07-Mbp chromosome and a 63-kbp plasmid. The results of genomic analyses suggested that C. tyrobutyricum produces butyrate from butyryl-coenzyme A (butyryl-CoA) through acetate reassimilation by CoA transferase, differently from Clostridium acetobutylicum, which uses the phosphotransbutyrylase-butyrate kinase pathway; this was validated by reverse transcription-PCR (RT-PCR) of related genes, protein expression levels, in vitro CoA transferase assay, and fed-batch fermentation. In addition, the changes in protein expression levels during the course of batch fermentations on glucose were examined by shotgun proteomics. Unlike C. acetobutylicum, the expression levels of proteins involved in glycolytic and fermentative pathways in C. tyrobutyricum did not decrease even at the stationary phase. Proteins related to energy conservation mechanisms, including Rnf complex, NfnAB, and pyruvate-phosphate dikinase that are absent in C. acetobutylicum, were identified. Such features explain why this organism can produce butyric acid to a much higher titer and better tolerate toxic metabolites. This study presenting the complete genome sequence, global protein expression profiles, and genome-based metabolic characteristics during the batch fermentation of C. tyrobutyricum will be valuable in designing strategies for metabolic engineering of this strain. IMPORTANCE: Bio-based production of chemicals from renewable biomass has become increasingly important due to our concerns on climate change and other environmental problems. C. tyrobutyricum has been used for efficient butyric acid production. In order to further increase the performance and expand the capabilities of this strain toward production of other chemicals, metabolic engineering needs to be performed. For this, better understanding on the metabolic and physiological characteristics of this bacterium at the genome level is needed. This work reporting the results of complete genomic and proteomic analyses together with new insights on butyric acid biosynthetic pathway and energy conservation will allow development of strategies for metabolic engineering of C. tyrobutyricum for the bio-based production of various chemicals in addition to butyric acid.

Exploring the proteomic characteristics of the Escherichia coli B and K-12 strains in different cellular compartments.

Escherichia coli, one of the well-characterized prokaryotes, has been the most widely used bacterial host in scientific studies and industrial applications. Many different strains have been developed for the widespread use of E. coli in biotechnology, and selecting an ideal host to produce a specific protein of interest is a critical step in developing a production process. The E. coli B and K-12 strains are among the most frequently used bacterial hosts for the production of recombinant proteins as well as small-molecule metabolites such as amino acids, biofuels, carboxylic acids, diamines, and others. However, both strains have distinctive differences in genotypic and phenotypic attributes, and their behaviors can still be unpredictable at times, especially while expressing a recombinant protein. Therefore, in this review, an in-depth analysis of the physiological behavior on the proteomic level was performed, wherein the particularly distinct proteomic differences between the E. coli B and K-12 strains were investigated in the four distinctive cellular compartments. Interesting differences in the proteins associated with key cellular properties including cell growth, protein production and quality, cellular tolerance, and motility were observed between the two representative strains. The resulting enhancement of knowledge regarding host physiology that is summarized herein is expected to contribute to the acceleration of strain improvements and optimization for biotechnology-related processes.

An efficient bacterial surface display system based on a novel outer membrane anchoring element from the Escherichia coli protein YiaT.

In a bacterial surface display system, the display of a successful recombinant protein is highly dependent on the choice of anchoring motif. In this study, we developed an efficient Escherichia coli display system using novel anchoring motifs derived from the protein YiaT. To determine the best surface-anchoring motif, full-length YiaT and two of its C-terminal truncated forms, cut at the R181 and R232 sites, were evaluated. Two industrial enzymes, a lipase from Pseudomonas fluorescens SIK W1 and an α-amylase from Bacillus subtilis, were used as the target proteins for display. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blot, immunofluorescence microscopy and whole-cell enzyme activity measurements confirmed the expression of the fusion proteins on the E. coli surface. Using YiaTR181 or YiaTR232 as the anchoring motif, the fusion proteins showed very high enzyme activities and did not exert any adverse effects on either cell growth or the outer membrane integrity. Additionally, these fusion proteins were suitable for displaying proteins of large molecular size in an active form. Compared with the previous anchoring motifs FadL and OprF, YiaTR181 and YiaTR232 had approximately 10-fold and 20-fold higher enzyme activities, respectively. These results suggest that YiaT can be used as an E. coli anchoring motif to efficiently display various enzymes; hence, this system could be employed in a variety of biotechnological and industrial applications.

Proteomic analyses of the phase transition from acidogenesis to solventogenesis using solventogenic and non-solventogenic Clostridium acetobutylicum strains.

The fermentation carried out by the solvent-producing bacterium, Clostridium acetobutylicum, is characterized by two distinct phases: acidogenic and solventogenic phases. Understanding the cellular physiological changes occurring during the phase transition in clostridial fermentation is important for the enhanced production of solvents. To identify protein changes upon entry to stationary phase where solvents are typically produced, we herein analyzed the proteomic profiles of the parental wild type C. acetobutylicum strains, ATCC 824, the non-solventogenic strain, M5 that has lost the solventogenic megaplasmid pSOL1, and the synthetic simplified alcohol forming strain, M5 (pIMP1E1AB) expressing plasmid-based CoA-transferase (CtfAB) and aldehyde/alcohol dehydrogenase (AdhE1). A total of 68 protein spots, corresponding to 56 unique proteins, were unambiguously identified as being differentially present after the phase transitions in the three C. acetobutylicum strains. In addition to changes in proteins known to be involved in solventogenesis (AdhE1 and CtfB), we identified significant alterations in enzymes involved in sugar transport and metabolism, fermentative pathway, heat shock proteins, translation, and amino acid biosynthesis upon entry into the stationary phase. Of these, four increased proteins (AdhE1, CAC0233, CtfB and phosphocarrier protein HPr) and six decreased proteins (butyrate kinase, ferredoxin:pyruvate oxidoreductase, phenylalanyl-tRNA synthetase, adenylosuccinate synthase, pyruvate kinase and valyl-tRNA synthetase) showed similar patterns in the two strains capable of butanol formation. Interestingly, significant changes of several proteins by post-translational modifications were observed in the solventogenic phase. The proteomic data from this study will improve our understanding on how cell physiology is affected through protein levels patterns in clostridia.

Comparison of the large-scale periplasmic proteomes of the Escherichia coli K-12 and B strains.

Escherichia coli typically secretes many proteins into the periplasmic space, and the periplasmic proteins have been used for the secretory production of various proteins by the biotechnology industry. However, the identity of all of the E. coli periplasmic proteins remains unknown. Here, high-resolution periplasmic proteome reference maps of the E. coli K-12 and B strains were constructed and compared. Of the 145 proteins identified by tandem mass spectrometry, 61 proteins were conserved in the two strains, whereas 11 and 12 strain-specific proteins were identified for the E. coli K-12 and B strains, respectively. In addition, 27 proteins exhibited differences in intensities greater than 2-fold between the K-12 and B strains. The periplasmic proteins MalE and OppA were the most abundant proteins in the two E. coli strains. Distinctive differences between the two strains included several proteins that were caused by genetic variations, such as CybC, FliC, FliY, KpsD, MglB, ModA, and Ybl119, hydrolytic enzymes, particularly phosphatases, glycosylases, and proteases, and many uncharacterized proteins. Compared to previous studies, the localization of many proteins, including 30 proteins for the K-12 strain and 53 proteins for the B strain, was newly identified as periplasmic. This study identifies the largest number of proteins in the E. coli periplasm as well as the dynamics of these proteins. Additionally, these findings are summarized as reference proteome maps that will be useful for studying protein secretion and may provide new strategies for the enhanced secretory production of recombinant proteins.

Expression of a lipase on the cell-surface of Escherichia coli using the OmpW anchoring motif and its application to enantioselective reactions.

Microbial-surface display is the expression of proteins or peptides on the surface of cells by fusing an appropriate protein as an anchoring motif. Here, the outer membrane protein W (OmpW) was selected as a fusion partner for functional expression of Pseudomonas fluorescence SIK W1 lipase (TliA) on the cell-surface of Escherichia coli. Localization of the truncated OmpW-TliA fusion protein on the cell-surface was confirmed by immunoblotting and functional assay of lipase activity. Enantioselective hydrolysis of rac-phenylethyl butanoate by the displayed lipase resulted in optically active (R)-phenyl ethanol with 96% enantiomeric excess and 44% of conversion in 5 days. Thus, a small outer membrane protein OmpW, is a useful anchoring motif for displaying an active enzyme of ~50 kDa on the cell-surface and the surface-displayed lipase can be employed as an enantioselective biocatalyst in organic synthesis.

Isolation of a potential anchoring motif based on proteome analysis of Escherichia coli and its use for cell surface display.

For bacterial cell surface display, the target protein needs to be linked to an anchoring motif, and it is essential to choose an appropriate anchoring motif for efficient and stable display of the protein on the cell surface. To isolate a potential anchoring motif that would allow a stable and enhanced display of target proteins on the surface of an Escherichia coli host, we analyzed the outer membrane proteome of E. coli. On the basis of this proteomic analysis, the outer membrane protein X (OmpX), which has a small, monomeric ß-barrel structure and is highly expressed, was selected as a potential anchoring motif. The role of OmpX as an anchoring motif for cell surface display was demonstrated using three important industrial enzymes: endoxylanase, lipase, and alkaline phosphatase. Two different positions (Lys(122), Val(160)) in the extracellular loops of OmpX were examined for C-terminal fusion, and the biological activities and localization of the displayed enzymes were analyzed. All three enzymes examined were efficiently displayed on the E. coli cell surface with high activity. These results reveal that the use of OmpX as an anchoring motif is an efficient method to display functional enzymes on the surface of an E. coli host.

Comparative multi-omics systems analysis of Escherichia coli strains B and K-12.

BACKGROUND: Elucidation of a genotype-phenotype relationship is critical to understand an organism at the whole-system level. Here, we demonstrate that comparative analyses of multi-omics data combined with a computational modeling approach provide a framework for elucidating the phenotypic characteristics of organisms whose genomes are sequenced. RESULTS: We present a comprehensive analysis of genome-wide measurements incorporating multifaceted holistic data - genome, transcriptome, proteome, and phenome - to determine the differences between Escherichia coli B and K-12 strains. A genome-scale metabolic network of E. coli B was reconstructed and used to identify genetic bases of the phenotypes unique to B compared with K-12 through in silico complementation testing. This systems analysis revealed that E. coli B is well-suited for production of recombinant proteins due to a greater capacity for amino acid biosynthesis, fewer proteases, and lack of flagella. Furthermore, E. coli B has an additional type II secretion system and a different cell wall and outer membrane composition predicted to be more favorable for protein secretion. In contrast, E. coli K-12 showed a higher expression of heat shock genes and was less susceptible to certain stress conditions. CONCLUSIONS: This integrative systems approach provides a high-resolution system-wide view and insights into why two closely related strains of E. coli, B and K-12, manifest distinct phenotypes. Therefore, systematic understanding of cellular physiology and metabolism of the strains is essential not only to determine culture conditions but also to design recombinant hosts.

Comparative analysis of envelope proteomes in Escherichia coli B and K-12 strains.

Recent genome comparisons of E. coli B and K-12 strains have indicated that the makeup of the cell envelopes in these two strains is quite different. Therefore, we analyzed and compared the envelope proteomes of E. coli BL21(DE3) and MG1655. A total of 165 protein spots, including 62 nonredundant proteins, were unambiguously identified by two-dimensional gel electrophoresis and mass spectrometry. Of these, 43 proteins were conserved between the two strains, whereas 4 and 16 strain-specific proteins were identified only in E. coli BL21(DE3) and MG1655, respectively. Additionally, 24 proteins showed more than 2-fold differences in intensities between the B and K-12 strains. The reference envelope proteome maps showed that E. coli envelope mainly contained channel proteins and lipoproteins. Interesting proteomic observations between the two strains were as follows: (i) B produced more OmpF porin with a larger pore size than K-12, indicating an increase in the membrane permeability; (ii) B produced higher amounts of lipoproteins, which facilitates the assembly of outer membrane beta-barrel proteins; and (iii) motility- (FliC) and chemotaxis-related proteins (CheA and CheW) were detected only in K-12, which showed that E. coli B is restricted with regard to migration under unfavorable conditions. These differences may influence the permeability and integrity of the cell envelope, showing that E. coli B may be more susceptible than K-12 to certain stress conditions. Thus, these findings suggest that E. coli K-12 and its derivatives will be more favorable strains in certain biotechnological applications, such as cell surface display or membrane engineering studies.

Genome-wide identification of the subcellular localization of the Escherichia coli B proteome using experimental and computational methods.

Escherichia coli K-12 and B strains have most widely been employed for scientific studies as well as industrial applications. Recently, the complete genome sequences of two representative descendants of E. coli B strains, REL606 and BL21(DE3), have been determined. Here, we report the subproteome reference maps of E. coli B REL606 by analyzing cytoplasmic, periplasmic, inner and outer membrane, and extracellular proteomes based on the genome information using experimental and computational approaches. Among the total of 3487 spots, 651 proteins including 410 non-redundant proteins were identified and characterized by 2-DE and LC-MS/MS; they include 440 cytoplasmic, 45 periplasmic, 50 inner membrane, 61 outer membrane, and 55 extracellular proteins. In addition, subcellular localizations of all 4205 ORFs of E. coli B were predicted by combined computational prediction methods. The subcellular localizations of 1812 (43.09%) proteins of currently unknown function were newly assigned. The results of computational prediction were also compared with the experimental results, showing that overall precision and recall were 92.16 and 92.16%, respectively. This work represents the most comprehensive analyses of the subproteomes of E. coli B, and will be useful as a reference for proteome profiling studies under various conditions. The complete proteome data are available online (http://ecolib.kaist.ac.kr).

Understanding and engineering of microbial cells based on proteomics and its conjunction with other omics studies.

The abilities of microorganisms to produce a wide variety of products ranging from human therapeutics to chemicals and to tolerate or detoxify exogenous stresses such as toxic compounds and pollutants are of great importance in fundamental and applied research. Proteomics has become an indispensable tool for large-scale protein analyses and can be used to understand the resulting physiological changes and uncover the mechanisms responsible for the cellular processes under various genetic and environmental conditions. Recent development of a multi-omic approach that combines proteomics with one or more of other omics is allowing us to better understand cellular physiology and metabolism at the systems-wide level, and consequently paving a way toward more efficient metabolic engineering. In this review, we describe the use of proteomics and its combination with other omics to broaden our knowledge on microorganisms in the field of bioscience and biotechnology. With the increasing interest in practical applications, the strategies of employing proteomics for the successful metabolic engineering of microorganisms toward the enhanced production of desired products as well as the approaches taken to identify novel bacterial components are reviewed with corresponding examples.

A systems biology analysis of metastatic melanoma using in-depth three-dimensional protein profiling.

Melanoma is an excellent model to study molecular mechanisms of tumor progression because melanoma usually develops through a series of architecturally and phenotypically distinct stages that are progressively more aggressive, culminating in highly metastatic cells. In this study, we used an in-depth, 3-D protein level, comparative proteome analysis of two genetically, very closely related melanoma cell lines with low- and high-metastatic potentials to identify proteins and key pathways involved in tumor progression. This proteome comparison utilized fluorescent tagging of cell lysates followed by microscale solution IEF prefractionation and subsequent analysis of each fraction on narrow-range 2-D gels. LC-MS/MS analysis of gel spots exhibiting significant abundance changes identified 110 unique proteins. The majority of observed abundance changes closely correlate with biological processes central to cancer progression, such as cell death and growth and tumorigenesis. In addition, the vast majority of protein changes mapped to six cellular networks, which included known oncogenes (JNK, c-myc, and N-myc) and tumor suppressor genes (p53 and transforming growth factor-ß) as critical components. These six networks showed substantial connectivity, and most of the major biological functions associated with these pathways are involved in tumor progression. These results provide novel insights into cellular pathways implicated in melanoma metastasis.

Optimization of enantioselective synthesis of methyl (R)-2-chloromandelate by whole cells of Saccharomyces cerevisiae.

Methyl (R)-2-chloromandelate, a key intermediate in the synthesis of clopidogrel, was obtained by the reduction of methyl-2-chlorobenzoylformate using whole cells of Saccharomyces cerevisiae. A 100% conversion and 96.1% of enantiomeric excess (ee) value was obtained when 17 methyl-2-chlorobenzoylformate/l was reacted with 8 g S. cerevisiae/l and 83 g glucose/l at pH 7.

Extracellular proteome of Aspergillus terreus grown on different carbon sources.

Extracellular proteins of filamentous fungi are important for biomedical and biotechnological applications. Aspergillus terreus not only comprises an important class of organisms that have significant commercial relevance to the biotechnology industry, but also is an emerging fungal pathogen. However, no information is available on the extracellular proteome of A. terreus. Thus, we analyzed the extracellular proteomes of A. terreus under different culture conditions using sucrose, glucose, or starch as a main carbon source. A total of 82 protein spots including 39 unique proteins was successfully identified by 2-DE and nano-LC-MS/MS. Of these, 12 proteins were detected in the presence of at least two different carbon sources, whereas 16 proteins were unique to sucrose-, 3 to glucose-, and 8 to starch-grown A. terreus. Most of the proteins with known functions are hydrolytic enzymes, such as hydrolases, glycosylases and proteases, some of which include potential allergens. Both oryzin and a predicted protein (ATEG_07481) were the most abundant in all three media. Particularly, oryzin was highly secreted in high concentration sucrose medium. These proteomic data will be useful for studying protein secretion in further detail, and finding fusion partners for the extracellular production of homologous or heterologous proteins in A. terreus.