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Metabolome- and genome-scale model analyses for engineering of Aureobasidium pullulans to enhance polymalic acid and malic acid production from sugarcane molasses
Jun Feng1 na1,
Jing Yang1 na1,
Wenwen Yang1,
Jie Chen2,3,
Min Jiang4 &
Xiang Zou ORCID: orcid.org/0000-0002-5875-12241
Biotechnology for Biofuels volume 11, Article number: 94 (2018) Cite this article
Polymalic acid (PMA) is a water-soluble biopolymer with many attractive properties for food and pharmaceutical applications mainly produced by the yeast-like fungus Aureobasidium pullulans. Acid hydrolysis of PMA, resulting in release of the monomer l-malic acid (MA), which is widely used in the food and chemical industry, is a competitive process for producing bio-based platform chemicals.
In this study, the production of PMA and MA from sucrose and sugarcane molasses by A. pullulans was studied in shake flasks and bioreactors. Comparative metabolome analysis of sucrose- and glucose-based fermentation identified 81 intracellular metabolites and demonstrated that pyruvate from the glycolysis pathway may be a key metabolite affecting PMA synthesis. In silico simulation of a genome-scale metabolic model (iZX637) further verified that pyruvate carboxylase (pyc) via the reductive tricarboxylic acid cycle strengthened carbon flux for PMA synthesis. Therefore, an engineered strain, FJ-PYC, was constructed by overexpressing the pyc gene, which increased the PMA titer by 15.1% compared with that from the wild-type strain in a 5-L stirred-tank fermentor. Sugarcane molasses can be used as an economical substrate without any pretreatment or nutrient supplementation. Using fed-batch fermentation of FJ-PYC, we obtained the highest PMA titers (81.5, 94.2 g/L of MA after hydrolysis) in 140 h with a corresponding MA yield of 0.62 g/g and productivity of 0.67 g/L h.
We showed that integrated metabolome- and genome-scale model analyses were an effective approach for engineering the metabolic node for PMA synthesis, and also developed an economical and green process for PMA and MA production from renewable biomass feedstocks.
Polymalic acid (PMA) is a water-soluble biopolymer composed of l-malic acid (MA) monomers and is mainly produced by the yeast-like fungus Aureobasidium pullulans [1]. PMA has free carboxyl groups, making it simple to perform chemical modifications and create various derivatives or carrier-linked pro-drugs. Due to its unique properties, including high water solubility, biocompatibility, and biodegradability, PMA has attracted an increasing attention as a drug carrier or biomaterial in the past few years and is expected to have applications in the preparation of various polymeric micelles, microparticles, nanoconjugates, and nanoparticles for drug delivery systems [2,3,4,5]. PMA also has the potential to be used as a nano-imaging agent for safe and noninvasive diagnosis in the clinical setting [6]. In addition, its monomer MA can be easily generated from PMA via acid hydrolysis. Like other dicarboxylic acids, MA is also an important organic acid and is regarded as a C4 platform chemical in the food and pharmaceutical industries, for which there is a strong global market of over 600,000 tons/year, with an annual growth rate of 4% [7, 8].
Inexpensive renewable feedstocks and media for fermentation, as well as high PMA titers with good productivity are also important constraints in developing industrial PMA fermentation processes. The previous studies have demonstrated fermentative production of PMA from low-cost substrates, such as sweet potato [9], corncob [10, 11], soy molasses [12], and wheat straw [13]. However, starchy and lignocellulosic feedstocks must first be subjected to thermochemical, dilute acid, or enzymatic treatments to release fermentable sugars; these limitations must be overcome to achieve an economically competitive process, including enhanced strain tolerance and fermentation performance in the presence of inhibitors released from dilute acid-pretreated lignocellulose [8].
In this study, we aimed to develop an economical and environmentally friendly bioprocess for PMA and MA production from sugarcane molasses as a feedstock. Sugarcane molasses is the main by-product of sugar production and contains ~ 50% reducing sugars, mainly sucrose and some glucose and fructose; in addition, sugarcane molasses can be used directly in fermentation without requiring any pretreatment [14, 15], and its price is ~ 40% lower than corn (starch and dextrose) based on the fermentable sugar content according to the current market prices. Therefore, sugarcane molasses is considered a relatively inexpensive and renewable biomass feedstocks for biorefinery applications due to its rich sugar content and cost-effectiveness. As omics analysis developing rapidly over the last decade, metabolome analysis has been employed to characterize intracellular metabolic states for understanding cell metabolism and improving the production of target metabolites [16]. Metabolomics has been a powerful tool for understanding the intracellular metabolism with broad range of applications in various fields, including medical science [17], synthetic biology [18], medicine [19], plant systems [20], and microbial systems [21]. In this study, we evaluated the performance of the strain Aureobasidium pullulans for the utilization of different sugars, including sucrose, fructose, and glucose contained in sugarcane molasses, and compared the metabolic processes of sucrose- and glucose-based fermentation through metabolome analysis. In silico simulation of a genome-scale model of A. pullulans was further verified, and the metabolic node containing pyruvate carboxylase via the reductive tricarboxylic acid (TCA) cycle was engineered to enhance PMA production. Finally, direct sugarcane molasses fermentation, without any pretreatment or nutrient supplementation, was developed for PMA production. This work will be beneficial for the development of an economical and green process for PMA and bio-based MA production from renewable biomass feedstocks.
Strains, media, and culture conditions
The strain A. pullulans CCTCC M2012223 was isolated by our laboratory and can be obtained from the China Center for Type Culture Collection (Wuhan, China). This strain was maintained on potato dextrose agar slants. The seed culture medium contained 60-g/L glucose, 2-g/L NH4NO3, 0.1-g/L KH2PO4, 0.1-g/L MgSO4, 0.1-g/L ZnSO4, 0.5-g/L KCl, and 20-g/L CaCO3. The seed culture was grown in a 500-mL shake flask containing 50-mL liquid medium and incubated at 25 °C on a rotary shaker (180 rpm) for 2 days. The fermentation medium contained 90-g/L sugar (glucose, sucrose, xylose, arabinose, or fructose), 2-g/L NH4NO3, 0.1-g/L KH2PO4, 0.1-g/L MgSO4, 0.1-g/L ZnSO4, 0.5-g/L KCl, and 20-g/L CaCO3. Escherichia coli DH5α cells were employed for routine DNA manipulations. E. coli was grown in LB medium (5 g/L yeast extract, 10 g/L tryptone, 10 g/L NaCl, pH 7.0) at 37 °C, and kanamycin or ampicillin (50 mg/L) was added when required. Agrobacterium tumefaciens AGL1 was grown on YEB medium (10-g/L yeast extract, 5-g/L tryptone, 5-g/L sucrose, 0.5-g/L MgSO4·7H2O, pH 7.0) at 28 °C, and kanamycin or carbenicillin (50 mg/L) was added when required.
Sampling, quenching, and extraction of intracellular metabolites
The cells of strain A. pullulans CCTCC M2012223 were collected after culturing for 48 and 72 h with glucose- and sucrose-based fermentation, respectively, and intracellular metabolism was quenched by immediately adding five volumes of prechilled 60% (v/v) methanol. After quenching at − 40 °C, cells were pelleted in a centrifuge (4000×g, 4 °C, 10 min). The pellets were washed with phosphate-buffered saline (pH 7.4), frozen in liquid nitrogen, and stored at − 80 °C. Next, 60 mg per sample with six biological replicates was ball-milled into a fine powder under frozen conditions with 1 mL of extraction mix consisting of deionized water (50%), methanol (50%), and the internal standards nonadecanoic acid (0.2 mg/mL) and isotope alanine (10 mM), and then lyophilized. The samples were derivatized by vortexing with 60 μL of 15-mg/mL methoxypyridine hydrochloride for 30 s and then incubating for 2 h at 37 °C. The samples were then trimethylsilylated by adding 60-μL bis(trimethylsilyl) trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane and incubating for 90 min at 37 °C. Subsequently, an extract containing all intracellular metabolites was collected via centrifugation at 12,000×g for 10 min at 4 °C [22].
Metabolite detection with gas chromatography–mass spectrometry (GC–MS)
GC–MS analysis was performed to detect the metabolites in the samples. Briefly, 1 μL sample was injected into an Agilent 7890A/5975C GC–MS system (Agilent Technologies, CA, USA) with a 20:1 split injection ratio. The system was equipped with an HP-5MS capillary column (5% phenyl methyl silox: 30 m × 250 μm i.d., 0.25 μm; Agilent J&W Scientific, Folsom, CA, USA). The inlet temperature was maintained at 280 °C, the interface temperature was 150 °C, and the ion source temperature was 250 °C. The GC temperature program was as follows: hold at 70 °C for 2 min, ramp at 10 °C/min to 300 °C, and a final hold at 300 °C for 5 min. Helium was used as carrier gas at a constant flow rate of 1 mL/min, with a total run time of 30 min. MS conditions were as follows: electron ionization (EI); full-scan mode (m/z 35–780); electron energy, 70 eV [23]. The chromatographic differences between sample groups were obvious, and the retention time was reproducible and stable, demonstrating the reliability of metabolomics analysis.
Data processing and statistical analysis
XCMS software was used to identify and quantify mass spectral peaks. Data preprocessing, including raw data filtering, peak detection, alignment, normalization, and identification, was performed automatically. Metabolites were identified by comparison of mass spectra with the National Institution of Standards and Technology (NIST), Wiley metabolome, Golm metabolome (GM), and in-house databases (Additional file 1: Table S1, red color) [24]. Six independent experiments were performed for each conclusion. Pattern recognition methods based on principal component analysis (PCA) and orthogonal-partial least squares-discriminant analysis (OPLS-DA) were then carried out using SIMCA-P V13.0 (Umetrics AB, Umea, Sweden). All variables were unit variance scaled before PCA and OPLS-DA. Hierarchical clustering analysis was carried out using R software (version 3.1.3) to visualize and group metabolite profiles. A list of variance metabolites that contributed mostly to the model was generated from the P values obtained using Student's t tests (P < 0.05) and variable importance in the projection values (> 1). In addition, the false discovery rate (FDR) significance criterion was performed with an FDR limit of 0.05 to avoid false-positive results in analyzing differential metabolite correlations. Metabolic pathways were constructed according to pathway analysis of the Metaboanalyst and KEGG metabolic databases [25, 26].
In silico analysis of a genome-scale metabolic model
The reconstructed genome-scale metabolic network (iZX637) of A. pullulans, containing 1347 reactions and 1133 metabolites, was used for constraint-based flux analyses [27] to determine the most effective route for maximizing PMA production and minimizing the influence of biomass accumulation. The synthesis of PMA is closely related to MA synthesis in A. pullulans. Therefore, three routes of MA synthesis, the oxidative branches of the TCA cycle, the reductive branches of the TCA cycle, and the glyoxylate shunt, were examined. In silico, constrained reactions of the oxidative branches of the TCA cycle contained oxoglutarate dehydrogenase, aconitate hydratase, fumarase, succinate dehydrogenase, and succinate-CoA ligase. Constrained reactions of the reductive branches of the TCA cycle contained malate dehydrogenase, and constrained reactions of the glyoxylate shunt contained fumarase, isocitrate lyase, and glyoxylate lyase. For in silico simulation, the reaction fluxes corresponding to one route of MA synthesis (the oxidative branches of the TCA cycle, the reductive branches of the TCA cycle, or the glyoxylate shunt) were constrained from 0 to 140 mmol/gDW/h (the reaction fluxes of other two routes of MA synthesis were set to 0 gDW/h), and the cell growth rate was constrained from 0 to 1 gDW/h. The specific PMA synthesis rate was then maximized as an objective function and the substrate uptake rates for glucose in the simulation were constrained by 10 mmol/gDW/h. The specific PMA synthesis rate at different flux values of the cell growth rate and route of MA synthesis were plotted on the Z-axis of the flux profile graph. In addition, because the reductive branches of the TCA cycle were assumed to be located only in the cytoplasm in silico, the flux of malate dehydrogenase located in the mitochondria was set to 0 mmol/gDW/h in simulation of three routes. The basic tools used for the model analysis were flux balance analysis (FBA) [28]. GLPK (freeware) (http://www.gnu.org/software/glpk/) was used as a linear programming solver. The COBRA Toolbox 2.0 [29] was used for calculation and analysis in the MATLAB environment.
Gene cloning and expression in the host strain
The DNA of A. pullulans was extracted using a TIANamp Yeast DNA Kit (TIANGEN, China) and was used as a template to amplify the pyruvate carboxylase (pyc, EC: 6.4.1.1) gene. First, one 3.748-kb pyc fragment was amplified using SmaI-pyc-S and MunI-pyc-A, as listed in Additional file 1: Table S1. Second, the pyc fragment obtained was double digested with SmaI and MunI and then ligated into plasmid pBARGPE1, which was also treated with SmaI and EcoRI (MunI isocaudarner) to generate the plasmid pBARGPE1-pyc, which contained a gpdA promoter (PgpdA) and a trpC terminator (TtrpC) flanking the pyc gene. The PgpdA-pyc-TtrpC cassette was amplified by primers PgpdA.pyc.TtrpC-S and PgpdA.pyc.TtrpC-A. The PgpdA-pyc-TtrpC cassette was cloned into the EcoRI site of the pK2-PtrpC-hyg-TtrpC plasmid using a One Step Cloning Kit (Vazyme, USA), forming the plasmid pK2-hyg-pyc for A. tumefaciens-mediated transformation (ATMT), as described in our previous study [30]. T-DNA insertion sites were analyzed using a Genome Walking Kit (TaKaRa, Japan) in ATMT-derived clones. Polymerase chain reaction (PCR) primers were used to amplify the T-DNA flanking sequence (Additional file 1: Table S1).
Batch and fed-batch fermentation in a 5-L stirred-tank fermentor
Batch fermentation kinetics were studied in a 5-L stirred-tank fermentor (Shanghai Baoxing Co., Ltd., China) containing 3.5 L of fermentation media (with glucose as carbon resource) or only diluted sugarcane molasses. The fermentation medium was inoculated with 350 mL of the seed culture grown in a shake flask for 48 h and operated at 25 °C with agitation and aeration at 400–700 rpm and 0.8 vvm, respectively. During fermentation, the agitation was controlled to maintain the dissolved oxygen level at greater than 20%. For sugarcane molasses fermentation, the crude sugarcane molasses (containing ~ 420 g/kg of total sugar and 14.29 ± 0.27 g/kg of total nitrogen) were diluted by water to ~ 90 g/L total sugar. For fed-batch fermentation, when the total sugar concentration was lower than 30 g/L, sugarcane molasses (approximately 270-g/L reducing sugars) was continuously added to maintain the total sugar concentration at approximately 10–20 g/L. Broth samples were collected periodically for the analysis of residual sugar, biomass, and PMA titers. All trials were performed in triplicate.
Quantitative reverse transcription (RT)-PCR
Total RNA was extracted using a Fungal RNA Kit (Omega, USA) and reverse transcribed into cDNA using reverse transcriptase (TIANGEN). The quantitative PCR assay was performed using the SYBR Green method (FastQuant RT Super Mix; TIANGEN). Primers for the pyc gene are shown in Additional file 1: Table S1. The gene encoding β-actin was used as the reference gene.
Biomass was determined by the dry cell weight method. Prior to measurement, excessive CaCO3 was eliminated from the broth by adding 1-M HCl. The cell suspension was centrifuged at 4000×g and then dried overnight at 105 °C [31]. PMA was analyzed by centrifuging the fermentation broth and the resulting supernatant was mixed with an equal volume of 2-M H2SO4 in an incubator at 85 °C for 8 h. The hydrolyzed PMA sample was analyzed by high-performance liquid chromatography (Agilent 1260, USA) using a Spursil C18-EP organic acid column at 40 °C and eluted with 5-mM H2SO4 at a rate of 0.6 mL/min to determine its malic acid content [32]. The glucose concentration was measured using a fully automatic residual sugar analyzer (Biology Institute of Shandong Academy of Sciences, China).
Evaluation of carbon sources in shake-flask culture
Sugarcane molasses is the main by-product of sugar production, and mainly comprises sucrose and some glucose and fructose. In this study, fermentation with different carbon sources in shake-flask culture was investigated (Fig. 1). Of the carbon sources used, sucrose and fructose showed superior PMA production of 28.30 ± 2.58 and 22.58 ± 1.08 g/L, respectively, which was higher than that (20.38 ± 2.32 g/L) from glucose as the carbon source. Moreover, five-carbon sugar (arabinose and xylose) fermentation was also superior to that with glucose for PMA production. In our previous study, a novel PMA-producing strain, A. pullulans YJ6-11, was also found to utilize xylose as a superior carbon source compared with glucose [33]. The fermentation kinetics of sucrose and glucose in shake-flask culture are shown in Fig. 2. A total of 33.91 ± 3.70 g/L PMA (~ 38.98 g/L MA after hydrolysis) were produced from ~ 90 g/L sucrose in 96 h via batch fermentation; this was increased by 40.7% compared with that of glucose (24.10 ± 3.30 g/L). These results showed that sucrose present in sugarcane molasses was the most suitable sugar for PMA biosynthesis. However, the underlying factors responsible for the differences between sucrose and glucose were still unclear.
Effects of carbon sources on cell growth and PMA production in shake-flask. Values are the means and standard deviations of three independent experiments. *P < 0.05, **P < 0.01
Batch fermentation of PMA production from glucose or sucrose in shake-flask culture. a Biomass and b PMA production. Glucose: line with blue circles; sucrose: line with red squares. The values shown represent the means of three independent experiments, and the error bars represent standard deviations of three values
Analysis of key metabolites and pathways associated with PMA biosynthesis
To explain the differences in metabolites of A. pullulans, the metabolic states of sucrose- and glucose-based fermentation were compared by metabolomics analysis. As shown in Additional file 2: Table S2, in total, 81 intracellular metabolites were identified and divided into eight categories. These groups mainly included organic acids, amino acids, and sugar metabolism that contained 21, 15, and 14 metabolites, respectively. Among these metabolites, compared with culture at 48 h, 13 and 3 differential metabolites were increased at 72 h in glucose and sucrose, respectively. 27 and 30 differentially abundant metabolites from glucose versus sucrose after culture for 48 and 72 h, respectively, were validated. Of these metabolites, both at 48 and 72 h, 16 in sucrose fermentation, including fructose, ribose, glycolic acid, oleic acid, and heptanoic acid, were higher than that of glucose (Additional files 3 and 4: Figure S1 and Table S3). These results indicated that the differences of metabolites were varied in the different sugar fermentation. Moreover, compared with glucose fermentation at 48 h, 13 differential metabolites were increased at 72 h. Notably, at 72 h, citric acid was increased by 1.84-fold, whereas other intermediate metabolites associated with the TCA cycle (such as pyruvic acid, succinic acid, and fumaric acid) did not differ significantly at 48 h (Additional file 4: Table S3). However, at 72 h, MA was decreased by 1.04-fold compared with that at 48 h, which was negative correlated with citric acid (Fig. 3). This may induce a decrease in PMA biosynthesis, because accumulation of citric acid resulted in carbon flux that was not efficiently channeled to the objective product. Pyruvate is a key node in central carbon metabolism and plays a major role in pathways related to organic acid synthesis [34]. The carbon flux can be channeled into different products through pyruvate, including succinate [35], lactate [36], and malate [37]. The reductive TCA cycle has been identified as the most efficient pathway for many products, such as succinate [38] and malate [37]. Thus, the accumulation of citric acid also indicated that metabolic flux could not effectively direct pyruvate to PMA synthesis or the high-efficiency PMA synthesis route in native metabolic regulation. Therefore, the enhanced carboxylation of pyruvate to oxaloacetate, catalyzed by pyruvate carboxylase, may be an important target for improving PMA synthesis.
Key metabolite changes in PMA biosynthesis based on comparative metabolome analysis. Suc sucrose, Glc glucose, Fru fructose, G6P glucose 6-phosphate, F6P fructose-6-phosphate, FBP fructose 1,6-bisphosphate, DHAP dihydroxyacetone phosphate, G3P glyceraldehyde 3-phosphate, 6PGC gluconate 6-phosphate, RU5P ribulose 5-phosphate, XU5P xylulose 5-phosphate, R5P ribose 5-phosphate, E4P erythrose 4-phosphate, S7P sedoheptulose 7-phosphate, PEP phosphoenolpyruvate, PYR pyruvate, OAA oxaloacetic acid, AcCoA acetyl-CoA, Cit citrate, Icit isocitrate, αKg α-oxoglutarate, SucCoA succinyl-CoaA, Succ succinate, Fum fumarate, Mal malate, Glx glyoxylate, Trp tryptophan, Tyr tyrosine, Phe phenylalanine, Gly glycine, Ser serine, Met methionine, Cys cysteine, Asn asparagine, Asp aspartic acid, Thr threonine, Ala alanine, Val valine, Ile isoleucine, Leu leucine, Glu glutamic acid, Arg arginine, Lys lysine, Gln glutamine, Pro proline. The content of metabolites in A. pullulans CTCC2012223 cultured in glucose for 48 h was used as control and defined as 1.00; the contents of the other samples were used to calculate the fold change with the following formula: fold change = log2(R), R = B/A, A is the control, and B is the treated sample
In addition, pyruvate is also a major component in pathways related to amino acid metabolism. Amino acids are crucial to microorganism metabolism and have roles in synthesis of proteins (e.g., leucine, threonine, and phenylalanine), regulation of gene expression (e.g., arginine and leucine), osmoregulation (e.g., citrulline and proline), control of enzyme activity (e.g., alanine and leucine), transamination (e.g., aspartate, alanine, and glutamate), and ammonia detoxification (e.g., citrulline, arginine, and glutamate) [39]. As shown in Fig. 3, amino acids present at higher levels, particularly leucine and proline, could be directly or indirectly transformed from pyruvate and α-oxoglutarate [40]. Most amino acids were observed at higher levels in sucrose than in glucose because of the abundant precursors in the glycolytic pathway and TCA cycle. The redox balance is maintained by modifying the distribution of the metabolic flux of pyruvate [41].
In addition, when sucrose was used as the carbon source, relative concentration of trehalose was significantly decreased 2.37- and 2.70-fold at 48 and 72 h, respectively, compared with that when glucose was used as the carbon source (Additional file 4: Table S3). Accumulation of trehalose in yeasts has been shown to be an important mechanism mediating tolerance against adverse stress conditions, and trehalose functions as a reserve carbohydrate that can be synthesized when the exogenous energy exceeds the cellular needs for growth and biosynthesis [42, 43]. These results suggested that glucose as a rapidly metabolizable carbon source can induce a regulatory response (e.g., osmotic stress) to restore excess exogenous energy, which would cause some of the carbon source to be used for other processes, not PMA synthesis, thereby decreasing PMA titers.
PMA is synthesized from MA, which is an intermediate in the TCA cycle in aerobic metabolism. Genome-scale flux sensitivity analyses were performed to determine the most effective route for maximizing PMA synthesis. Three metabolic routes involved in MA synthesis, including oxidative branches of the TCA cycle, reductive branches of the TCA cycle, and glyoxylate shunt, were simulated using the genome-scale metabolic model (iZX637). As shown in Fig. 4, the optimal PMA synthesis rate, using glucose or sucrose as the carbon source, reached 15 mmol/gDW/h (indicated by an arrow), increasing the reductive TCA malate synthesis rate to 15 mmol/gDW/h and the biomass growth rate to 0 mmol/gDW/h. When the carbon flux turned to oxidative branches of the TCA cycle and the glyoxylate shunt, the optimal PMA synthesis rates reached only as high as 12.89 mmol/gDW/h (sucrose as a carbon source: 12.58 mmol/gDW/h) and 14.67 mmol/gDW/h (sucrose as a carbon source: 14.33 mmol/gDW/h) with corresponding malate synthesis rates of 13 and 8 mmol/gD/h, respectively. In addition, the malate synthesis rate of the corresponding route constantly increased, resulting in a negative influence on the optimal PMA synthesis rate. When the malate synthesis rates of the reductive TCA route, oxidative TCA route, and glyoxylate shunt reached 120, 37, and 37 mmol/gDW/h, respectively, the PMA synthesis rate and biomass growth rate were reduced to 0 mmol/gDW/h (glucose or sucrose as a carbon source). Furthermore, the PMA synthesis rate decreased gradually as the biomass synthesis rate increased for all simulation results. These results showed that PMA and biomass synthesis showed a competitive relationship. However, in the reductive TCA route, biomass synthesis could be maintained with a relatively high PMA synthesis rate compared with the other two routes, regardless of whether glucose or sucrose was used as the carbon source. In our previous study, we found that high levels of the nitrogen source favored cell growth but decreased PMA synthesis [44]. Therefore, the efficiency of the PMA synthesis route should balance cell growth and PMA formation. In our previous study, in silico analysis of the carbon flux distribution and changes in PMA synthesis rates showed that, in the context of a high PMA synthesis rate, a large amount of carbon flux was transformed into pyruvate and channeled into the reductive TCA route by pyruvate carboxylase [27]. These in silico results showed that the reductive TCA route, as the optimal PMA synthesis route, showed a relatively high optimal PMA synthesis rate and mitigated the competitive relationship between PMA and biomass synthesis. In addition, the simulation results also showed that the reductive TCA route required a relatively high route flux (the reductive TCA malate synthesis rate to 15 mmol/gDW/h), yielding a high PMA synthesis rate (the optimal PMA synthesis rate to 15 mmol/gDW/h). Therefore, pyruvate carboxylase (pyc) in the reductive TCA route was selected as the target to be overexpressed to improve PMA yield and productivity.
Evaluation of malate synthesis route for PMA production based on genome-scale metabolic simulation. a Schematic of simulation analysis in different malate synthesis route. b X- and Y-axes show malate synthesis route flux (mmol/gDW/h) and PMA production rate (mmol/gDW/h), respectively. The Z-axis represents the cell growth rate (gDW/h). The color gradient represents the value according to the scale shown in the box
Moreover, according to the simulation result from sucrose and glucose as carbon sources, sucrose was not dominant compared with glucose as a carbon source for the optimal PMA synthesis rate and cell growth by A. pullulans strains in the conversion efficiency of metabolic pathways. This could be explained by the observations that the reaction of sucrose hydrolysis exhibited no energy consumption or carbon loss in silico.
Overexpression of the pyc gene in A. pullulans
To evaluate the effects of the pyc gene on PMA biosynthesis, the endogenous pyc gene was amplified using the genomic DNA of A. pullulans as a template (Additional file 3: Figure S2). The PgpdA promoter of Aspergillus nidulans, which directs the constitutively high expression of the glyceraldehyde-3-phosphate dehydrogenase (gpd) gene, can be used to induce high levels of constitutive expression of genes of interest or marker genes and to drive the overexpression of various genes in fungi [45,46,47]. Therefore, the PgpdA promoter of A. nidulans was used to constitute the pyc cassette by cloning into pBARGPE1 (Additional file 3: Figure S3). In our previous study, we developed a simple and efficient system for genetic transformation of A. pullulans using A. tumefaciens [30]. Therefore, A. tumefaciens carrying the pK2-hyg-pyc binary plasmid was used to transform A. pullulans. Because ATMT-mediated integration is random, five different ATMT-derived clones were tested for PMA production in shake-flask culture. The PMA titers of five clones compared with the wild-type strain were improved from 3.5 to 7.1% (Additional file 3: Figure S4). The differences among mutants were due to random integration, causing interruption of gene function by insertion of T-DNA, and were not caused by intrinsic factors [30]. This result indicated that the increased PMA titer was mainly dependent on pyc gene overexpression. Thus, we selected the highest PMA producer (E10 strain; as called FJ-PYC) for further analysis of the sequences flanking the T-DNA in the genome. As shown in Additional file 3: Figure S5, the hygromycin B resistance cassette and overexpression pyc cassette were inserted into the noncoding regions of g4940.t1 and g4941.t1 on the A. pullulans genome. The insertions did not disrupt the g4940.t1 and g4941.t1 open reading frames. Furthermore, we found that the expression level of the pyc gene in strain E-10 was increased by 9.5-fold compared with that in the wild-type strain (Fig. 5). These results also revealed that the promoter PgpdA was an effective tool for regulating gene expression in A. pullulans.
Expression level of the pyc gene in the engineered strain E10. Each column was calculated with three parallel experiments. ***P < 0.001 versus the wild-type strain
Subsequently, the engineered strain E10 was tested with glucose as the carbon source in a 5-L stirred-tank fermentor. As shown in Fig. 6, about 36.2 g/L of PMA was produced via batch fermentation, with a PMA productivity of 0.53 g/L h, which was increased by 15.1 and 12.7% compared with the control, respectively. This result indicated that overexpression of the pyc gene could improve the production of PMA, which was consistent with the results of in silico simulation showing that strengthening of the reductive TCA route resulted in increased carbon flux toward PMA synthesis.
Batch fermentation of PMA production with the engineered strain E10 in a stirred-tank fermentor. All the data were derived from two independent experiments
Batch and fed-batch fermentation with sugarcane molasses
Sugarcane molasses is a low-cost by-product of sugar production and mainly contains sucrose, with some fructose and glucose. To develop an economical fermentation process, sugarcane molasses without any pretreatment or nutrient supplementation was employed for PMA production by FJ-PYC strain in a 5-L stirred-tank fermentor. As shown in Fig. 7a, during the early fermentation stage, sucrose was rapidly consumed and transformed into glucose and fructose, and glucose was then used for cell growth and PMA synthesis compared with fructose. This phenomenon, called glucose-mediated carbon catabolite repression (CCR), is widely observed in bacteria and yeasts [48,49,50]. About 31.5-g/L PMA (36.5-g/L MA after hydrolysis) was produced from ~ 95-g/L mixed sugar (61.8-g/L sucrose, 18.5-g/L glucose, and 15.3-g/L fructose from sugarcane molasses) in 60 h via batch fermentation, with an MA yield of 0.44 g/g. Moreover, without adding any other media, the productivity of PMA (0.53 g/L h) was the same as that from glucose fermentation. In fed-batch fermentation (Fig. 7b), a high final PMA titer of 81.5 g/L (94.2-g/L MA after hydrolysis) was achieved in 140 h with a corresponding MA yield of 0.62 g/g and productivity of 0.67 g/L h. As shown in Table 1, compared with other renewable feedstocks, PMA fermentation from sugarcane molasses by strain FJ-PYC resulted in the highest titer and productivity. Nevertheless, the titer and productivity could be further increased by continuous fed operation, because the PMA synthesis rate maintained a high speed. In addition, the fed-batch fermentation with FJ-PYC resulted in an overall MA yield of 0.62 g/g from sugarcane molasses without nitrogen supplementation, which was the same as the highest yield from sugarcane juice [49]. However, sugarcane juice contained only ~ 15% reducing sugars and relatively fewer inhibitors, requiring it to be condensed for high-sugar PMA fermentation and dramatically decreasing its economic efficiency. These results indicated that the strain FJ-PYC exhibited extremely high adaptation and tolerance against sugarcane molasses.
Fermentation kinetics of PMA production from sugarcane molasses in a stirred-tank fermentor. a Batch fermentation; b fed-batch fermentation. PMA production is reported as malic acid content after acid hydrolysis of PMA. All the data were derived from two independent experiments
Table 1 PMA production from various biomass substrates by different strains of A. pullulans
In this study, different carbon sources were evaluated, and sugarcane molasses was assessed as a potential feedstock for economic PMA and MA production. Among the different carbon sources examined in this study, sucrose was found to be the optimal carbon source for PMA biosynthesis. Metabolomics analysis of sucrose- and glucose-based fermentation identified a total of 81 intracellular metabolites and showed that pyruvate from the glycolysis pathway may be a key metabolite affecting PMA synthesis. In silico analysis of a genome-scale metabolic model (iZX637) verified that the pyc gene via the reductive TCA cycle was a target affecting PMA synthesis. Consistent with this, overexpression of the pyc gene in A. pullulans strain FJ-PYC increased the PMA titer by 15.1% compared with the control.
Moreover, sugarcane molasses could be directly utilized by this recombinant strain without any pretreatment or nutrient supplementation, producing 31.5-g/L PMA (36.5-g/L MA) with a high-level PMA productivity of 0.53 g/L h in batch fermentation. In fed-batch fermentation, compared with the other renewable feedstock, the highest final PMA titer of 81.5 g/L (94.2-g/L MA after hydrolysis) was achieved in 140 h, with a corresponding MA yield of 0.62 g/g and productivity of 0.67 g/L h. This study demonstrated the great potential of sugarcane molasses for the economical production of PMA and MA on the industrial scale.
PMA:
polymalic acid
l-malic acid
pyc :
pyruvate carboxylase gene
TCA:
tricarboxylicacid
GEM:
genome scale metabolic model
high-performance liquid chromatography
BSTFA:
bis(trimethylsilyl) trifluoroacetamide
GC–MS:
gas chromatography–mass spectrometry
EI:
electron ionization
NIST:
National Institution of Standards and Technology
GM:
Golm Metabolome
PCA:
OPLS-DA:
orthogonal-partial least squares-discriminant analysis
FDR:
false discovery rate
PgpdA :
gpdA promoter
TtrpC :
trpC terminator
ATMT:
A. tumefaciens-mediated transformation
PCR:
gpd :
glyceraldehyde-3-phosphate dehydrogenase gene
CCR:
carbon catabolite repression
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FJ and ZX designed and carried out the model simulations and fermentations. YJ and JC evaluated effects of carbon sources on cell growth and PMA production different carbon sources and analyzed the data of metabolome. FJ and YJ drafted the manuscript. YWW assisted in shake-flask fermentation. ZX and MJ supervised the project and revised the manuscript. All authors read and approved the final manuscript.
This study was supported in part by Grants from the National Natural Science Foundation of China (Grant No. 31571816), National High Technology Research and Development Program of China (863 Program) (2015AA021005), Chongqing Social and People's Livelihood Guarantee Special Program (cstc2016shmszx80075), and State Key Laboratory of Materials-Oriented Chemical Engineering (Nanjing Tech University), China (KL15-10).
Availability of supporting data
The GEM iZX637 during the current study is available in the PubMed repository https://doi.org/10.1016/j.gene.2016.12.034.The authors declare that all other data supporting the findings of this study are available within the article its Additional files.
The authors consent for publication.
Publisher's Note
Jun Feng and Jing Yang contributed equally to this work
College of Pharmaceutical Sciences, Chongqing Engineering Research Center for Pharmaceutical Process and Quality Control, Southwest University, 2 Tian Sheng Road, Beibei, Chongqing, 400715, People's Republic of China
Jun Feng, Jing Yang, Wenwen Yang & Xiang Zou
Wuhan Sunhy Biology Co., Ltd, Wuhan, 430074, People's Republic of China
Jie Chen
School of Chemical Engineering & Pharmacy, Wuhan Institute of Technology, Wuhan, 430205, People's Republic of China
State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, 211816, People's Republic of China
Min Jiang
Jun Feng
Wenwen Yang
Xiang Zou
Correspondence to Xiang Zou.
Additional file 1: Table S1.
List of strains, plasmids, and primers.
Identified metabolites by comparison of mass spectra.
Additional file 3: Figure S1.
The statistical numbers of differential metabolites between groups. Red bars represent the numbers of relative concentration increased metabolites and blue bars represent the numbers of relative concentration decreased metabolites. Figure S2. PCR amplification of the endogenous pyc gene using the genomic DNA of A. pullulans. Figure S3. Construction of the pyc cassette by cloning into the plasmid pBARGPE1 with the PgpdA promoter. Figure S4. Batch fermentation of different ATMT-derived clones with glucose as carbon source in shake flask. Figure S5. Analysis of sequences flanking to T-DNA in genome of the strain E10.
The changes of differential metabolites in glucose- and sucrose-based fermentation.
Feng, J., Yang, J., Yang, W. et al. Metabolome- and genome-scale model analyses for engineering of Aureobasidium pullulans to enhance polymalic acid and malic acid production from sugarcane molasses. Biotechnol Biofuels 11, 94 (2018). https://doi.org/10.1186/s13068-018-1099-7
Aureobasidium pullulans
Pyruvate carboxylase


text:
Frankenstein ou o Prometeu Moderno (Frankenstein: or the Modern Prometheus, no original em inglês), mais conhecido simplesmente por Frankenstein, é um romance de terror gótico com inspirações do movimento romântico, de autoria de Mary Shelley, escritora britânica nascida em Londres. É considerada a primeira obra de ficção científica da história. O romance relata a história de Victor Frankenstein, um estudante de ciências naturais que constrói um monstro em seu laboratório. Mary Shelley escreveu a história quando tinha apenas 19 anos, entre 1816 e 1817, e a obra foi primeiramente publicada em 1818, sem crédito para  a autora na primeira edição. Atualmente costuma-se considerar a versão revisada da terceira edição do livro, publicada em 1831, como a definitiva.

O romance obteve grande sucesso e gerou todo um novo gênero de horror, tendo grande influência na literatura e cultura popular ocidental. O aclamado autor de literatura de terror Stephen King considerou Frankenstein um dos três grandes clássicos do gênero, sendo os outros dois Drácula e Strange Case of Dr Jekyll and Mr Hyde. A obra está em domínio público e está disponível gratuitamente na Internet em língua inglesa.

Enredo
O romance é narrado através de cartas escritas pelo capitão Robert Walton para sua irmã enquanto ele está ao comando de uma expedição náutica que busca achar uma passagem para o Pólo Norte. O navio sob o comando do capitão Walton fica preso quando o mar se congela, e a tripulação avista a criatura de Victor Frankenstein viajando em um trenó puxado por cães. A seguir o mar se agita, liberando o navio, e em uma balsa de gelo avistam o moribundo doutor Victor Frankenstein. Ao ser recolhido, Frankenstein passa a narrar sua história ao capitão Walton, que a reproduz nas cartas a irmã. A história do capitão Walton é chamada de narrativa moldura (às vezes também narrativa quadro), onde uma história contém outra.

Victor Frankenstein começa contando de sua infância em Genebra como filho de um aristocrata suíço e adolescência como estudante autodidata dedicado e talentoso. Neste ponto ele apresenta Elizabeth, criada como irmã adotiva, e Henry Clerval, seu amigo para a vida toda. Frankenstein interessa-se pelas ciências naturais, e acaba estudando livros de mestres alquimistas, especialmente Cornélio Agripa, Paracelso e Albertus Magnus até os 17 anos de idade, quando seus pais enviam-no para estudar na Universidade de Ingolstadt, na Alemanha. Porém, antes da partida sua mãe vem a falecer.

Ao chegar em Ingolstadt o jovem Victor procura seus futuros mestres, que condenam fortemente o tempo de estudo dedicado aos mestres alquimistas, e apresentam-lhe as modernas ciências naturais. Empenhado em descobrir os mistérios da criação, Victor estuda febrilmente e acaba encontrando o segredo da geração da vida, o qual se recusa a detalhar ao seu interlocutor, o capitão Walton.

Frankenstein então dedica-se a criar um ser humano gigantesco, sacrificando o contato com a família e a própria saúde, e após dois anos obtém sucesso. Porém, Victor enoja-se com sua criação, e abandona-a, fugindo. É encontrado por seu amigo Clerval, que viera a Ingolstadt estudar. Exausto, sucumbe à febre, sendo cuidado por seu amigo pelos meses seguintes, até seu restabelecimento.

Victor Frankenstein recebe uma carta de seu pai relatando o assassinato de William, o seu irmão mais novo, e pedindo a sua volta. Ao chegar em Genebra, é informado que Justine, uma criada muito querida da casa dos Frankenstein, é acusada do crime, sendo encontrada com ela a jóia que o menino levava antes de desaparecer, e que não estava junto ao cadáver. Mesmo assim Victor está convencido de que Justine é inocente, e o verdadeiro culpado é a sua criatura. Porém as evidências contra ela são fortes e Justine é condenada a morte e executada pelo crime. Frankenstein passa a se sentir culpado por ter criado o monstro, e o segredo e a culpa passaram a lhe torturar.

Lutando contra o desespero, o doutor Frankenstein resolve escalar o Monte Branco. Durante a subida, é encontrado por sua criatura, que é surpreendentemente articulada e eloquente. O monstro conta sua história, narrando como fugiu do laboratório de Frankenstein para uma floresta próxima, onde aprendeu a comer frutas e vegetais, e a usar o fogo. Porém, ao encontrar seres humanos era sempre escorraçado e agredido, então eventualmente esconde-se no depósito de lenha anexo a uma cabana. Lá, observa através de frestas na parede a vida de uma família pobre de ex-nobres, afeiçoando-se a eles e ajudando-os em segredo. A família consistia de um pai cego e um casal de irmãos. Aprende a língua e a escrita  espionando as aulas que davam à noiva árabe do irmão, e encontra livros onde aprende sobre a vida e a virtude. Após longo tempo toma coragem para se apresentar a família, e consegue conversar com o pai cego, mas quando os filhos chegam e o veem junto ao pai também escorraçam o monstro, e fogem para sempre da cabana. A criatura torna-se amargurada e resolve procurar seu criador, cujo diário descobrira no bolso do casaco que levou do laboratório na noite da fuga. Durante a travessia é sempre agredido pelos humanos.

Ao chegar em Genebra encontra o irmão mais novo de Victor, William, e assassina-o, incriminando depois Justine. Ao terminar sua história, o monstro exige a promessa de que Frankenstein construa uma fêmea para ele, prometendo por sua vez deixar a humanidade em paz e ir viver com a sua noiva nas selvas sul-americanas. Caso o cientista se recusasse, o monstro promete fazê-lo passar por tormentos inimagináveis. Extremamente contrariado, Frankenstein concorda, e ao voltar para Genebra torna-se noivo de Elizabeth, partindo com Clerval para a Inglaterra, a fim de cumprir a sua promessa.

Na Grã-Bretanha, Frankenstein, após passar por Londres, onde havia os mais recentes avanços das ciências naturais e algumas cidades da Escócia vai para uma das ilhas do arquipélago das Orkneys, onde começa a construir a fêmea. Entretanto, ele muda de ideia, temendo criar uma raça de monstros que pudesse se virar não só contra ele, mas contra toda a raça humana. Após fazer várias considerações, Frankenstein decide que ele tem que sofrer as conseqüências por seus atos e não a humanidade, destruindo a criatura incompleta. O monstro acompanha o ato, e jura se vingar. Em seguida assassina Clerval. Frankenstein chega a ser acusado do crime, mas é inocentado por possuir um forte álibi. Seu pai vem lhe buscar e ambos retornam à Suíça.

Mesmo devastado pela culpa e pela tristeza, Victor casa-se com Elizabeth e no mesmo dia sai para viajar em lua de mel. Na noite de núpcias, fica vigiando a casa, temendo um ataque da criatura contra ele, mas o monstro ataca Elizabeth e a estrangula. Victor volta a Genebra, e com a notícia da morte de Elizabeth, seu pai adoece e morre em seguida. Jurando vingança, o criador passa a perseguir a criatura, que o leva através de uma longa caçada em direção ao norte, prosseguindo pelos mares congelados, onde eventualmente são avistados pelo capitão Walton e sua tripulação.

O navio dos exploradores fica preso no gelo, e Victor, já bastante doente, acaba morrendo. O capitão Walton então surpreende a criatura na cabine, no leito de morte de Frankenstein, pranteando seu criador. Ela diz para Walton que não havia mais o que temer pois seus crimes terminaram com a morte de Frankestein, prometendo ir ao extremo Norte e lá ela cometeria seu suicídio, trazendo paz aos humanos.

Origens

Em 1815 o Monte Tambora na ilha de Sumbawa, na atual Indonésia, entrou em erupção. Como consequência, um milhão e meio de toneladas de poeira foram lançadas na atmosfera, bloqueando a luz solar, deixando o ano de 1816 sem verão no hemisfério norte.

Neste ano, Mary Shelley, então com 19 anos e ainda com o nome de solteira Mary Wollstonecraft Godwin, e seu futuro marido, Percy Bysshe Shelley, foram passar o verão a beira do Lago Léman, onde também se encontrava o amigo e escritor Lord Byron. Forçados a ficar confinados por vários dias em ambiente fechado pelo clima hostil anormal para a época e local, os três e mais outro hóspede, o também escritor John Polidori, passavam o tempo lendo uns para os outros historias de horror, principalmente histórias de fantasmas alemãs traduzidas para o francês.

Eventualmente Lord Byron propôs que os quatro escrevessem, cada um, uma história de fantasmas.
Byron escreveu um conto que usaria em parte mais tarde na conclusão de seu poema Mazzepa. Inspirado por outro fragmento de história de Byron desta época, Polidori mais tarde escreveria o romance "O Vampiro", que seria a primeira história ocidental contendo o vampiro como conhecemos hoje, e que décadas depois inspiraria Bram Stoker no seu Drácula. Porém, passados vários dias, Mary Shelley ainda não conseguira criar uma história. Eventualmente ela veio a ter uma visão sobre um estudante dando vida a uma criatura. Essa visão tornou-se a base da história de Frankenstein, a qual Mary Shelley veio a desenvolver em um romance, encorajada pelo seu futuro marido.

Desta forma, é curioso notar que o Frankenstein e o Vampiro vieram a ter sua gênese literária na mesma ocasião.

Shelley relatou sua versão da gênese da história no prefácio à terceira edição de seu romance.

O nome da criatura
Embora a cultura popular tenha associado o nome Frankenstein à criatura, esta não é nomeada por Mary Shelley. Ela é referida como "criatura", "monstro", "demônio", "desgraçado" por sua criadora. Após o lançamento do filme Frankenstein em 1931 o público passou a chamar assim a criatura. Isso foi adotado mais tarde em outros filmes. Alguns argumentam que o monstro é, de certa forma, um "filho" de Victor, e portanto pode ser chamado pelo mesmo sobrenome.

Frankenstein é o antigo nome de uma antiga cidade na Silésia, local de origem da família Frankenstein. Mary Shelley teria conhecido um membro desta família, o que possivelmente influenciou sua criação.

Amarelo
Ao contrário da forma como se tornou conhecida no cinema, a criatura de Frankenstein não era verde e sim amarela, como a própria autora o descreve no capítulo 5 da obra: "(...) Sua pele amarela mal cobria o relevo dos músculos e das artérias que jaziam por baixo; seus cabelos eram corrido e de um negro lustoso; seus dentes eram alvos como pérolas. Todas essas exuberâncias, porém, não formavam senão um contraste horrível com seus olhos desmaiados, quase da mesma cor acinzentada das órbitas onde se cravavam, e com a pele encarquilhada e os lábios negros e retos. (...)"

Edições

Mary Shelley completou o romance em 1817 e Frankenstein ou o moderno Prometeu foi publicado em 1 de janeiro  de 1818 por uma pequena editora de Londres, a Lackington, Hughes, Harding, Mavor & Jones, após ter sido rejeitada por duas outras editoras. A publicação não continha o nome da autora, somente um prefácio escrito por Percy Bysshe Shelley, seu noivo, e uma dedicatória a William Godwin, seu pai. A primeira edição foi feita em três volumes e teve impressas somente 500 cópias.

Apesar das críticas desfavoráveis, a edição teve um sucesso de público quase imediato. Ficou bastante conhecida, principalmente através de adaptações para o teatro, e a obra foi traduzida para o francês.

Em 2021,um exemplar da primeira edição do livro quebrou o recorde mundial para uma obra impressa escrita por uma mulher, ao ser leiloada por 1,17 milhão de dólares.

A segunda edição de Frankenstein foi publicada em 11 de agosto de 1823 em dois volumes, desta vez com o crédito como autora para Mary Shelley.

Em 31 de outubro de 1831 a editora Henry Colburn & Richard Bentley lançou a primeira edição popular em um volume. Esta edição foi significativamente revisada por Mary Shelley, e continha um novo e longo prefácio escrito por ela, relatando a gênese da história. Esta edição é a mais conhecida e mais usada como base para traduções.

Temas
Frankenstein aborda diversos temas ao longo do texto, sendo o mais gritante a relação de criatura e criador, com óbvias implicações religiosas. Uma influência notável na obra é o poema Paraíso Perdido de John Milton, que aborda a criação do homem e sua subseqüente queda. A influência torna-se explícita tanto através da epígrafe que cita três versos do poema, quanto aparecendo diretamente em Frankenstein: é um dos livros que a criatura lê.

A queda, ou a ruína, está bastante presente no livro de Shelley, que traça a destruição física e moral de Victor Frankenstein, e é aludida não só nas citações de Paraíso Perdido, como no próprio título da obra: O Moderno Prometeu. Prometeu é um personagem da mitologia grega, um titã que, ao roubar o segredo do fogo, o qual era reservado aos deuses, para doá-lo a humanidade, é severamente punido por Zeus. O paralelo com a trajetória de Victor Frankenstein é direto, e o livro deixa claro que o segredo da criação da vida a partir de matéria inanimada é de natureza divina.

O poder exercido pela humanidade sobre a Natureza através da ciência e da tecnologia é outro tema principal da obra, e encaixa-se no espírito da época, o estágio inicial da Revolução Industrial.

Outros temas são abordados com menos ênfase. A amizade verdadeira é tratada, com o Capitão Walton desejando tornar-se amigo de Victor, e Victor elaborando sobre ela ao se referir a sua amizade com Clerval.

Preconceito, ingratidão e injustiça também estão presentes. A criatura é sempre julgada por sua aparência, e agredida antes de ter uma chance de se defender. Em um episódio, o monstro salva uma garotinha inconsciente e, ao tentar devolvê-la para seu pai, é baleado e acusado de tê-la agredido. A inveja também aparece, ao subverter os bons sentimentos iniciais do monstro.

A expressão do sublime através da grandiosidade da Natureza é um tema caro ao Romantismo, e aparece em Frankenstein nas descrições das grandes planícies de gelo e das paisagens da Europa.

Por fim, a inevitabilidade do destino, tema muito desenvolvido na literatura clássica, é constantemente aludida ao longo do romance, que é uma obra que se presta a múltiplas interpretações e leituras.

Aparência
Um dos aspectos a serem analisados no livro é a questão da aparência. No livro, Victor Frankenstein jamais se inteirou do paradeiro de sua criação, tendo o primeiro contato somente após a iniciativa por parte da criatura, que tentava dar fim a sua solidão causada por sua figura apavorante. O trecho do primeiro encontro após o abandono da criatura demonstra a repulsa e desprezo pela aparência do monstro:Sua estatura, à medida que se aproximava, parecia exceder o normal. Senti-me perturbado. Uma névoa passou-me pelos olhos, e fui acometido de vertigem, mas não tardou que o sopro gelado das montanhas me refizesse. Tão logo a forma chegou à distância de ser distinguida, percebi — oh! visão odiosa e aterradora! — que era a aberração humana que eu criara. Impelido pela cólera e o horror, dispus-me, assim que estivesse ao meu alcance, a empenhar-me em luta mortal com ele. O demônio aproximou-se, ofegante. Havia em sua expressão um misto de amargura, malignidade e desdém, que o tornava ainda mais hediondo à contemplação humana. Não me detive, porém, a observá-lo. A surpresa tinha-me, a princípio, embargado a fala, e quando a recobrei foi para derramar sobre ele a torrente de meu ódio e repulsa.A relação conturbada entre a criatura e o criador gira em torno da busca por vingança. O pavor gerado pela aparência do monstro leva a seu abandono, que, por vez, leva ao desejo de compensar a falta de afeto e consideração através do derramamento de sangue.

A abordagem romântica tem o horrível e a repulsa como característica. O horror como fonte de apreciação da beleza no gênero romântico alterou o padrão de estética da época. Dessa forma, o horror passou a integrar o entendimento do belo. A beleza do horrível reflete-se na criatura de Frankenstein, que concentra características desarmônicas e repulsivas.

A dinâmica central da relação opressor e oprimido origina-se na família, sendo depois encaminhada para a sociedade. A tentativa do monstro em adquirir conhecimento não foi suficiente para possibilitar seu convívio social. Quando o cego De Lacey conversa com a criatura, não há qualquer atitude que demonstre desconforto ou repulsa por parte do ancião. Em um dos trechos do diálogo entre os dois personagens, o "demônio" reflete sobre o preconceito:Mas infelizmente têm preconceitos contra mim. Não que eu seja de má natureza. Minha vida até aqui tem sido inofensiva e, até certo ponto, benéfica. Mas um preconceito fatal lhes obscurece os olhos e, onde seria de esperar que eles vissem um amigo solidário e sensível, nada mais lhes aparece do que... um monstro, na acepção da palavra.A percepção da criatura sobre sua própria existência é fortemente influenciada pelo tratamento recebido desde sua criação. Percebe-se, então, como vilão, limitado à sua condição marginal. A sua aparência repugnante leva ao indagamento "Devo eu ser visto como o único criminoso, quando toda a humanidade pecou contra mim?". Começa a perceber que pouco importava a maneira como se portava perante os demais seres humanos, se com compaixão ou violência, pois nenhum benefício lograria de sua bondade, posta em segundo plano devido à sua aparência grotesca.

Adaptações
O romance foi primeiramente adaptado para o teatro, e posteriormente para um grande número de mídias, incluindo rádio, televisão e cinema, além de quadrinhos.

A primeira adaptação para o cinema foi feita pelos Edison Studios em 1910. Foi produzida por Thomas Edison e trazia Charles Ogle no papel da criatura. Uma das mais famosas transposições do romance para as telas é a realizada no filme Frankenstein (1931) pela Universal Pictures, dirigida por James Whale, com Boris Karloff como o Monstro. Esta adaptação deu a aparência mais conhecida do monstro, com uma cabeça chata, eletrodos no pescoço e movimentos pesados e desajeitados (apesar do livro descrever a criatura como extremamente ágil). Este filme tornou-se um clássico do cinema.

Um grande número de continuações seguiram-se, mas desta vez divergindo bastante da história narrada no romance. Em 1943 o personagem foi vivido por Bela Lugosi em Frankenstein Encontra o Lobisomem. Já em 1969 foi a vez de Peter Cushing estrelar a versão do diretor Terence Fisher que levou o título de Frankenstein tem que ser Destruído. Na década de 1980 o personagem voltaria em dois filmes: Frankenstein do diretor James Ormerod e Gothic de Ken Russell.

Em 1994 foi lançada uma adaptação cinematográfica dirigida por Kenneth Branagh de nome Mary Shelley's Frankenstein, com o próprio Branagh no papel de Victor Frankenstein, Robert De Niro como a criatura e Helena Bonham Carter como Elizabeth. Esta versão foi a que mais se aproximou da versão original, tendo a história, tempo e características idênticas ao livro e mínimas alterações. Em 2004 a criatura apareceu no filme Van Helsing, dirigido por Stephen Sommers.

As representações do Monstro e sua história têm variado bastante, de uma simples máquina de matar sem capacidade de reflexão a uma criatura trágica e plenamente articulada, o que seria mais próximo do retratado no livro.

O romance Frankenstein ainda serviu como inspiração para o filme Edward Mãos de Tesoura (1990), de Tim Burton o qual inclui a participação de Johnny Depp como Edward.

Em Novembro de 2015 uma nova versão foi lançada intitulada como Victor Frankenstein, com James McAvoy e Daniel Radcliffe como protagonistas.

Em 1974, Mel Brooks, lança O Jovem Frankenstein (Young Frankestein) filme de comédia que faz paródia com a história. No filme, Gene Wilder é o neto de Victor Frankestein, e cria o monstro (Peter Boyle).

Em 2018, a escola de samba Beija-Flor apresentou o enredo "Monstro é aquele que não sabe amar. Os filhos abandonados da pátria que os pariu" que foi baseado no romance que estava completando 200 anos.

Ligações externas

 Frankenstein: A New Reality!
 Texto integral da obra no original em inglês na Online Literature Library
 Frankenstein e o Espectro do Desejo - Richard Miskolci

Livros de 1818
Livros do Reino Unido
Livros de horror

Romances epistolares
Literatura gótica
Livros de ficção científica
Romances de ficção científica da década de 1810
Obras de Mary Shelley
Livros censurados
!Mais Teoria da História na Wiki (Mulheres)
!Mais Teoria da História na Wiki (Wikiconcurso de edição)

redpajama_set_name: RedPajamaWikipedia


My Thoughts on Turning 19.
It's crazy to think that when I started this blog I was sixteen about to turn seventeen. Here I am, spending yet another year on Porcelain Princess. Although 19 is not exactly a "landmark' birthday, I intend on filling this year with productivity and positivity.
Making this year important, I'm choosing 3 topics for this years resolutions. The first one has to do with school. I'm starting college in about a week, so I would like to push myself to get out of my comfort zone. I'm attending a conservatory that offers so many opportunities in the entertainment business and it would be silly of me to hold myself back. I'm going to challenge myself to get out of my comfort zone when meeting new people, being the first to sign up for things, etc.
My next resolution has to do with actual relationships. I want to continue to grow and build healthy relationships with the people in my life. Along with the previous goal, I would like to step outside of my bubble and make new friends. I love the friends that I have, but I've been in the same mix for the past four years and it'd be nice to meet someone new.
My last resolution has to do with new opportunities and importance. Not only do I want to put a lot of my focus into relationships and my career, I would like to put more effort into the blog. This has been a major creative outlet for me for the past two years and I would to really work hard to improve the quality AND quantity of the posts. I want to post on a schedule, have a strong team, and start a little community here on the page!
19 really doesn't speak to me the way 17 (or 18) did. 19, to me, is cross between 18 part two or "I'm about to turn 20." I was actually talking to a teacher I had a few years ago and I didn't even say that I was 19. I said "Yeah, I'm almost 20." 19 feels like a crossroads, not an actual year. It feels confusing and pointless. My goal is to work so hard that I prove myself wrong. Maybe 19 is a useful year. That's what I would like it to become. I want 19 to become a landmark year, just like the others.
You're a Cute Little Strawberry!
Previous Out with the Old. In with the New.


Adding a grenade launcher to a G36 replica has always been an expensive proposition, as previously, one of the only proper AG36 launchers available was made by Star, and cost about $300. S&T has manufactured an affordable AG36, complete with the proper handguard and flip up leaf sight for about half that price, and today, I'll be reviewing it! For my testing purposes, I'll have it installed on a WE G36C, with extended inner and outer barrels. Keep reading for more info on this gorgeous 40mm launcher!
I obtained this launcher through Evike, who has it available HERE, priced at $160.00. This means it is eligible for Evike's free shipping through UPS Ground, which got the gun to me in two days. This launcher includes everything that you need to mount it up on your G36 model, however some models may require slight modification to fit properly.
The AG36 is a 40mm grenade launcher, and this specific model is designed specifically for G36 replicas. The launcher is hard mounted to a modified handguard that serves as the mounting system to secure it to your gun. S&T also includes a side mounted flip up leaf sight assembly which is a fairly faithful replica of the real steel sight assembly. It uses a unique side opening breach assembly which allows for easy loading of your choice of 40mm shell.
S&T packed the AG36 in a pretty secure cardboard box complete with high density foam liner to keep everything safe and secure. The box even comes complete with a carry handle, making the box useful even after you receive it. I was really impressed with the overall look and feel of the launcher once I pulled it out of the package, as it seemed very well built and felt great in your hand.
Along with the launcher assembly, S&T includes a basic manual which includes an exploded parts diagram. It has mounting instructions as well, but really, this thing is a no brainer to mount up.
The AG36 is comprised of both nylon polymer as well as metal for stability and strength. It has the same metal content as the real launcher, with the launcher receiver itself and barrel being made of metal and the upper handguard and pistol grip being made of nylon polymer. It mounts to your G36 replica using your existing handguard pin system, which works quite well for keeping it securely mounted up.
Unlike most other launchers, the AG36 has an integrated pistol grip, which helps you maintain good control of your replica. It is positioned lower and farther back than you would normally mount a foregrip, so it's not in the optimum position, but it works quite well regardless.
In front of the grip, you'll find the trigger unit, which houses the barrel release button as well. The trigger pull largely depends on the shell that you use, as some shells require a lot of force to fire, but the angle of the trigger lever helps make the pull as easy as possible.
In front of the trigger unit, you'll find the barrel release button. Upon pushing the button, the barrel will pop open and rotate a few degrees to the left, allowing you to load the launcher. This side opening breach is what makes this thing so easy to use.
At the rear of the launcher, you'll find the ambidextrous safety switch, which blocks the trigger unit from being pulled back. It's a basic H&K style switch, which keeps the look consistent throughout the gun.
Above the controls, you'll find the most important part of the launcher, the frame and barrel, which are both made of metal. The frame serves as the mounting point between the controls and the handguard, and it houses the barrel assembly. The barrel is a fairly complex design, featuring a few stepped down portions eliminating in the large 40mm muzzle. It is held at the front by a large pin with a spring to flip the barrel out when you push the release button. In the rear, it locks in place using a large diameter pin that locks into a semi circle shaped cut-out at the bottom of the barrel.
The launcher attaches to your gun using the attached handguard, which is basically a modified version of the full length G36 handguard. There is still some room inside to mount a battery if you are using this on an AEG. This is where you may need to modify it, depending on the gun used, as some lower locking posts and pin holes are positioned slightly differently in different guns. It fit perfectly on my WE G36C, but the pin hole was misaligned on my SRC.
One of the nice features of this launcher is the side mounted leaf sight assembly. It can be removed if you don't want to use it by simply taking out the two screws holding it in. The front and rear portions flip up when in use, and the rear sight has multiple aiming positions for different ranges. With some practice, you can certainly use these markings for airsoft use.
Overall, the externals of this thing are really quite impressive, certainly when considering the price point. There is a minimum amount of overmolding and seam lines, and it feels great in your hand when mounted up on the gun. When fitted, there is absolutely zero wobble, creaking, or other detrimental issues.
S&T actually had some markings laser engraved on the barrel and receiver of the launcher, including caliber and various proof marks. The markings add to the realistic look of the launcher, and frankly, they look just plain good.
Being that this is a single shot 40mm launcher, it doesn't use magazines, instead relying on a single 40mm shell for it to fire. I tested it with Madbull, CA, ICS, S-Thunder, and King Arms shells without issue. Due to the side loading breach, you can use longer shells than normal M203 launchers, as you have a straight shot into the breach. The shells were a little bit tricky to remove at first, as there was some manufacturing residue inside the breach causing them to stick, but a quick wipe with some hot water removed it, and the shells popped in and out with ease.
There's no performance to measure with this thing, as again, all of your firing force depends on the shell that you use. It does have a fairly long barrel, so launching other projectiles like nerf rounds or 40mm rounds from Milsim Labs works quite well.
Being that the function of this thing is to simply push the button on the rear of the shell, the internals are quite simple. The trigger is connected to a basic firing pin bar using a lever system. Everything inside is made of metal, so it should be quite durable.
As far as modifications go, you can certainly mount up a rail to the vent holes on the side of the handguard if you want to run a light or laser. For my use, I've left it stock, as I love the futuristic look it gives my G36. I extended the inner and outer barrels on my WE G36C so that the flashhider just peeks out of the handguard. My goal was to build something similar to the G36 used by Norwegian special forces, and I think I got pretty close.
Proper AG36 launcher for your G36!
I love the look of mid-length G36 replicas with underbarrel grenade launchers installed, and with the S&T AG36, you can easily and relatively inexpensively make one yourself! Before, if you wanted to get an AG36, you would be paying about $300, but this is available at about half the price. Overall, yes, this thing makes my gun bulky and heavy, but it looks amazing, and the additional 40mm firepower is certainly formidable!


The Apple Watch Series 3 with GPS + cellular starts at $399.
The Apple Watch Series 3 is finally upon us, and with it, comes perhaps the most sought-after feature smart watch aficionados have wanted from on day one: cellular connectivity.
Unveiled at Apple's (AAPL) event on Tuesday, the cellular version of the Series 3 starts at $399, while the non-cellular version begins at $329. Both flavors will be available for pre-order on September 15 and ship on September 22.
But how does the Series 3 stack up against the Series 2, and is it potentially worth an upgrade?
In some ways, the Series 3 is more of the same. If you were hoping for an all-new design this year, you may be somewhat disappointed: the Series 3 still has the same display and look as the Series 2, for instance. Battery life is also the same as last year: up to 18 hours in-between charges.
In fact, the only way folks can tell the difference is if you own one of the two new colors available: a "new gold" color that looks like a paler take on the now discontinued "rose gold," or the much pricier $1,299 dark gray ceramic. The knob, or dial, on the cellular versions of the watch are also red — a small change only eagle-eyed Apple fans may notice.
But if you're more concerned about speed or having a cellular connection, the Series 3 outshines the Series 2. The Series 3 uses a new dual-core S3 chip that Apple says is 70% faster than last year's version — something borne out during my hands-on time with the watch on Tuesday. The bump up in speed was instantly noticeable: apps like Mail and Workout loaded much quicker. It was also a snap to swap digital watch faces, which could sometimes be a sluggish experience on the Series 2.
As for that LTE cellular connection, it was hard to gauge just how reliable the connection was during our limited time with the Series 3, though map data loaded quickly as we swiped and tapped around the watch.
But the cellular connection should help the Series 3 finally become its own standalone device. Although I've enjoyed using previous Apple Watches, I always felt like my Apple Watch Series 2 was just an extension of my iPhone — and for the most part, it was. Stray too far from that phone, and you lose access to things like messages, Facebook (FB), Twitter (TWTR), Instagram, Uber and Lyft because all that information is crunched on your phone and fed to the watch.
Not so with the Series 3. Assuming that LTE connection is solid, the watch should become that much more useful for people like me who want the option of leaving their phone at home while they go for a long run, or even if you just want to leave your phone in the house while you mow the lawn. The cellular connection also means you can listen to 40 million songs streamed directly from Apple Music — a nice bonus if you regularly use Apple's popular music service. Spotify or Pandora customers, however, will have to stream music off their phones — at least for now.
For more thoughts on the Apple Watch Series 3, come back to Yahoo Finance for our review.


text:
Tenebrae sings Will Todd - Choral at Cadogan
Labels: Choral at Cadogan, concert review
The chamber choir Tenebrae directed by Nigel Short made their appearance in the Choral at Cadogan series at the Cadogan Hall on 5 April 2013 accompanied by the English Chamber Orchestra in a programme which celebrated the music of Will Todd. Todd's music was performed with a pair of works, selected by Todd, which reflected his influences: Bernstein's Chichester Psalms and Elgar's part-song There is sweet music.
Though the concert was centred round the composer Will Todd, anyone unfamiliar with his work had to rely solely on a forward by him in the programme without a full biography to illuminate you further. I have to admit that this was the first time I was hearing any of Todd's music live.
In the forward Todd talked of his being excited by Bernstein's Chichester Psalms and of their folk/jazz energy. Tenebrae performed them in the version for harp, percussion and organ, which meant of course relying on an electronic organ, the Cadogan Hall not having one of its own. The organ sound was acceptable, but by no means completely convincing and it had that slightly dead quality which some electronic organs can have, not helped of course by the Cadogan Hall acoustic. The performance of Chichester Psalms was generally rather proper and correct. Only sporadically did Short and the choir generate the sort of energy and enthusiasm that the work needs, and the whole performance felt a little score-bound.
The men's opening entry in the first movement was very under-powered, the women had a nice bright focussed sound but the whole movement took some time to develop, and it was all a bit po-faced. Bernstein might have written the piece for English cathedral usage, but the material is based on fragments from his Broadway work and any performance needs to acknowledge this cross-over. The solo in the second movement was taken by counter-tenor David Allsop. Not the boy treble, which I prefer, but Allsop sang beautifully with a fine, controlled sense of line. The men's interruption with Lamah rag'shu goyim was brilliantly crisp and brisk with a nice edge to it. The final movement was performed with a nice sense of line and lovely tone, but was a bit understated and lacked swing. It reflected the general performance, beautifully sung but rather correct and understated.
The first Will Todd piece followed, The Lord is my Shepherd, accompanied by strings and harp. This is a movement from Todd's Te Deum of 2009 (a further movement from the Te Deum was sung in the second half). Todd's setting of the Te Deum mixes the Latin text with other texts, though this was not explained in the programme so one might have puzzled at where a setting of the 23rd Psalm fitted into things. It was originally sung by children's chorus and is a attractive, melodic piece which acquired some rhythmic interest in the middle section.
Next came Elgar's lovely part-song, There is Sweet Music, which is a setting of words by Tennyson and shows Elgar experimenting as the women's parts are in a different key to the men's. Elgar uses this harmonic tension to create some magical effects, and Tenebrae's performance was beautifully done, though I found the men perhaps a little too rugged with not enough bloom on their tone.
The final item in the first half was another piece of Will Todd, this time Stay with me Lord for choir, strings and harp. The work sets words by Padre Pio, in an English version by Todd's regular collaborator Ben Dunwell and was premiered in 2008 by the Sixteen, in a version for choir and piano. It starts with a lovely melodic line for the women, finely sung with clear tone, around which the work develops. Todd's style in these pieces was generally melody based, with harmonic and rhythmic interest moving around the basic melody line. The piece had some very affecting moments.
The second half consisted of five of Todd's pieces, so this means that I need to address a fundamental problem. There are times when a critic finds himself out of sympathy with the music being performed. What to do? I could simply say that Todd's music is lyrical, attractive and personable. Or I could say that his default position seems to be to write music so blandly accessible that is veers dangerously close to easy listening and had my companion making comments about music for TV adverts. The former is true to an extent, the latter a value judgement which did rather reflect my thoughts during the concert.
The second half opened with Todd's 2006 piece, Among Angels setting a short text by Ben Dunwell. Here Todd placed a series of rather sinuous melodies against some fascinating choral textures, including glissandi and murmuring. That we may love again comes from the 2009 Te Deum and sets a text by Ben Dunwell, a plea for forgiveness from the horrors of war. There were some lovely moments, with some fascinating textures and I particularly loved the way he introduced a bass drum. But for me, Todd's melodic response, plangently pleasing though it was, did not get to the heart of the text. Here I must also address another aspect of the performance, that of diction. The choir's diction was not always clear, Short and the singers seemed to be rather more concerned with beauties of line and tone so that words in the English pieces were only patchily audible, which was a great shame.
Todd's setting of the Latin text Vidi Speciosam (from the Song of Songs), was written in 2012 and is for unaccompanied choir. Here, though his writing is still recognisably his own, his harmonic language approached that of the popular American choral composers Morten Lauridsen and Eric Whitacre. Tenebrae were fully at home here and the choral textures were quite simply ravishing.
The earliest of Todd's pieces in the programme was the rather effective little miniature Man unkind, words by Ben Dunwell, from their 1996 oratorio St. Cuthbert which was first performed at Durham Cathedral. The final work in the programme was the newest, dating from 2013, Blessed Art Thou. Here, after a rapturous opening, we finally had some lively, up-tempo music to send us on our way.
As an encore, Tenebrae performed a lovely little unaccompanied piece by Todd, My Lord has come, a carol from 2011.
Todd is undoubtedly a highly gifted melodist and every piece in the programme had an attractive melodic line, but his style seemed to be too eager to please and too lacking in dramatic edge and danger for my own tastes. But I have to confess that I seemed to be in the minority and the music received an enthusiastic response from both audience and choir. I also thought that there was a little too much of Todd's music in the concert and that the second half would have benefited from the omission of two pieces and their replacement by contrasting material.
Whilst the Bernstein which opened the concert seemed a little too correct, the performances of Todd's music from Tenebrae and the English Chamber Orchestra were superb. Todd's music relies for its effect very much on the upper voices being able to sustain his lovely melodic lines, and Tenebrae did this to perfection. The choir clearly relished Todd's music and produced some ravishing choral textures.
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Posted by Unknown at Friday, April 05, 2013
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Veronique Gens and Julius Drake in Duparc, Poulenc...
Alessio Bax plays Mozart Piano Concertos nos 24 & ...
The first piano in England
Graham Johnson to receive Wigmore Hall medal
CLoSer - Poulenc and Paris
Modern tides in East London
BBC Music Magazine Awards
Celebrations at the LSO
Coming up at the Handel House Museum
Songs about words
The perils of opera travel
Giles Swayne - Stations of the Cross
Radio 3 Composer of the Week - Joseph Horovitz and...
Summer at the Royal Academy
Opera Awards
The Sixteen - Choral Pilgrimage 2013
Poeme d'un jour - Ailyn Perez and Iain Burnside
Charles Jennens - the man behind Handel's Messiah
Rediscover the English Oboe tonight
AyseDeniz Gokcin
Music up close
Making musical fireworks - The Opera Group at the ...
The Ashby left behind
A day with Bach - Bach Marathon with John Eliot Ga...
Songs of the Sea, Songs of Travel - Anthony Michae...
In case you missed it - March on Planet Hugill
Sunken Garden teaser
Spring and Summer in Smith Square
St John's Passion: Bach at the Barbican
Locus genii
Bach and beyond - April to June highlight at Kings...

redpajama_set_name: RedPajamaCommonCrawl


Lemi Shine Pacs Review: I'm Loving Them!
Here's my review of Lemi Shine Pacs, the dishwasher detergent additive for hard water, which helps remove film, spots and build up.
I don't know about you, but in my area we have extremely hard water. Most people around here have water softeners, but we don't (at least not yet -- we're definitely considering it).
Because of that we've got quite a bit of hard water build up in our dishwasher, and recently it actually caused some mechanical problems with it.
We paid money to get it fixed, but were told by the repairman we needed to be cleaning our dishwasher more often, so then began my quest to find a good dishwasher cleaner.
In the process of this I found Lemi Shine, which has an appliance cleaner which I absolutely loved.
So then I began to learn more about their products and low and behold they have a dishwasher detergent additive that you add to each load of dishes you run in your dishwasher to combat hard water spots and build up.
The company was kind enough to send me a couple of bags of these pacs to try, as well as a couple of containers of the original powder. They asked me to try it out and share how it worked for me with you, and I have to say I've not been disappointed.
I've now been using the pacs for about a week, using one with each load of the dishwasher. Since this is also the first full week since school let out, and all the kids have been home all week my dishwasher has gotten quite the work out already, with me doing one load at least per day, sometimes more.
The directions say to add the pac into the main detergent tray, and then add the dishwasher detergent itself to the prewash tray, as you can see I did in the picture to the right.
The directions also recommend using powder detergent when using this product, which works out fine since that is what I typically use anyway.
After using about eight of the packs so far, I'm already noticing some nice changes to the look of my dishwasher itself, plus my plates, bowls and glasses.
As you can see from the picture of the detergent tray above, despite several cleanings lots of hard water has build up on the dishwasher itself, including in and around the detergent tray.
It is not all gone yet by any means, but I have noticed it is getting better looking and shiner inside the machine itself after each load now (see picture to the left). Although with the amount of build up in there I bet it will take at least one bag full of pacs, maybe both, to really get it all cleared out.
What I'm really surprised by is how much shinier all my plates, bowls and glasses look after just these few uses. I don't think I realized how much hard water build up was on them since they all looked the same, but as I put away some of my bowls this afternoon from the dishwasher I noticed how much shinier the ones I was putting away were than the ones still in the cabinet (that hadn't run through the dishwasher recently).
If you suffer from hard water like I do, I definitely recommend trying this product. Do a run through or two with the machine cleaner and then beginning using this additive along with your normal dishwasher detergent in each load and hard water film and spots will hopefully no longer be as much of a problem for you. I know they're not for me!
Lots of stores carry this product. I personally have seen everything from the line (including the new pacs) at my local Walmart.
FTC Disclosure: Lemi Shine provided me with this product for my use, as well as compensated me for the time to use and review this product. However, all opinions and thoughts about this product are my own, and have not been influenced by the company.


What Is So Rare As A Day In June? Pilsner's Picks!
Featuring a rendition of "On The Good Ship Lollipop" that definitely won't remind you of Shirley Temple!
This is a another in a series of Tank portraits.
This print represents an A9 Cruiser Mk I tank in North Africa with the 7th Armoured Division (The Desert Rats), 1940.
What's so special about Macchi? Not only their Schneider Trophy fame and world's speed records but also a large family of flying boat fighters and light bombers.
Cap'n's Cabaret #29: Honolulu Luau!
Aloha! Hail and welcome to the paradise isle of the Pacific, the home of King Kamehameha and birthplace of Hula, Surfing, and Luau! Yes, beautiful Honolulu, Hawaii, land of rainbows and pineapples, a string of emeralds in the middle of the mighty Pacific!
It's been so much fun I swear we must have lost three weeks along the way!
Back in 2005 I created archiveclassicmovies.com to post my old movies. Since that time I've added almost 500 old movies, serials, shorts, and cartoons. With that much content it's been difficult to update, and poor ACM has really been neglected the past couple of years. I've finally developed a CMS that will let me more easily update my content, so I'm going to retire ACM, which will become dieselpunkindustries.com.
The Garage is proud to present: the MG T-Series!
Magazines come in two basic types, single or double stack, meaning rounds are stacked in a single row or slightly staggered, doubling capacity and creating wider magazine. This is model has been standared for pistols, submachine guns and rifles for 70+ years, with the occasional "stick magazine" aberration.
I don't know how good it's going to be but from seeing the visuals I have to say HOLY CRAP!!! If nothing else the movie looks beautiful.
This will be a short one. Meet a Bristol monoplane built in 1922 but looking a good ten years "younger".
Why I love old time radio, a short video.