RESUMO
INTRODUCTION: During an oral food challenge (OFC), there is a risk of adverse reactions, including anaphylaxis. Therefore, the physician should carefully conduct the OFC. This study aimed to evaluate the OFC results in individuals with low levels of egg white (EW)- and ovomucoid (OVM)-specific immunoglobulin E (sIgE) and the safety of a hen's egg (HE) OFC in these individuals. METHODS: A total of 2,058 individuals with low EW- or OVM-sIgE underwent HE-OFC at two institutions in Kumamoto prefecture, located in the western area of Japan, between January 1, 2017, and December 31, 2021, within 1 year of recorded sIgE measurements. The ImmunoCAP systems were used to measure sIgEs. The HE-OFC test was performed according to the 2017 Food Allergy Guidelines in an open and unblinded method. RESULTS: Five hundred and one individuals (24.3%) had low EW-sIgE levels (class 2 or lower), and 926 (45.0%) had low OVM-sIgE levels (class 2 or lower). Individuals with low EW-sIgE had lower total IgE and OVM-sIgE than did those with high EW-sIgE (greater than class 2). Those with low OVM-sIgE had lower total IgE and EW-sIgE than did those with high OVM-sIgE (greater than class 2). Among the individuals with low EW-sIgE, 86.4% (433/501 cases) passed the OFC without symptoms. Among the individuals with low OVF-sIgE, 82.6% (765/926 cases) passed the OFC without symptoms. CONCLUSION: More than 80% of individuals with suspected IgE-dependent HE allergy and low levels of EW- or OVM-specific IgE were able to consume at least a small amount of HE. As the OFC results are independent of the loading dose in cases with low EW- or OVM-sIgE, a medium-dose HE-OFC may be performed safely in individuals with no history of anaphylaxis.
Assuntos
Anafilaxia , Hipersensibilidade a Ovo , Humanos , Feminino , Animais , Clara de Ovo/efeitos adversos , Hipersensibilidade a Ovo/diagnóstico , Ovomucina/efeitos adversos , Galinhas , Imunoglobulina E , AlérgenosRESUMO
To realize lignocellulose-based bioeconomy, efficient conversion of xylose into valuable chemicals by microbes is necessary. Xylose oxidative pathways that oxidize xylose into xylonate can be more advantageous than conventional xylose assimilation pathways because of fewer reaction steps without loss of carbon and ATP. Moreover, commodity chemicals like 3,4-dihydroxybutyrate and 3-hydroxybutyrolactone can be produced from the intermediates of xylose oxidative pathway. However, successful implementations of xylose oxidative pathway in yeast have been hindered because of the secretion and accumulation of xylonate which is a key intermediate of the pathway, leading to low yield of target product. Here, high-yield production of 3,4-dihydroxybutyrate from xylose by engineered yeast was achieved through genetic and environmental perturbations. Specifically, 3,4-dihydroxybutyrate biosynthetic pathway was established in yeast through deletion of ADH6 and overexpression of yneI. Also, inspired by the mismatch of pH between host strain and key enzyme of XylD, alkaline fermentations (pH ≥ 7.0) were performed to minimize xylonate accumulation. Under the alkaline conditions, xylonate was re-assimilated by engineered yeast and combined product yields of 3,4-dihydroxybutyrate and 3-hydroxybutyrolactone resulted in 0.791 mol/mol-xylose, which is highest compared with previous study. These results shed light on the utility of the xylose oxidative pathway in yeast.
Assuntos
Saccharomyces cerevisiae , Xilose , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Xilose/metabolismo , Engenharia Metabólica/métodos , FermentaçãoRESUMO
Aromatic secondary metabolites are widely used in various industries, including the nutraceutical, dietary supplement, and pharmaceutical industries. Their production currently relies on plant extraction. Microbe-based processes have recently attracted attention as sustainable alternatives to plant-based processes. We previously showed that the yeast Pichia pastoris (Komagataella phaffii) is an optimal host for producing aromatic secondary metabolites. Additionally, titers of resveratrol, an aromatic secondary metabolite, increased by 156 % when glycerol was used as a carbon source instead of glucose. However, the mechanisms by which glycerol resulted in higher production has remained unclear. In this study, we aimed to elucidate how P. pastoris produces higher levels of aromatic secondary metabolites from glycerol than from glucose. Titers of p-coumarate, naringenin, and resveratrol increased by 103 %, 118 %, and 157 %, respectively, in natural complex media containing glycerol compared with that in media containing glucose. However, the titers decreased in minimal synthetic medium without amino acids, indicating that P. pastoris cells used the amino acids only when glycerol was the carbon source. Fermentation with the addition of single amino acids showed that resveratrol titers from glycerol varied depending on the amino acid supplemented. In particular, addition of aspartate or tryptophan into the medium improved resveratrol titers by 146 % and 156 %, respectively. These results suggest that P. pastoris could produce high levels of aromatic secondary metabolites from glycerol with enhanced utilization of specific amino acids. This study provides a basis for achieving high-level production of aromatic secondary metabolites by P. pastoris. KEY POINTS: ⢠P. pastoris can produce high levels of aromatic metabolites from glycerol ⢠P. pastoris cells use amino acids only when glycerol is the carbon source ⢠Aromatic metabolite titers from glycerol increase with amino acids utilization.
Assuntos
Glicerol , Pichia , Glicerol/metabolismo , Pichia/genética , Pichia/metabolismo , Aminoácidos/metabolismo , Resveratrol/metabolismo , Carbono/metabolismo , Glucose/metabolismo , Metanol/metabolismo , Proteínas Recombinantes/metabolismoRESUMO
Recombinant yeast strains that display heterologous amylolytic enzymes on their cell surface via the glycosylphosphatidylinositol (GPI)-anchoring system are considered as promising biocatalysts for direct ethanol production from starchy materials. For the effective hydrolysis of these materials, the ratio optimization of multienzyme activity displayed on the cell surface is important. In this study, we have presented a ratio control system of multienzymes displayed on the yeast cell surface by using different GPI-anchoring domains. The novel gene cassettes for the cell-surface display of Streptococcus bovis α-amylase and Rhizopus oryzae glucoamylase were constructed using the Saccharomyces cerevisiae SED1 promoter and two different GPI-anchoring regions derived from Saccharomyces cerevisiae SED1 or SAG1. These gene cassettes were integrated into the Saccharomyces cerevisiae genome in different combinations. Then, the cell-surface α-amylase and glucoamylase activities and ethanol productivity of these recombinant strains were evaluated. The combinations of the gene cassettes of these enzymes affected the ratio of cell-surface α-amylase and glucoamylase activities and ethanol productivity of the recombinant strains. The highest ethanol productivity from raw starch was achieved by the strain harboring one α-amylase gene cassette carrying the SED1-anchoring region and two glucoamylase gene cassettes carrying the SED1-anchoring region (BY-AASS/GASS/GASS). This strain yielded 22.5 ± 0.6 g/L of ethanol from 100 g/L of raw starch in 120 h of fermentation.
Assuntos
Técnicas de Visualização da Superfície Celular/métodos , Etanol/metabolismo , Glucana 1,4-alfa-Glucosidase/metabolismo , Rhizopus/enzimologia , Saccharomyces cerevisiae/enzimologia , Streptococcus bovis/enzimologia , alfa-Amilases/metabolismo , Clonagem Molecular , Expressão Gênica , Glucana 1,4-alfa-Glucosidase/genética , Mutagênese Insercional , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rhizopus/genética , Saccharomyces cerevisiae/genética , Amido/metabolismo , Streptococcus bovis/genética , alfa-Amilases/genéticaRESUMO
Feeder-free culture of human induced pluripotent stem (hiPS) cells is necessary for their clinical application to avoid adverse effects of foreign proteins. hiPS cells were cultured with combinations of activin (A), CHIR99021 (C), basic fibroblast growth factor (F), and leukemia inhibitory factor (L) under feeder-free conditions. Culture was terminated after 12 passages or when the cell morphology changed from pluripotency. Pluripotency was analyzed by alkaline phosphatase (ALP) staining and immunostaining with antibodies to Oct3/4, Nanog, SSEA4, and TRA-1-60. SB431542 (SB), an activin inhibitor, was added to the culture, and the morphology of the cells was observed. hiPS cells cultured with A, AC, and ACL after 12 passages were positive for ALP staining. Oct3/4 was positive in hiPS cells cultured with A, AC, and ACL. hiPS cells were positive for Nanog when cultured with A and AC; however, Nanog signal was weaker in cells cultured with ACL. SSEA4 was positive in hiPS cells cultured with A and AC but almost negative in those cultured with ACL. hiPS cells were positive for TRA-1-60 when cultured with A, AC, and ACL. hiPS cells lose their undifferentiated morphology at six passages when cultured with A + SB, five passages with AC + SB, and nine passages with ACL. We conclude that feeder-free culture of hiPS cells requires A or AC to maintain pluripotency.
Assuntos
Ativinas/farmacologia , Técnicas de Cultura de Células , Células Alimentadoras , Células-Tronco Pluripotentes Induzidas/metabolismo , Piridinas/farmacologia , Pirimidinas/farmacologia , Ativinas/antagonistas & inibidores , Fosfatase Alcalina/análise , Animais , Antígenos de Superfície/análise , Benzamidas/farmacologia , Colágeno , Dioxóis/farmacologia , Combinação de Medicamentos , Fator 2 de Crescimento de Fibroblastos/farmacologia , Proteínas de Homeodomínio/análise , Humanos , Laminina , Fator Inibidor de Leucemia/farmacologia , Camundongos , Proteína Homeobox Nanog , Fator 3 de Transcrição de Octâmero/análise , Proteoglicanas/análise , Antígenos Embrionários Estágio-Específicos/análiseRESUMO
BACKGROUND/AIMS: To clarify the usefulness of screening ultrasonography (US) to diagnose gastric and colorectal cancer, patient records were analyzed retrospectively. METHODOLOGY: Ultrasonography was performed for patients with abdominal symptoms. They were then subjected to computed tomography (CT) when diagnosed with gastric cancer, colorectal cancer, or bowel obstruction. Patient records were analyzed retrospectively after final diagnosis of gastric cancer or colorectal cancer by endoscopy, surgery or necropsy. RESULTS: Twelve patients were diagnosed with colorectal cancer and six with gastric cancer. The detailed structure of colorectal cancer was visible as wall thickening with US, while cancer was often illustrated as a mass by CT. Loss of stratification was clear with US in 11 patients. US demonstrated wall thickening in 10 patients and a mass in 1 patient, while CT demonstrated wall thickening in 3 patients and a mass in 8 patients. The structure of colorectal cancer was more obvious when using US than when using CT. One patient demonstrated focal wall thickening with US, but this was not detected by CT. CONCLUSIONS: US is useful for diagnosis of gastric cancer and colorectal cancer. US produces more detailed findings in colorectal cancer than CT.
Assuntos
Neoplasias Colorretais/diagnóstico por imagem , Programas de Rastreamento/métodos , Neoplasias Gástricas/diagnóstico por imagem , Idoso , Idoso de 80 Anos ou mais , Análise de Variância , Neoplasias Colorretais/patologia , Endoscopia Gastrointestinal , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Valor Preditivo dos Testes , Estudos Retrospectivos , Neoplasias Gástricas/patologia , Tomografia Computadorizada por Raios X , UltrassonografiaRESUMO
Recombination of biosynthetic gene clusters including those of non-ribosomal peptide synthetases (NRPSs) is essential for understanding the mechanisms of biosynthesis. Due to relatively huge gene cluster sizes ranging from 10 to 150 kb, the prevalence of sequence repeats, and inability to clearly define optimal points for manipulation, functional characterization of recombinant NRPSs with maintained activity has been hindered. In this study, we introduce a simple yet rapid approach named "Seamed Express Assembly Method (SEAM)" coupled with Ordered Gene Assembly in Bacillus subtilis (OGAB) to reconstruct fully functional plipastatin NRPS. This approach is enabled by the introduction of restriction enzyme sites as seams at module borders. SEAM-OGAB is then first demonstrated by constructing the ppsABCDE NRPS (38.4 kb) to produce plipastatin, a cyclic decapeptide in B. subtilis. The introduced amino acid level seams do not hinder the NRPS function and enable successful production of plipastatin at a commensurable titer. It is challenging to modify the plipastatin NRPS gene cluster due to the presence of three long direct-repeat sequences; therefore, this study demonstrates that SEAM-OGAB can be readily applied towards the recombination of various NRPSs. Compared to previous NRPS gene assembly methods, the advantage of SEAM-OGAB is that it readily enables the shuffling of NRPS gene modules, and therefore, chimeric NRPSs can be rapidly constructed for the production of novel peptides. This chimeric assembly application of SEAM-OGAB is demonstrated by swapping plipastatin NRPS and surfactin NRPS modules to produce two novel lipopeptides in B. subtilis.
Assuntos
Bacillus subtilis , Peptídeo Sintases , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Peptídeo Sintases/metabolismo , Sequência de Bases , Lipopeptídeos/genéticaRESUMO
Conventional analysis of microbial bioproducers requires the extraction of metabolites from liquid cultures, where the culturing steps are time consuming and greatly limit throughput. To break through this barrier, the current study aims to directly evaluate microbial bioproduction colonies by way of supercritical fluid extraction-supercritical fluid chromatography-triple quadrupole mass spectrometry (SFE-SFC-MS/MS). The online SFE-SFC-MS/MS system offers great potential for high-throughput analysis due to automated metabolite extraction without any need for pretreatment. This is the first report of SFE-SFC-MS/MS as a method for direct colony screening, as demonstrated in the high-throughput screening of (-)-limonene bioproducers. Compared with conventional analysis, the SFE-SFC-MS/MS system enables faster and more convenient screening of highly productive strains.
Assuntos
Cromatografia com Fluido Supercrítico , Espectrometria de Massas em Tandem , Espectrometria de Massas em Tandem/métodos , Limoneno , Cromatografia com Fluido Supercrítico/métodos , Cromatografia Líquida , Ensaios de Triagem em Larga Escala/métodosRESUMO
Insulin-like growth factor (IGF)-I is up-regulated in pancreatic cancer tissues. Pancreatic cancer cell lines were analyzed in serum-free media as a model of the fibrous tissues that these cells often invade. Pancreatic cancer surgical specimens were immunostained with anti-IGF-I receptor (IGF-IR)ß antibody. The growth of pancreatic cancer cells in serum-free media was also analyzed. Cell lysates were analyzed for protein by western blot analysis. Cells cultured in the presence of picropodophyllin (PPP), LY294002, or PD98059, were subjected to cell proliferation and scratch assays. In addition, BrdU uptake and apoptosis were analyzed in these cells. IGF-IRß was detected in pancreatic cancer cells invading fibrous tissues. NOR-P1 grew most rapidly in serum-free media. The concentrations of IGF-I and IGF-II in the media were higher in NOR-P1 than the other cell lines. Cell proliferation in NOR-P1 cells was enhanced by IGF-I or IGF-II treatment more than in MIA-Paca2 or PK-1 cells. PPP, LY294002, and PD98059 suppressed proliferation and motility of NOR-P1 cells and inhibited BrdU uptake, while PPP induced apoptosis. IGF-IRß may be a potential therapeutic target to inhibit invasion of pancreatic cancer.
Assuntos
Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Receptor IGF Tipo 1/metabolismo , Linhagem Celular Tumoral , Meios de Cultura Livres de Soro , Desoxiuridina/farmacologia , Humanos , Fator de Crescimento Insulin-Like I/metabolismo , Fator de Crescimento Insulin-Like I/farmacologia , Fator de Crescimento Insulin-Like II/metabolismo , Fator de Crescimento Insulin-Like II/farmacologiaAssuntos
Desastres , Terremotos , Hiperamonemia/etiologia , Doença da Deficiência de Ornitina Carbomoiltransferase/complicações , Doença da Deficiência de Ornitina Carbomoiltransferase/psicologia , Estresse Psicológico/etiologia , Adulto , Feminino , Humanos , Hiperamonemia/diagnóstico , Hiperamonemia/terapia , Japão , Doença da Deficiência de Ornitina Carbomoiltransferase/terapia , Estresse Psicológico/fisiopatologiaRESUMO
Hereditary tyrosinemia type 1 (HT1) is an autosomal recessive disorder caused by a defect in fumarylacetoacetate hydroxylase (FAH) encoded by the FAH gene. Patients with HT1 disorder present with increased blood tyrosine, succinyl acetoacetate, and succinyl acetone levels, and develop clinical manifestations including liver failure, kidney tubular dysfunction, growth failure, rickets, pseudo-porphyric crises, and hepatocellular carcinoma. We encountered two siblings with HT1. Among the siblings, the elder brother developed acute liver failure with coagulopathy at the age of 2 months and was rescued by liver transplantation (LT) following combination therapy with continuous hemodiafiltration and plasma exchange. The younger sister was followed up from the prenatal period for signs of HT1 due to prior history of the condition in her sibling. She was initially considered a carrier of HT1 owing to the lack of overt signs of the disease and negative urine screening for succinyl acetone (SA). She was eventually diagnosed with HT1 because of liver disorder at 9 months of age, associated with a positive urine SA result. Her disease state was controlled by treatment with nitisinone (NTBC). DNA analysis of both siblings identified heterozygous status for a previously reported FAH pathogenic allele (c.782C > T) and a novel likely pathogenic variant (c.688C.G). The siblings have stable lives with no developmental delay or impaired growth. NTBC treatment is effective in preventing the progression of liver and kidney diseases. However, even in cases treated without LT, clinicians should follow up the clinical outcomes over long term, as patients may require LT when developing complications, such as hepatocellular carcinoma.
RESUMO
Bioactive plant-based secondary metabolites such as stilbenoids, flavonoids, and benzylisoquinoline alkaloids (BIAs) are produced from l-tyrosine (l-Tyr) and have a wide variety of commercial applications. Therefore, building a microorganism with high l-Tyr productivity (l-Tyr chassis) is of immense value for large-scale production of various aromatic compounds. The aim of this study was to develop an l-Tyr chassis in the nonconventional yeast Pichia pastoris (Komagataella phaffii) to produce various aromatic secondary metabolites (resveratrol, naringenin, norcoclaurine, and reticuline). Overexpression of feedback-inhibition insensitive variants of 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (ARO4K229L) and chorismate mutase (ARO7G141S) enhanced l-Tyr titer from glycerol in P. pastoris. These engineered P. pastoris strains increased the titer of resveratrol, naringenin, and norcoclaurine by 258, 244, and 3400%, respectively, after expressing the corresponding heterologous pathways. The titer of resveratrol and naringenin further increased by 305 and 249%, resulting in yields of 1825 and 1067 mg/L, respectively, in fed-batch fermentation, which is the highest titer from glycerol reported to date. Furthermore, the resveratrol-producing strain accumulated intermediates in the shikimate pathway. l-Tyr-derived aromatic compounds were produced using crude glycerol byproducts from biodiesel fuel (BDF) production. Constructing an l-Tyr chassis is a promising strategy to increase the titer of various aromatic secondary metabolites and P. pastoris is an attractive host for high-yield production of l-Tyr-derived aromatic compounds from glycerol.
Assuntos
Glicerol , Engenharia Metabólica , Glicerol/metabolismo , Engenharia Metabólica/métodos , Pichia/genética , Pichia/metabolismo , Resveratrol/metabolismo , Saccharomycetales , Tirosina/metabolismoRESUMO
mRNA-based vaccines have been used globally to eradicate the coronavirus-disease 2019 (COVID-19) pandemic. Vaccine efficacy and adverse reactions depend on immune responses, such as proinflammatory cytokine production and lymphocyte activation. We conducted a prospective cohort study to investigate relationships among specific antibody titers, adverse reactions, proinflammatory cytokine production, and immune-regulatory microRNA (miRNA) levels in serum extracellular vesicles (EVs) after COVID-19 vaccination (BNT162b2). Local adverse reactions after the second dose, such as local pain and swelling, were less correlated with those of systemic symptoms, such as fever and muscle pain, whereas serum TNF-α levels were associated with systemic adverse reactions and with specific antibody titers. Interestingly, EV miR-92a-2-5p levels in sera were negatively correlated with degrees of adverse reactions, and EV miR-148a levels were associated with specific antibody titers. Our data suggest a potential of circulating EV miRNAs as biomarkers for vaccine efficacy and adverse reactions.
RESUMO
Engineering the microbial production of secondary metabolites is limited by the known reactions of correctly annotated enzymes. Therefore, the machine learning discovery of specialized enzymes offers great potential to expand the range of biosynthesis pathways. Benzylisoquinoline alkaloid production is a model example of metabolic engineering with potential to revolutionize the paradigm of sustainable biomanufacturing. Existing bacterial studies utilize a norlaudanosoline pathway, whereas plants contain a more stable norcoclaurine pathway, which is exploited in yeast. However, committed aromatic precursors are still produced using microbial enzymes that remain elusive in plants, and additional downstream missing links remain hidden within highly duplicated plant gene families. In the current study, machine learning is applied to predict and select plant missing link enzymes from homologous candidate sequences. Metabolomics-based characterization of the selected sequences reveals potential aromatic acetaldehyde synthases and phenylpyruvate decarboxylases in reconstructed plant gene-only benzylisoquinoline alkaloid pathways from tyrosine. Synergistic application of the aryl acetaldehyde producing enzymes results in enhanced benzylisoquinoline alkaloid production through hybrid norcoclaurine and norlaudanosoline pathways.
Assuntos
Alcaloides , Benzilisoquinolinas , Benzilisoquinolinas/metabolismo , Aprendizado de Máquina , Engenharia Metabólica , Plantas/genética , Plantas/metabolismoRESUMO
BACKGROUND/AIMS: Patients with abdominal symptoms and leukocytosis or elevated C-reactive protein were subjected to abdominal ultrasonography (US) for correct diagnosis. METHODOLOGY: Patients with abdominal symptoms, leukocytosis and elevated C-reactive protein were enrolled. Those with abnormal liver enzymes or radiographs were excluded since either of them might be a clue to proper diagnosis, such as hepatobiliary diseases or bowel obstruction. RESULTS: Total number of patients was 38. Number of acute diverticulitis, colitis, acute appendicitis and enteritis were 8, 7, 7 and 6, respectively. One patient with pelvis tumor and 2 with colon cancer were successfully diagnosed with abdominal US. Colon cancers were confirmed and pelvis tumor was diagnosed as ovarian squamous cell carcinoma with surgical specimens. Sensitivity and specificity were 100% (95% CI: 44-100%) and 97.1% (95% CI: 85-99%), respectively. CONCLUSIONS: Our data clearly recommended that abdominal US be performed carefully for patients with abdominal symptoms and leukocytosis or elevated CRP since potentially they had malignancies.
Assuntos
Abdome/diagnóstico por imagem , Proteína C-Reativa/análise , Leucocitose/diagnóstico por imagem , Humanos , Sensibilidade e Especificidade , UltrassonografiaRESUMO
Nitrogen is essential for the biosynthesis of various molecules in cells, such as amino acids and nucleotides, as well as several types of lipids and sugars. Cyanobacteria can assimilate several forms of nitrogen, including nitrate, ammonium, and urea, and the physiological and genetic responses to these nitrogen sources have been studied previously. However, the metabolic changes in cyanobacteria caused by different nitrogen sources have not yet been characterized. This study aimed to elucidate the influence of nitrate and ammonium on the metabolic profiles of the cyanobacterium Synechocystis sp. strain PCC 6803. When supplemented with NaNO3 or NH4Cl as the nitrogen source, Synechocystis sp. PCC 6803 grew faster in NH4Cl medium than in NaNO3 medium. Metabolome analysis indicated that some metabolites in the CBB cycle, glycolysis, and TCA cycle, and amino acids were more abundant when grown in NH4Cl medium than NaNO3 medium. 15N turnover rate analysis revealed that the nitrogen assimilation rate in NH4Cl medium was higher than in NaNO3 medium. These results indicate that the mechanism of nitrogen assimilation in the GS-GOGAT cycle differs between NaNO3 and NH4Cl. We conclude that the amounts and biosynthetic rate of cyanobacterial metabolites varies depending on the type of nitrogen.
RESUMO
Data-driven engineering of microbes has been demonstrated for the sustainable production of high-performance chemicals. Metabolic profiling analysis is essential to increase the productivity of target compounds. However, improvement of comprehensive analysis methodologies is required for the high demands of metabolic engineering. Therefore, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) based methodology was designed and applied to cover a wide target range with high precision. Ion-pair free separation of metabolites on a pentafluorophenyl propyl column enabled high-precision quantification of 113 metabolites. The method was further evaluated for high reproducibility and robustness. Target analytes consisted of primary metabolites and intermediate metabolites for microbial production of high-performance chemicals. 95 metabolites could be detected with high reproducibility of peak area (intraday data: CV<15%), and 53 metabolites could be sensitively determined within a wide dynamic linear range (3-4 orders of magnitude). The developed system was further applied to the metabolomic analysis of various prokaryotic and eukaryotic microorganisms. Differences due to culture media and metabolic phenotypes could be observed when comparing the metabolomes of conventional and non-conventional yeast. Furthermore, almost all Kluyveromyces marxianus metabolites could be detected with moderate reproducibility (CV<40%, among independent extractions), where 41 metabolites were detected with very high reproducibility (CV<15%). In addition, the accuracy was validated via a spike-and-recovery test,and 78 metabolites were detected with analyte recovery in the 80-120% range. Together these results establish ion-pair free metabolic profiling as a comprehensive and precise tool for data-driven bioengineering applications.
Assuntos
Metabolômica , Espectrometria de Massas em Tandem , Cromatografia Líquida , Kluyveromyces , Reprodutibilidade dos TestesRESUMO
Ultrasound, which refers to frequencies above the audible limit of human hearing, is a candidate for inducing resistance to pathogens in plants. We revealed that aerial ultrasound of 40.5 kHz could induce disease resistance in tomatoes and rice when the plants were irradiated with ultrasound of ca. 100 dB for 2 weeks during nursery season and reduced the incidence of Fusarium wilt and blast diseases, respectively, when plants were inoculated with pathogen 0 or 1 week after terminating irradiation. Disease control efficacy was also observed with ultrasound at frequencies of 19.8 and 28.9 kHz. However, cabbage yellows and powdery mildew on lettuce were not suppressed by ultrasound irradiation. No significant positive or negative effect on growth was observed in tomato and rice plants. RT-qPCR showed that the expression of PR1a involved in the salicylic acid (SA) signaling pathway was upregulated in the ultrasound-irradiated tomato.
RESUMO
Previous studies have utilized monoamine oxidase (MAO) and L-3,4-dihydroxyphenylalanine decarboxylase (DDC) for microbe-based production of tetrahydropapaveroline (THP), a benzylisoquinoline alkaloid (BIA) precursor to opioid analgesics. In the current study, a phylogenetically distinct Bombyx mori 3,4-dihydroxyphenylacetaldehyde synthase (DHPAAS) is identified to bypass MAO and DDC for direct production of 3,4-dihydroxyphenylacetaldehyde (DHPAA) from L-3,4-dihydroxyphenylalanine (L-DOPA). Structure-based enzyme engineering of DHPAAS results in bifunctional switching between aldehyde synthase and decarboxylase activities. Output of dopamine and DHPAA products is fine-tuned by engineered DHPAAS variants with Phe79Tyr, Tyr80Phe and Asn192His catalytic substitutions. Balance of dopamine and DHPAA products enables improved THP biosynthesis via a symmetrical pathway in Escherichia coli. Rationally engineered insect DHPAAS produces (R,S)-THP in a single enzyme system directly from L-DOPA both in vitro and in vivo, at higher yields than that of the wild-type enzyme. However, DHPAAS-mediated downstream BIA production requires further improvement.
Assuntos
Descarboxilases de Aminoácido-L-Aromático/metabolismo , Escherichia coli/metabolismo , Proteínas de Insetos/metabolismo , Engenharia Metabólica/métodos , Tetra-Hidropapaverolina/metabolismo , Ácido 3,4-Di-Hidroxifenilacético/análogos & derivados , Ácido 3,4-Di-Hidroxifenilacético/metabolismo , Motivos de Aminoácidos/genética , Animais , Descarboxilases de Aminoácido-L-Aromático/química , Descarboxilases de Aminoácido-L-Aromático/genética , Descarboxilases de Aminoácido-L-Aromático/isolamento & purificação , Bombyx , Dopamina/metabolismo , Proteínas de Insetos/química , Proteínas de Insetos/genética , Proteínas de Insetos/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Relação Estrutura-AtividadeRESUMO
In the original version of this Article, the abbreviation of 3,4-dihydroxyphenylacetaldehyde synthase presented in the first paragraph of the Discussion section was given incorrectly as DYPAA. The correct abbreviation for this enzyme is DHPAAS. This error has been corrected in both the PDF and HTML versions of the Article.