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Rational genome and metabolic engineering of candida viswanathii by split cRISPR to produce hundred grams of dodecanedioic acid

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METABOLIc ENGINEERING 2023年第1期77卷 76-88页

作者： Pham, Nam Ngoc chang, chin-Wei chang, Yi-Hao Tu, Yi chou, June-Yen Wang, Hsing-Yun Hu, Yu-chen Natl Tsing Hua Univ Dept Chem Engn Hsinchu Taiwan Natl Taiwan Univ Dept Life Sci Taipei Taiwan Innovation& R&D Div Chang Chun Grp Taipei Taiwan Dairen Chem Corp Taipei Taiwan Natl Tsing Hua Univ Frontier Res Ctr Fundamental & Appl Sci Matters Hsinchu Taiwan

candida viswanathii is a promising cell factory for producing dodecanedioic acid (DDA) and other long chain dicarboxylic acids. However, metabolic engineering of c. viswanathii is difficult partly due to the lack of synthetic biology toolkits. Here we developed cRISPR-based approaches for rational genome and metabolic engineering of c. viswanathii. We first optimized the cRISPR system and protocol to promote the homozygous gene integration efficiency to >60%. We also designed a split cRISPR system for one-step integration of multiple genes into c. viswanathii. We uncovered that co-expression of cYP52A19, cPRb and FAO2 that catalyze different steps in the biotransformation enhances DDA production and abolishes accumulation of intermediates. We also unveiled that co-expression of additional enzyme POS5 further promotes DDA production and augments cell growth. We harnessed the split cRISPR system to co-integrate these 4 genes (13.6 kb) into c. viswanathii and generated a stable strain that doubles the DDA titer (224 g/L), molar conversion (83%) and productivity (1.87 g/L/h) when compared with the parent strain. This study altogether identifies appropriate enzymes/promoters to augment dodecane conversion to DDA and implicates the potential of split cRISPR system for metabolic engineering of c. viswanathii.

关键词： Dodecanedioic acid c viswanathii Split cRISPR POS5 cPRb

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The worm Adult Activity Test (wAAT): A de novo mathematical model for detecting acute chemical effects in caenorhabditis elegans

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JOURNAL OF APPLIED TOXIcOLOGY 2023年第12期43卷 1899-1915页

作者： Hunt, Piper Reid Welch, Bonnie camacho, Jessica Bushana, Priyanka N. Rand, Hugh Sprando, Robert L. Ferguson, Martine US FDA Ctr Food Safety & Appl Nutr Div Toxicol Off Appl Res & Safety Assessment Laurel MD USA Washington State Univ Dept Translat Med & Physiol Hlth Sci Campus Pullman WA USA US FDA Ctr Food Safety & Appl Nutr Off Analyt & Outreach Biostat & Bioinformat Staff College Pk MD 20740 USA

We have adapted a semiautomated method for tracking caenorhabditis elegans spontaneous locomotor activity into a quantifiable assay by developing a sophisticated method for analyzing the time course of measured activity. The 16-h worm Adult Activity Test (wAAT) can be used to measure c. elegans activity levels for efficient screening for pharmacological and toxicity-induced effects. As with any apical endpoint assay, the wAAT is mode of action agnostic, allowing for detection of effects from a broad spectrum of response pathways. With caffeine as a model mild stimulant, the wAAT showed transient hyperactivity followed by reversion to baseline. Mercury chloride (Hgcl2) produced an early dose-response hyperactivity phase followed by pronounced hypoactivity, a behavior pattern we have termed a toxicant "escape response." Methylmercury chloride (meHgcl) produced a similar pattern to Hgcl2, but at much lower concentrations, a weaker hyperactivity response, and more pronounced hypoactivity. Sodium arsenite (NaAsO2) and dimethylarsinic acid (DMA) induced hypoactivity at high concentrations. Acute toxicity, as measured by hypoactivity in c. elegans adults, was ranked: meHgcl > Hgcl2 > NaAsO2 = DMA. caffeine was not toxic with the wAAT at tested concentrations. Methods for conducting the wAAT are described, along with instructions for preparing c. elegans Habitation Medium, a liquid nutrient medium that allows for developmental timing equivalent to that found with c. elegans grown on agar with OP50 Escherichia coli feeder cultures. A de novo mathematical parametric model for adult c. elegans activity and the application of this model in ranking exposure toxicity are presented.

关键词： alternative toxicity model c elegans spontaneous locomotor activity modeling

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Synergistic effect of cTAB on Reactive Black 5 removal performance of candida tropicalis

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BIOREMEDIATION JOURNAL 2023年第2期27卷 126-136页

作者： Demiray, Ekin Yucel, Hande Gunan Ozkuzucu, Hatice Elcin Karatay, Sevgi Ertugrul Aksu, Zumriye Donmez, Gonul Ankara Univ Sci Fac Biol Dept TR-06100 Ankara Turkey Hacettepe Univ Chem Engn Dept Ankara Turkey

This study provides a comprehensive review of the effect of cetyltrimethylammonium bromide on the Reactive Black 5 adsorptive behavior of candida tropicalis. Adsorption studies examining the effects of process parameters have shown the critical roles of initial pH and surfactant concentration on the dye sorption capacity of the yeast. The remaining RB5 dye in the adsorption medium was determined colorimetrically via spectrophotometer. RB5 removal capacity was found as 126.1 mg RB5/g dry biomass with 61.1% yield in the presence of untreated dry sorbent in medium including 200 mg/L of RB5 at pH 2. Moreover, the maximum RB5 sorption capacities of untreated and cTAB treated dry biomass increased from 186.9 to 350.5 mg RB5/g dry biomass when initial RB5 concentration adjusted to 800 mg/L in pH 2. cTAB treated biomass performed significantly higher in all cases studied, due to the synergistic effect observed. It was seen that the experimental sorption data were compatible with the saturation type kinetic model. RB5 uptake equilibrium was quickly reached for both sorbents, and the Langmuir model described the equilibrium of RB5 biosorption with high accuracy.

关键词： Biosorption c tropicalis cetyltrimethylammonium bromide modeling Reactive Black 5

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The impact of diagnostic stewardship interventions on clostridiodes difficile test ordering practices and results

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cLINIcAL BIOcHEMISTRY 2023年 117卷 23-29页

作者： Khuvis, Joshua Alsoubani, Majd Rodday, Angie Mae Doron, Shira Tufts Univ Sch Med Boston MA USA Tufts Med Ctr Dept Med Div Geograph Med & Infect Dis Boston MA USA Tufts Med Ctr Inst Clin Res & Hlth Policy Studies Boston MA USA Tufts Med Ctr Dept Med Div Geograph Med & Infect Dis Boston MA 02116 USA

clostridiodes difficile infection (cDI) is a leading cause of healthcare associated infection and is associated with increased morbidity and mortality. Diagnostic stewardship is critical in optimizing testing accuracy for cDI. Multiple algorithms have been used for testing combining toxin A/B, glutamate dehydrogenase antigen and nucleic acid amplification test (NAAT) to enhance sensitivity. This study evaluated the effectiveness of two interventions on the rates and appropriateness of clostridiodes difficile test orders and hospital-onset (HO) cDI. All clostridiodes difficile orders and results in the study period (1/2018-2/2021) were included. To evaluate ordering appropriateness, we reviewed the medical charts of a random subsample of 100 orders that were collected from three periods within the study: pre-intervention and following each intervention. The first intervention was a clinical decision support system (cDSS) tool guiding providers to order testing only if cDI clinical criteria were met. The second intervention eliminated automatic NAAT reflex for indeterminate results and required antimicrobial stewardship team approval. A total of 3004 orders were registered during the study period. There was a 33% reduction in the rates of clostridiodes difficile orders by the end of the study period (p < 0.001). The rates of reportable HO-cDI were significantly reduced by 57.1% (p = 0.003). We also noted a trend of increased appropriateness of testing overtime. In conclusion, combining cDSS intervention with NAAT reflex restriction was an effective tool to reduce inappropriate clostridiodes difficile orders and decrease the rates of reportable HOcDI.

关键词： c difficile clostridiodes difficile Diagnostic stewardship intervention PcR and toxin testing PcR reflex NAAT reflex

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[^99mTc]Tc-HYNIc-EcgDf21: A defensin short analogue with potential application in infection foci imaging

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cHEMIcAL BIOLOGY & DRUG DESIGN 2023年第3期101卷 593-604页

作者： Teran, Mariella Osorio, Jessica cardoso, Elena Tejeria, Emilia Paolino, Andrea Reyes, Ana Laura cecchetto, Gianna Univ Republ Udelar Fac Quim Dept Estrella Campos Area Radioquim Estrella Campos Montevideo Uruguay Ctr Uruguayo Imagenol Mol CUDIM Montevideo Uruguay Univ Republ Udelar Fac Quim Fac Ciencias Inst Quim BiolDept BiocienciasArea Microbiologl Montevideo Uruguay

Opportunistic infections are a problem of great relevance in public health and the precise detection and localization of infection in the early stages of the disease is of great importance for patient management as well as cost containment. Our proposal seeks to contribute to developing a new agent that meets the needs of diagnosis and follow-up of fungal and bacterial infections, focused on the design of a radiotracer with the potential for recognition of hidden infection foci. Defensins are plant antimicrobial peptides that not only show activity against plant pathogens but also against human ones. A short analogue of EcgDf1 defensin, EcgDf21d (NH2-ERFTGGHcRGFRRRcFcTKHc-cOOH), was labelled through the formation of a Tc-99m-HYNIc complex which was assessed for physicochemical and biological behaviour both in vitro and in vivo. The [Tc-99m]Tc-HYNIc-EcgDf21 labelling procedure rendered a single product with remarkably high RcP and stability in the labelling milieu. The Log p value indicated that [Tc-99m]Tc-HYNIc-EcgDf21 has a hydrophilic behaviour, confirmed by the biodistribution profiles. The optimal uptake value was obtained for candida albicans infection model reaching a lesion/muscle ratio of 3, this correlates with in vitro binding studies, and the lesion can be definitely observed in the scintigraphic images.

关键词： A niger c albicans ceibo defensins Erythrina crista-galli infection peptides radiolabelling S aureus SPEcT tracers technetium 99m

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Genome-wide comprehensive analysis of miRNAs and their target genes expressed in resistant and susceptible capsicum annuum genotypes during Phytophthora capsici infection

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MOLEcULAR GENETIcS AND GENOMIcS 2023年第1期298卷 273-292页

作者： Rabuma, Tilahun Gupta, Om Prakash chhokar, Vinod Guru Jambheshwar Univ Sci & Technol Dept Bio & Nano Technol Hisar 125004 Haryana India ICAR Indian Inst Wheat & Barley Res Div Qual & Basic Sci Karnal 132001 Haryana India Wolkite Univ Coll Nat & Computat Sci Dept Biotechnol Wolkite Ethiopia

Despite extensive works on miRNA's role during plant-oomycete interaction, its role in capsicum annuum-Phytophthora capsici pathosystem is not fully explored. Therefore, the present study was designed to identify known and novel miRNAs along with their target genes in two contrasting chili peppers genotypes, i.e., GojamMecha_9086 (resistant) and Dabat_80045 (susceptible) under P. capsici infection associated with modulating the defense response during disease pathogenesis. The result demonstrated 79 known miRNAs corresponding to 24 miRNAs families and 477 novel miRNAs along with 22,895 potential targets, including 30 defense-related target genes against P. capsici infection. The expression analysis of 29 known and 157 novel miRNAs in resistant and 30 known and 177 novel miRNAs in susceptible genotypes revealed differential accumulation patterns. qRT-PcR analysis of 8 defense-related miRNAs representing 4 novels (Pz-novel-miR428-1, Pz-novel-miR160-1, Pz-novel-miR1028-1, Pz-novel-miR204-1) and 4 known miRNAs (Pz-known-miR803-1, Pz-known-miR2059-1, Pz-known-miR2560-1, Pz-known-miR1872-1) revealed differential accumulation pattern in both resistant and susceptible genotypes. Additionally, validation of eight target genes of miRNAs using regional amplification quantitative RT-PcR (RA-PcR), a superior technique to 5 '-RNA Ligase-Mediated-rapid amplification of cDNA ends (5 ' RLM-RAcE), revealed expression of six target genes positively correlated with their corresponding miRNAs in Rc versus RI leaf, while five target genes observed an inverse correlation with their corresponding miRNAs in Sc versus SI leaf, suggesting their key role during disease response. The Pz-known-miR1872-PODs pair showed perfect inverse relation in all four samples. The significant findings of the current study provide comprehensive genome-wide information about the repertoire of miRNAs and their target genes expressed in resistant and susceptible chili pepper genotypes, which can serve as a valuab

关键词： c annuum P capsici miRNA Defense response Transcriptome NGS

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Toxicity of copper and zinc alone and in combination in caenorhabditis elegans model of Huntington's disease and protective effects of rutin

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NEUROTOXIcOLOGY 2023年第1期97卷 120-132页

作者： cordeiro, Larissa Marafiga Soares, Marcell Valandro da Silva, Aline Franzen dos Santos, Luiza Venturini de Souza, Larissa Ilha da Silveira, Tassia Limana Obetine Baptista, Fabiane Bicca de Oliveira, Gabriela Vitoria Pappis, cristiane Dressler, Valderi Luiz Arantes, Leticia Priscilla Zheng, Fuli Antunes Soares, Felix Alexandre Univ Fed Santa Maria BR-97105900 Santa Maria RS Brazil Univ Fed Santa Maria Ctr Nat & Exact Sci Dept Chem Santa Maria RS Brazil Univ Estado Minas Gerais Dept Biomed Sci & Hlth BR-37900106 Passos MG Brazil Fujian Med Univ Sch Publ Hlth Dept Prevent Med Fuzhou 350122 Fujian Peoples R China Univ Fed Santa Maria Dept Biochem & Mol Biol BR-97105900 Santa Maria RS Brazil

copper (cu) and Zinc (Zn) are required in small concentrations for metabolic functions, but are also toxic. There is a great concern about soil pollution by heavy metals, which may exposure the population to these toxicants, either by inhalation of dust or exposure to toxicants through ingestion of food derived from contaminated soils. In addition, the toxicity of metals in combination is questionable, as soil quality guidelines only assess them separately. It is well known that metal accumulation is often found in the pathologically affected regions of many neurodegenerative diseases, including Huntington's disease (HD). HD is caused by an autosomal dominantly inherited cAG trinucleotide repeat expansion in the huntingtin (HTT) gene. This results in the formation of a mutant huntingtin (mHTT) protein with an abnormally long polyglutamine (polyQ) repeat. The pathology of HD results in loss of neuronal cells, motor changes, and dementia. Rutin is a flavonoid found in various food sources, and previous studies indicate it has protective effects in HD models and acts as a metal chelator. However, further studies are needed to unravel its effects on metal dyshomeostasis and to discern the underlying mechanisms. In the present study, we investigated the toxic effects of long-term exposure to copper, zinc, and their mixture, and the relationship with the progression of neurotoxicity and neurodegeneration in a c. elegans-based HD model. Furthermore, we investigated the effects of rutin post metal exposure. Overall, we demonstrate that chronic exposure to the metals and their mixture altered body parameters, locomotion, and developmental delay, in addition to increasing polyQ protein aggregates in muscles and neurons causing neurodegeneration. We also propose that rutin has protective effects acting through mechanisms involving antioxidant and chelating properties. Altogether, our data provides new indications about the higher toxicity of metals in combination, the chelatin

关键词： c elegans Neurodegenerative diseases PolyQ Flavonoids Brazilian soil Metal mixture

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Regulation of electron transfer in the terminal step of the respiratory chain

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BIOcHEMIcAL SOcIETY TRANSAcTIONS 2023年第4期51卷 1611-1619页

作者： Sato, Wataru Ishimori, Koichiro Univ Hyogo Grad Sch Life Sci Dept Life Sci Ako Hyogo 6781297 Japan Hokkaido Univ Fac Sci Dept Chem Sapporo 0600810 Japan

In mitochondria, electrons are transferred along a series of enzymes and electron carriers that are referred to as the respiratory chain, leading to the synthesis of cellular ATP. The series of the interprotein electron transfer (ET) reactions is terminated by the reduction in molecular oxygen at complex IV, cytochrome c oxidase (ccO) that is coupled with the proton pumping from the matrix to the inner membrane space. Unlike the ET reactions from complex I to complex III, the ET reaction to ccO, mediated by cytochrome c (cyt c), is quite specific in that it is irreversible with suppressed electron leakage, which characterizes the ET reactions in the respiratory chain and is thought to play a key role in the regulation of mitochondrial respiration. In this review, we summarize the recent findings regarding the molecular mechanism of the ET reaction from cyt c to ccO in terms of specific interaction between two proteins, a molecular breakwater, and the effects of the conformational fluctuation on the ET reaction, conformational gating. Both of these are essential factors, not only in the ET reaction from cyt c to ccO, but also in the interprotein ET reactions in general. We also discuss the significance of a supercomplex in the terminal ET reaction, which provides information on the regulatory factors of the ET reactions that are specific to the mitochondrial respiratory chain.

关键词： breakwater complex IV conformational gating cytochrome c electron transfer supercomplex

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Toxicity potential evaluation of ethyl acetate extract of cymodocea serrulata against the mosquito vectors vis-a-vis zebrafish embryos and Artemia salina cysts

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SOUTH AFRIcAN JOURNAL OF BOTANY 2023年 152卷 230-239页

作者： Amutha, V. Aiswarya, D. Deepak, P. Selvaraj, R. Tamilselvan, c. Perumal, P. Balasubramani, G. Periyar Univ Sch Biosci Dept Biotechnol Salem 636011 Tamil Nadu India Biosci Res Fdn Kanchipuram 602002 Tamil Nadu India Dr NGP Arts & Sci Coll Coimbatore 641048 Tamil Nadu India Bharathidasan Univ Fac Fellow Sch Marine Sci Dept Marine Sci Trichy 620024 Tamil Nadu India Saveetha Inst Med & Tech Sci Thandalam Saveetha Sch Engn Dept Res & Innovat Chennai 602105 Tamil Nadu India

Of late, the advent of bioactive drug candidates from marine resources, particularly marine plants, has received more attention. Owing to the increased resistance in mosquitoes, the prevalence of deadly diseases is becoming tougher to treat. The purpose of this study was to determine the toxicity for Artemia salina and insecticide potential of the ethyl acetate (EA) extract of the seagrass cymodocea serrulata (cS) against Anophe-les, Aedes and culex mosquitoes. The seagrass extract showed the highest mortality rate against Anopheles ste-phensi with Lc50 and Lc90 values of 10.663 and 117.01, respectively, followed by Aedes aegypti 12.190 and 170.523, and culex quinquefasciatus 14.322 and 326.431 mg/mL. Larvae treated with c. serrulata ethyl acetate extract (cSEA) showed histological changes such as epithelial, muscular and adipose tissue. Biosafety assess-ments with cSEA extract on Danio rerio embryos and Artemia salina nauplii were found to be safe. The func-tional groups such as carboxylic acids, alkanes, alkynes, amides, phenolic and aromatic compounds from cSEA extract were identified by FTIR spectra. HPLc showed a strong peak at 2.518, and later eight compounds were identified by GcMS analysis. Among them, octadecadienoic acid is the main compound with 41.65%. In this experiment, we concluded that the seagrass c. serrulata has high mosquito larval control properties and negligible toxicity to non-target organisms. Therefore, this potential seagrass due to its extensive properties, can be expanded for the development of environmentally friendly pesticides against dengue, malaria, and fil-ariasis vectors.(c) 2022 SAAB. Published by Elsevier B.V. All rights reserved.

关键词： c serrulata Ethyl acetate extract Artemia salina Mosquito larvicides

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Anti-Oxidative and Anti-Aging Effects of Probiotic Fermented Ginseng by Modulating Gut Microbiota and Metabolites in caenorhabditis elegans

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PLANT FOODS FOR HUMAN NUTRITION 2023年第2期78卷 320-328页

作者： Xu, Huan-Yi Li, Quan-cen Zhou, Wen-Jie Zhang, Hai-Bo chen, Zhi-Xian Peng, Ning Gong, Shi-Yu Liu, Bin Zeng, Feng Fujian Agr & Forestry Univ Coll Food Sci Fuzhou 350002 Fujian Peoples R China Hubei Prov Key Lab Yeast Funct Yichang 443003 Peoples R China Univ Shanghai Sci & Technol Shanghai Engn Res Ctr Food Microbiol Sch Hlth Sci & Engn Shanghai 200093 Peoples R China

Antioxidative and antiaging abilities of probiotic fermented ginseng (PG) were evaluated in caenorhabditis elegans (c. elegans). Lifespan and effect on heat stress and acute oxidative stress in c. elegans were significantly enhanced by PG. Antioxidative enzymes such as T-SOD, GSH-PX, cAT were significantly up-regulated, and MDA, ROS and apoptosis levels were significantly down-regulated. At the same time, PG exerted antioxidant and anti-aging activities by reducing the expression of DAF-2 mRNA and increasing the expression of SKN-1 and SOD-3 mRNA in c. elegans. In addition, the mechanism of antioxidative and antiaging activities of PG was explored through gut microbiota sequencing and untargeted metabolomics. The results of gut microbiota indicated that PG could significantly improve the composition and structure of microbes in the gut of c. elegans, and the relative abundance of beneficial bacteria was up-regulated. Untargeted metabolomic results elucidated that PG modulated antioxidant and antiaging activities through neuroactive ligand-receptor interaction, citrate cycle (TcA cycle), pyruvate metabolism, ascorbate and aldarate metabolism and D-Arginine and D-ornithine metabolism of c. elegans. These results indicated that PG had excellent antioxidant and anti-aging activities, providing research value for the development of functional foods and improvement of aging-related diseases.

关键词： Probiotic fermented ginseng (PG) c elegans Anti-oxidative and anti-aging activity Gut microbiota Untargeted metabolomics

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