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1.
Commun Biol ; 4(1): 1027, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34471192

RESUMO

Lignin has significant potential as an abundant and renewable source for commodity chemicals yet remains vastly underutilized. Efforts towards engineering a biochemical route to the valorization of lignin are currently limited by the lack of a suitable heterologous host for the production of lignin-degrading enzymes. Here, we show that expression of fungal genes in Nicotiana benthamiana enables production of members from seven major classes of enzymes associated with lignin degradation (23 of 35 tested) in soluble form for direct use in lignin activity assays. We combinatorially characterized a subset of these enzymes in the context of model lignin dimer oxidation, revealing that fine-tuned coupling of peroxide-generators to peroxidases results in more extensive C-C bond cleavage compared to direct addition of peroxide. Comparison of peroxidase isoform activity revealed that the extent of C-C bond cleavage depends on peroxidase identity, suggesting that peroxidases are individually specialized in the context of lignin oxidation. We anticipate the use of N. benthamiana as a platform to rapidly produce a diverse array of fungal lignin-degrading enzymes will facilitate a better understanding of their concerted role in nature and unlock their potential for lignin valorization, including within the plant host itself.


Assuntos
Genes Fúngicos , Lignina/metabolismo , Nicotiana/metabolismo , Peroxidases/química , Plantas Geneticamente Modificadas/metabolismo , Peroxidases/genética , Peroxidases/metabolismo
2.
Metab Eng ; 30: 1-6, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25887638

RESUMO

Here we report recombinant expression and activity of several type I fatty acid synthases that can function in parallel with the native Escherichia coli fatty acid synthase. Corynebacterium glutamicum FAS1A was the most active in E. coli and this fatty acid synthase was leveraged to produce oleochemicals including fatty alcohols and methyl ketones. Coexpression of FAS1A with the ACP/CoA-reductase Maqu2220 from Marinobacter aquaeolei shifted the chain length distribution of fatty alcohols produced. Coexpression of FAS1A with FadM, FadB, and an acyl-CoA-oxidase from Micrococcus luteus resulted in the production of methyl ketones, although at a lower level than cells using the native FAS. This work, to our knowledge, is the first example of in vivo function of a heterologous fatty acid synthase in E. coli. Using FAS1 enzymes for oleochemical production have several potential advantages, and further optimization of this system could lead to strains with more efficient conversion to desired products. Finally, functional expression of these large enzyme complexes in E. coli will enable their study without culturing the native organisms.


Assuntos
Proteínas de Bactérias/biossíntese , Corynebacterium glutamicum/genética , Escherichia coli/metabolismo , Ácido Graxo Sintases/biossíntese , Ácidos Graxos/biossíntese , Marinobacter/genética , Micrococcus luteus/genética , Proteínas de Bactérias/genética , Corynebacterium glutamicum/enzimologia , Escherichia coli/genética , Ácido Graxo Sintases/genética , Ácidos Graxos/genética , Marinobacter/enzimologia , Micrococcus luteus/enzimologia
3.
PLoS One ; 7(8): e43566, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22952707

RESUMO

Natural variation in DNA sequence contributes to individual differences in quantitative traits. While multiple studies have shown genetic control over gene expression variation, few additional cellular traits have been investigated. Here, we investigated the natural variation of NADPH oxidase-dependent hydrogen peroxide (H(2)O(2) release), which is the joint effect of reactive oxygen species (ROS) production, superoxide metabolism and degradation, and is related to a number of human disorders. We assessed the normal variation of H(2)O(2) release in lymphoblastoid cell lines (LCL) in a family-based 3-generation cohort (CEPH-HapMap), and in 3 population-based cohorts (KORA, GenCord, HapMap). Substantial individual variation was observed, 45% of which were associated with heritability in the CEPH-HapMap cohort. We identified 2 genome-wide significant loci of Hsa12 and Hsa15 in genome-wide linkage analysis. Next, we performed genome-wide association study (GWAS) for the combined KORA-GenCord cohorts (n = 279) using enhanced marker resolution by imputation (>1.4 million SNPs). We found 5 significant associations (p<5.00×10-8) and 54 suggestive associations (p<1.00×10-5), one of which confirmed the linked region on Hsa15. To replicate our findings, we performed GWAS using 58 HapMap individuals and ∼2.1 million SNPs. We identified 40 genome-wide significant and 302 suggestive SNPs, and confirmed genome signals on Hsa1, Hsa12, and Hsa15. Genetic loci within 900 kb from the known candidate gene p67phox on Hsa1 were identified in GWAS in both cohorts. We did not find replication of SNPs across all cohorts, but replication within the same genomic region. Finally, a highly significant decrease in H(2)O(2) release was observed in Down Syndrome (DS) individuals (p<2.88×10-12). Taken together, our results show strong evidence of genetic control of H(2)O(2) in LCL of healthy and DS cohorts and suggest that cellular phenotypes, which themselves are also complex, may be used as proxies for dissection of complex disorders.


Assuntos
Peróxido de Hidrogênio/química , Adulto , Idoso , Linhagem Celular Tumoral , Estudos de Coortes , Ligação Genética , Genoma Humano , Estudo de Associação Genômica Ampla , Humanos , Peróxido de Hidrogênio/metabolismo , Recém-Nascido , Pessoa de Meia-Idade , Modelos Genéticos , NADPH Oxidases/metabolismo , Fenótipo , Polimorfismo de Nucleotídeo Único , Espécies Reativas de Oxigênio , Análise de Sequência de DNA
4.
Genome Res ; 19(8): 1471-9, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19581486

RESUMO

Recent studies have demonstrated extensive transcriptional activity across the human genome, a substantial fraction of which is not associated with any functional annotation. However, very little is known regarding the post-transcriptional processes that operate within the different classes of RNA molecules. To characterize the post-transcriptional properties of expressed sequences from human chromosome 21 (HSA21), we separated RNA molecules from three cell lines (GM06990, HeLa S3, and SK-N-AS) according to their ribosome content by sucrose gradient fractionation. Polyribosomal-associated RNA and total RNA were subsequently hybridized to genomic tiling arrays. We found that approximately 50% of the transcriptional signals were located outside of annotated exons and were considered as TARs (transcriptionally active regions). Although TARs were observed among polysome-associated RNAs, RT-PCR and RACE experiments revealed that approximately 40% were likely to represent nonspecific cross-hybridization artifacts. Bioinformatics discrimination of TARs according to conservation and sequence complexity allowed us to identify a set of high-confidence TARs. This set of TARs was significantly depleted in the polysomes, suggesting that it was not likely to be involved in translation. Analysis of polysome representation of RefSeq exons showed that at least 15% of RefSeq transcripts undergo significant post-transcriptional regulation in at least two of the three cell lines tested. Among the regulated transcripts, enrichment analysis revealed an over-representation of genes involved in Alzheimer's disease (AD), including APP and the BACE1 protease that cleaves APP to produce the pathogenic beta 42 peptide. We demonstrate that the combination of RNA fractionation and tiling arrays is a powerful method to assess the transcriptional and post-transcriptional properties of genomic regions.


Assuntos
Cromossomos Humanos Par 21/genética , Perfilação da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Transcrição Gênica/genética , Regiões 5' não Traduzidas/genética , Fracionamento Celular/métodos , Linhagem Celular Transformada , Linhagem Celular Tumoral , Centrifugação com Gradiente de Concentração , Genômica/métodos , Células HeLa , Humanos , Polirribossomos/metabolismo , RNA/genética , RNA/isolamento & purificação , RNA/metabolismo , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa
5.
Reprod Toxicol ; 28(4): 536-41, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19549566

RESUMO

Some steps of the assisted reproduction techniques, such as superovulation, may interfere with imprinting reprogramming. In the present study, superovulation was induced in the mouse and its possible effects on the differentially methylated domains of 2 paternally (H19 and Gtl2) and 3 maternally (Peg1, Snrpn and Peg3) imprinted genes were tested in the male offspring over 2 generations. The CpGs methylation status was analyzed by pyro- and bisulfite sequencing. In liver, skeletal muscle and tail, no effect of superovulation could be observed. In the sperm, however, a significant 6% decrease in the number of methylated CpGs of H19 and significant 2.8- and 7.0-fold increases in those of Peg1 and Snrpn, respectively were observed following superovulation. The changes were still present in the H19 and Snrpn genes of the second generation offspring. This suggests that superovulation in the mother transgenerationally affects the offspring sperm methylation pattern.


Assuntos
Metilação de DNA , Impressão Genômica , Espermatozoides/metabolismo , Superovulação/genética , Animais , Ilhas de CpG , Masculino , Camundongos , Camundongos Endogâmicos , Proteínas Centrais de snRNP
6.
Nucleic Acids Res ; 36(21): 6918-25, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18978018

RESUMO

Variation in cellular gene expression levels has been shown to be inherited. Expression is controlled at transcriptional and post-transcriptional levels. Internal ribosome entry sites (IRES) are used by viruses to bypass inhibition of cap-dependent translation, and by eukaryotic cells to control translation under conditions when protein synthesis is inhibited. We aimed at identifying genomic determinants of variability in IRES-mediated translation of viral [Encephalomyocarditis virus (EMCV)] and cellular IRES [X-linked inhibitor-of-apoptosis (XIAP) and c-myc]. Bicistronic lentiviral constructs expressing two fluorescent reporters were used to transduce laboratory and B lymphoblastoid cell lines [15 CEPH pedigrees (n = 205) and 50 unrelated individuals]. IRES efficiency varied according to cell type and among individuals. Control of IRES activity has a significant genetic component (h(2) of 0.47 and 0.36 for EMCV and XIAP, respectively). Quantitative linkage analysis identified a suggestive locus (LOD 2.35) on chromosome 18q21.2, and genome-wide association analysis revealed of a cluster of SNPs on chromosome 3, intronic to the FHIT gene, marginally associated (P = 5.9E-7) with XIAP IRES function. This study illustrates the in vitro generation of intermediate phenotypes by using cell lines for the evaluation of genetic determinants of control of elements such as IRES.


Assuntos
Regiões 5' não Traduzidas , Biossíntese de Proteínas , Locos de Características Quantitativas , Linhagem Celular , Vírus da Encefalomiocardite/genética , Ligação Genética , Estudo de Associação Genômica Ampla , Humanos , Proteínas Proto-Oncogênicas c-myc/genética , RNA Viral/química , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética
7.
Am J Hum Genet ; 81(2): 252-63, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17668376

RESUMO

Down syndrome (DS) is characterized by extensive phenotypic variability, with most traits occurring in only a fraction of affected individuals. Substantial gene-expression variation is present among unaffected individuals, and this variation has a strong genetic component. Since DS is caused by genomic-dosage imbalance, we hypothesize that gene-expression variation of human chromosome 21 (HSA21) genes in individuals with DS has an impact on the phenotypic variability among affected individuals. We studied gene-expression variation in 14 lymphoblastoid and 17 fibroblast cell lines from individuals with DS and an equal number of controls. Gene expression was assayed using quantitative real-time polymerase chain reaction on 100 and 106 HSA21 genes and 23 and 26 non-HSA21 genes in lymphoblastoid and fibroblast cell lines, respectively. Surprisingly, only 39% and 62% of HSA21 genes in lymphoblastoid and fibroblast cells, respectively, showed a statistically significant difference between DS and normal samples, although the average up-regulation of HSA21 genes was close to the expected 1.5-fold in both cell types. Gene-expression variation in DS and normal samples was evaluated using the Kolmogorov-Smirnov test. According to the degree of overlap in expression levels, we classified all genes into 3 groups: (A) nonoverlapping, (B) partially overlapping, and (C) extensively overlapping expression distributions between normal and DS samples. We hypothesize that, in each cell type, group A genes are the most dosage sensitive and are most likely involved in the constant DS traits, group B genes might be involved in variable DS traits, and group C genes are not dosage sensitive and are least likely to participate in DS pathological phenotypes. This study provides the first extensive data set on HSA21 gene-expression variation in DS and underscores its role in modulating the outcome of gene-dosage imbalance.


Assuntos
Síndrome de Down/genética , Dosagem de Genes , Variação Genética , Aneuploidia , Linhagem Celular , Transformação Celular Viral , Cromossomos Humanos Par 21 , Fibroblastos , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Linfócitos , Reação em Cadeia da Polimerase Via Transcriptase Reversa
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