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1.
Biochemistry ; 59(16): 1630-1639, 2020 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-32250597

RESUMO

α/ß hydrolases make up a large and diverse protein superfamily. In natural product biosynthesis, cis-acting thioesterase α/ß hydrolases can terminate biosynthetic assembly lines and release products by hydrolyzing or cyclizing the biosynthetic intermediate. Thioesterases can also act in trans, removing aberrant intermediates and restarting stalled biosynthesis. Knockout of this "editing" function leads to reduced product titers. The borrelidin biosynthetic gene cluster from Streptomyces parvulus Tü4055 contains a hitherto uncharacterized stand-alone thioesterase, borB. In this work, we demonstrate that purified BorB cleaves acyl substrates with a preference for propionate, which supports the hypothesis that it is also an editing thioesterase. The crystal structure of BorB shows a wedgelike hydrophobic substrate binding crevice that limits substrate length. To investigate the structure-function relationship, we made chimeric BorB variants using loop regions from characterized homologues with different specificities. BorB chimeras slightly reduced activity, arguing that the modified region is a not major determinant of substrate preference. The structure-function relationships described here contribute to the process of elimination for understanding thioesterase specificity and, ultimately, engineering and applying trans-acting thioesterases in biosynthetic assembly lines.


Assuntos
Proteínas de Bactérias/química , Ácido Graxo Sintases/química , Streptomyces/enzimologia , Tioléster Hidrolases/química , Proteínas de Bactérias/genética , Domínio Catalítico , Ácido Graxo Sintases/genética , Cinética , Família Multigênica , Engenharia de Proteínas , Especificidade por Substrato , Tioléster Hidrolases/genética
2.
PLoS One ; 14(1): e0210243, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30653574

RESUMO

Aspergillus niger and other filamentous fungi are widely used in industry, but efficient genetic engineering of these hosts remains nascent. For example, while molecular genetic tools have been developed, including CRISPR/Cas9, facile genome engineering of A. niger remains challenging. To address these challenges, we have developed a simple Cas9-based gene targeting method that provides selectable, iterative, and ultimately marker-free generation of genomic deletions and insertions. This method leverages locus-specific "pop-out" recombination to suppress off-target integrations. We demonstrated the effectiveness of this method by targeting the phenotypic marker albA and validated it by targeting the glaA and mstC loci. After two selection steps, we observed 100% gene editing efficiency across all three loci. This method greatly reduces the effort required to engineer the A. niger genome and overcomes low Cas9 transformations efficiency by eliminating the need for extensive screening. This method represents a significant addition to the A. niger genome engineering toolbox and could be adapted for use in other organisms. It is expected that this method will impact several areas of industrial biotechnology, such as the development of new strains for the secretion of heterologous enzymes and the discovery and optimization of metabolic pathways.


Assuntos
Aspergillus niger/genética , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Genoma Fúngico/genética , Marcação de Genes , Genômica
3.
mBio ; 10(3)2019 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-31064836

RESUMO

Despite intensive study for 50 years, the biochemical and genetic links between lysine metabolism and central metabolism in Pseudomonas putida remain unresolved. To establish these biochemical links, we leveraged random barcode transposon sequencing (RB-TnSeq), a genome-wide assay measuring the fitness of thousands of genes in parallel, to identify multiple novel enzymes in both l- and d-lysine metabolism. We first describe three pathway enzymes that catabolize l-2-aminoadipate (l-2AA) to 2-ketoglutarate (2KG), connecting d-lysine to the TCA cycle. One of these enzymes, P. putida 5260 (PP_5260), contains a DUF1338 domain, representing a family with no previously described biological function. Our work also identified the recently described coenzyme A (CoA)-independent route of l-lysine degradation that results in metabolization to succinate. We expanded on previous findings by demonstrating that glutarate hydroxylase CsiD is promiscuous in its 2-oxoacid selectivity. Proteomics of selected pathway enzymes revealed that expression of catabolic genes is highly sensitive to the presence of particular pathway metabolites, implying intensive local and global regulation. This work demonstrated the utility of RB-TnSeq for discovering novel metabolic pathways in even well-studied bacteria, as well as its utility a powerful tool for validating previous research.IMPORTANCEP. putida lysine metabolism can produce multiple commodity chemicals, conferring great biotechnological value. Despite much research, the connection of lysine catabolism to central metabolism in P. putida remained undefined. Here, we used random barcode transposon sequencing to fill the gaps of lysine metabolism in P. putida We describe a route of 2-oxoadipate (2OA) catabolism, which utilizes DUF1338-containing protein P. putida 5260 (PP_5260) in bacteria. Despite its prevalence in many domains of life, DUF1338-containing proteins have had no known biochemical function. We demonstrate that PP_5260 is a metalloenzyme which catalyzes an unusual route of decarboxylation of 2OA to d-2-hydroxyglutarate (d-2HG). Our screen also identified a recently described novel glutarate metabolic pathway. We validate previous results and expand the understanding of glutarate hydroxylase CsiD by showing that can it use either 2OA or 2KG as a cosubstrate. Our work demonstrated that biological novelty can be rapidly identified using unbiased experimental genetics and that RB-TnSeq can be used to rapidly validate previous results.


Assuntos
Aptidão Genética , Lisina/metabolismo , Pseudomonas putida/enzimologia , Pseudomonas putida/genética , Redes e Vias Metabólicas
4.
ACS Chem Biol ; 13(8): 2261-2268, 2018 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-29912551

RESUMO

In the search for molecular machinery for custom biosynthesis of valuable compounds, the modular type I polyketide synthases (PKSs) offer great potential. In this study, we investigate the flexibility of BorM5, the iterative fifth module of the borrelidin synthase, with a panel of non-native priming substrates in vitro. BorM5 differentially extends various aliphatic and substituted substrates. Depending on substrate size and substitution BorM5 can exceed the three iterations it natively performs. To probe the effect of methyl branching on chain length regulation, we engineered a BorM5 variant capable of incorporating methylmalonyl- and malonyl-CoA into its intermediates. Intermediate methylation did not affect overall chain length, indicating that the enzyme does not to count methyl branches to specify the number of iterations. In addition to providing regulatory insight about BorM5, we produced dozens of novel methylated intermediates that might be used for production of various hydrocarbons or pharmaceuticals. These findings enable rational engineering and recombination of BorM5 and inform the study of other iterative modules.


Assuntos
Policetídeo Sintases/metabolismo , Streptomyces/enzimologia , Clonagem Molecular , Escherichia coli/genética , Álcoois Graxos/metabolismo , Malonil Coenzima A/metabolismo , Metilação , Policetídeo Sintases/genética , Engenharia de Proteínas , Streptomyces/genética , Streptomyces/metabolismo , Especificidade por Substrato
6.
Nat Cell Biol ; 17(8): 1049-61, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26147250

RESUMO

The TOR (target of rapamycin) kinase limits longevity by poorly understood mechanisms. Rapamycin suppresses the mammalian TORC1 complex, which regulates translation, and extends lifespan in diverse species, including mice. We show that rapamycin selectively blunts the pro-inflammatory phenotype of senescent cells. Cellular senescence suppresses cancer by preventing cell proliferation. However, as senescent cells accumulate with age, the senescence-associated secretory phenotype (SASP) can disrupt tissues and contribute to age-related pathologies, including cancer. MTOR inhibition suppressed the secretion of inflammatory cytokines by senescent cells. Rapamycin reduced IL6 and other cytokine mRNA levels, but selectively suppressed translation of the membrane-bound cytokine IL1A. Reduced IL1A diminished NF-κB transcriptional activity, which controls much of the SASP; exogenous IL1A restored IL6 secretion to rapamycin-treated cells. Importantly, rapamycin suppressed the ability of senescent fibroblasts to stimulate prostate tumour growth in mice. Thus, rapamycin might ameliorate age-related pathologies, including late-life cancer, by suppressing senescence-associated inflammation.


Assuntos
Interleucina-1alfa/metabolismo , Neoplasias da Próstata/enzimologia , Serina-Treonina Quinases TOR/metabolismo , Animais , Anti-Inflamatórios/farmacologia , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células , Senescência Celular , Relação Dose-Resposta a Droga , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Regulação Neoplásica da Expressão Gênica , Humanos , Mediadores da Inflamação/metabolismo , Interleucina-1alfa/genética , Interleucina-6/metabolismo , Masculino , Camundongos SCID , Mitoxantrona/farmacologia , NF-kappa B/metabolismo , Fenótipo , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Interferência de RNA , RNA Mensageiro/metabolismo , Sirolimo/metabolismo , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/genética , Fatores de Tempo , Transcrição Gênica , Transfecção , Carga Tumoral , Regulação para Cima , Ensaios Antitumorais Modelo de Xenoenxerto
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