RESUMO
Clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas9 is an effector protein that targets invading DNA and plays a major role in the prokaryotic adaptive immune system. Although Streptococcus pyogenes CRISPR-Cas9 has been widely studied and repurposed for applications including genome editing, its origin and evolution are poorly understood. Here, we investigate the evolution of Cas9 from resurrected ancient nucleases (anCas) in extinct firmicutes species that last lived 2.6 billion years before the present. We demonstrate that these ancient forms were much more flexible in their guide RNA and protospacer-adjacent motif requirements compared with modern-day Cas9 enzymes. Furthermore, anCas portrays a gradual palaeoenzymatic adaptation from nickase to double-strand break activity, exhibits high levels of activity with both single-stranded DNA and single-stranded RNA targets and is capable of editing activity in human cells. Prediction and characterization of anCas with a resurrected protein approach uncovers an evolutionary trajectory leading to functionally flexible ancient enzymes.
Assuntos
Sistemas CRISPR-Cas , Endonucleases , Firmicutes , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Endonucleases/genética , Endonucleases/metabolismo , Edição de Genes , Firmicutes/enzimologia , Firmicutes/genética , RNA Guia de Sistemas CRISPR-CasRESUMO
BACKGROUND: PrsA is an extracytoplasmic folding catalyst essential in Bacillus subtilis. Overexpression of the native PrsA from B. subtilis has repeatedly lead to increased amylase yields. Nevertheless, little is known about how the overexpression of heterologous PrsAs can affect amylase secretion. RESULTS: In this study, the final yield of five extracellular alpha-amylases was increased by heterologous PrsA co-expression up to 2.5 fold. The effect of the overexpression of heterologous PrsAs on alpha-amylase secretion is specific to the co-expressed alpha-amylase. Co-expression of a heterologous PrsA can significantly reduce the secretion stress response. Engineering of the B. licheniformis PrsA lead to a further increase in amylase secretion and reduced secretion stress. CONCLUSIONS: In this work we show how heterologous PrsA overexpression can give a better result on heterologous amylase secretion than the native PrsA, and that PrsA homologs show a variety of specificity towards different alpha-amylases. We also demonstrate that on top of increasing amylase yield, a good PrsA-amylase pairing can lower the secretion stress response of B. subtilis. Finally, we present a new recombinant PrsA variant with increased performance in both supporting amylase secretion and lowering secretion stress.
Assuntos
Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Proteínas de Bactérias/metabolismo , Lipoproteínas/metabolismo , Proteínas de Membrana/metabolismo , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , alfa-Amilases/metabolismo , Proteínas de Bactérias/genética , Lipoproteínas/genética , Proteínas de Membrana/genética , Engenharia Metabólica , Peptidilprolil Isomerase de Interação com NIMA/genética , Dobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , alfa-Amilases/químicaRESUMO
The field of systems biology has been rapidly developing in the past decade. However, the data produced by "omics" approaches is lagging behind the requirements of this field, especially when it comes to absolute abundances of membrane proteins. In the present study, a novel approach for large-scale absolute quantification of this challenging subset of proteins has been established and evaluated using osmotic stress management in the Gram-positive model bacterium Bacillus subtilis as proof-of-principle precedent. Selected membrane proteins were labeled using a SNAP-tag, which allowed us to visually inspect the enrichment of the membrane fraction by immunoassays. Absolute membrane protein concentrations were determined via shotgun proteomics by spiking crude membrane extracts of chromosomally SNAP-tagged and wild-type B. subtilis strains with protein standards of known concentration. Shotgun data was subsequently calibrated by targeted mass spectrometry using SNAP as an anchor protein, and an enrichment factor was calculated in order to obtain membrane protein copy numbers per square micrometer. The presented approach enabled the accurate determination of physiological changes resulting from imposed hyperosmotic stress, thereby offering a clear visualization of alterations in membrane protein arrangements and shedding light on putative membrane complexes. This straightforward and cost-effective methodology for quantitative proteome studies can be implemented by any research group with mass spectrometry expertise. Importantly, it can be applied to the full spectrum of physiologically relevant conditions, ranging from environmental stresses to the biotechnological production of small molecules and proteins, a field heavily relying on B. subtilis secretion capabilities.
