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TetR-like regulator BP1026B_II1561 controls aromatic amino acid biosynthesis and intracellular pathogenesis in Burkholderia pseudomallei [ChIP-seq]

ABSTRACT: TetR-like regulator BP1026B_II1561 controls aromatic amino acid biosynthesis and intracellular pathogenesis in Burkholderia pseudomallei [ChIP-seq]

PROVIDER: PRJNA1141961 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR30040494.fastq.gz	Fastqsanger.gz
	SRR30040495.fastq.gz	Fastqsanger.gz

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Project description:Its characteristic rose-like aroma makes phenylethanol a popular ingredient in foods, beverages and cosmetics. Microbial production of phenylethanol currently relies on whole-cell bioconversion of phenylalanine with yeasts that harbor an Ehrlich pathway for phenylalanine catabolism. Complete biosynthesis of phenylethanol from a cheap carbon source such as glucose provides an economically attractive alternative for phenylalanine bioconversion. In this study, a Synthetic Genetic Array screening was applied to identify genes involved in regulation of phenylethanol synthesis in Saccharomyces cerevisiae. The screen focused on transcriptional regulation of ARO10, which encodes the major decarboxylase involved in conversion of phenylpyruvate to phenylethanol. A deletion in ARO8, which encodes an aromatic amino acid transaminase, was found to cause a transcriptional upregulation of ARO10 during growth with ammonium sulfate as the sole nitrogen source. Physiological characterization revealed that the aro8M-oM-^AM-^D mutation led to substantial changes in the absolute and relative intracellular concentrations of amino acids. Moreover, deletion of ARO8 led to de novo production of phenylethanol during growth on a glucose synthetic medium with ammonium as the sole nitrogen source. The aro8 mutation also stimulated phenylethanol production when combined with other, previously documented mutations that deregulate aromatic amino acid biosynthesis in S. cerevisiae. The resulting engineered S. cerevisiae strain produced over 3 mM of phenylethanol from glucose during growth on a simple synthetic medium. The strong impact of a transaminase deletion on intracellular amino acid concentrations opens new possibilities for yeast-based production of amino acid-derived products. The goal of the present study was to identify genes that influence the transcriptional (de)repression of the Ehrlich pathway during growth with ammonium as the nitrogen source. With the aid of Synthetic Genetic Array technology, we constructed a strain collection in which deletions in the non-essential genes in the S. cerevisiae genome were combined with a reporter plasmid comprising the ARO10 promoter fused to a reporter gene (egfp) encoding a fluorescent reporter protein. After screening by flow cytometry, deletion of ARO8 led to a deregulated expression from the ARO10 promoter. The impact of this deletion was further studied by transcriptome and intracellular metabolite analyses. Furthermore, phenylethanol production was measured in strains that combined the aro8 mutation with mutations that were previously shown to deregulate aromatic amino acid biosynthesis.

Project description:Burkholderia pseudomallei (Bp) causes the tropical disease melioidosis that afflicts an estimated 165,000 people each year. Bp is a facultative intracellular pathogen that transits through distinct intracellular stages including attachment to host cells, invasion through the endocytic pathway, escape from the endosome, replication in the cytoplasm, generation of protrusions towards neighboring cells, and host cell fusion allowing Bp infection to spread without exiting the intracellular environment. We have identified a TetR-like transcriptional regulator, BP1026B_II1561, that is up-regulated during the late stages of infection as Bp protrudes toward neighboring cells. We have characterized BP1026B_II1561 and determined that it has a role in pathogenesis. A deletional mutant of BP1026B_II1561 is attenuated in RAW264.7 macrophage and BALB/c mouse models of infection. Using RNA-seq, we found that BP1026B_II1561 controls secondary metabolite biosynthesis, fatty acid degradation, and propanoate metabolism. In addition, we identified that BP1026B_II1561 directly controls expression of an outer membrane porin and genes in the shikimate biosynthetic pathway using ChIP-seq. Transposon mutants of genes within the BP1026B_II1561 regulon show defects during intracellular replication in RAW264.7 cells confirming the role of this transcriptional regulator and the pathways it controls in pathogenesis. BP1026B_II1561 also up-regulates the majority of the enzymes in shikimate and tryptophan biosynthetic pathways, suggesting their importance for Bp physiology. To investigate this, we tested fluorinated analogs of anthranilate and tryptophan, intermediates and products of the shikimate and tryptophan biosynthetic pathways, respectively, and showed inhibition of Bp growth at nanomolar concentrations. The expression of these pathways by BP1026b_II1561 and during intracellular infection combined with the inhibition of Bp growth by fluorotryptophan/anthranilate highlights these pathways as potential targets for therapeutic intervention against melioidosis. In the present study, we have identified BP1026B_II1561 as a critical transcriptional regulator for Bp pathogenesis and partially characterized its role during host cell infection.

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TetR-like regulator BP1026B_II1561 controls aromatic amino acid biosynthesis and intracellular pathogenesis in Burkholderia pseudomallei [ChIP-seq]

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