Identification of an unusual type II thioesterase in the dithiolopyrrolone antibiotics biosynthetic pathway

Zhai, Ying; Bai, Silei; Liu, Jingjing; Yang, Liyuan; Han, Li; Huang, Xueshi; He, Jialin

doi:10.1016/j.bbrc.2016.03.105

Cited by 11 publications

(14 citation statements)

References 31 publications

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“…Using a flow cell (see Materials and Methods; 0.5 mL/min DMEM), supplements of 10 mM pyruvate and then 10 mM lactate were used to set the fully oxidized and fully reduced limits of the sensor. This strategy has been widely employed ,, because intracellular lactate dehydrogenase catalyzes a rapid equilibration of lactate/pyruvate and NADH/NAD + pools: …”

Section: Resultsmentioning

confidence: 99%

“…Using a flow cell (see Materials and Methods; 0.5 mL/min DMEM), supplements of 10 mM pyruvate and then 10 mM lactate were used to set the fully oxidized and fully reduced limits of the sensor. This strategy has been widely employed 11,23,68 The data in Figure 11A shows a collection of cells in the flow cell, and the average of 5 experiments (see Materials and Methods) is shown in panel B. In each case the lactate wash was replaced with DMEM medium alone to assess the steady state of the intracellular NAD + /NADH pool, prior to a return to the limiting oxidized state upon reintroduction of pyruvate.…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

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Designing Flavoprotein-GFP Fusion Probes for Analyte-Specific Ratiometric Fluorescence Imaging

Hudson

Caplan²,

Thorpe

2018

Biochemistry

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The development of genetically encoded fluorescent probes for analyte-specific imaging has revolutionized our understanding of intracellular processes. Current classes of intracellular probes depend on the selection of binding domains that either undergo conformational changes on analyte binding or can be linked to thiol redox chemistry. Here we have designed novel probes by fusing a flavoenzyme, whose fluorescence is quenched on reduction by the analyte of interest, with a GFP domain to allow for rapid and specific ratiometric sensing. Two flavoproteins, Escherichia coli thioredoxin reductase and Saccharomyces cerevisiae lipoamide dehydrogenase, were successfully developed into thioredoxin and NAD/NADH specific probes, respectively, and their performance was evaluated in vitro and in vivo. A flow cell format, which allowed dynamic measurements, was utilized in both bacterial and mammalian systems. In E. coli the first reported intracellular steady-state of the cytoplasmic thioredoxin pool was measured. In HEK293T mammalian cells, the steady-state cytosolic ratio of NAD/NADH induced by glucose was determined. These genetically encoded fluorescent constructs represent a modular approach to intracellular probe design that should extend the range of metabolites that can be quantitated in live cells.

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“…Using a flow cell (see Materials and Methods; 0.5 mL/min DMEM), supplements of 10 mM pyruvate and then 10 mM lactate were used to set the fully oxidized and fully reduced limits of the sensor. This strategy has been widely employed ,, because intracellular lactate dehydrogenase catalyzes a rapid equilibration of lactate/pyruvate and NADH/NAD + pools: …”

Section: Resultsmentioning

confidence: 99%

“…Using a flow cell (see Materials and Methods; 0.5 mL/min DMEM), supplements of 10 mM pyruvate and then 10 mM lactate were used to set the fully oxidized and fully reduced limits of the sensor. This strategy has been widely employed 11,23,68 The data in Figure 11A shows a collection of cells in the flow cell, and the average of 5 experiments (see Materials and Methods) is shown in panel B. In each case the lactate wash was replaced with DMEM medium alone to assess the steady state of the intracellular NAD + /NADH pool, prior to a return to the limiting oxidized state upon reintroduction of pyruvate.…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

Designing Flavoprotein-GFP Fusion Probes for Analyte-Specific Ratiometric Fluorescence Imaging

Hudson

Caplan²,

Thorpe

2018

Biochemistry

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“…(Yang et al, 2007). The potent dithiolopyrrolone antibiotic thiomarinol A was also readily detected in the cotton-containing cultures of P. luteoviolacea 2ta16 and was undetectable in standard cultures as seen in Figure 2 (p-value 6.0 × 10 −4 , deemed significant) The thiomarinols are produced by a number of Pseudoalteromonas strains including P. luteoviolacea 2ta16 (Maansson et al, 2016;Murphy et al, 2014;Shiozawa et al, 1993) and although there has been considerable investigation of their biosynthesis (Dunn, Wever, Economou, Bowers, & Li, 2015;Qin, Huang, Yu, & Deng, 2013;Zhai et al, 2016), to the best of our knowledge there have not been reports on the regulation of this biosynthetic pathway or exploration of thiomarinol chemical ecology. Using cotton scaffolds to stimulate molecule production may facilitate examination of these unanswered questions.…”

Section: Pseudoalteromonas Luteoviolacea 2ta16mentioning

confidence: 98%

Culturing marine bacteria from the genus Pseudoalteromonas on a cotton scaffold alters secondary metabolite production

et al. 2018

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The discovery of secondary metabolites from marine microorganisms is beset by numerous challenges including difficulties cultivating and subsequently eliciting expression of biosynthetic genes from marine microbes in the laboratory. In this paper, we describe a method of culturing three species from the marine bacterial genus Pseudoalteromonas using cotton scaffold supplemented liquid media. This simple cultivation method was designed to mimic the natural behavior of some members of the genus wherein they form epibiotic/symbiotic associations with higher organisms such as sponges and corals or attach to solid structures as a biofilm. Our scaffolded cultivation is highly effective at stimulating an attachment/biofilm phenotype and causes large changes to metabolite profiles for the microbes investigated. Metabolite changes include alteration to the production levels of known molecules such as violacein, thiomarinol A, and the alterochromide and prodiginine families of molecules. Finally and critically, our technique stimulates the production of unknown compounds that will serve as leads for future natural product discovery. These results suggest our cultivation approach could potentially be used as a general strategy for the activation of silent gene clusters in marine microbes to facilitate access to their full natural product biosynthetic capacity.

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“…The comparison of the aut gene cluster with holomycin ( hlm ) gene cluster from S. clavuligerus showed the aut gene cluster lacks the hlmK gene which encodes a distinctive type II thioesterase. The production of dithiolopyrrolone in S. albus was greatly improved through the co-expression of hlmK (Zhai et al, 2016). …”

Section: Streptomyces Albus As a Heterologous Hostmentioning

confidence: 99%

Streptomycetes: Surrogate hosts for the genetic manipulation of biosynthetic gene clusters and production of natural products

Nepal

Wang

2019

Biotechnology Advances

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Due to the worldwide prevalence of multidrug-resistant pathogens and high incidence of diseases such as cancer, there is an urgent need for the discovery and development of new drugs. Nearly half of the FDA-approved drugs are derived from natural products that are produced by living organisms, mainly bacteria, fungi, and plants. Commercial development is often limited by the low yield of the desired compounds expressed by the native producers. In addition, recent advances in whole genome sequencing and bioinformatics have revealed an abundance of cryptic biosynthetic gene clusters within microbial genomes. Genetic manipulation of clusters in the native host is commonly used to awaken poorly expressed or silent gene clusters, however, the lack of feasible genetic manipulation systems in many strains often hinders our ability to engineer the native producers. The transfer of gene clusters into heterologous hosts for expression of partial or entire biosynthetic pathways is an approach that can be used to overcome this limitation. Heterologous expression also facilitates the chimeric fusion of different biosynthetic pathways, leading to the generation of “unnatural” natural products. The genus Streptomyces is especially known to be a prolific source of drugs/antibiotics, its members are often used as heterologous expression hosts. In this review, we summarize recent applications of Streptomyces species, S. coelicolor, S. lividans, S. albus, S. venezuelae and S. avermitilis, as heterologous expression systems.

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Identification of an unusual type II thioesterase in the dithiolopyrrolone antibiotics biosynthetic pathway

Cited by 11 publications

References 31 publications

Designing Flavoprotein-GFP Fusion Probes for Analyte-Specific Ratiometric Fluorescence Imaging

Designing Flavoprotein-GFP Fusion Probes for Analyte-Specific Ratiometric Fluorescence Imaging

Culturing marine bacteria from the genus Pseudoalteromonas on a cotton scaffold alters secondary metabolite production

Streptomycetes: Surrogate hosts for the genetic manipulation of biosynthetic gene clusters and production of natural products

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