Oxidative Pathways of Deoxyribose and Deoxyribonate Catabolism

Price, Morgan N.; J, Ray; Iavarone, Anthony T.; Carlson, Hans K.; Ryan, Elizabeth; Malmstrom, Rex R.; Arkin, Adam P.; Deutschbauer, Adam M.

doi:10.1128/msystems.00297-18

Cited by 29 publications

(29 citation statements)

References 43 publications

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“…While aromatic catabolism has been extensively studied in P. putida , genes implicated in these pathways are still being discovered [15]. The genes involved in the first steps of coumarate catabolism reside in an operon with a putative acyl-CoA dehydrogenase ( PP_3354 ) and a putative beta-ketothiolase ( PP_3355 ), which have been proposed to be involved in an alternative catabolic pathway [16].…”

Section: Resultsmentioning

confidence: 99%

Leveraging host metabolism for bisdemethoxycurcumin production in Pseudomonas putida

Incha

Thompson

Blake-Hedges

et al. 2019

Preprint

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31Pseudomonas putida is a saprophytic bacterium with robust carbon metabolisms and 32 strong solvent tolerance making it an attractive host for metabolic engineering and 33 bioremediation. Due to its diverse carbon metabolisms, its genome encodes an array of proteins 34 and enzymes that can be readily applied to produce valuable products. In this work we sought to 35 identify design principles and bottlenecks in the production of type III polyketide synthase 36 (T3PKS)-derived compounds in P. putida. T3PKS products are widely used as nutraceuticals and 37 medicines and often require aromatic starter units, such as coumaroyl-CoA, which is also an 38 intermediate in the native coumarate catabolic pathway of P. putida. Using a randomly barcoded 39 transposon mutant (RB-TnSeq) library, we assayed gene functions for a large portion of aromatic 40 catabolism, confirmed known pathways, and proposed new annotations for two aromatic 41 transporters. The tetrahydroxynapthalene synthase of Streptomyces coelicolor (RppA), a 42 microbial T3PKS, was then used to rapidly assay growth conditions for increased T3PKS 43 product accumulation. The feruloyl/coumaroyl CoA synthetase (Fcs) of P. putida was used to 44 supply coumaroyl-CoA for the curcuminoid synthase (CUS) of Oryza sativa, a plant T3PKS. We 45 identified that accumulation of coumaroyl-CoA in this pathway results in extended growth lag 46 times in P. putida. Deletion of the second step in coumarate catabolism, the enoyl-CoA 47 hydratase lyase (Ech), resulted in 'drop-in' production of the type III polyketide 48 bisdemethoxycurcumin. 49 1 INTRODUCTION 50 Secondary metabolites of fungi, plants, and bacteria have been used as medicines and 51 supplements for millennia [1]. Compounds such as naringenin, raspberry ketone, resveratrol, and 52 curcumin are widely used as nutraceuticals and are biosynthesized through similar pathways 53 3 [2,3]. Commercially, these chemicals are either extracted directly from plants or produced 54 synthetically, as in the case of raspberry ketone [4]. Renewable microbial production of these 55 compounds will decrease reliance on agriculture and fossil fuel-derived chemical synthesis. The 56 biosynthesis of these compounds (naringenin, raspberry ketone, resveratrol, and curcumin) relies 57 on a class of enzymes called type III polyketide synthases (T3PKSs). T3PKSs carry out iterative 58 Claisen condensation reactions typically with coenzyme A (CoA)-based starter and extender 59 units, both of which vary widely among these enzymes [5]. In the case of the 60 tetrahydroxynapthalene synthase of Streptomyces coelicolor (RppA) the starter and extender 61 units are simply malonyl-CoA, while in many plant T3PKSs the starter unit is a 62 phenylpropenoyl-CoA thioester, usually derived from ferulate, coumarate, or cinnamate [6,7]. 63Coumarate and ferulate are components of lignin found in lignocellulosic hydrolysate 64 (LH), which has been proposed for use as a renewable feedstock for biocatalysis [8,9]. However, 65 the limited capabilities of commonl...

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Section: Resultsmentioning

confidence: 99%

Leveraging host metabolism for bisdemethoxycurcumin production in Pseudomonas putida

Incha

Thompson

Blake-Hedges

et al. 2019

Preprint

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“…6). Previous work has reported potential routes of capsaicin catabolism (37,38), but current LC-time of flight (TOF)-MS analysis of spent medium could not detect specific masses consistent with predicted degradation products, such as vanillylamine, vanillin, or vanillate ( Fig. S2A).…”

Section: Figmentioning

confidence: 94%

Fungal Seed Pathogens of Wild Chili Peppers Possess Multiple Mechanisms To Tolerate Capsaicinoids

Adams

Zimmerman

Fenstermacher

et al. 2020

Appl Environ Microbiol

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The wild chili pepper Capsicum chacoense produces the spicy defense compounds known as capsaicinoids, including capsaicin and dihydrocapsaicin, which are antagonistic to the growth of fungal pathogens. Compared to other microbes, fungi isolated from infected seeds of C. chacoense possess much higher levels of tolerance of these spicy compounds, having their growth slowed but not entirely inhibited. Previous research has shown capsaicinoids inhibit microbes by disrupting ATP production by binding NADH dehydrogenase in the electron transport chain (ETC) and, thus, throttling oxidative phosphorylation (OXPHOS). Capsaicinoids may also disrupt cell membranes. Here, we investigate capsaicinoid tolerance in fungal seed pathogens isolated from C. chacoense. We selected 16 fungal isolates from four ascomycete genera (Alternaria, Colletotrichum, Fusarium, and Phomopsis). Using relative growth rate as a readout for tolerance, fungi were challenged with ETC inhibitors to infer whether fungi possess alternative respiratory enzymes and whether effects on the ETC fully explained inhibition by capsaicinoids. In all isolates, we found evidence for at least one alternative NADH dehydrogenase. In many isolates, we also found evidence for an alternative oxidase. These data suggest that wild-plant pathogens may be a rich source of alternative respiratory enzymes. We further demonstrate that these fungal isolates are capable of the breakdown of capsaicinoids. Finally, we determine that the OXPHOS theory may describe a weak primary mechanism by which dihydrocapsaicin, but not capsaicin, slows fungal growth. Our findings suggest that capsaicinoids likely disrupt membranes, in addition to energy poisoning, with implications for microbiology and human health. IMPORTANCE Plants make chemical compounds to protect themselves. For example, chili peppers produce the spicy compound capsaicin to inhibit pathogen damage and animal feeding. In humans, capsaicin binds to a membrane channel protein, creating the sensation of heat, while in microbes, capsaicin limits energy production by binding respiratory enzymes. However, some data suggest that capsaicin also disrupts membranes. Here, we studied fungal pathogens (Alternaria, Colletotrichum, Fusarium, and Phomopsis) isolated from a wild chili pepper, Capsicum chacoense. By measuring growth rates in the presence of antibiotics with known respiratory targets, we inferred that wild-plant pathogens might be rich in alternative respiratory enzymes. A zone of clearance around the colonies, as well as liquid chromatography-mass spectrometry data, further indicated that these fungi can break down capsaicin. Finally, the total inhibitory effect of capsaicin was not fully explained by its effect on respiratory enzymes. Our findings lend credence to studies proposing that capsaicin may disrupt cell membranes, with implications for microbiology, as well as human health.

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“…The BarSeq_P2 primer contains the tag that is used for demultiplexing by Illumina software, and the new Barseq3_P1 primer contained an additional sequence to verify that it came from the expected sample. The new Barseq3_P1 primer contains the same sequence as BarSeq_P1 reported earlier with 1-4 N's (which varies with the index) followed by the reverse (not the reverse complement) of the 6-nucleotide index sequence [251]. This modification to earlier BarSeq PCR protocol was done to eliminate the barcode bleed-through problem in sequencing and also to aid in cluster and sample discrimination on the HiSeq4000.…”

Section: Barseq Of Rb-tnseq and Dub-seq Pooled Fitness Assay Samplesmentioning

confidence: 99%

High-throughput mapping of the phage resistance landscape in E. coli

et al. 2020

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Bacteriophages (phages) are critical players in the dynamics and function of microbial communities and drive processes as diverse as global biogeochemical cycles and human health. Phages tend to be predators finely tuned to attack specific hosts, even down to the strain level, which in turn defend themselves using an array of mechanisms. However, to date, efforts to rapidly and comprehensively identify bacterial host factors important in phage infection and resistance have yet to be fully realized. Here, we globally map the host genetic determinants involved in resistance to 14 phylogenetically diverse double-stranded DNA phages using two model Escherichia coli strains (K-12 and BL21) with known sequence divergence to demonstrate strain-specific differences. Using genome-wide lossof-function and gain-of-function genetic technologies, we are able to confirm previously described phage receptors as well as uncover a number of previously unknown host factors that confer resistance to one or more of these phages. We uncover differences in resistance factors that strongly align with the susceptibility of K-12 and BL21 to specific phage. We also identify both phage-specific mechanisms, such as the unexpected role of cyclic-di-GMP in host sensitivity to phage N4, and more generic defenses, such as the overproduction of colanic acid capsular polysaccharide that defends against a wide array of phages. Our results indicate that host responses to phages can occur via diverse cellular mechanisms. Our systematic and high-throughput genetic workflow to characterize phage-host interaction determinants can be extended to diverse bacteria to generate datasets that allow predictive models of how phage-mediated selection will shape bacterial phenotype and evolution. The results of this study and future efforts to map the phage resistance landscape will lead to new insights into the coevolution of hosts and their phage, which can

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Oxidative Pathways of Deoxyribose and Deoxyribonate Catabolism

Cited by 29 publications

References 43 publications

Leveraging host metabolism for bisdemethoxycurcumin production in Pseudomonas putida

Leveraging host metabolism for bisdemethoxycurcumin production in Pseudomonas putida

Fungal Seed Pathogens of Wild Chili Peppers Possess Multiple Mechanisms To Tolerate Capsaicinoids

High-throughput mapping of the phage resistance landscape in E. coli

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