A metabolic pathway for catabolizing levulinic acid in bacteria

Rand, Jacqueline M.; Pisithkul, Tippapha; Clark, Ryan L.; Thiede, Joshua M.; Mehrer, Christopher R.; Agnew, Daniel E.; Campbell, Candace E.; Markley, Andrew L.; Price, Morgan N.; J, Ray; Wetmore, Kelly M.; Suh, Yumi; Arkin, Adam P.; Deutschbauer, Adam M.; Amador‐Noguez, Daniel; Pfleger, Brian F.

doi:10.1038/s41564-017-0028-z

Cited by 86 publications

(101 citation statements)

References 60 publications

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“…To assay for genes involved in coumarate and related metabolisms, we grew a randomly barcoded transposon mutant (RB-TnSeq) library of P. putida KT2440 in minimal medium with a variety of different aromatic compounds often found in LH (p-coumarate, ferulate, benzoate, p-hydroxybenzoate, protocatechuate, vanillin, vanillate, phenylacetate) and glucose as sole carbon sources. The fitness of each gene was calculated by comparing the abundance of barcodes before versus after growth selection, using barcode sequencing (BarSeq) [17,18]. Negative values indicate that the gene was important for growth in that condition.…”

Section: Resultsmentioning

confidence: 99%

“…The robust catabolic pathways of P. putida , while useful for producing valuable molecules from diverse carbon sources, can also serve as an obstacle to achieving high product titers as it can often metabolize the desired products [26]. Given recent advances in gene editing techniques [27–31] and our ability to rapidly assay for gene function with transposon site sequencing [17,18,32], engineering non-model hosts like P. putida for industrial applications has become less challenging.…”

Section: Discussionmentioning

confidence: 99%

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Leveraging host metabolism for bisdemethoxycurcumin production in Pseudomonas putida

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Thompson

Blake-Hedges

et al. 2019

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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%

Section: Discussionmentioning

confidence: 99%

Leveraging host metabolism for bisdemethoxycurcumin production in Pseudomonas putida

Incha

Thompson

Blake-Hedges

et al. 2019

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“…To improve GapMind, we tested it on 35 diverse bacteria that can make all 20 amino acids and for which we have large-scale genetic data from pools of transposon mutants (Price, Wetmore, et al 2018;Liu et al 2019;Rand et al 2017) . The bacteria are listed in supplementary table S1.…”

Section: Expanding Gapmind's Database Using Genetic Datamentioning

confidence: 99%

“…We also considered whether serine might be formed from glycine: although glycine is usually formed from serine, it might also form by the glycine cleavage reaction in reverse, which may be thermodynamically feasible if one-carbon substrates such as formate reach high concentrations (see Appendix 2). However, genes from the glycine cleavage system were not important for the growth of either strain of D. vulgaris in minimal media (V. V. Trotter, personal communication; data of (Price, Wetmore, et al 2018;Liu et al 2019;Rand et al 2017) ). Also, a metabolic labeling study suggests that D. vulgaris…”

Section: Many Gaps In Amino Acid Biosynthesis In Diverse Prokaryotesmentioning

confidence: 99%

GapMind: Automated annotation of amino acid biosynthesis

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Deutschbauer

Arkin

2019

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“…Pseudomonas simiae WCS417 and Paraburkholderia bryophila 376MFSha3.1 were provided by Jeff Dangl (University of North Carolina). Pools of barcoded transposon mutant of Pseudomonas simiae WCS417, Pseudomonas putida KT2440 , Burkholderia phytofirmans PsJN, and Klebsiella michiganensis M5al were described previously (Price et al 2018;Rand et al 2017) . Each mutant has a transposon insertion that includes kanR (providing kanamycin resistance) as well as a 20-nucleotide barcode flanked by common priming sites.…”

Section: Strains and Growth Mediamentioning

confidence: 99%

Oxidative pathways of deoxyribose and deoxyribonate catabolism

Price

Iavarone

et al. 2017

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High-throughput genetics is a powerful approach to identify new genes involved in bacterial carbon catabolism. Here, we used genome-wide fitness assays to identify a novel pathway for 2-deoxy-D-ribose catabolism in Pseudomonas simiae WCS417. The genes that were important for deoxyribose utilization but not in other conditions included two putative dehydrogenases, a lactonase, a β-keto acid cleavage enzyme, and a glycerate kinase. We propose that deoxyribose is oxidized twice to 2-deoxy-3-keto-D-ribonoate (a β-keto acid) before cleavage to D-glycerate, which is phosphorylated and enters lower glycolysis. We purified the two dehydrogenases and reconstituted the enzymatic pathway for the conversion of deoxyribose to 2-deoxy-3-keto-D-ribonoate in vitro .

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A metabolic pathway for catabolizing levulinic acid in bacteria

Cited by 86 publications

References 60 publications

Leveraging host metabolism for bisdemethoxycurcumin production in Pseudomonas putida

Leveraging host metabolism for bisdemethoxycurcumin production in Pseudomonas putida

GapMind: Automated annotation of amino acid biosynthesis

Oxidative pathways of deoxyribose and deoxyribonate catabolism

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