scholarly journals Host engineering for improved valerolactam production in Pseudomonas putida

2019 ◽  
Author(s):  
Mitchell G. Thompson ◽  
Luis E. Valencia ◽  
Jacquelyn M. Blake-Hedges ◽  
Pablo Cruz-Morales ◽  
Alexandria E. Velasquez ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
Carbon Source ◽  
Pseudomonas Putida ◽  
Aromatic Compounds ◽  
Sole Carbon Source ◽  
Carbon Sources ◽  
Shotgun Proteomics ◽  
Rich Media ◽  
Amide Hydrolase

ABSTRACTPseudomonas putida is a promising bacterial chassis for metabolic engineering given its ability to metabolize a wide array of carbon sources, especially aromatic compounds derived from lignin. However, this omnivorous metabolism can also be a hindrance when it can naturally metabolize products produced from engineered pathways. Herein we show that P. putida is able to use valerolactam as a sole carbon source, as well as degrade caprolactam. Lactams represent important nylon precursors, and are produced in quantities exceeding one million tons per year[1]. To better understand this metabolism we use a combination of Random Barcode Transposon Sequencing (RB-TnSeq) and shotgun proteomics to identify the oplBA locus as the likely responsible amide hydrolase that initiates valerolactam catabolism. Deletion of the oplBA genes prevented P. putida from growing on valerolactam, prevented the degradation of valerolactam in rich media, and dramatically reduced caprolactam degradation under the same conditions. Deletion of oplBA, as well as pathways that compete for precursors L-lysine or 5-aminovalerate, increased the titer of valerolactam from undetectable after 48 hours of production to ~90 mg/L. This work may serve as a template to rapidly eliminate undesirable metabolism in non-model hosts in future metabolic engineering efforts.

scholarly journals Cinnamic Acid, an Autoinducer of Its Own Biosynthesis, Is Processed via Hca Enzymes in Photorhabdus luminescens

2008 ◽  
Vol 74 (6) ◽  
pp. 1717-1725 ◽  
Author(s):  
Sabina Chalabaev ◽  
Evelyne Turlin ◽  
Sylvie Bay ◽  
Christelle Ganneau ◽  
Emma Brito-Fravallo ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Carbon Source ◽  
Cinnamic Acid ◽  
Aromatic Compounds ◽  
Sole Carbon Source ◽  
Biosynthetic Pathway ◽  
Phenylalanine Ammonia Lyase ◽  
Carbon Sources ◽  
Degradation Pathway ◽  
Photorhabdus Luminescens

ABSTRACT Photorhabdus luminescens, an entomopathogenic bacterium and nematode symbiont, has homologues of the Hca and Mhp enzymes. In Escherichia coli, these enzymes catalyze the degradation of the aromatic compounds 3-phenylpropionate (3PP) and cinnamic acid (CA) and allow the use of 3PP as sole carbon source. P. luminescens is not able to use 3PP and CA as sole carbon sources but can degrade them. Hca dioxygenase is involved in this degradation pathway. P. luminescens synthesizes CA from phenylalanine via a phenylalanine ammonia-lyase (PAL) and degrades it via the not-yet-characterized biosynthetic pathway of 3,5-dihydroxy-4-isopropylstilbene (ST) antibiotic. CA induces its own synthesis by enhancing the expression of the stlA gene that codes for PAL. P. luminescens bacteria release endogenous CA into the medium at the end of exponential growth and then consume it. Hca dioxygenase is involved in the consumption of endogenous CA but is not required for ST production. This suggests that CA is consumed via at least two separate pathways in P. luminescens: the biosynthesis of ST and a pathway involving the Hca and Mhp enzymes.

scholarly journals Studies on poly-3-hydroxyoctanoate biosynthesis by a consortium of microorganisms

2016 ◽  
Vol 27 (1) ◽  
pp. 44-47 ◽  
Author(s):  
Mihaela Carmen Eremia ◽  
Irina Lupescu ◽  
Mariana Vladu ◽  
Maria Petrescu ◽  
Gabriela Savoiu ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Carbon Source ◽  
Pseudomonas Putida ◽  
Sole Carbon Source ◽  
Bacterial Biomass ◽  
Fermentation Medium ◽  
Energy Reserve ◽  
Bacterial Strains ◽  
Sodium Octanoate ◽  
Intracellular Energy

Abstract Polyhydroxyalcanoates (PHAs) are specifically produced by a wide variety of bacteria, as an intracellular energy reserve in the form of homo- and copolymers of [R]-β-hydroxyalkanoic acids, depending on the C source used for microorganism growth, when the cells are grown under stressing conditions. In this paper we present microbiological accumulation of poly-3-hydroxyoctanoate (PHO) by using a consortium of bacterial strains, Pseudomonas putida and Bacillus subtilis, in a rate of 3:1, grown on a fermentation medium based on sodium octanoate as the sole carbon source. The experiments performed in the above mentioned conditions led to the following results: from 18.70 g sodium octanoate (7.72 g/L in the fermentation medium) used up during the bioprocess, 3.93-3.96 g/L dry bacterial biomass and 1.834 - 1.884 g/L PHA, containing 85.83 - 86.8% PHO, were obtained.

scholarly journals Comparative metabolomics of Phialemonium curvatum as an omnipotent fungus cultivated on crude palm oil versus glucose

2020 ◽  
Vol 19 (1) ◽  
Author(s):  
Arief Izzairy Zamani ◽  
Susann Barig ◽  
Sarah Ibrahim ◽  
Hirzun Mohd. Yusof ◽  
Julia Ibrahim ◽  
...  
Keyword(s):  
Carbon Source ◽  
Organic Acids ◽  
Vegetable Oil ◽  
Carbon Metabolism ◽  
Sole Carbon Source ◽  
Carbon Sources ◽  
Tca Cycle ◽  
Central Carbon Metabolism ◽  
Targeted Metabolomics ◽  
Central Carbon

Abstract Background Sugars and triglycerides are common carbon sources for microorganisms. Nonetheless, a systematic comparative interpretation of metabolic changes upon vegetable oil or glucose as sole carbon source is still lacking. Selected fungi that can grow in acidic mineral salt media (MSM) with vegetable oil had been identified recently. Hence, this study aimed to investigate the overall metabolite changes of an omnipotent fungus and to reveal changes at central carbon metabolism corresponding to both carbon sources. Results Targeted and non-targeted metabolomics for both polar and semi-polar metabolites of Phialemonium curvatum AWO2 (DSM 23903) cultivated in MSM with palm oil (MSM-P) or glucose (MSM-G) as carbon sources were obtained. Targeted metabolomics on central carbon metabolism of tricarboxylic acid (TCA) cycle and glyoxylate cycle were analysed using LC–MS/MS-TripleQ and GC–MS, while untargeted metabolite profiling was performed using LC–MS/MS-QTOF followed by multivariate analysis. Targeted metabolomics analysis showed that glyoxylate pathway and TCA cycle were recruited at central carbon metabolism for triglyceride and glucose catabolism, respectively. Significant differences in organic acids concentration of about 4- to 8-fold were observed for citric acid, succinic acid, malic acid, and oxaloacetic acid. Correlation of organic acids concentration and key enzymes involved in the central carbon metabolism was further determined by enzymatic assays. On the other hand, the untargeted profiling revealed seven metabolites undergoing significant changes between MSM-P and MSM-G cultures. Conclusions Overall, this study has provided insights on the understanding on the effect of triglycerides and sugar as carbon source in fungi global metabolic pathway, which might become important for future optimization of carbon flux engineering in fungi to improve organic acids production when vegetable oil is applied as the sole carbon source.

scholarly journals Bacterial Degradation of N,N-Diethyl-m-Toluamide (DEET): Cloning and Heterologous Expression of DEET Hydrolase

2007 ◽  
Vol 73 (9) ◽  
pp. 3105-3108 ◽  
Author(s):  
Giomar Rivera-Cancel ◽  
Daniela Bocioaga ◽  
Anthony G. Hay
Keyword(s):  
Carbon Source ◽  
Heterologous Expression ◽  
Pseudomonas Putida ◽  
Sole Carbon Source ◽  
Bacterial Degradation ◽  
Ring Cleavage

ABSTRACT Pseudomonas putida DTB grew aerobically with N,N-diethyl-m-toluamide (DEET) as a sole carbon source, initially breaking it down into 3-methylbenzoate and diethylamine. The former was further metabolized via 3-methylcatechol and meta ring cleavage. A gene from DTB, dthA, was heterologously expressed and shown to encode the ability to hydrolyze DEET into 3-methylbenzoate and diethylamine.

scholarly journals The Pseudomonas putida Crc Global Regulator Controls the Expression of Genes from Several Chromosomal Catabolic Pathways for Aromatic Compounds

2004 ◽  
Vol 186 (5) ◽  
pp. 1337-1344 ◽  
Author(s):  
Gracia Morales ◽  
Juan Francisco Linares ◽  
Ana Beloso ◽  
Juan Pablo Albar ◽  
José Luis Martínez ◽  
...  
Keyword(s):  
Pseudomonas Putida ◽  
Carbon Metabolism ◽  
Aromatic Compounds ◽  
Carbon Sources ◽  
Signal Transduction Pathway ◽  
Catabolic Repression ◽  
Catabolic Pathways ◽  
Expression Of Genes ◽  
Proteome Profile

ABSTRACT The Crc protein is involved in the repression of several catabolic pathways for the assimilation of some sugars, nitrogenated compounds, and hydrocarbons in Pseudomonas putida and Pseudomonas aeruginosa when other preferred carbon sources are present in the culture medium (catabolic repression). Crc appears to be a component of a signal transduction pathway modulating carbon metabolism in pseudomonads, although its mode of action is unknown. To better understand the role of Crc, the proteome profile of two otherwise isogenic P. putida strains containing either a wild-type or an inactivated crc allele was compared. The results showed that Crc is involved in the catabolic repression of the hpd and hmgA genes from the homogentisate pathway, one of the central catabolic pathways for aromatic compounds that is used to assimilate intermediates derived from the oxidation of phenylalanine, tyrosine, and several aromatic hydrocarbons. This led us to analyze whether Crc also regulates the expression of the other central catabolic pathways for aromatic compounds present in P. putida. It was found that genes required to assimilate benzoate through the catechol pathway (benA and catBCA) and 4-OH-benzoate through the protocatechuate pathway (pobA and pcaHG) are also negatively modulated by Crc. However, the pathway for phenylacetate appeared to be unaffected by Crc. These results expand the influence of Crc to pathways used to assimilate several aromatic compounds, which highlights its importance as a master regulator of carbon metabolism in P. putida.

scholarly journals Nitrobenzoates and Aminobenzoates Are Chemoattractants for Pseudomonas Strains

2004 ◽  
Vol 70 (1) ◽  
pp. 285-292 ◽  
Author(s):  
Rebecca E. Parales
Keyword(s):  
Pseudomonas Putida ◽  
Aromatic Compounds ◽  
Carbon Sources ◽  
Chemotactic Response ◽  
Aromatic Acids ◽  
Wide Range ◽  
Catechol 1,2 Dioxygenase ◽  
Sense And Respond ◽  
Chemotaxis System ◽  
Broad Specificity

ABSTRACT Three Pseudomonas strains were tested for the ability to sense and respond to nitrobenzoate and aminobenzoate isomers in chemotaxis assays. Pseudomonas putida PRS2000, a strain that grows on benzoate and 4-hydroxybenzoate by using the β-ketoadipate pathway, has a well-characterized β-ketoadipate-inducible chemotactic response to aromatic acids. PRS2000 was chemotactic to 3- and 4-nitrobenzoate and all three isomers of aminobenzoate when grown under conditions that induce the benzoate chemotactic response. P. putida TW3 and Pseudomonas sp. strain 4NT grow on 4-nitrotoluene and 4-nitrobenzoate by using the ortho (β-ketoadipate) and meta pathways, respectively, to complete the degradation of protocatechuate derived from 4-nitrotoluene and 4-nitrobenzoate. However, based on results of catechol 1,2-dioxygenase and catechol 2,3-dioxygenase assays, both strains were found to use the β-ketoadipate pathway for the degradation of benzoate. Both strains were chemotactic to benzoate, 3- and 4-nitrobenzoate, and all three aminobenzoate isomers after growth with benzoate but not succinate. Strain TW3 was chemotactic to the same set of aromatic compounds after growth with 4-nitrotoluene or 4-nitrobenzoate. In contrast, strain 4NT did not respond to any aromatic acids when grown with 4-nitrotoluene or 4-nitrobenzoate, apparently because these substrates are not metabolized to the inducer (β-ketoadipate) of the chemotaxis system. The results suggest that strains TW3 and 4NT have a β-ketoadipate-inducible chemotaxis system that responds to a wide range of aromatic acids and is quite similar to that present in PRS2000. The broad specificity of this chemotaxis system works as an advantage in strains TW3 and 4NT because it functions to detect diverse carbon sources, including 4-nitrobenzoate.

scholarly journals The foliar endophytePhialocephala scopiformisDAOMC 229536 secretes enzymes supporting growth on wood as sole carbon source

2018 ◽  
Author(s):  
Jennifer M. Bhatnagar ◽  
Grzegorz Sabat ◽  
Daniel Cullen
Keyword(s):  
Carbon Source ◽  
Aromatic Compounds ◽  
Glycoside Hydrolase ◽  
Pinus Contorta ◽  
Sole Carbon Source ◽  
Oxidative Degradation ◽  
Cellobiose Dehydrogenase ◽  
Lytic Polysaccharide Monooxygenases ◽  
Polysaccharide Monooxygenases ◽  
Conifer Needle

AbstractThe conifer needle endophyte,Phialocephala scopiformis, was cultivated in media containing groundPinus contortawood as sole carbon source. After five and seven days growth, concentrated extracellular fluids were subjected to LC-MS/MS analyses. A total of 590 proteins were identified of which 99 were assigned to glycoside hydrolase families within the Carbohydrate Active Enzyme (CAzyme) system. Multiple isozymes of exo-and endo-acting cellulases were among the most abundant proteins, and oxidative degradation of cellulose was supported by the presence of lytic polysaccharide monooxygenases, glucooligosaccharide oxidase and cellobiose dehydrogenase. Oxidoreductases were also plentiful and included GMC oxidoreductases, alcohol dehydrogenases, laccases, copper radical oxidases, tyrosinases and catalase. The expression and diversity of extracellular oxidoreductases indicates a capacity to metabolize alcohols and aromatic compounds.

scholarly journals Isolation and characterization of a novel Sphingobium yanoikuyae strain variant that uses biohazardous saturated hydrocarbons and aromatic compounds as sole carbon sources

F1000Research ◽  
2020 ◽  
Vol 9 ◽  
pp. 767
Author(s):  
Mautusi Mitra ◽  
Kevin Manoap-Anh-Khoa Nguyen ◽  
Taylor Wayland Box ◽  
Jesse Scott Gilpin ◽  
Seth Ryan Hamby ◽  
...  
Keyword(s):  
Carbon Source ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Aromatic Compounds ◽  
Agar Medium ◽  
Carbon Sources ◽  
Future Research ◽  
Rrna Gene ◽  
Saturated Hydrocarbons ◽  
Isolation And Characterization

Background: Green micro-alga, Chlamydomonas reinhardtii (a Chlorophyte), can be cultured in the laboratory heterotrophically or photo-heterotrophically in Tris-Phosphate-Acetate (TAP) medium, which contains acetate as the carbon source. Chlamydomonas can convert acetate in the TAP medium to glucose via the glyoxylate cycle, a pathway present in many microbes and higher plants. A novel bacterial strain, CC4533, was isolated from a contaminated TAP agar medium culture plate of a Chlamydomonas wild type strain. In this article, we present our research on the isolation, and biochemical and molecular characterizations of CC4533. Methods: We conducted several microbiological tests and spectrophotometric analyses to biochemically characterize CC4533. The 16S rRNA gene of CC4533 was partially sequenced for taxonomic identification. We monitored the growth of CC4533 on Tris-Phosphate (TP) agar medium (lacks a carbon source) containing different sugars, aromatic compounds and saturated hydrocarbons, to see if CC4533 can use these chemicals as the sole source of carbon. Results: CC4533 is a Gram-negative, non-enteric yellow pigmented, aerobic, mesophilic bacillus. It is alpha-hemolytic and oxidase-positive. CC4533 can ferment glucose, sucrose and lactose, is starch hydrolysis-negative, resistant to penicillin, polymyxin B and chloramphenicol. CC4533 is sensitive to neomycin. Preliminary spectrophotometric analyses indicate that CC4533 produces b-carotenes. NCBI-BLAST analyses of the partial 16S rRNA gene sequence of CC4533 show 99.55% DNA sequence identity to that of Sphingobium yanoikuyae strain PR86 and S. yanoikuyae strain NRB095. CC4533 can use cyclo-chloroalkanes, saturated hydrocarbons present in car motor oil, polyhydroxyalkanoate, and mono- and poly-cyclic aromatic compounds, as sole carbon sources for growth. Conclusions: Taxonomically, CC4533 is very closely related to the alpha-proteobacterium S. yanoikuyae, whose genome has been sequenced. Future research is needed to probe the potential of CC4533 for environmental bioremediation. Whole genome sequencing of CC4533 will confirm if it is a novel strain of S. yanoikuyae or a new Sphingobium species.

Saccharomyces cerevisiae glucose signalling regulator Mth1p regulates the organellar Na+/H+ exchanger Nhx1p

2010 ◽  
Vol 432 (2) ◽  
pp. 343-352 ◽  
Author(s):  
Keiji Mitsui ◽  
Masafumi Matsushita ◽  
Hiroshi Kanazawa
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Carbon Source ◽  
Sole Carbon Source ◽  
Glucose Sensor ◽  
Ph Regulation ◽  
Gene Knockout ◽  
Carbon Sources ◽  
Acidic Ph ◽  
In Cells

Organelle-localized NHEs (Na+/H+ exchangers) are found in cells from yeast to humans and contribute to organellar pH regulation by exporting H+ from the lumen to the cytosol coupled to an H+ gradient established by vacuolar H+-ATPase. The mechanisms underlying the regulation of organellar NHEs are largely unknown. In the present study, a yeast two-hybrid assay identified Mth1p as a new binding protein for Nhx1p, an organellar NHE in Saccharomyces cerevisiae. It was shown by an in vitro pull-down assay that Mth1p bound to the hydrophilic C-terminal half of Nhx1p, especially to the central portion of this region. Mth1p is known to bind to the cytoplasmic domain of the glucose sensor Snf3p/Rgt2p and also functions as a negative transcriptional regulator. Mth1p was expressed in cells grown in a medium containing galactose, but was lost (possibly degraded) when cells were grown in medium containing glucose as the sole carbon source. Deletion of the MTH1 gene increased cell growth compared with the wild-type when cells were grown in a medium containing galactose and with hygromycin or at an acidic pH. This resistance to hygromycin or acidic conditions was not observed for cells grown with glucose as the sole carbon source. Gene knockout of NHX1 increased the sensitivity to hygromycin and acidic pH. The increased resistance to hygromycin was reproduced by truncation of the Mth1p-binding region in Nhx1p. These results implicate Mth1p as a novel regulator of Nhx1p that responds to specific extracellular carbon sources.

scholarly journals Outer membrane vesicles catabolize lignin-derived aromatic compounds in Pseudomonas putida KT2440

2020 ◽  
Vol 117 (17) ◽  
pp. 9302-9310 ◽  
Author(s):  
Davinia Salvachúa ◽  
Allison Z. Werner ◽  
Isabel Pardo ◽  
Martyna Michalska ◽  
Brenna A. Black ◽  
...  
Keyword(s):  
Pseudomonas Putida ◽  
Outer Membrane ◽  
Aromatic Compounds ◽  
Value Added ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Pseudomonas Putida Kt2440 ◽  
Rich Media

Lignin is an abundant and recalcitrant component of plant cell walls. While lignin degradation in nature is typically attributed to fungi, growing evidence suggests that bacteria also catabolize this complex biopolymer. However, the spatiotemporal mechanisms for lignin catabolism remain unclear. Improved understanding of this biological process would aid in our collective knowledge of both carbon cycling and microbial strategies to valorize lignin to value-added compounds. Here, we examine lignin modifications and the exoproteome of three aromatic–catabolic bacteria: Pseudomonas putida KT2440, Rhodoccocus jostii RHA1, and Amycolatopsis sp. ATCC 39116. P. putida cultivation in lignin-rich media is characterized by an abundant exoproteome that is dynamically and selectively packaged into outer membrane vesicles (OMVs). Interestingly, many enzymes known to exhibit activity toward lignin-derived aromatic compounds are enriched in OMVs from early to late stationary phase, corresponding to the shift from bioavailable carbon to oligomeric lignin as a carbon source. In vivo and in vitro experiments demonstrate that enzymes contained in the OMVs are active and catabolize aromatic compounds. Taken together, this work supports OMV-mediated catabolism of lignin-derived aromatic compounds as an extracellular strategy for nutrient acquisition by soil bacteria and suggests that OMVs could potentially be useful tools for synthetic biology and biotechnological applications.

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