scholarly journals Engineering Pseudomonas putida S12 for Efficient Utilization of d-Xylose and l-Arabinose

2008 ◽  
Vol 74 (16) ◽  
pp. 5031-5037 ◽  
Author(s):  
Jean-Paul Meijnen ◽  
Johannes H. de Winde ◽  
Harald J. Ruijssenaars
Keyword(s):  
Growth Rate ◽  
Pseudomonas Putida ◽  
Glucose Dehydrogenase ◽  
Xylose Isomerase ◽  
Dry Weight ◽  
High Yield ◽  
Efficient Production ◽  
Primary Metabolism ◽  
Dead End ◽  
Solvent Tolerant

ABSTRACT The solvent-tolerant bacterium Pseudomonas putida S12 was engineered to utilize xylose as a substrate by expressing xylose isomerase (XylA) and xylulokinase (XylB) from Escherichia coli. The initial yield on xylose was low (9% [g CDW g substrate−1], where CDW is cell dry weight), and the growth rate was poor (0.01 h−1). The main cause of the low yield was the oxidation of xylose into the dead-end product xylonate by endogenous glucose dehydrogenase (Gcd). Subjecting the XylAB-expressing P. putida S12 to laboratory evolution yielded a strain that efficiently utilized xylose (yield, 52% [g CDW g xylose−1]) at a considerably improved growth rate (0.35 h−1). The high yield could be attributed in part to Gcd inactivity, whereas the improved growth rate may be connected to alterations in the primary metabolism. Surprisingly, without any further engineering, the evolved d-xylose-utilizing strain metabolized l-arabinose as efficiently as d-xylose. Furthermore, despite the loss of Gcd activity, the ability to utilize glucose was not affected. Thus, a P. putida S12-derived strain was obtained that efficiently utilizes the three main sugars present in lignocellulosic hydrolysate: glucose, xylose, and arabinose. This strain will form the basis for a platform host for the efficient production of biochemicals from renewable feedstock.

scholarly journals Effect of Organic Solvents on the Yield of Solvent-Tolerant Pseudomonas putida S12

1999 ◽  
Vol 65 (6) ◽  
pp. 2631-2635 ◽  
Author(s):  
Sonja Isken ◽  
Antoine Derks ◽  
Petra F. G. Wolffs ◽  
Jan A. M. de Bont
Keyword(s):  
Growth Rate ◽  
Pseudomonas Putida ◽  
Organic Solvents ◽  
Critical Concentration ◽  
Bacterial Membrane ◽  
Maximal Growth ◽  
Wild Type ◽  
Active Efflux ◽  
Maximal Growth Rate ◽  
Solvent Tolerant

ABSTRACT Solvent-tolerant microorganisms are useful in biotransformations with whole cells in two-phase solvent-water systems. The results presented here describe the effects that organic solvents have on the growth of these organisms. The maximal growth rate of Pseudomonas putida S12, 0.8 h−1, was not affected by toluene in batch cultures, but in chemostat cultures the solvent decreased the maximal growth rate by nearly 50%. Toluene, ethylbenzene, propylbenzene, xylene, hexane, and cyclohexane reduced the biomass yield, and this effect depended on the concentration of the solvent in the bacterial membrane and not on its chemical structure. The dose response to solvents in terms of yield was linear up to an approximately 200 mM concentration of solvent in the bacterial membrane, both in the wild type and in a mutant lacking an active efflux system for toluene. Above this critical concentration the yield of the wild type remained constant at 0.2 g of protein/g of glucose with increasing concentrations of toluene. The reduction of the yield in the presence of solvents is due to a maintenance higher by a factor of three or four as well as to a decrease of the maximum growth yield by 33%. Therefore, energy-consuming adaptation processes as well as the uncoupling effect of the solvents reduce the yield of the tolerant cells.

scholarly journals Establishment of Oxidative d-Xylose Metabolism in Pseudomonas putida S12

2009 ◽  
Vol 75 (9) ◽  
pp. 2784-2791 ◽  
Author(s):  
Jean-Paul Meijnen ◽  
Johannes H. de Winde ◽  
Harald J. Ruijssenaars
Keyword(s):  
Pseudomonas Putida ◽  
Caulobacter Crescentus ◽  
Glucose Dehydrogenase ◽  
Dry Weight ◽  
Maximum Growth ◽  
Maximum Growth Rate ◽  
Xylose Utilization ◽  
Catabolic Pathway ◽  
Xylose Metabolism ◽  
Semialdehyde Dehydrogenase

ABSTRACT The oxidative d-xylose catabolic pathway of Caulobacter crescentus, encoded by the xylXABCD operon, was expressed in the gram-negative bacterium Pseudomonas putida S12. This engineered transformant strain was able to grow on d-xylose as a sole carbon source with a biomass yield of 53% (based on g [dry weight] g d-xylose−1) and a maximum growth rate of 0.21 h−1. Remarkably, most of the genes of the xylXABCD operon appeared to be dispensable for growth on d-xylose. Only the xylD gene, encoding d-xylonate dehydratase, proved to be essential for establishing an oxidative d-xylose catabolic pathway in P. putida S12. The growth performance on d-xylose was, however, greatly improved by coexpression of xylXA, encoding 2-keto-3-deoxy-d-xylonate dehydratase and α-ketoglutaric semialdehyde dehydrogenase, respectively. The endogenous periplasmic glucose dehydrogenase (Gcd) of P. putida S12 was found to play a key role in efficient oxidative d-xylose utilization. Gcd activity not only contributes to d-xylose oxidation but also prevents the intracellular accumulation of toxic catabolic intermediates which delays or even eliminates growth on d-xylose.

scholarly journals Engineering of Solvent-Tolerant Pseudomonas putida S12 for Bioproduction of Phenol from Glucose

2005 ◽  
Vol 71 (12) ◽  
pp. 8221-8227 ◽  
Author(s):  
Nick J. P. Wierckx ◽  
Hendrik Ballerstedt ◽  
Jan A. M. de Bont ◽  
Jan Wery
Keyword(s):  
Pseudomonas Putida ◽  
High Throughput Screening ◽  
Random Mutagenesis ◽  
Batch Process ◽  
Shake Flask Culture ◽  
Efficient Production ◽  
Fed Batch ◽  
Tyrosine Phenol Lyase ◽  
Solvent Tolerant ◽  
Biphasic Medium

ABSTRACT Efficient bioconversion of glucose to phenol via the central metabolite tyrosine was achieved in the solvent-tolerant strain Pseudomonas putida S12. The tpl gene from Pantoea agglomerans, encoding tyrosine phenol lyase, was introduced into P. putida S12 to enable phenol production. Tyrosine availability was a bottleneck for efficient production. The production host was optimized by overexpressing the aroF-1 gene, which codes for the first enzyme in the tyrosine biosynthetic pathway, and by random mutagenesis procedures involving selection with the toxic antimetabolites m-fluoro-dl-phenylalanine and m-fluoro-l-tyrosine. High-throughput screening of analogue-resistant mutants obtained in this way yielded a P. putida S12 derivative capable of producing 1.5 mM phenol in a shake flask culture with a yield of 6.7% (mol/mol). In a fed-batch process, the productivity was limited by accumulation of 5 mM phenol in the medium. This toxicity was overcome by use of octanol as an extractant for phenol in a biphasic medium-octanol system. This approach resulted in accumulation of 58 mM phenol in the octanol phase, and there was a twofold increase in the overall production compared to a single-phase fed batch.

scholarly journals Refactoring the upper sugar metabolism ofPseudomonas putidafor co-utilization of disaccharides, pentoses, and hexoses

2018 ◽  
Author(s):  
Pavel Dvořák ◽  
Víctor de Lorenzo
Keyword(s):  
Glucose Dehydrogenase ◽  
Sugar Metabolism ◽  
Xylose Isomerase ◽  
Pentose Phosphate ◽  
Soil Bacterium ◽  
Biochemical Network ◽  
Xylose Metabolism ◽  
Native Proteins ◽  
Dead End ◽  
A Genome

AbstractGiven its capacity to tolerate stress, NAD(P)H/ NAD(P) balance, and increased ATP levels, the platform strainPseudomonas putidaEM42, a genome-edited derivative of the soil bacteriumP. putidaKT2440, can efficiently host a suite of harsh reactions of biotechnological interest. Because of the lifestyle of the original isolate, however, the nutritional repertoire ofP. putidaEM42 is centered largely on organic acids, aromatic compounds and some hexoses (glucose and fructose). To enlarge the biochemical network ofP. putidaEM42 to include disaccharides and pentoses, we implanted heterologous genetic modules for D-cellobiose and D-xylose metabolism into the enzymatic complement of this strain. Cellobiose was actively transported into the cells through the ABC complex formed by native proteins PP1015-PP1018. The knocked-in β-glucosidase BglC fromThermobifida fuscacatalyzed intracellular cleavage of the disaccharide to D-glucose, which was then channelled to the default central metabolism. Xylose oxidation to the dead end product D-xylonate was prevented by by deleting thegcdgene that encodes the broad substrate range quinone-dependent glucose dehydrogenase. Intracellular intake was then engineered by expressing theEscherichia coliproton-coupled symporter XylE. The sugar was further metabolized by the products ofE. coli xylA(xylose isomerase) andxylB(xylulokinase) towards the pentose phosphate pathway. The resultingP. putidastrain co-utilized xylose with glucose or cellobiose to complete depletion of the sugars. These results not only show the broadening of the metabolic capacity of a soil bacterium towards new substrates, but also promoteP. putidaEM42 as a platform for plug-in of new biochemical pathways for utilization and valorization of carbohydrate mixtures from lignocellulose processing.

scholarly journals Bioproduction of p-Hydroxystyrene from Glucose by the Solvent-Tolerant Bacterium Pseudomonas putida S12 in a Two-Phase Water-Decanol Fermentation

2008 ◽  
Vol 75 (4) ◽  
pp. 931-936 ◽  
Author(s):  
Suzanne Verhoef ◽  
Nick Wierckx ◽  
R. G. Maaike Westerhof ◽  
Johannes H. de Winde ◽  
Harald J. Ruijssenaars
Keyword(s):  
Pseudomonas Putida ◽  
Value Added ◽  
Batch Cultivation ◽  
Volumetric Productivity ◽  
Acid Decarboxylase ◽  
Efficient Production ◽  
Second Phase ◽  
Fed Batch ◽  
Solvent Tolerant ◽  
Fed Batch Cultivation

ABSTRACT Two solvent-tolerant Pseudomonas putida S12 strains, originally designed for phenol and p-coumarate production, were engineered for efficient production of p-hydroxystyrene from glucose. This was established by introduction of the genes pal and pdc encoding l-phenylalanine/l-tyrosine ammonia lyase and p-coumaric acid decarboxylase, respectively. These enzymes allow the conversion of the central metabolite l-tyrosine into p-hydroxystyrene, via p-coumarate. Degradation of the p-coumarate intermediate was prevented by inactivating the fcs gene encoding feruloyl-coenzyme A synthetase. The best-performing strain was selected and cultivated in the fed-batch mode, resulting in the formation of 4.5 mM p-hydroxystyrene at a yield of 6.7% (C-mol of p-hydroxystyrene per C-mol of glucose) and a maximum volumetric productivity of 0.4 mM h−1. At this concentration, growth and production were completely halted due to the toxicity of p-hydroxystyrene. Product toxicity was overcome by the application of a second phase of 1-decanol to extract p-hydroxystyrene during fed-batch cultivation. This resulted in a twofold increase of the maximum volumetric productivity (0.75 mM h−1) and a final total p-hydroxystyrene concentration of 21 mM, which is a fourfold improvement compared to the single-phase fed-batch cultivation. The final concentration of p-hydroxystyrene in the water phase was 1.2 mM, while a concentration of 147 mM (17.6 g liter−1) was obtained in the 1-decanol phase. Thus, a P. putida S12 strain producing the low-value compound phenol was successfully altered for the production of the toxic value-added compound p-hydroxystyrene.

Effect of rhizobacteria strains on the induction of resistance in barley genotypes against Cochliobolus sativus

2020 ◽  
Vol 13 (2) ◽  
pp. 83-92 ◽  
Author(s):  
A. Adam
Keyword(s):  
Plant Growth ◽  
Pseudomonas Putida ◽  
Growth Parameters ◽  
Disease Incidence ◽  
Dry Weight ◽  
Cochliobolus Sativus ◽  
Resistance Level ◽  
Wet Weight ◽  
Number Of Leaves ◽  
Barley Genotypes

SummaryEnhancement of the resistance level in plants by rhizobacteria has been proven in several pathosystems. This study investigated the ability of four rhizobacteria strains (Pseudomonas putida BTP1 and Bacillus subtilis Bs2500, Bs2504 and Bs2508) to promote the growth in three barley genotypes and protect them against Cochliobolus sativus. Our results demonstrated that all tested rhizobacteria strains had a protective effect on barley genotypes Arabi Abiad, Banteng and WI2291. However, P. putida BTP1 and B. subtilis Bs2508 strains were the most effective as they reduced disease incidence by 53 and 38% (mean effect), respectively. On the other hand, there were significant differences among the rhizobacteria-treated genotypes on plant growth parameters, such as wet weight, dry weight, plant height and number of leaves. Pseudomonas putida BTP1 strain was the most effective as it significantly increased plant growth by 15-32%. In addition, the susceptible genotypes Arabi Abiad and WI2291 were the most responsive to rhizobacteria. This means that these genotypes have a high potential for increase of their resistance against the pathogen and enhancement of plant growth after the application of rhizobacteria. Consequently, barley seed treatment with the tested rhizobacteria could be considered as an effective biocontrol method against C. sativus.

BOTANICAL COMPOSITION AND PRODUCTIVITY OF PERENNIAL GRASSES AGROPHYTOCENOSES WITH TETRAPLOID MEADOW CLOVER

1970 ◽  
pp. 59-62
Author(s):  
N. I. Kasatkina ◽  
Zh. S. Nelyubina
Keyword(s):  
Dry Weight ◽  
Single Species ◽  
Lotus Corniculatus ◽  
Specific Weight ◽  
Biological Properties ◽  
Phleum Pratense ◽  
Perennial Grasses ◽  
High Yield ◽  
Botanical Composition ◽  
The Third

The biological properties of plants, their mutual relations under different growth conditions and at different periods of their life, must be known for obtaining highly productive agrophytocenoses with participation of a meadow clover (Trifolium pratense L.). Botanical composition and fodder productivity of perennial grasses in agrocenoses with participation of meadow tetraploid clover Kudesnik were studied in 2014-2017. It was revealed that in the first and second years of use the agrophytocenosis, the yield of green mass was formed due to meadow tetraploid clover, the share of its participation in the first mowing was at level of 71-87% and 64-97% respectively. Specific weight of clover in multispecies agrocenoses considerably decreased by the third year of use: in the first mowing up to 32-68%, in the second - up to 8-52%. At the same time, the percentage of long-term herbaceous grasses increased: meadow timothy (Phleum pratense L.) - up to 34-54%, eastern galega (Galéga orientális Lam.) - up to 33%, changeable alfalfa (Medicago x varia Martyn) - up to 22-54%, lotus corniculatus (Lotus corniculatus L.) - up to 14-19%. The proportion of weed admixture in single-species clover planting was 12%, in agrocenoses - 2-14%. The grass mixtures clover + timothy and clover + alfalfa + timothy were less infested by weeds. High yield of dry weight of single-species sowing of meadow tetraploid clover was obtained in the first two years of use - 7.8 and 6.5 tons / ha, respectively. By the third year of use, the productivity of clover has decreased to 2.9 t / ha. On average, for three years of use, the highest yield (6.2-6.3 t / ha) was formed by agrocenoses meadow tetraploid clover + meadow timothy and meadow tetraploid clover + changeable alfalfa + meadow timothy.

scholarly journals Vegetative and Reproductive Response to Fruit Load in Two Jojoba (Simmondsia chinensis) Cultivars

Agronomy ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 889
Author(s):  
Aviad Perry ◽  
Noemi Tel-Zur ◽  
Arnon Dag
Keyword(s):  
Vegetative Growth ◽  
Accumulation Rate ◽  
Dry Weight ◽  
High Yield ◽  
Lower Amount ◽  
Simmondsia Chinensis ◽  
Alternate Bearing ◽  
Fruit Removal ◽  
Growing Period ◽  
Fruit Load

Jojoba (Simmondsia chinensis) is a wax crop cultivated mainly in arid and semi-arid regions. This crop has been described as an alternate-bearing plant, meaning that it has a high-yield year (“on-year”) followed by a low-yield year (“off-year”). We investigated the effect of fruit load on jojoba’s vegetative and reproductive development. For two consecutive years, we experimented with two high-yielding cultivars—Benzioni and Hazerim—which had opposite fruit loads, i.e., one was under an on-year load, while the other was under an off-year load simultaneously. We found that removing the developing fruit from the shoot during an off-year promotes further vegetative growth in the same year, whereas in an on-year, this action has no effect. Moreover, after fruit removal in an on-year, there was a delay in vegetative growth renewal in the consecutive year, suggesting that the beginning of the growing period is dependent on the previous year’s yield load. We found that seed development in the 2018 season started a month earlier than in the 2017 season in both cultivars, regardless of fruit load. This early development was associated with higher wax content in the seeds. Hence, the wax accumulation rate, as a percentage of dry weight, was affected by year and not by fruit load. However, on-year seeds stopped growing earlier than off-year seeds, resulting in smaller seeds and an overall lower amount of wax per seed.

scholarly journals Additive-Enhanced Exfoliation for High-Yield 2D Materials Production

Nanomaterials ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 601
Author(s):  
Dinh-Tuan Nguyen ◽  
Hsiang-An Ting ◽  
Yen-Hsun Su ◽  
Mario Hofmann ◽  
Ya-Ping Hsieh
Keyword(s):  
Large Scale ◽  
2D Materials ◽  
Transition Metal Dichalcogenides ◽  
Spectroscopic Characterization ◽  
High Yield ◽  
Nanometer Scale ◽  
Efficient Production ◽  
Device Performance ◽  
Metal Dichalcogenides ◽  
Scale Deposition

The success of van-der-Waals electronics, which combine large-scale-deposition capabilities with high device performance, relies on the efficient production of suitable 2D materials. Shear exfoliation of 2D materials’ flakes from bulk sources can generate 2D materials with low amounts of defects, but the production yield has been limited below industry requirements. Here, we introduce additive-assisted exfoliation (AAE) as an approach to significantly increase the efficiency of shear exfoliation and produce an exfoliation yield of 30%. By introducing micrometer-sized particles that do not exfoliate, the gap between rotor and stator was dynamically reduced to increase the achievable shear rate. This enhancement was applied to WS2 and MoS2 production, which represent two of the most promising 2D transition-metal dichalcogenides. Spectroscopic characterization and cascade centrifugation reveal a consistent and significant increase in 2D material concentrations across all thickness ranges. Thus, the produced WS2 films exhibit high thickness uniformity in the nanometer-scale and can open up new routes for 2D materials production towards future applications.

scholarly journals Characterizing the Anoxic Phenotype of Pseudomonas putida Using a Bioelectrochemical System

2019 ◽  
Vol 2 (2) ◽  
pp. 26 ◽  
Author(s):  
Bin Lai ◽  
Anh Nguyen ◽  
Jens Krömer
Keyword(s):  
Pseudomonas Putida ◽  
High Yield ◽  
Gas Transfer ◽  
Bioelectrochemical System ◽  
Terminal Electron Acceptor ◽  
Industrial Fermentation ◽  
Electrochemical Technology ◽  
Poor Understanding ◽  
Cultivation Technology ◽  
Obligate Aerobe

Industrial fermentation in aerobic processes is plagued by high costs due to gas transfer limitations and substrate oxidation to CO2. It has been a longstanding challenge to engineer an obligate aerobe organism, such as Pseudomonas putida, into an anaerobe to facilitate its industrial application. However, the progress in this field is limited, due to the poor understanding of the constraints restricting its anoxic phenotype. In this paper, we provide a methodological description of a novel cultivation technology for P. putida under anaerobic conditions, using the so-called microbial electrochemical technology within a bioelectrochemical system. By using an electrode as the terminal electron acceptor (mediated via redox chemicals), glucose catabolism could be activated without oxygen present. This (i) provides an anoxic-producing platform for sugar acid production at high yield and (ii) more importantly, enables systematic and quantitative characterizations of the phenotype of P. putida in the absence of molecular oxygen. This unique electrode-based cultivation approach offers a tool to understand and in turn engineer the anoxic phenotype of P. putida and possibly also other obligate aerobes.

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