Comparison of mcl-Poly(3-hydroxyalkanoates) synthesis by different Pseudomonas putida strains from crude glycerol: citrate accumulates at high titer under PHA-producing conditions

ABSTRACT Enzymes are powerful biocatalysts capable of performing specific chemical transformations under mild conditions, yet as catalysts they remain subject to the laws of thermodynamics, namely, that they cannot catalyze chemical reactions beyond equilibrium. Here we report the phenomenon and application of using extracytosolic enzymes and medium conditions, such as pH, to catalyze metabolic pathways beyond their intracellular catalytic limitations. This methodology, termed “integrated bioprocessing” because it integrates intracellular and extracytosolic catalysis, was applied to a lactonization reaction in Pseudomonas putida for the economical and high-titer biosynthesis of 4-valerolactone from the inexpensive and renewable source levulinic acid. Mutant paraoxonase I (PON1) was expressed in P. putida, shown to export from the cytosol in Escherichia coli and P. putida using an N-terminal sequence, and demonstrated to catalyze the extracytosolic and pH-dependent lactonization of 4-hydroxyvalerate to 4-valerolactone. With this production system, the titer of 4-valerolactone was enhanced substantially in acidic medium using extracytosolically expressed lactonase versus an intracellular lactonase: from <0.2 g liter−1 to 2.1 ± 0.4 g liter−1 at the shake flask scale. Based on these results, the production of 4-hydroxyvalerate and 4-valerolactone was examined in a 2-liter bioreactor, and titers of 27.1 g liter−1 and 8.2 g liter−1 for the two respective compounds were achieved. These results illustrate the utility of integrated bioprocessing as a strategy for enabling production from novel metabolic pathways and enhancing product titers.

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Crude glycerol as feedstock for the sustainable production of p-hydroxybenzoate by Pseudomonas putida S12

New Biotechnology ◽

10.1016/j.nbt.2013.08.006 ◽

2014 ◽

Vol 31 (1) ◽

pp. 114-119 ◽

Cited By ~ 16

Author(s):

Suzanne Verhoef ◽

Nisi Gao ◽

Harald J. Ruijssenaars ◽

Johannes H. de Winde

Keyword(s):

Pseudomonas Putida ◽

Crude Glycerol ◽

Sustainable Production

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A simple strategy to effectively produce d-lactate in crude glycerol-utilizing Escherichia coli

Biotechnology for Biofuels ◽

10.1186/s13068-019-1615-4 ◽

2019 ◽

Vol 12 (1) ◽

Cited By ~ 2

Author(s):

Yao-De Wang ◽

Jin-Yi Liao ◽

Chung-Jen Chiang ◽

Yun-Peng Chao

Keyword(s):

Escherichia Coli ◽

Batch Fermentation ◽

Crude Glycerol ◽

High Titer ◽

Fed Batch ◽

High Productivity ◽

Simple Strategy ◽

Production Scheme ◽

Fed Batch Fermentation ◽

Fermentation Strategy

Abstract Background Fed-batch fermentation has been conventionally implemented for the production of lactic acid with a high titer and high productivity. However, its operation needs a complicated control which increases the production cost. Results This issue was addressed by simplifying the production scheme. Escherichia coli was manipulated for its glycerol dissimilation and d-lactate synthesis pathways and then subjected to adaptive evolution under high crude glycerol. Batch fermentation in the two-stage mode was performed by controlling the dissolved oxygen (DO), and the evolved strain deprived of poxB enabled production of 100 g/L d-lactate with productivity of 1.85 g/L/h. To increase productivity, the producer strain was further evolved to improve its growth rate on crude glycerol. The fermentation was performed to undergo the aerobic growth with low substrate, followed by the anaerobic production with high substrate. Moreover, the intracellular redox of the strain was balanced by fulfillment of the anaerobic respiratory chain with nitrate reduction. Without controlling the DO, the microbial fermentation resulted in the homofermentative production of d-lactate (ca. 0.97 g/g) with a titer of 115 g/L and productivity of 3.29 g/L/h. Conclusions The proposed fermentation strategy achieves the highest yield based on crude glycerol and a comparable titer and productivity as compared to the approach by fed-batch fermentation. It holds a promise to sustain the continued development of the crude glycerol-based biorefinery.

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High-titer production of monomeric hydroxyvalerates from levulinic acid in Pseudomonas putida

Journal of Biotechnology ◽

10.1016/j.jbiotec.2008.09.002 ◽

2009 ◽

Vol 139 (1) ◽

pp. 61-67 ◽

Cited By ~ 28

Author(s):

Collin H. Martin ◽

Kristala L. Jones Prather

Keyword(s):

Pseudomonas Putida ◽

Levulinic Acid ◽

High Titer

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Inhibitorentwicklung nach früher hoher Exposition und Hirnblutung

Hämostaseologie ◽

10.1055/s-0037-1619091 ◽

2010 ◽

Vol 30 (S 01) ◽

pp. S115-S118

Author(s):

C. Moorthi ◽

A. Bade ◽

C. Niekrens ◽

G. Auerswald ◽

K. Haubold

Keyword(s):

High Titer ◽

Cerebral Haemorrhage ◽

High Dose ◽

Haemophilia A ◽

Severe Haemophilia ◽

Intron 22 Inversion ◽

Neurosurgical Intervention

SummarySevere haemophilia A was diagnosed postpartum in a newborn. The mother was known as a conductor (intron 22 inversion) and an uncle had a persistently high titer inhibitor after failed ITI.Due to a cephalhaematoma, a high-dose pdFVIII substitution was given within the first days after birth. At the age of six month a severe cerebral haemorrhage occurred, making a high-dose pdFVIII substitution and neurosurgical intervention necessary. Several days later a porth-a-cath-system was implanted. The development of a high titer inhibitor occured six days later, an ITI was started according to the Bonn Protocol. Initially rFVIIa was given in addition to the pdFVIII substitution. Seven days after the beginning of treatment the inhibitor was no longer detectable. At monthly intervals the FVIII dosage was reduced until the dosage complied with a prophylaxis in severe haemophilia A.The duration of the ITI was nine months. A total of 30 mg rFVIIa and 276 000 IU pdFVIII were used; costs in total: 280 173.60 Euro.

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Pesticides Residues P-Nitrophenol Degrading Pseudomonas putida from Cultivated Vegetables Soil Samples of Chikhli Tahsil of Buldana District

International Journal of Scientific Research ◽

10.15373/22778179/may2013/4 ◽

2012 ◽

Vol 2 (5) ◽

pp. 9-10

Author(s):

Waghode S. M Waghode S. M ◽

◽

A. M. Garode A. M. Garode

Keyword(s):

Pseudomonas Putida ◽

Soil Samples ◽

Pesticides Residues

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Enzimaktivitások és a fluoreszkáló pszeudomonasz csíraszámok változása a fehér lóhere (Trifolium repens L.) rizoszférájában sókezelés (NaCl) hatására

Agrokémia és Talajtan ◽

10.1556/agrokem.53.2004.3-4.11 ◽

2004 ◽

Vol 53 (3-4) ◽

pp. 367-376 ◽

Cited By ~ 2

Author(s):

A. A. Khalif ◽

H. Abdorhim ◽

Hosam E. H. T. Bayoumi ◽

Anna Füzy ◽

Mihály Kecskés

Keyword(s):

Pseudomonas Putida ◽

Trifolium Repens ◽

Trifolium Repens L

Üvegházi körülmények között savanyú barna erdotalajban nevelt fehér here (Trifolium repens L.) növények rizoszférájának sókezelés hatására bekövetkezo változását ellenoriztük. Megvizsgáltuk a különbözo sókoncentrációknak (0, 0,2, 0,4, 0,6 és 0,8 tömeg %) a baktériumnépesség összetételére és a különbözo talajenzimek aktivitására gyakorolt hatását. Megállapítottuk, hogy a talaj sótartalma közvetlenül befolyásolta a rizoszférában található fluoreszkáló pszeudomonaszok csíraszámát. A legsurubb populáció a 0,2% NaCl-ot tartalmazó talajban volt mérheto, ahol a fluoreszkáló pszeudomonaszok között a Pseudomonas putida és a P. fluorescens fordultak elo a legnagyobb számban. A pszeudomonaszok ily módon jól tolerálják a talaj magas NaCl-tartalmát, és gyökérkolonizáló tevékenységet képesek kifejteni a magas NaCl-tartalmú talajban is. A sókoncentráció növelésével kezdetben (a 0,2-0,4%-os tartományban) jelentosen növekedett a dehidrogenáz, kataláz, és ureáz enzimek aktivitása. A proteáz enzimek aktivitásmaximuma a 0,1-0,2% NaCl-koncentráció tartományba esett. A 0,4%-nál magasabb koncentrációkban a kontrollhoz hasonló mértékure csökkent mind a négy enzim aktivitása, és a baktériumok száma is. A foszfatáz- és a b-glükozidáz-tevékenység viszont a NaCl-dózis növelése következtében a koncentrációval arányosan, jelentosen csökkent a kontrollhoz viszonyítva. Feltételezésünk szerint az enzimaktivitások változását is a sókezelés hatására bekövetkezo mikrobióta összetételének megváltozása okozta.

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