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

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.

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

Applied and Environmental Microbiology ◽

10.1128/aem.02186-08 ◽

2008 ◽

Vol 75 (4) ◽

pp. 931-936 ◽

Cited By ~ 87

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.

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pH-stat fed-batch process to enhance the production of cis, cis-muconate from benzoate by Pseudomonas putida KT2440-JD1

Biotechnology Progress ◽

10.1002/btpr.709 ◽

2011 ◽

Vol 28 (1) ◽

pp. 85-92 ◽

Cited By ~ 41

Author(s):

Jozef B. J. H. van Duuren ◽

Dorien Wijte ◽

Bianka Karge ◽

Vítor A.P. Martins dos Santos ◽

Yu Yang ◽

...

Keyword(s):

Pseudomonas Putida ◽

Batch Process ◽

Fed Batch ◽

Pseudomonas Putida Kt2440 ◽

Ph Stat

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In situ phenol removal from fed-batch fermentations of solvent tolerant Pseudomonas putida S12 by pertraction

Biochemical Engineering Journal ◽

10.1016/j.bej.2010.11.002 ◽

2011 ◽

Vol 53 (3) ◽

pp. 245-252 ◽

Cited By ~ 11

Author(s):

Louise Heerema ◽

Nick Wierckx ◽

Mark Roelands ◽

Jan Henk Hanemaaijer ◽

Earl Goetheer ◽

...

Keyword(s):

Pseudomonas Putida ◽

Phenol Removal ◽

Fed Batch ◽

Solvent Tolerant

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Development of a simple and high-yielding fed-batch process for the production of porcine circovirus type 2 virus-like particle subunit vaccine

AMB Express ◽

10.1186/s13568-019-0880-8 ◽

2019 ◽

Vol 9 (1) ◽

Author(s):

Wenlong Cao ◽

Hui Cao ◽

Xiaoping Yi ◽

Yingping Zhuang

Keyword(s):

Cell Density ◽

Batch Culture ◽

Subunit Vaccine ◽

Batch Process ◽

Porcine Circovirus Type ◽

Porcine Circovirus Type 2 ◽

Porcine Circovirus ◽

Efficient Production ◽

Fed Batch

Abstract The cap protein is encoded by the orf2 gene of porcine circovirus type 2 (PCV2) has the main antigen epitope of PCV2 and can form virus-like particles (VLPs), which are expressed in insect cells. PCV2-VLPs can effectively inhibit PCV2 replication as a subunit vaccine. In this study, a robust and reliable fed-batch process was successfully developed for the production of PCV2-VLPs by Sf9 cells. The feeding solution, feeding strategy, and cell density at infection were optimized to maximize the final PCV2-VLPs production yields. The cell density at infection and the volumetric PCV2-VLPs production reached 12 × 106 cells/mL and 110 mg/L, respectively, which yielded 3- and 3.6-fold enhancements compared to the batch culture. The PCV2-VLPs produced in fed-batch culture were not different from the PCV2-VLPs produced in a batch culture in an immunity test. A highly efficient production process was produced for PCV2-VLPs subunit vaccines, which could provide an effective means for the industrial production of PCV2 vaccines.

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

Applied and Environmental Microbiology ◽

10.1128/aem.00924-08 ◽

2008 ◽

Vol 74 (16) ◽

pp. 5031-5037 ◽

Cited By ~ 63

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.

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Mutations in Genes Involved in the Flagellar Export Apparatus of the Solvent-Tolerant Pseudomonas putida DOT-T1E Strain Impair Motility and Lead to Hypersensitivity to Toluene Shocks

Journal of Bacteriology ◽

10.1128/jb.183.14.4127-4133.2001 ◽

2001 ◽

Vol 183 (14) ◽

pp. 4127-4133 ◽

Cited By ~ 25

Author(s):

Ana Segura ◽

Estrella Duque ◽

Ana Hurtado ◽

Juan L. Ramos

Keyword(s):

Pseudomonas Putida ◽

Mutant Strain ◽

Type Strain ◽

Wild Type Strain ◽

Random Mutagenesis ◽

Luria Bertani ◽

Wild Type ◽

Knockout Mutant ◽

Flagellar Proteins ◽

Solvent Tolerant

ABSTRACT Pseudomonas putida DOT-T1E is a solvent-tolerant strain able to grow in the presence of 1% (vol/vol) toluene in the culture medium. Random mutagenesis with mini-Tn5-′phoA-Km allowed us to isolate a mutant strain (DOT-T1E-42) that formed blue colonies on Luria-Bertani medium supplemented with 5-bromo-4-chloro-3-indolylphosphate and that, in contrast to the wild-type strain, was unable to tolerate toluene shocks (0.3%, vol/vol). The mutant strain exhibited patterns of tolerance or sensitivity to a number of antibiotics, detergents, and chelating agents similar to those of the wild-type strain. The mutation in this strain therefore seemed to specifically affect toluene tolerance. Cloning and sequencing of the mutation revealed that the mini-Tn5-′phoA-Km was inserted within the fliPgene, which is part of the fliLMNOPQRflhBA cluster, a set of genes that encode flagellar structure components. FliP is involved in the export of flagellar proteins, and in fact, theP. putida fliP mutant was nonmotile. The finding that, after replacing the mutant allele with the wild-type one, the strain recovered the wild-type pattern of toluene tolerance and motility unequivocally assigned FliP a function in solvent resistance. An flhB knockout mutant, another gene component of the flagellar export apparatus, was also nonmotile and hypersensitive to toluene. In contrast, a nonpolar mutation at the fliLgene, which encodes a cytoplasmic membrane protein associated with the flagellar basal body, yielded a nonmotile yet toluene-resistant strain. The results are discussed regarding a possible role of the flagellar export apparatus in the transport of one or more proteins necessary for toluene tolerance in P. putida DOT-T1E to the periplasm.

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Directed Evolution of P450 Fatty Acid Decarboxylases via High-Throughput Screening Towards Improved Catalytic Activity

10.26434/chemrxiv.9791162 ◽

2019 ◽

Author(s):

Huifang Xu ◽

Weinan Liang ◽

Linlin Ning ◽

Yuanyuan Jiang ◽

Wenxia Yang ◽

...

Keyword(s):

Catalytic Activity ◽

Fatty Acid ◽

Directed Evolution ◽

High Throughput ◽

High Throughput Screening ◽

Colorimetric Detection ◽

Screening Method ◽

Random Mutagenesis ◽

Direct Production ◽

Consumption Activity

P450 fatty acid decarboxylases (FADCs) have recently been attracting considerable attention owing to their one-step direct production of industrially important 1-alkenes from biologically abundant feedstock free fatty acids under mild conditions. However, attempts to improve the catalytic activity of FADCs have met with little success. Protein engineering has been limited to selected residues and small mutant libraries due to lack of an effective high-throughput screening (HTS) method. Here, we devise a catalase-deficient Escherichia coli host strain and report an HTS approach based on colorimetric detection of H2O2-consumption activity of FADCs. Directed evolution enabled by this method has led to effective identification for the first time of improved FADC variants for medium-chain 1-alkene production from both DNA shuffling and random mutagenesis libraries. Advantageously, this screening method can be extended to other enzymes that stoichiometrically utilize H2O2 as co-substrate.

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Production of Rhamnolipid Biosurfactant from Fed Batch Culture by Pseudomonas aeruginosa using Multiple Substrates

Current Nutrition & Food Science ◽

10.2174/1573401314666181107100127 ◽

2020 ◽

Vol 16 (6) ◽

pp. 928-933

Author(s):

Jujjavarapu S. Eswari

Keyword(s):

Pseudomonas Aeruginosa ◽

Batch Process ◽

Time Interval ◽

Fed Batch ◽

Multiple Substrates ◽

Surface Active Agents ◽

Limiting Nutrients ◽

Limiting Nutrient ◽

Surface Active ◽

Rhamnolipid Biosurfactant

Objective: Biosurfactants are the surface active agents which are used for the reduction of surface and interfacial tensions of liquids. Rhamnolipids are the surfactants produced by Pseudomonas aeruginosa. It requires minimum nutrition for its growth as it can also grow in distilled water. The rhamnolipids produced by Pseudomonas aeruginosa are extra-cellular glycolipids consisting of L-rhamnose and 3-hydroxyalkanoic acid. Methods: The fed-batch method for the rhamnolipid production is considered in this study to know the influence of the carbon, nitrogen, phosphorous substrates as growth-limiting nutrients. Pulse feeding is employed for limiting nutrient addition at particular time interval to obtain maximum rhamnolipid formation from Pseudomonas aeruginosa compared with the batch process. Results: Out of 3 fed batch strategies constant glucose fed batch strategy shows best and gave maximum rhamnolipid concentration of 0.134 g/l.

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Evolutionary design of optimal surface topographies for biomaterials

Scientific Reports ◽

10.1038/s41598-020-78777-2 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Aliaksei Vasilevich ◽

Aurélie Carlier ◽

David A. Winkler ◽

Shantanu Singh ◽

Jan de Boer

Keyword(s):

High Throughput Screening ◽

Random Mutagenesis ◽

Material Design ◽

Fitness Assessment ◽

Surface Topographies ◽

Optimal Surface ◽

Design Production ◽

Brute Force Approach ◽

Survival Of The Fittest ◽

Selection Of

AbstractNatural evolution tackles optimization by producing many genetic variants and exposing these variants to selective pressure, resulting in the survival of the fittest. We use high throughput screening of large libraries of materials with differing surface topographies to probe the interactions of implantable device coatings with cells and tissues. However, the vast size of possible parameter design space precludes a brute force approach to screening all topographical possibilities. Here, we took inspiration from Nature to optimize materials surface topographies using evolutionary algorithms. We show that successive cycles of material design, production, fitness assessment, selection, and mutation results in optimization of biomaterials designs. Starting from a small selection of topographically designed surfaces that upregulate expression of an osteogenic marker, we used genetic crossover and random mutagenesis to generate new generations of topographies.

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