scholarly journals Production of the Chiral Compound (R)-3-Hydroxybutyrate by a Genetically Engineered Methylotrophic Bacterium

2010 ◽  
Vol 76 (16) ◽  
pp. 5585-5591 ◽  
Author(s):  
Tina Hölscher ◽  
Uta Breuer ◽  
Lorenz Adrian ◽  
Hauke Harms ◽  
Thomas Maskow
Keyword(s):  
Citric Acid Cycle ◽  
Sole Source ◽  
Genetically Engineered ◽  
Methylotrophic Bacterium ◽  
Cultivation Conditions ◽  
Chiral Compound ◽  
Batch Cultures ◽  
Alternative Pathways ◽  
Intracellular Degradation ◽  
Methylobacterium Rhodesianum

ABSTRACT In this study, a methylotrophic bacterium, Methylobacterium rhodesianum MB 126, was used for the production of the chiral compound (R)-3-hydroxybutyrate (R-3HB) from methanol. R-3HB is formed during intracellular degradation of the storage polymer (R)-3-polyhydroxybutyrate (PHB). Since the monomer R-3HB does not accumulate under natural conditions, M. rhodesianum was genetically modified. The gene (hbd) encoding the R-3HB-degrading enzyme, R-3HB dehydrogenase, was inactivated in M. rhodesianum. The resulting hbd mutant still exhibited low growth rates on R-3HB as the sole source of carbon and energy, indicating the presence of alternative pathways for R-3HB utilization. Therefore, transposon mutagenesis was carried out with the hbd mutant, and a double mutant unable to grow on R-3HB was obtained. This mutant was shown to be defective in lipoic acid synthase (LipA), resulting in an incomplete citric acid cycle. Using the hbd lipA mutant, we produced 3.2 to 3.5 mM R-3HB in batch and 27 mM (2,800 mg liter−1) in fed-batch cultures. This was achieved by sequences of cultivation conditions initially favoring growth, then PHB accumulation, and finally PHB degradation.

Accumulation of poly(3-hydroxybutyric acid) and overproduction of exopolysaccharides in a mutant of a methylotrophic bacterium

1995 ◽  
Vol 41 (13) ◽  
pp. 55-59 ◽  
Author(s):  
Uta Breuer ◽  
Jörg-Uwe Ackermann ◽  
Wolfgang Babel
Keyword(s):  
Ammonium Phosphate ◽  
Sole Source ◽  
Chemical Mutagenesis ◽  
Methylotrophic Bacterium ◽  
Wild Type ◽  
Hydroxybutyric Acid ◽  
Serine Pathway ◽  
Methylobacterium Rhodesianum

The pink-pigmented facultatively methylotrophic bacterium Methylobacterium rhodesianum MB 126 is able to grow on methanol as the sole source of carbon and energy. Under certain conditions, e.g., limitation of ammonium, phosphate, or oxygen, carbon from methanol is channeled into poly(3-hydroxybutyric acid) (PHB) whereas other polymers or metabolites are hardly overproduced. A mutant of this strain, which we isolated after chemical mutagenesis, is impaired in its ability to synthesize PHB. Under the conditions mentioned above, the mutant still accumulated PHB, but in the absence of ammonium it simultaneously synthesized PHB and a considerable amount of an exopolysaccharide. This phenomenon was surprising insofar as the wild type did not produce exopolysaccharide in such amounts. An attempt was made to elucidate and discuss the possible reasons for these findings.Key words: methylotrophy, serine pathway bacteria, PHB, exopolysaccharides.

scholarly journals Methylobacterium populi sp. nov., a novel aerobic, pink-pigmented, facultatively methylotrophic, methane-utilizing bacterium isolated from poplar trees (Populus deltoides×nigra DN34)

2004 ◽  
Vol 54 (4) ◽  
pp. 1191-1196 ◽  
Author(s):  
Benoit Van Aken ◽  
Caroline M. Peres ◽  
Sharon Lafferty Doty ◽  
Jong Moon Yoon ◽  
Jerald L. Schnoor
Keyword(s):  
Populus Deltoides ◽  
Novel Species ◽  
Phylogenetic Analyses ◽  
Sole Source ◽  
Methylotrophic Bacterium ◽  
Carbon Source Utilization ◽  
Methylobacterium Extorquens ◽  
Poplar Trees ◽  
Methylobacterium Rhodesianum ◽  
Bacterium Strain

A pink-pigmented, aerobic, facultatively methylotrophic bacterium, strain BJ001T, was isolated from internal poplar tissues (Populus deltoides×nigra DN34) and identified as a member of the genus Methylobacterium. Phylogenetic analyses showed that strain BJ001T is related to Methylobacterium thiocyanatum, Methylobacterium extorquens, Methylobacterium zatmanii and Methylobacterium rhodesianum. However, strain BJ001T differed from these species in its carbon-source utilization pattern, particularly its use of methane as the sole source of carbon and energy, an ability that is shared with only one other member of the genus, Methylobacterium organophilum. In addition, strain BJ001T is the only member of the genus Methylobacterium to be described as an endophyte of poplar trees. On the basis of its physiological, genotypic and ecological properties, the isolate is proposed as a member of a novel species of the genus Methylobacterium, Methylobacterium populi sp. nov. (type strain, BJ001T=ATCC BAA-705T=NCIMB 13946T).

scholarly journals Functions of the Membrane-Associated and Cytoplasmic Malate Dehydrogenases in the Citric Acid Cycle ofEscherichia coli

2000 ◽  
Vol 182 (24) ◽  
pp. 6892-6899 ◽  
Author(s):  
Michel E. van der Rest ◽  
Christian Frank ◽  
Douwe Molenaar
Keyword(s):  
Citric Acid ◽  
Malic Enzyme ◽  
Citric Acid Cycle ◽  
Wild Type ◽  
Batch Cultures ◽  
Acid Cycle ◽  
E Coli ◽  
Link Type ◽  
Phosphoenolpyruvate Synthase ◽  
Rates Of Growth

ABSTRACT Oxidation of malate to oxaloacetate in Escherichia colican be catalyzed by two enzymes: the well-known NAD-dependent malate dehydrogenase (MDH; EC 1.1.1.37 ) and the membrane-associated malate:quinone-oxidoreductase (MQO; EC 1.1.99.16 ), encoded by the genemqo (previously called yojH). Expression of themqo gene and, consequently, MQO activity are regulated by carbon and energy source for growth. In batch cultures, MQO activity was highest during exponential growth and decreased sharply after onset of the stationary phase. Experiments with the β-galactosidase reporter fused to the promoter of the mqo gene indicate that its transcription is regulated by the ArcA-ArcB two-component system. In contrast to earlier reports, MDH did not repressmqo expression. On the contrary, MQO and MDH are active at the same time in E. coli. For Corynebacterium glutamicum, it was found that MQO is the principal enzyme catalyzing the oxidation of malate to oxaloacetate. These observations justified a reinvestigation of the roles of MDH and MQO in the citric acid cycle of E. coli. In this organism, a defined deletion of the mdh gene led to severely decreased rates of growth on several substrates. Deletion of the mqo gene did not produce a distinguishable effect on the growth rate, nor did it affect the fitness of the organism in competition with the wild type. To investigate whether in an mqo mutant the conversion of malate to oxaloacetate could have been taken over by a bypass route via malic enzyme, phosphoenolpyruvate synthase, and phosphenolpyruvate carboxylase, deletion mutants of the malic enzyme genessfcA and b2463 (coding for EC 1.1.1.38 and EC1.1.1.40 , respectively) and of the phosphoenolpyruvate synthase (EC2.7.9.2 ) gene pps were created. They were introduced separately or together with the deletion of mqo. These studies did not reveal a significant role for MQO in malate oxidation in wild-type E. coli. However, comparing growth of themdh single mutant to that of the double mutant containingmdh and mqo deletions did indicate that MQO partly takes over the function of MDH in an mdh mutant.

scholarly journals Batch cultivation of non-transgenic males suitable for SIT programs

2019 ◽  
Author(s):  
Siba Das ◽  
Maciej Maselko ◽  
Ambuj Upadhyay ◽  
Michael J. Smanski
Keyword(s):  
Field Performance ◽  
Mass Rearing ◽  
Batch Cultivation ◽  
Genetically Engineered ◽  
Sex Selection ◽  
Batch Cultures ◽  
Detection Assay ◽  
Heterogametic Sex ◽  
Genetic Constructs ◽  
Transgene Detection

AbstractThe field performance of Sterile Insect Technique (SIT) is improved by sex-sorting and releasing only the sterile males. This can be accomplished by resource-intensive separation of males from females by morphology. Alternatively, sex-ratio biasing genetic constructs can be used to selectively kill one sex without the need for manual or automated sorting, but the resulting genetically engineered (GE) control agents would be subject to additional governmental regulation. Here we describe and demonstrate a method for the batch production of non-GE males that is applicable for sex-selective production of males suitable for genetic biocontrol programs. This method could be applied to generate the heterogametic sex (XY, or WZ) in any organism with chromosomal sex determination. We observed up to 100% sex-selection with batch cultures of more than 103 individuals. Using a stringent transgene detection assay, we demonstrate the potential of mass rearing of transgene free males.

scholarly journals Genetic engineering of sex chromosomes for batch cultivation of non-transgenic, sex-sorted males

PLoS Genetics ◽  
2020 ◽  
Vol 16 (11) ◽  
pp. e1009180
Author(s):  
Siba R. Das ◽  
Maciej Maselko ◽  
Ambuj Upadhyay ◽  
Michael J. Smanski
Keyword(s):  
Field Performance ◽  
Batch Cultivation ◽  
Genetically Engineered ◽  
Sex Selection ◽  
Batch Cultures ◽  
Genetic Method ◽  
Detection Assay ◽  
Heterogametic Sex ◽  
Genetic Constructs ◽  
Transgene Detection

The field performance of Sterile Insect Technique (SIT) is improved by sex-sorting and releasing only sterile males. This can be accomplished by resource-intensive separation of males from females by morphology. Alternatively, sex-ratio biasing genetic constructs can be used to selectively remove one sex without the need for manual or automated sorting, but the resulting genetically engineered (GE) control agents would be subject to additional governmental regulation. Here we describe and demonstrate a genetic method for the batch production of non-GE males. This method could be applied to generate the heterogametic sex (XY, or WZ) in any organism with chromosomal sex determination. We observed up to 100% sex-selection with batch cultures of more than 103 individuals. Using a stringent transgene detection assay, we demonstrate the potential of mass production of transgene free males.

scholarly journals Naphthalene Degradation and Incorporation of Naphthalene-Derived Carbon into Biomass by the ThermophileBacillus thermoleovorans

2000 ◽  
Vol 66 (2) ◽  
pp. 518-523 ◽  
Author(s):  
E. Annweiler ◽  
H. H. Richnow ◽  
G. Antranikian ◽  
S. Hebenbrock ◽  
C. Garms ◽  
...  
Keyword(s):  
Carbon Balance ◽  
Sole Source ◽  
Degradation Pathway ◽  
Gas Chromatography Mass Spectrometry ◽  
Aerobic Bacterium ◽  
Batch Cultures ◽  
Naphthalene Degradation ◽  
Thermophilic Conditions ◽  
Bacillus Thermoleovorans ◽  
Isotope Analyses

ABSTRACT The thermophilic aerobic bacterium Bacillus thermoleovorans Hamburg 2 grows at 60°C on naphthalene as the sole source of carbon and energy. In batch cultures, an effective substrate degradation was observed. The carbon balance, including naphthalene, metabolites, biomass, and CO2, was determined by the application of [1-13C]naphthalene. The incorporation of naphthalene-derived carbon into the bulk biomass as well as into specified biomass fractions such as fatty acids and amino acids was confirmed by coupled gas chromatography-mass spectrometry (GC-MS) and isotope analyses. Metabolites were characterized by GC-MS; the established structures allow tracing the degradation pathway under thermophilic conditions. Apart from typical metabolites of naphthalene degradation known from mesophiles, intermediates such as 2,3-dihydroxynaphthalene, 2-carboxycinnamic acid, and phthalic and benzoic acid were identified for the pathway of this bacterium. These compounds indicate that naphthalene degradation by the thermophilicB. thermoleovorans differs from the known pathways found for mesophilic bacteria.

Degradation of acrylamide by immobilized cells of a Pseudomonas sp. and Xanthomonas maltophilia

1993 ◽  
Vol 39 (2) ◽  
pp. 207-212 ◽  
Author(s):  
Mohamed S. Nawaz ◽  
Wirt Franklin ◽  
Carl E. Cerniglia
Keyword(s):  
Acrylic Acid ◽  
Calcium Alginate ◽  
Specific Activity ◽  
Immobilized Cells ◽  
Sole Source ◽  
Pseudomonas Sp ◽  
Batch Cultures ◽  
Xanthomonas Maltophilia ◽  
Carbon And Nitrogen ◽  
Hydrolysis Of

Two bacterial isolates capable of utilizing acrylamide as the sole source of carbon and nitrogen were isolated from herbicide-contaminated soil samples and identified as Pseudomonas sp. and Xanthomonas maltophilia. Batch cultures of Pseudomonas sp. and X. maltophilia completely degraded 62.8 mM acrylamide to acrylic acid and ammonia in 24 and 48 h, respectively. Pseudomonas sp. And X. maltophilia, when immobilized in calcium alginate, markedly increased the rate of degradation of acrylamide over batch cultures. Cells of the isolates immobilized in calcium alginate degraded acrylamide to acrylic acid and ammonia in less than 6 h. Initial metabolism of acrylamide by immobilized cells of Pseudomonas sp. followed by inoculation with nonimmobilized cells after 6 h totally removed acrylamide and its metabolites in 72 h. A similar procedure with X. maltophilia resulted in the total metabolism of acrylamide in 96 h. An inducible, intracellular amidase was responsible for the hydrolysis of acrylamide to acrylic acid and ammonia. The specific activity of Pseudomonas sp. amidase was higher than the specific activity of X. maltophilia amidase.Key words: acrylamide, biodegradation, immobilization.

scholarly journals Predator-Specific Enrichment of Actinobacteria from a Cosmopolitan Freshwater Clade in Mixed Continuous Culture

2001 ◽  
Vol 67 (5) ◽  
pp. 2145-2155 ◽  
Author(s):  
Jakob Pernthaler ◽  
Thomas Posch ◽  
Karel S̆imek ◽  
Jaroslav Vrba ◽  
Annelie Pernthaler ◽  
...  
Keyword(s):  
Continuous Cultivation ◽  
Sole Source ◽  
Taxonomic Composition ◽  
Community Members ◽  
Bacterial Populations ◽  
Batch Cultures ◽  
Gram Positive Bacteria ◽  
Freshwater Bacteria ◽  
Freshwater Habitats ◽  
Average Size

ABSTRACT We investigated whether individual populations of freshwater bacteria in mixed experimental communities may exhibit specific responses to the presence of different bacterivorous protists. In two successive experiments, a two-stage continuous cultivation system was inoculated with nonaxenic batch cultures of the cryptophyteCryptomonas sp. Algal exudates provided the sole source of organic carbon for growth of the accompanying microflora. The dynamics of several 16S rRNA-defined bacterial populations were followed in the experimental communities. Although the composition and stability of the two microbial communities differed, numerous members of the first assemblage could again be detected during the second experiment. The introduction of a size-selectively feeding mixotrophic nanoflagellate (Ochromonas sp.) always resulted in an immediate bloom of a single phylotype population of members of the classActinobacteria (Ac1). These bacteria were phylogenetically affiliated with an uncultured lineage of gram-positive bacteria that have been found in freshwater habitats only. The Ac1 cells were close to the average size of freshwater bacterioplankton and significantly smaller than any of the other experimental community members. In contrast, no increase of the Ac1 population was observed in vessels exposed to the bacterivorous ciliate Cyclidium glaucoma. However, when the Ochromonas sp. was added after the establishment of C. glaucoma, the proportion of population Ac1 within the microbial community rapidly increased. Populations of a beta proteobacterial phylotype related to an Aquabacteriumsp. decreased relative to the total bacterial communities following the addition of either predator, albeit to different extents. The community structure of pelagic microbial assemblages can therefore be influenced by the taxonomic composition of the predator community.

scholarly journals Isolation and Initial Characterization of a Bacterial Consortium Able To Mineralize Fluorobenzene

2002 ◽  
Vol 68 (1) ◽  
pp. 102-105 ◽  
Author(s):  
M.F. Carvalho ◽  
C.C.T. Alves ◽  
M.I.M. Ferreira ◽  
P. De Marco ◽  
P.M.L. Castro
Keyword(s):  
Sole Source ◽  
Bacterial Consortium ◽  
Aerobic Biodegradation ◽  
Fluoride Ions ◽  
16S Rrna Analysis ◽  
Batch Cultures ◽  
Selective Enrichment ◽  
Microbial Strains ◽  
First Time

ABSTRACT Fluorinated compounds are known to be more resistant to microbial degradation than other halogenated chemicals. A microbial consortium capable of aerobic biodegradation of fluorobenzene (FB) as the sole source of carbon and energy was isolated by selective enrichment from sediments collected in a drain near an industrial site. A combination of three microbial strains recovered from the enriched consortium was shown to be necessary for complete FB mineralization. Two of the strains (F1 and F3) were classified by 16S rRNA analysis as belonging to the Sphingobacterium/Flavobacterium group, while the third (F4) falls in the β-Proteobacteria group, clustering with Alcaligenes species. Strain F4 was consistently found in the liquid cultures in a much greater proportion than strains F1 and F3 (86:8:6 for F4, F1, and F3, respectively). Stoichiometric release of fluoride ions was measured in batch and fed-batch cultures. In batch cultures, the consortium was able to use FB up to concentrations of 400 mg liter−1 and was able to utilize a range of other organic compounds, including 4-fluorophenol and 4-fluorobenzoate. To our knowledge this is the first time biodegradation of FB as a sole carbon source has been reported.

scholarly journals PARAMETERS OPTIMIZATION FOR INCREASED INTRACELLULAR INULINASE ACTIVITY OF A YEAST STRAIN

2019 ◽  
pp. 62-70
Author(s):  
Lyudmila Kabaivanova ◽  
Adriana Goushterova ◽  
Mariya Brazkova ◽  
Petar Grozdanov ◽  
Elena Chorukova ◽  
...  
Keyword(s):  
Yeast Strain ◽  
Sole Source ◽  
Parameters Optimization ◽  
Activity Assay ◽  
Cultivation Conditions ◽  
Inulinase Activity ◽  
Cultivation Process ◽  
Favorable Conditions ◽  
Extracellular Activity ◽  
Selection Of

This study reveals the selection of a yeast strain, possessing inulinase activity and finding the optimal conditions of cultivation. Intra- and extracellular activity assay was performed after cultivation on media, containing inulin as a sole source of carbon. Optimization of the cultivation conditions was carried out for establishing the favorable conditions for biosynthesis of inulinase. Modifying the physicochemical and nutritional parameters of a cultivation process lead to major improvement of the enzyme activity. Highest intra- and extracellular inulinase activity was registered when 1.5% inulin was used, 5 % inoculum, temperature 28°C, pH=6.5 and agitation of 200rpm. The selected strain Kluyveromyces sp. C showed higher values for the intracellular inulinase activity, making it suitable for immobilization and further use. Key words: Kluyveromyces sp., inulinase activity, parameters optimization

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