scholarly journals Cloning of Escherichia coli lacZ and lacY Genes and Their Expression in Gluconobacter oxydans and Acetobacter liquefaciens

2002 ◽  
Vol 68 (5) ◽  
pp. 2619-2623 ◽  
Author(s):  
Hesham E. Mostafa ◽  
Knut J. Heller ◽  
Arnold Geis
Keyword(s):  
Escherichia Coli ◽  
Acetic Acid ◽  
Gluconobacter Oxydans ◽  
Acetic Acid Bacteria ◽  
Broad Host Range ◽  
Transformation Protocol ◽  
Ethanol Medium ◽  
Range Vector ◽  
Growth Ability ◽  
Broad Host Range Vector

ABSTRACT An efficient transformation protocol for Gluconobacter oxydans and Acetobacter liquefaciens strains was developed by preparation of electrocompetent cells grown on yeast extract-ethanol medium. Plasmid pBBR122 was used as broad-host-range vector to clone the Escherichia coli lacZY genes in G. oxydans and A. liquefaciens. Although both lac genes were functionally expressed in both acetic acid bacteria, only a few transformants were able to grow on lactose. However, this ability strictly depended on the presence of a plasmid expressing both lac genes. Mutations in the plasmids and/or in the chromosome were excluded as the cause of growth ability on lactose.

scholarly journals Microbial Production of Glyceric Acid, an Organic Acid That Can Be Mass Produced from Glycerol

2009 ◽  
Vol 75 (24) ◽  
pp. 7760-7766 ◽  
Author(s):  
Hiroshi Habe ◽  
Yuko Shimada ◽  
Toshiharu Yakushi ◽  
Hiromi Hattori ◽  
Yoshitaka Ano ◽  
...  
Keyword(s):  
Acetic Acid ◽  
Growth Parameters ◽  
Operating Time ◽  
Enantiomeric Excess ◽  
Gluconobacter Oxydans ◽  
Acetic Acid Bacteria ◽  
Culture Broth ◽  
Glyceric Acid ◽  
Bacterial Strains ◽  
Biotechnological Production

ABSTRACT Glyceric acid (GA), an unfamiliar biotechnological product, is currently produced as a small by-product of dihydroxyacetone production from glycerol by Gluconobacter oxydans. We developed a method for the efficient biotechnological production of GA as a target compound for new surplus glycerol applications in the biodiesel and oleochemical industries. We investigated the ability of 162 acetic acid bacterial strains to produce GA from glycerol and found that the patterns of productivity and enantiomeric GA compositions obtained from several strains differed significantly. The growth parameters of two different strain types, Gluconobacter frateurii NBRC103465 and Acetobacter tropicalis NBRC16470, were optimized using a jar fermentor. G. frateurii accumulated 136.5 g/liter of GA with a 72% d-GA enantiomeric excess (ee) in the culture broth, whereas A. tropicalis produced 101.8 g/liter of d-GA with a 99% ee. The 136.5 g/liter of glycerate in the culture broth was concentrated to 236.5 g/liter by desalting electrodialysis during the 140-min operating time, and then, from 50 ml of the concentrated solution, 9.35 g of GA calcium salt was obtained by crystallization. Gene disruption analysis using G. oxydans IFO12528 revealed that the membrane-bound alcohol dehydrogenase (mADH)-encoding gene (adhA) is required for GA production, and purified mADH from G. oxydans IFO12528 catalyzed the oxidation of glycerol. These results strongly suggest that mADH is involved in GA production by acetic acid bacteria. We propose that GA is potentially mass producible from glycerol feedstock by a biotechnological process.

scholarly journals A tunable l-arabinose-inducible expression plasmid for the acetic acid bacterium Gluconobacter oxydans

2020 ◽  
Vol 104 (21) ◽  
pp. 9267-9282
Author(s):  
Philipp Moritz Fricke ◽  
Tobias Link ◽  
Jochem Gätgens ◽  
Christiane Sonntag ◽  
Maike Otto ◽  
...  
Keyword(s):  
Acetic Acid ◽  
Stationary Phase ◽  
Gene Knockout ◽  
Glucose Dehydrogenase ◽  
Gluconobacter Oxydans ◽  
Acetic Acid Bacteria ◽  
Acetic Acid Bacterium ◽  
Basal Expression ◽  
Fold Induction ◽  
Membrane Bound

Abstract The acetic acid bacterium (AAB) Gluconobacter oxydans incompletely oxidizes a wide variety of carbohydrates and is therefore used industrially for oxidative biotransformations. For G. oxydans, no system was available that allows regulatable plasmid-based expression. We found that the l-arabinose-inducible PBAD promoter and the transcriptional regulator AraC from Escherichia coli MC4100 performed very well in G. oxydans. The respective pBBR1-based plasmids showed very low basal expression of the reporters β-glucuronidase and mNeonGreen, up to 480-fold induction with 1% l-arabinose, and tunability from 0.1 to 1% l-arabinose. In G. oxydans 621H, l-arabinose was oxidized by the membrane-bound glucose dehydrogenase, which is absent in the multi-deletion strain BP.6. Nevertheless, AraC-PBAD performed similar in both strains in the exponential phase, indicating that a gene knockout is not required for application of AraC-PBAD in wild-type G. oxydans strains. However, the oxidation product arabinonic acid strongly contributed to the acidification of the growth medium in 621H cultures during the stationary phase, which resulted in drastically decreased reporter activities in 621H (pH 3.3) but not in BP.6 cultures (pH 4.4). These activities could be strongly increased quickly solely by incubating stationary cells in d-mannitol-free medium adjusted to pH 6, indicating that the reporters were hardly degraded yet rather became inactive. In a pH-controlled bioreactor, these reporter activities remained high in the stationary phase (pH 6). Finally, we created a multiple cloning vector with araC-PBAD based on pBBR1MCS-5. Together, we demonstrated superior functionality and good tunability of an AraC-PBAD system in G. oxydans that could possibly also be used in other AAB. Key points • We found the AraC-PBADsystem from E. coli MC4100 was well tunable in G. oxydans. •  In the absence of AraC orl-arabinose, expression from PBADwas extremely low. • This araC-PBADsystem could also be fully functional in other acetic acid bacteria.

A broad-host-range vector system for cloning and translational lacZ fusion analysis

Plasmid ◽  
1988 ◽  
Vol 19 (3) ◽  
pp. 175-188 ◽  
Author(s):  
Tsu-Ning Tai ◽  
Wendy A. Havelka ◽  
Samuel Kaplan
Keyword(s):  
Host Range ◽  
Lacz Fusion ◽  
Vector System ◽  
Broad Host Range ◽  
Range Vector ◽  
Fusion Analysis ◽  
Broad Host Range Vector

Monitoring Azospirillum-wheat interactions using the gfp and gusA genes constitutively expressed from a new broad-host range vector

2002 ◽  
Vol 97 (3) ◽  
pp. 243-252 ◽  
Author(s):  
Humberto J.O Ramos ◽  
Lauren D.B Roncato-Maccari ◽  
Emanuel M Souza ◽  
Juliana R.L Soares-Ramos ◽  
Mariangela Hungria ◽  
...  
Keyword(s):  
Host Range ◽  
Broad Host Range ◽  
Range Vector ◽  
Broad Host Range Vector

scholarly journals Gene Targeting in Gram-Negative Bacteria by Use of a Mobile Group II Intron (“Targetron”) Expressed from a Broad-Host-Range Vector

2007 ◽  
Vol 73 (8) ◽  
pp. 2735-2743 ◽  
Author(s):  
Jun Yao ◽  
Alan M. Lambowitz
Keyword(s):  
Host Range ◽  
Gene Targeting ◽  
Inducible Promoter ◽  
Broad Host Range ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Efficient Gene ◽  
Range Vector ◽  
Group Ii ◽  
Broad Host Range Vector

ABSTRACT Mobile group II introns (“targetrons”) can be programmed for insertion into virtually any desired DNA target with high frequency and specificity. Here, we show that targetrons expressed via an m-toluic acid-inducible promoter from a broad-host-range vector containing an RK2 minireplicon can be used for efficient gene targeting in a variety of gram-negative bacteria, including Escherichia coli, Pseudomonas aeruginosa, and Agrobacterium tumefaciens. Targetrons expressed from donor plasmids introduced by electroporation or conjugation yielded targeted disruptions at frequencies of 1 to 58% of screened colonies in the E. coli lacZ, P. aeruginosa pqsA and pqsH, and A. tumefaciens aopB and chvI genes. The development of this broad-host-range system for targetron expression should facilitate gene targeting in many bacteria.

scholarly journals Isolation of dihydroxyacetone-producing acetic acid bacteria in Vietnam

2016 ◽  
Vol 19 (4) ◽  
pp. 31-38
Author(s):  
Huong Thi Lan Vu ◽  
Oanh Thi Kim Nguyen ◽  
Van Thi Thu Bui ◽  
Uyen Thi Tu Bui ◽  
Nghiep Dai Ngo ◽  
...  
Keyword(s):  
Acetic Acid ◽  
Gluconobacter Oxydans ◽  
Acetic Acid Bacteria ◽  
Rrna Gene ◽  
16S Rrna Gene Sequences ◽  
Group Iii ◽  
Group I ◽  
Routine Identification ◽  
Group Ii ◽  
Potential Use

Sixty-six acetic acid bacteria (AAB) were isolated from fourty-five flowers and fruits collected in Hochiminh City, Vietnam. Of the sixty-six, thirty-one isolates were selected as dihydroxyacetone (DHA)-producing AAB based on the reaction with Fehling’s solution and grouped into three groups by routine identification with phenotypic features. Group I composed of fourteen isolates and was assigned to the genus Acetobacter, Group II composed of thirteen isolates and was assigned to the genus Gluconobacter and Group III was the remaining four isolates and was assigned to the genus Gluconacetobacter. Ten isolates among the thirteen isolates of Group II gave a larger amount of DHA (22.2–26.0 mg/mL) than Gluconobacter oxydans NBRC 14819T (19.8 mg/mL), promising for the potential use in producing DHA. In phylogenetic analysis based on 16S rRNA gene sequences, six isolates of the ten potential DHA producers were suggested to be candidates for new taxa in the genus Gluconobacter.

scholarly journals Characterization and inactivation of the membrane-bound polyol dehydrogenase in Gluconobacter oxydans DSM 7145 reveals a role in meso-erythritol oxidation

Microbiology ◽  
2010 ◽  
Vol 156 (6) ◽  
pp. 1890-1899 ◽  
Author(s):  
Jörn Voss ◽  
Armin Ehrenreich ◽  
Wolfgang Liebl
Keyword(s):  
Acetic Acid ◽  
Gluconobacter Oxydans ◽  
Acetic Acid Bacteria ◽  
Second Phase ◽  
Growth Substrate ◽  
Whole Cell ◽  
Polyol Dehydrogenase ◽  
Membrane Bound ◽  
Two Stages ◽  
Substrate Spectrum

The growth of Gluconobacter oxydans DSM 7145 on meso-erythritol is characterized by two stages: in the first stage, meso-erythritol is oxidized almost stoichiometrically to l-erythrulose according to the Bertrand–Hudson rule. The second phase is distinguished from the first phase by a global metabolic change from membrane-bound meso-erythritol oxidation to l-erythrulose assimilation with concomitant accumulation of acetic acid. The membrane-associated erythritol-oxidizing enzyme was found to be encoded by a gene homologous to sldA known from other species of acetic acid bacteria. Disruption of this gene in the genome of G. oxydans DSM 7145 revealed that the membrane-bound polyol dehydrogenase not only oxidizes meso-erythritol but also has a broader substrate spectrum which includes C3–C6 polyols and d-gluconate and supports growth on these substrates. Cultivation of G. oxydans DSM 7145 on different substrates indicated that expression of the polyol dehydrogenase was not regulated, implying that the production of biomass of G. oxydans to be used as whole-cell biocatalysts in the biotechnological conversion of meso-erythritol to l-erythrulose, which is used as a tanning agent in the cosmetics industry, can be conveniently carried out with glucose as the growth substrate.

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