Cloning and Heterologous Expression of the Gene Encoding a Family 7 Glycosyl Hydrolase fromPenicillium funiculosum

2004 ◽  
pp. 170-193 ◽  
Author(s):  
Yat-Chen Chou ◽  
William S. Adney ◽  
Stephen R. Decker ◽  
John O. Baker ◽  
Glenna Kunkel ◽  
...  
Keyword(s):  
Heterologous Expression ◽  
Glycosyl Hydrolase ◽  
Gene Encoding

scholarly journals Functional Characterization of Corynebacterium alkanolyticum β-Xylosidase and Xyloside ABC Transporter in Corynebacterium glutamicum

2015 ◽  
Vol 81 (12) ◽  
pp. 4173-4183 ◽  
Author(s):  
Akira Watanabe ◽  
Kazumi Hiraga ◽  
Masako Suda ◽  
Hideaki Yukawa ◽  
Masayuki Inui
Keyword(s):  
Heterologous Expression ◽  
Corynebacterium Glutamicum ◽  
Abc Transporter ◽  
Binding Protein ◽  
Functional Characterization ◽  
Product Inhibition ◽  
Gene Encoding ◽  
Content Type ◽  
Industrial Strains ◽  
Efficient Fermentation

ABSTRACTTheCorynebacterium alkanolyticumxylEFGDgene cluster comprises thexylDgene that encodes an intracellular β-xylosidase next to thexylEFGoperon encoding a substrate-binding protein and two membrane permease proteins of a xyloside ABC transporter. Cloning of the cluster revealed a recombinant β-xylosidase of moderately high activity (turnover forp-nitrophenyl-β-d-xylopyranoside of 111 ± 4 s−1), weak α-l-arabinofuranosidase activity (turnover forp-nitrophenyl-α-l-arabinofuranoside of 5 ± 1 s−1), and high tolerance to product inhibition (Kifor xylose of 67.6 ± 2.6 mM). Heterologous expression of the entire cluster under the control of the strong constitutivetacpromoter in theCorynebacterium glutamicumxylose-fermenting strain X1 enabled the resultant strain X1EFGD to rapidly utilize not only xylooligosaccharides but also arabino-xylooligosaccharides. The ability to utilize arabino-xylooligosaccharides depended oncgR_2369, a gene encoding a multitask ATP-binding protein. Heterologous expression of the contiguousxylDgene in strain X1 led to strain X1D with 10-fold greater β-xylosidase activity than strain X1EFGD, albeit with a total loss of arabino-xylooligosaccharide utilization ability and only half the ability to utilize xylooligosaccharides. The findings suggest some inherent ability ofC. glutamicumto take up xylooligosaccharides, an ability that is enhanced by in the presence of a functionalxylEFG-encoded xyloside ABC transporter. The finding thatxylEFGimparts nonnative ability to take up arabino-xylooligosaccharides should be useful in constructing industrial strains with efficient fermentation of arabinoxylan, a major component of lignocellulosic biomass hydrolysates.

scholarly journals Sequencing and Heterologous Expression of an Epimerase and Two Lyases from Iminodisuccinate-Degrading Bacteria

2006 ◽  
Vol 72 (4) ◽  
pp. 2824-2828 ◽  
Author(s):  
Bettina Bäuerle ◽  
Željko Cokesa ◽  
Silvia Hofmann ◽  
Paul-Gerhard Rieger
Keyword(s):  
Amino Acid ◽  
Agrobacterium Tumefaciens ◽  
Heterologous Expression ◽  
Bacterial Strain ◽  
Amino Acid Sequences ◽  
Complexing Agent ◽  
Recombinant Enzymes ◽  
Gene Encoding ◽  
Degrading Bacteria

ABSTRACT Recently, degradation of all existing epimers of the complexing agent iminodisuccinate (IDS) in the bacterial strain Agrobacterium tumefaciens BY6 was proven to depend on an epimerase and a C-N lyase (Cokesa et al., Appl. Environ. Microbiol. 70:3941-3947, 2004). In the bacterial strain Ralstonia sp. strain SLRS7, a corresponding C-N lyase is responsible for the initial degradation step (Cokesa et al., Biodegradation 15:229-239, 2004). The ite gene, encoding the IDS-transforming epimerase, and the genes icl B and icl S, encoding the IDS-converting BY6-lyase and SLRS7-lyase, respectively, were cloned and sequenced. The epimerase gene encodes a protein with a predicted subunit molecular mass of 47.6 kDa. The highest degree of epimerase amino acid sequence identities was found with proteins of unknown function, indicating a novel protein. For the lyases, the deduced amino acid sequences show high similarity to enzymes of the fumarase II family. A classification into a new subfamily within the enzyme family is proposed. The subunit molecular masses of the lyases were calculated to be 54.4 and 54.7 kDa, respectively. In Agrobacterium tumefaciens BY6, the ite gene was on an approximately 180-kb circular plasmid, whereas the icl B gene was chromosomal like the corresponding icl S gene in Ralstonia sp. strain SLRS7. Heterologous expression in Escherichia coli and subsequent purification revealed recombinant enzymes with in vitro activity similar to that of the corresponding enzymes from the wild-type strains.

scholarly journals Fungal α-arabinofuranosidases of glycosyl hydrolase families 51 and 54 show a dual arabinofuranosyl- and galactofuranosyl-hydrolyzing activity

2012 ◽  
Vol 393 (8) ◽  
pp. 767-775 ◽  
Author(s):  
Boris Tefsen ◽  
Ellen L. Lagendijk ◽  
Joohae Park ◽  
Michiel Akeroyd ◽  
Doreen Schachtschabel ◽  
...  
Keyword(s):  
Cell Wall ◽  
Enzyme Activity ◽  
Molecular Modeling ◽  
Aspergillus Niger ◽  
Glycosyl Hydrolase ◽  
Hydrolase Activity ◽  
Gene Encoding ◽  
A Cell ◽  
Or Gene

Abstract Aspergillus niger possesses a galactofuranosidase activity, however, the corresponding enzyme or gene encoding this enzyme has never been identified. As evidence is mounting that enzymes exist with affinity for both arabinofuranose and galactofuranose, we investigated the possibility that α-l-arabinofuranosidases, encoded by the abfA and abfB genes, are responsible for the galactofuranosidase activity of A. niger. Characterization of the recombinant AbfA and AbfB proteins revealed that both enzymes do not only hydrolyze p-nitrophenyl-α-l-arabinofuranoside (pNp-α-Araf) but are also capable of hydrolyzing p-nitrophenyl-β-d-galactofuranoside (pNp-β-Galf). Molecular modeling of the AbfB protein with pNp-β-Galf confirmed the possibility for AbfB to interact with this substrate, similarly as with pNp-α-Araf. We also show that galactomannan, a cell wall compound of A. niger, containing β-linked terminal and internal galactofuranosyl moieties, can be degraded by an enzyme activity that is present in the supernatant of inulin-grown A. niger. Interestingly, purified AbfA and AbfB did not show this hydrolyzing activity toward A. nigergalactomannan. In summary, our studies demonstrate that AbfA and AbfB, α-l-arabinofuranosidases from different families, both contain a galactofuranose (Galf)-hydrolyzing activity. In addition, our data support the presence of a Galf-hydrolase activity expressed by A. niger that is capable of degrading fungal galactomannan.

scholarly journals Role of Glutamine Synthetase in Nitrogen Metabolite Repression in Aspergillus nidulans

2001 ◽  
Vol 183 (20) ◽  
pp. 5826-5833 ◽  
Author(s):  
Soula Margelis ◽  
Cletus D'Souza ◽  
Anna J. Small ◽  
Michael J. Hynes ◽  
Thomas H. Adams ◽  
...  
Keyword(s):  
Amino Acid ◽  
Glutamine Synthetase ◽  
Heterologous Expression ◽  
Aspergillus Nidulans ◽  
Essential Amino Acid ◽  
Gene Encoding ◽  
Nitrogen Metabolite Repression ◽  
Link Type ◽  
Wide Range

ABSTRACT Glutamine synthetase (GS), EC 6.3.1.2 , is a central enzyme in the assimilation of nitrogen and the biosynthesis of glutamine. We have isolated the Aspergillus nidulans glnA gene encoding GS and have shown that glnA encodes a highly expressed but not highly regulated mRNA. Inactivation of glnA results in an absolute glutamine requirement, indicating that GS is responsible for the synthesis of this essential amino acid. Even when supplemented with high levels of glutamine, strains lacking a functionalglnA gene have an inhibited morphology, and a wide range of compounds have been shown to interfere with repair of the glutamine auxotrophy. Heterologous expression of the prokaryotic Anabaena glnA gene from the A. nidulans alcA promoter allowed full complementation of the A. nidulans glnAΔ mutation. However, the A. nidulans fluG gene, which encodes a protein with similarity to prokaryotic GS, did not replace A. nidulans glnA function when similarly expressed. Our studies with theglnAΔ mutant confirm that glutamine, and not GS, is the key effector of nitrogen metabolite repression. Additionally, ammonium and its immediate product glutamate may also act directly to signal nitrogen sufficiency.

scholarly journals Isolation, sequencing, and heterologous expression of the Paecilomyces variotii gene encoding S-hydroxymethylglutathione dehydrogenase (fldA)

2014 ◽  
Vol 99 (4) ◽  
pp. 1755-1763 ◽  
Author(s):  
Takuji Oka ◽  
Yuji Komachi ◽  
Kazufumi Ohshima ◽  
Yoichi Kawano ◽  
Kohsai Fukuda ◽  
...  
Keyword(s):  
Heterologous Expression ◽  
Paecilomyces Variotii ◽  
Gene Encoding
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