scholarly journals The Role of the STAS Domain in the Function and Biogenesis of a Sulfate Transporter as Probed by Random Mutagenesis

2006 ◽  
Vol 281 (32) ◽  
pp. 22964-22973 ◽  
Author(s):  
Nakako Shibagaki ◽  
Arthur R. Grossman
Keyword(s):  
Random Mutagenesis ◽  
Sulfate Transporter ◽  
Stas Domain

scholarly journals Enhanced production of Clavulanic acid by improving glycerol utilization using reporter-guided mutagenesis of an industrial Streptomyces clavuligerus strain

2021 ◽  
Author(s):  
Chang-Hun Shin ◽  
Hang Soo Cho ◽  
Hyung-Jin Won ◽  
Ho Jeong Kwon ◽  
Chan-Wha Kim ◽  
...  
Keyword(s):  
Clavulanic Acid ◽  
Wild Type Strain ◽  
Random Mutagenesis ◽  
Streptomyces Clavuligerus ◽  
Wild Type ◽  
Mutant Selection ◽  
Enhanced Production ◽  
Industrial Strains ◽  
Glycerol Utilization

Abstract Clavulanic acid (CA) produced by Streptomyces clavuligerus is a clinically important β-lactamase inhibitor. It is known that glycerol utilization can significantly improve cell growth and CA production of S. clavuligerus. We found that the industrial CA-producing S. clavuligerus strain OR generated by random mutagenesis consumes less glycerol than the wild-type strain; we then developed a mutant strain in which the glycerol utilization operon is overexpressed, as compared to the parent OR strain, through iterative random mutagenesis and reporter-guided selection. The CA production of the resulting S. clavuligerus ORUN strain was increased by approximately 31.3 per cent (5.21 ± 0.26 g/L) in a flask culture and 17.4 per cent (6.11 ± 0.36 g/L) in a fermenter culture, as compared to that of the starting OR strain. These results confirmed the important role of glycerol utilization in CA production and demonstrated that reporter-guided mutant selection is an efficient method for further improvement of randomly mutagenized industrial strains.

scholarly journals σ54 (σL) plays a central role in carbon metabolism in the industrially relevant Clostridium beijerinckii

2018 ◽  
Author(s):  
Rémi Hocq ◽  
Maxime Bouilloux-Lafont ◽  
Nicolas Lopes Ferreira ◽  
François Wasels
Keyword(s):  
Point Mutations ◽  
Random Mutagenesis ◽  
Value Added ◽  
Clostridium Beijerinckii ◽  
Phenotypic Traits ◽  
Wild Type ◽  
Genomic Comparison ◽  
Alcohol Synthesis

Microbial production of butanol and isopropanol, two high value-added chemicals, is naturally occurring in the solventogenic Clostridium beijerinckii DSM 6423. Despite its ancient discovery, the precise mechanisms controlling alcohol synthesis in this microorganism are poorly understood. In this work, an allyl alcohol tolerant strain obtained by random mutagenesis was characterized. This strain, designated as the AA mutant, shows a dominant production of acids, a severely diminished butanol synthesis capacity, and produces acetone instead of isopropanol. Interestingly, this solvent-deficient strain was also found to have a limited consumption of two carbohydrates and to be still able to form spores, highlighting its particular phenotype. Sequencing of the AA mutant revealed point mutations in several genes including CIBE_0767 (sigL), which encodes the σ54 sigma factor. Complementation with the wild-type sigL gene fully restored solvent production and sugar assimilation, demonstrating that σ54 plays a central role in regulating these pathways in C. beijerinckii DSM 6423. Genomic comparison with other strains further revealed that these functions are probably conserved among the C. beijerinckii strains. The importance of σ54 in C. beijerinckii was further assessed by the characterization of a sigL deletion mutant of the model strain NCIMB 8052 obtained with a CRISPR/Cas9 tool. The resulting mutant exhibited phenotypic traits similar to the AA strain, and was subsequently complemented with the sigL gene from either the wild type or the AA strains. The results of this experiment confirmed the crucial role of σ54 in the regulation of both solventogenesis and sugar consumption pathways in C. beijerinckii.

scholarly journals Localization of Slc26a9 and role of the STAS domain

2006 ◽  
Vol 20 (5) ◽  
Author(s):  
Aleksandra Sindic ◽  
Consuelo Plata ◽  
Caroline R. Sussman ◽  
David B. Mount ◽  
Min‐Hwang Chang ◽  
...  
Keyword(s):  
Stas Domain

scholarly journals In Vitro Generation of Novel Pyrimethamine Resistance Mutations in the Toxoplasma gondiiDihydrofolate Reductase

2001 ◽  
Vol 45 (4) ◽  
pp. 1271-1277 ◽  
Author(s):  
Mary G. Reynolds ◽  
Jung Oh ◽  
David S. Roos
Keyword(s):  
Potential Role ◽  
Opportunistic Infections ◽  
Potent Inhibitor ◽  
Random Mutagenesis ◽  
Protozoan Parasite ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Resistance Mutations

ABSTRACT Pyrimethamine is a potent inhibitor of dihydrofolate reductase and is widely used in the treatment of opportunistic infections caused by the protozoan parasite Toxoplasma gondii. In order to assess the potential role of dhfr sequence polymorphisms in drug treatment failures, we examined the dhfr-ts genes of representative isolates for T. gondii virulence types I, II, and III. These strains exhibit differences in their sensitivities to pyrimethamine but no differences in predicted dhfr-tsprotein sequences. To assess the potential for pyrimethamine-resistantdhfr mutants to emerge, three drug-sensitive variants of the T. gondii dhfr-ts gene (the wild-type T. gondii sequence and two mutants engineered to reflect polymorphisms observed in drug-sensitive Plasmodium falciparum) were subjected to random mutagenesis and transfected into either wild-type T. gondii parasites ordhfr-deficient Saccharomyces cerevisiae under pyrimethamine selection. Three resistance mutations were identified, at amino acid residues 25 (Trp→Arg), 98 (Leu→Ser), and 134 (Leu→His).

Rational engineering of a fluorescein-binding anticalin for improved ligand affinity

2005 ◽  
Vol 386 (11) ◽  
pp. 1097-1104 ◽  
Author(s):  
Sven Vopel ◽  
Hermine Mühlbach ◽  
Arne Skerra
Keyword(s):  
Protein Design ◽  
Random Mutagenesis ◽  
Quenching Effect ◽  
Ligand Affinity ◽  
Rational Engineering ◽  
Melting Temperatures ◽  
High Affinity ◽  
Rational Protein Design ◽  
Better Than

Abstract The anticalin FluA is an artificial lipocalin with novelspecificity for the fluorescein group, which was engineered from an insect bilin-binding protein by targeted random mutagenesis and selection. Based on the crystal structure of FluA, an attempt was made to improve the complementarity of its ligand pocket to fluorescein by rational protein design. Several side chains participating in sub-optimal interactions with the ligand were identified and replaced by residues that promised a better steric fit. As a result, the substitution of Ala45 by Ile and of Ser114 by Thr or Arg led to a tight affinity of ca. 1 nM, which is approximately 30-fold better than that of the parental anticalin. Similar to the original FluA, the improved version shows almost complete quenching of the bound ligand fluorescence. Interestingly, the quenching effect was significantly reduced when Trp129 was replaced by Tyr, thus supporting the previously postulated role of this residue, which closely packs against the bound ligand, for efficient electron transfer to the excited fluorescein. Circular dichroism spectra revealed that all variants investigated had retained the lipocalin fold. Corresponding thermal unfolding experiments confirmed similar folding stabilities, with melting temperatures ranging from 52.9 to 60.5°C (i.e., for the high-affinity variant).

Enhancement of lipase activity from Acinetobacter species SY-01 by random mutagenesis and the role of lipase-specific chaperone

2004 ◽  
Vol 35 (5) ◽  
pp. 377-384 ◽  
Author(s):  
Soo-Jin Han ◽  
Ji Hyung Chung ◽  
Chan Seong Cheong ◽  
Il Yup Chung ◽  
Ye Sun Han
Keyword(s):  
Lipase Activity ◽  
Random Mutagenesis ◽  
Acinetobacter Species
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