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Fulltext Development of a pyrG mutant of Aspergillus oryzae strain S1 as a host for the production of heterologous proteins

Ling SO, Storms R, Zheng Y, Rodzi MR, Mahadi NM, Illias RM, et al.

ScientificWorldJournal, 2013;2013:634317.
PMID: 24381522 DOI: 10.1155/2013/634317

The ease with which auxotrophic strains and genes that complement them can be manipulated, as well as the stability of auxotrophic selection systems, are amongst the advantages of using auxotrophic markers to produce heterologous proteins. Most auxotrophic markers in Aspergillus oryzae originate from chemical or physical mutagenesis that may yield undesirable mutations along with the mutation of interest. An auxotrophic A. oryzae strain S1 was generated by deleting the orotidine-5'-monophosphate decarboxylase gene (pyrG) by targeted gene replacement. The uridine requirement of the resulting strain GR6 pyrGΔ0 was complemented by plasmids carrying a pyrG gene from either Aspergillus nidulans or A. oryzae. β -Galactosidase expression by strain GR6 pyrGΔ0 transformed with an A. niger plasmid encoding a heterologous β -galactosidase was at least 150 times more than that obtained with the untransformed strain. Targeted gene replacement is thus an efficient way of developing auxotrophic mutants in A. oryzae and the auxotrophic strain GR6 pyrGΔ0 facilitated the production of a heterologous protein in this fungus.

Matched MeSH terms: Mutagenesis, Site-Directed/methods*
Combined Overlap Extension PCR Method for Improved Site Directed Mutagenesis

Hussain H, Chong NF

Biomed Res Int, 2016;2016:8041532.
PMID: 27995143

The combined overlap extension PCR (COE-PCR) method developed in this work combines the strengths of the overlap extension PCR (OE-PCR) method with the speed and ease of the asymmetrical overlap extension (AOE-PCR) method. This combined method allows up to 6 base pairs to be mutated at a time and requires a total of 40-45 PCR cycles. A total of eight mutagenesis experiments were successfully carried out, with each experiment mutating between two to six base pairs. Up to four adjacent codons were changed in a single experiment. This method is especially useful for codon optimization, where doublet or triplet rare codons can be changed using a single mutagenic primer set, in a single experiment.

Matched MeSH terms: Mutagenesis, Site-Directed/methods*
Manipulation of the conformation and enzymatic properties of T1 lipase by site-directed mutagenesis of the protein core

Wahab RA, Basri M, Rahman RN, Salleh AB, Rahman MB, Chor LT

Appl Biochem Biotechnol, 2012 Jun;167(3):612-20.
PMID: 22581079 DOI: 10.1007/s12010-012-9728-2

In silico and experimental investigations were conducted to explore the effects of substituting hydrophobic residues, Val, Met, Leu, Ile, Trp, and Phe into Gln 114 of T1 lipase. The in silico investigations accurately predicted the enzymatic characteristics of the mutants in the experimental studies and provided rationalization for some of the experimental observations. Substitution with Leu successfully improved the conformational stability and enzymatic characteristics of T1 lipase. However, replacement of Gln114 with Trp negatively affected T1 lipase and resulted in the largest disruption of protein stability, diminished lipase activity and inferior enzymatic characteristics. These results suggested that the substitution of a larger residue in a densely packed area of the protein core can have considerable effects on the structure and function of an enzyme. This is especially true when the residue is next to the catalytic serine as demonstrated with the Phe and Trp mutation.

Matched MeSH terms: Mutagenesis, Site-Directed/methods*
Use of Megaprimer and Overlapping Extension PCR (OE-PCR) to Mutagenize and Enhance Cyclodextrin Glucosyltransferase (CGTase) Function

Goh KM, Liew KJ, Chai KP, Illias RM

Methods Mol Biol, 2017;1498:385-396.
PMID: 27709591

Protein engineering is a very useful tool for probing structure-function relationships in proteins. Specifically, site-directed mutagenized proteins can provide useful insights into structural, binding and catalytic mechanisms of a protein, particularly when coupled with crystallization. In this chapter, we describe two protocols for performing site-directed mutagenesis of any protein-coding sequence, namely, megaprimer PCR and overlapping extension PCR (OE-PCR). We use as an example how these two SDM methods enhanced the function of a cyclodextrin glucosyltransferase (CGTase) from Bacillus lehensis strain G1.

Matched MeSH terms: Mutagenesis, Site-Directed/methods
Characterization of Chlamydomonas Ribulose-1,5-bisphosphate carboxylase/oxygenase variants mutated at residues that are post-translationally modified

Rasineni GK, Loh PC, Lim BH

Biochim Biophys Acta Gen Subj, 2017 Feb;1861(2):79-85.
PMID: 27816753 DOI: 10.1016/j.bbagen.2016.10.027

BACKGROUND: Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the chloroplast enzyme that fixes CO2 in photosynthesis, but the enzyme also fixes O2, which leads to the wasteful photorespiratory pathway. If we better understand the structure-function relationship of the enzyme, we might be able to engineer improvements. When the crystal structure of Chlamydomonas Rubisco was solved, four new posttranslational modifications were observed which are not present in other species. The modifications were 4-hydroxylation of the conserved Pro-104 and 151 residues, and S-methylation of the variable Cys-256 and 369 residues, which are Phe-256 and Val-369 in land plants. Because the modifications were only observed in Chlamydomonas Rubisco, they might account for the differences in kinetic properties between the algal and plant enzymes.
METHODS: Site-directed mutagenesis and chloroplast transformation have been used to test the essentiality of these modifications by replacing each of the residues with alanine (Ala). Biochemical analyses were done to determine the specificity factors and kinetic constants.
RESULTS: Replacing the modified-residues in Chlamydomonas Rubisco affected the enzyme's catalytic activity. Substituting hydroxy-Pro-104 and methyl-Cys-256 with alanine influenced Rubisco catalysis.
CONCLUSION: This is the first study on these posttranslationally-modified residues in Rubisco by genetic engineering. As these forms of modifications/regulation are not available in plants, the modified residues could be a means to modulate Rubisco activity.
GENERAL SIGNIFICANCE: With a better understanding of Rubisco structure-function, we can define targets for improving the enzyme.

Matched MeSH terms: Mutagenesis, Site-Directed/methods
Rapid cloning of disease-resistance genes in plants using mutagenesis and sequence capture

Steuernagel B, Periyannan SK, Hernández-Pinzón I, Witek K, Rouse MN, Yu G, et al.

Nat Biotechnol, 2016 Jun;34(6):652-5.
PMID: 27111722 DOI: 10.1038/nbt.3543

Wild relatives of domesticated crop species harbor multiple, diverse, disease resistance (R) genes that could be used to engineer sustainable disease control. However, breeding R genes into crop lines often requires long breeding timelines of 5-15 years to break linkage between R genes and deleterious alleles (linkage drag). Further, when R genes are bred one at a time into crop lines, the protection that they confer is often overcome within a few seasons by pathogen evolution. If several cloned R genes were available, it would be possible to pyramid R genes in a crop, which might provide more durable resistance. We describe a three-step method (MutRenSeq)-that combines chemical mutagenesis with exome capture and sequencing for rapid R gene cloning. We applied MutRenSeq to clone stem rust resistance genes Sr22 and Sr45 from hexaploid bread wheat. MutRenSeq can be applied to other commercially relevant crops and their relatives, including, for example, pea, bean, barley, oat, rye, rice and maize.

Matched MeSH terms: Mutagenesis, Site-Directed/methods*