Random Mutagenesis Method Research Articles

An overview of high-throughput screening systems for enantioselective enzymatic transformations.

The directed evolution of enantioselective enzymes (1,2) for use in organic synthesis constitutes an attractive alternative to traditional forms of asymmetric catalysis based on chiral transition metal complexes or catalytic antibodies. It involves the proper combination of molecular biological methods for random gene mutagenesis and gene expression coupled with appropriate high-throughput screening systems that allow the rapid determination of the enantiomeric purity of a chiral product. Typically, thousands of samples arising from the catalytic action of the evolved enzyme variants on a given substrate of interest need to be assayed within a reasonable time span, ideally within one day.

Directed evolution of selective enzymes and hybrid catalysts

The methods of directed evolution, developed in the 1990s, can be applied successfully to the creation of enantioselective enzymes for use in synthetic organic chemistry. The combination of appropriate molecular biological methods for random mutagenesis and expression coupled with high-throughput screening systems for the determination of ee-values forms the basis of this novel approach to asymmetric catalysis. The principle is illustrated by the dramatic enhancement of enantioselectivity of a lipase as the catalyst in the hydrolytic kinetic resolution of a chiral ester, the selectivity factor improving from E=1.1 to E=51. Reversal of enantioselectivity is also possible. Finally, the concept of directed evolution of selective hybrid catalysts has been delineated.

Laboratory method to study mutational effects on human erythrocyte spectrin tetramerization.

We have developed a laboratory method combining a random mutagenesis method and a yeast two-hybrid system to study effects of mutation on human erythrocyte spectrin tetramerization. A PCR-based procedure was used to generate random mutations in DNA fragments of the first 55 residues of alpha-spectrin. Each of the DNA fragments from random mutagenesis was fused with a DNA fragment of native spectrin consisting of residues 56 to 368 to give a DNA fragment of the first 368 residues in alpha-spectrin. The alpha-spectrin DNA fragment and a DNA fragment containing the last 449 residues in beta-spectrin were introduced into the yeast two-hybrid system for rapid screening of alpha- and beta-spectrin interaction. Yeast colonies with interacting alpha- and beta-peptides were blue, and those with non-interacting alpha- and beta-peptides were white. Six single amino acid mutations (R27G, Y35N, F38S, L49H, Y53N, and Y53C) and a double amino acid mutation (K16M, I24N) were identified from 8 white colonies, but no mutations were found in the DNA fragments of 14 blue colonies. Thus this simple laboratory method allows us to study effects of mutation on interactions of alpha- and beta-spectrin at the tetramerization site.

An antisense-based functional genomics approach for identification of genes critical for growth of Candida albicans.

Converting the complete genome sequence of Candida albicans into meaningful biological information will require comprehensive screens for identifying functional classes of genes. Most systems described so far are not applicable to C. albicans because of its difficulty with mating, its diploid nature, and the lack of functional random insertional mutagenesis methods. We examined artificial gene suppression as a means to identify gene products critical for growth of this pathogen; these represent new antifungal drug targets. To achieve gene suppression we combined antisense RNA inhibition and promoter interference. After cloning antisense complementary DNA (cDNA) fragments under control of an inducible GAL1 promoter, we transferred the resulting libraries to C. albicans. Over 2,000 transformant colonies were screened for a promoter-induced diminished-growth phenotype. After recovery of the plasmids, sequence determination of their inserts revealed the messenger RNA (mRNA) they inhibited or the gene they disrupted. Eighty-six genes critical for growth were identified, 45 with unknown function. When used in high-throughput screening for antifungals, the crippled C. albicans strains generated in this study showed enhanced sensitivity to specific drugs.

Efficient insertional mutagenesis in Streptococcus thermophilus

Bacteria have always been considered ideal organisms for genetic analysis. While this is true for some model organisms, like Escherichia coli, Bacillus subtilis and, more recently, Lactococcus lactis, genetic analysis of other organisms is often prevented by lack of valuable tools, like vectors, transposons and methods for transformation, gene inactivation and random insertional mutagenesis. This is the case of the moderately thermophilic bacterium Streptococcus thermophilus, an organism that, in spite of its widespread use for food fermentations, is only poorly characterized. We report here an insertional mutagenesis system that allows efficient random mutagenesis, easy characterization of the interrupted genes and construction of stable null mutations. This may become a powerful S. thermophilus tool for both genetic analysis and construction of ‘food-grade’ mutants of this biotechnologically relevant microorganism.

Random changes of amino acid residues with expected frequency by saturated point mutagenesis.

The yeast transcriptional activator protein, Gcn4p from Saccharomyces cerevisiae binds to the specific sequence in the promoters of many amino acid biosynthetic genes for general control. A new random saturation mutagenesis method was developed to isolate Gcn4p derivatives with only one or two mutations in the DNA binding domain without using radioactive isotope. This will be used to identify the amino acids of Gcn4p involved in protein-protein interactions. Saturation mutagenesis in the DNA binding domain of Gcn4p was performed using spiked degenerate oligonucleotides containing randomized codon bases designed specifically for only one or two base changes in the mutagenized area. These oligonucleotides were synthesized to have two flanking restriction enzyme sites for cloning to the appropriate vector. The 3' ends were mutually primed after hybridization via the palindromic sequences of the restriction enzyme sites. These molecules were then converted to double stranded DNA upon treatment with DNA polymerase. Here, a library collection of 100,680 in an altered Gcn4p pool was generated by cloning a mixed-base oligonucleotide in the place of the sequence coding for the DNA binding domains. The quality of the library was examined by DNA sequencing and found to be in good agreement with the expected statistical values. Calculated mutation frequency was 66% of mutant nucleotide rate and actual sequencing data revealed 68% mutant nucleotide rates from the sequenced library. Thus, among 21 mutants, 16 had one point mutations and 5 had two point mutations. This approach appears to be an effective and general tool for creating proteins with one or two amino acid change(s) in their molecules.

Enantioselective enzymes for organic synthesis created by directed evolution.

A new concept for the creation of enzymes displaying improved enantioselectivity in a given reaction is described; it is based on "evolution in the test tube". Accordingly, proper molecular biological methods for random mutagenesis, gene expression, and high-throughput screening systems for the rapid assay of enantioselectivity are combined. Several rounds of mutagenesis and screening are generally necessary in order to create mutant enzymes that show high degrees of enantioselectivity, as in the case of the lipase-catalyzed hydrolytic kinetic resolution of a chiral ester in which the original enantioselectivity of 2 % ee (E = 1) increases to > 90% ee (E = 25).

Random Changes of Amino Acid Residues with Expected Frequency by Saturated Point Mutagenesis

The yeast transcriptional activator protein, Gen4p from Saccharomyces cerevisiae binds to the specific sequence in the promoters of many amino acid biosynthetic genes for general control. A new random saturation mutagenesis method was developed to isolate Gen4p derivatives with only one or two mutations in the DNA binding domain without using radioactive isotope. This will be used to identify the amino acids of Gen4p involved in protein-protein interactions. Saturation mutagenesis in the DNA binding domain of Gen4p was performed using spiked degenerate oligonucleotides containing randomized codon bases designed specifically for only one or two base changes in the mutagenized area. These oligonucleotides were synthesized to have two flanking restriction enzyme sites for cloning to the appropriate vector. The 3′ ends were mutually primed after hybridization via the palindromic sequences of the restriction enzyme sites. These molecules were then converted to double stranded DNA upon treatment with DNA polymerase. Here, a library collection of 100,680 in an altered Gen4p pool was generated by cloning a mixed-base oligonucleotide in the place of the sequence coding for the DNA binding domains. The quality of the library was examined by DNA sequencing and found to be in good agreement with the expected statistical values. Calculated mutation frequency was 66% of mutant nucleotide rate and actual sequencing data revealed 68% mutant nucleotide rates from the sequenced library. Thus, among 21 mutants, 16 had one point mutantions and 5 had two point mutations. This approach appears to be an effective and general tool for creating proteins with one or two amino acid change(s) in their molecules.

Random mutagenesis by recombinational capture of PCR products in Bacillus subtilis and Acinetobacter calcoaceticus.

We describe a general method for random mutagenesis of cloned genes by error-prone PCR or DNA shuffling that eliminates the need for post-amplification subcloning following each cycle of mutagenesis. This method exploits the highly efficient and recombinogenic nature of DNA uptake during natural transformation in the Gram-positive bacterium Bacillus subtilis and the Gram-negative bacterium Acinetobacter calcoaceticus. Plasmid systems were designed that allow capture of PCR-amplified DNA fragments by marker-replacement recombination with a structurally similar helper plasmid resident in the transformation recipient. This recombination event simultaneously transfers the amplified sequences into the helper plasmid and restores the integrity of a drug resistance gene, thereby affording a direct selection for fragment capture. Although this strategy was sufficiently effective to permit recovery in B. subtilis of up to 10(3) transformants/microgram of PCR product, equivalent plasmid systems were approximately 100 times more efficient in A.calcoaceticus. Acinetobacter calcoaceticus also offers the advantage of essentially constitutive transformation competence in ordinary complex broth, such as LB, in contrast to two-step growth in semi-synthetic media required for optimal transformation of B.subtilis.

PCR-based random mutagenesis method using spiked oligonucleotides to randomize selected parts of a gene without any wild-type background.

PCR-based random mutagenesis method using spiked oligonucleotides to randomize selected parts of a gene without any wild-type background.

Mutagenesis and Chemical Rescue Indicate Residues Involved in β-Aspartyl-AMP Formation by Escherichia coli Asparagine Synthetase B

Site-directed mutagenesis and kinetic studies have been employed to identify amino acid residues involved in aspartate binding and transition state stabilization during the formation of beta-aspartyl-AMP in the reaction mechanism of Escherichia coli asparagine synthetase B (AS-B). Three conserved amino acids in the segment defined by residues 317-330 appear particularly crucial for enzymatic activity. For example, when Arg-325 is replaced by alanine or lysine, the resulting mutant enzymes possess no detectable asparagine synthetase activity. The catalytic activity of the R325A AS-B mutant can, however, be restored to about 1/6 of that of wild-type AS-B by the addition of guanidinium HCl (GdmHCl). Detailed kinetic analysis of the rescued activity suggests that Arg-325 is involved in stabilization of a pentacovalent intermediate leading to the formation beta-aspartyl-AMP. This rescue experiment is the second example in which the function of a critical arginine residue that has been substituted by mutagenesis is restored by GdmHCl. Mutation of Thr-322 and Thr-323 also produces enzymes with altered kinetic properties, suggesting that these threonines are involved in aspartate binding and/or stabilization of intermediates en route to beta-aspartyl-AMP. These experiments are the first to identify residues outside of the N-terminal glutamine amide transfer domain that have any functional role in asparagine synthesis.

Rational Molecular Design and Genetic Engineering of Herbicide Resistant Crops by Structure Modeling and Site-directed Mutagenesis of Acetohydroxyacid Synthase

Plants with specific resistance to a single class of herbicides have been genetically engineered by introduction of genes encoding rationally designed mutant acetohydroxyacid synthase (AHAS) enzymes. Suitable substitution mutations were identified from a three-dimensional model of an AHAS – inhibitor complex. The structural model was generated based on homology to pyruvate oxidase and an imidazolinone inhibitor was positioned in the proposed binding site using structure-activity data for this class of herbicide. Biochemical analysis of the mutant proteins expressed in Escherichia colienabled iterative optimization of the mutant genes. Expression of recombinant proteins in tobacco plants conferred resistance in vivo.The novel approach coupling molecular modeling and molecular biology has many advantages over traditional random mutagenesis and selection methods and will be crucial to the future development for environmentally safe and sustainable agricultural systems.

An Approach to Random Mutagenesis of DNA Using Mixtures of Triphosphate Derivatives of Nucleoside Analogues

We describe a new method for random mutagenesis of DNA based on the use of a mixture of triphosphates of nucleoside analogues. The method relies on DNA amplification in vitrowith Taqpolymerase and in the presence of the 5′-triphosphates of 6-(2-deoxy-β-D-ribofuranosyl)-3,4-dihy dro-8H-pyrimido-[4,5-C] [1,2]oxazin-7-one(dP) and of 8-oxo-2′deoxyguano- sine (8-oxodG). The newly synthesised triphosphate derivative of dP (dPTP) is an excellent substrate for Taqpolymerase ( K m=22 μM versus K m=9.5 μM for TTP); it is incorporated in place of TTP and, with a ∼fourfold lower efficiency, in place of dCTP. After 30 cycles of DNA amplification, equimolar mixtures of the four normal dNTPs and dPTP yield the following frequencies of the four transition mutations: A→G (4.4 × 10 −2), T→C (4.3 × 10 −2), G→A (1.1 × 10 −2) and C→T (1.0 × 10 −2). The triphosphate derivative of 8-oxodG (8-oxodGTP) is incorporated opposite template adenine and yields two transition mutations (A→C and T→G) at frequencies of 0.8 × 10 −2and 1.2 × 10 −2respectively. Reaction mixtures containing dPTP and 8-oxodGTP results in both dP and 8-oxodG-induced mutations and an extensive array of codon changes in the absence of insertions and deletions. The method described differs from previous mutagenesis procedures in three respects: (1) it enables very high frequencies of base substitutions (up to 1.9 × 10 −1), (2) it allows control of the mutational load viathe number of DNA amplification cycles and (3) it yields both transition and transversion mutations. The procedure may find application in the generation of libraries of DNA and protein mutants from which species with improved or novel activities may be selected.

Antibiotic resistance mechanisms of mutant EF-Tu species in Escherichia coli.

Analysis of antibiotic-resistant EF-Tu mutants has revealed a connection between resistance and structural elements that participate in the GTPase switching mechanism. Both random and site-directed mutagenesis methods have yielded sets of purified mutant EF-Tu resistant to kirromycin (kirT) or pulvomycin (pulT). All kirT mutations cluster in the interface of domain 1 and 3 of EF-Tu in its GTP-bound conformation, not in that of EF-Tu.GDP. Other evidence also suggests that kirromycin binds to the interface of wild-type EF-Tu, thereby jamming the GTPase switch. Various functional studies reveal two subsequent resistance mechanisms. The first hinders kirromycin binding to EF-Tu.GTP and the second occurs after GTP hydrolysis by rejection of bound kirromycin. All pulT mutations cluster in the three-domain junction interface of EF-Tu. GTP (which is an open hole in EF-Tu.GDP) and destabilize a salt-bridge network. Pulvomycin may bind nearby and overlap with tRNA binding. Mutations show that a D99-R230 salt bridge is not essential for the transduction of the GTPase switch signal from domain 1. In vivo and in vitro studies reveal that pulvomycin sensitivity is dominant over resistance. This demands a revision of the current view of the mechanism of pulvomycin inhibition of protein synthesis and may support a translation model with two EF-Tus on the ribosome. Several mutant EF-Tu species display altered behaviour towards aminoacyl-tRNA with interesting effects on translational accuracy. KirT EF-Tu(A375T) is able to reverse the streptomycin-dependent phenotype of a ribosomal protein S12 mutant strain to streptomycin sensitivity.

Discovery of new ligand binding pathways in myoglobin by random mutagenesis.

A random library of single amino acid mutants of myoglobin was generated using a highly efficient, single-base-misincorporation random mutagenesis method to discover new ligand-binding pathways in myoglobin. A surprisingly large fraction of the library exhibits ligand-binding kinetics that are substantially different from the wild-type protein. In addition to residues 45, 64 and 68, which comprise the best studied ligand-binding pathway single mutations of several other clusters of residues far away from that pathway are discovered which profoundly affect the ligand-binding kinetics. These results provide a new approach to explore the relationship between the fluctuations in protein structure and function.

Expression of cloned envelope protein genes from the flavivirus tick-borne encephalitis virus in mammalian cells and random mutagenesis by PCR.

The structural membrane proteins prM and E of the flavivirus tick-borne encephalitis (TBE) virus were expressed in mammalian cells for the purpose of probing the structure and molecular interactions of these proteins. Advantage was taken of the natural error frequency of the Taq polymerase used in the PCR amplification to generate a randomly mutated population of genes that were then cloned directly into plasmid expression vectors under the control of an SV40 promoter. Analysis of the mutation frequency by direct sequencing of 22 separate clones showed that the PCR produced mutations at a rate yielding an average of one to two amino acid changes per clone in the 496 amino acid long protein E. This is an ideal rate for assessing the importance of individual amino acid residues within protein domains, thus demonstrating the potential value of the PCR as a random mutagenesis method. Clones encoding wild-type prM and E proteins, and a truncated form of E, were also constructed by recombining portions of selected PCR clones. Transfection of COS-1 cells with these constructs resulted in expression of the prM and E proteins, which was demonstrated by indirect immunofluorescence using monoclonal antibodies (Mabs). The intracellular level of TBE virus antigen, measured in lysates of transfected cells by ELISA, reached approximately 25% of that found in virus-infected COS cells. Furthermore, it was shown by immunofluorescence using a panel of 19 anti-E Mabs that the antigenic structure of the expressed E proteins was nearly identical to that of E protein in infected cells, thus confirming the suitability of this model system as a tool for studying flavivirus protein structure.

Efficient random mutagenesis method with adjustable mutation frequency by use of PCR and dITP.

Efficient random mutagenesis method with adjustable mutation frequency by use of PCR and dITP.

Oligonucleotide-directed random mutagenesis using a high-efficiency procedure

A method for random mutagenesis using primers synthesized from four phosphoramidite solutiosn that have been “spiked” with a small amount of the other three residues is presented. The random oligonucleotides are used in an improved phosphorothioate mutagenesis technique that allows the entire procedure, including transformations, to be completed in 1 day. The phosphorothioate technique gives a high efficiency of mutagenesis from oligonucleotides with random mutations. A large proportion of the transformants analyzed by sequencing are found to contain random changes. The spiked oligonucleotides give truly random mutagenesis within the sequence defined by the primer. This is a very effective method for determining the functionally important amino acids in a protein when little information apart from the DNA sequence is known.

Antibody engineering using very long template-assembled oligonucleotides

We have previously constructed single-stranded DNA fragments up to 361 nucleotides long by the ligation of shorter synthetic fragments assembled on a closely homologous template and used them for site-directed mutagenesis. Here we have extended the utility of such long oligonucleotides for protein engineering in two ways. First oligonucleotides have been assembled on a template to which they show only relatively weak sequence homology (<70% identity), allowing conversion of genes encoding rather distantly related proteins. We have converted a gene encoding human light chain constant domain from the kappa to the lambda isotype, which share <40% amino acid identity. Second, libraries of random mutants were constructed within antibody variable domains with up to 20 targeted codons separated by up to 186 nucleotides. These libraries were created by template-directed assembly of degenerate oligonucleotides followed by PCR amplification and subcloning. Nucleotide sequence analysis of clones demonstrated that this is an effective method for the simultaneous random mutagenesis of several codons distant within the target gene. This should provide a useful tool for modifying the ligand binding properties of proteins, e.g., the antigen binding affinity and specificity of antibodies.

Gene-scrambling mutagenesis: generation and analysis of insertional mutations in the alginate regulatory region of Pseudomonas aeruginosa

A novel method for random mutagenesis of targeted chromosomal regions in Pseudomona aeruginosa was developed. This method can be used with a cloned DNA fragment of indefinite size that contains a putative gene of interest. Cloned DNA is digested to produce small fragments that are then randomly reassembled into long DNA inserts by using cosmid vectors and lambda packaging reaction. This DNA is then transferred into P. aeruginosa and forced into the chromosome via homologous recombination, producing in a single step a random set of insertional mutants along a desired region of the chromosome. Application of this method to extend the analysis of the alginate regulatory region, using a cloned 6.2-kb fragment with the algR gene and the previously uncharacterized flanking regions, produced several insertional mutations. One mutation was obtained in algR, a known transcriptional regulatory of mucoidy in P. aeruginosa. The null mutation of algR was generated in a mucoid derivative of the standard genetic strain PAO responsive to different environmental factors. This mutation was used to demonstrate that the algR gene product was not essential for the regulation of its promoters. Additional insertions were obtained in regions downstream and upstream of algR. A mutation that did not affect mucoidy was generated in a gene located 1 kb upstream of algR. This gene was transcribed in the direction opposite that of algR transcription and encoded a polypeptide of 47 kDa. Partial nucleotide sequence analysis revealed strong homology of its predicted gene product with the human and yeast argininosuccinate lyases. An insertion downstream of algR produced a strain showing reduced induction of mucoidy in response to growth on nitrate as the nitrogen source.