Cycles Of Mutagenesis Research Articles

Breeding of Saccharomyces cerevisiae with a High-Throughput Screening Strategy for Improvement of S-Adenosyl-L-Methionine Production.

S-adenosyl-l-methionine (SAM), a vital physiologically active substance in living organisms, is produced by fermentation over Saccharomyces cerevisiae. The main limitation in SAM production was the low biosynthesis ability of SAM in S. cerevisiae. The aim of this work is to breed an SAM-overproducing mutant through UV mutagenesis coupled with high-throughput selection. Firstly, a high-throughput screening method by rapid identification of positive colonies was conducted. White colonies on YND medium were selected as positive strains. Then, nystatin/sinefungin was chosen as a resistant agent in directed mutagenesis. After several cycles of mutagenesis, a stable mutant 616-19-5 was successfully obtained and exhibited higher SAM production (0.41g/L vs 1.39g/L). Furthermore, the transcript levels of the genes SAM2, ADO1, and CHO2 involved in SAM biosynthesis increased, while ergosterol biosynthesis genes in mutant 616-19-5 significantly decreased. Finally, building on the above work, S. cerevisiae 616-19-5 could produce 10.92 ± 0.2g/L SAM in a 5-L fermenter after 96h of fermentation, showing a 2.02-fold increase in the product yield compared with the parent strain. Paving the way of breeding SAM-overproducing strain has improved the good basis for SAM industrial production.

Forty years of directed evolution and its continuously evolving technology toolbox: A review of the patent landscape.

Generating functional protein variants with novel or improved characteristics has been a goal of the biotechnology industry and life sciences, for decades. Rational design and directed evolution are two major pathways to achieve the desired ends. While rational protein design approach has made substantial progress, the idea of using a method based on cycles of mutagenesis and natural selection to develop novel binding proteins, enzymes and structures has attracted great attention. Laboratory evolution of proteins/enzymes requires new tools and analytical approaches to create genetic diversity and identifying variants with desired traits. In this pursuit, construction of sufficiently large libraries of target molecules to search for improved variants and the need for new protocols to alter the properties of target molecules has been a continuing challenge in the directed evolution experiments. This review will discuss the in vivo and in vitro gene diversification tools, library screening or selection approaches, and artificial intelligence/machine-learning-based strategies to mutagenesis developed in the last 40 years to accelerate the natural process of evolution in creating new functional protein variants, optimization of microbial strains, and transformation of enzymes into industrial machines. Analyzing patent position over these techniques and mechanisms also constitutes an integral and distinctive part of this review. The aim is to provide an up-to-date resource/technology toolbox for research-based and pharmaceutical companies to discover the boundaries of competitor's intellectual property (IP) portfolio, their freedom-to-operate in the relevant IP landscape, and the need for patent due diligence analysis to rule out whether use of a particular patented mutagenesis method, library screening/selection technique falls outside the safe harbor of experimental use exemption. While so doing, we have referred to some recent cases that emphasize the significance of selecting a suitable gene diversification strategy in directed evolution experiments.

Breeding of Gluconobacter oxydans with high PQQ-dependent D-sorbitol dehydrogenase for improvement of 6-(N-hydroxyethyl)-amino-6-deoxy-α-L-sorbofuranose production

6-(N-hydroxyethyl)-amino-6-deoxy-L-sorbofuranose (6NSL), a key intermediate in the synthesis of miglitol, was produced from N-2-hydroxyethyl glucamine (NHEG) by biotransformation with whole cells of Gluconobacter oxydans. The main troubles in 6NSL production were the low activity of PQQ-dependent D-sorbitol dehydrogenase (mSLDH) in G. oxydans and the high cost of cell preparation. To solve these problems, a combined mutagenesis of 60Co-γ irradiation and microwave treatment with a high-throughput screening method by cultivation in a 96-well microtiter plate was conducted. After several cycles of mutagenesis, a stable mutant H-8 with high mSLDH activity was obtained, and the cell biomass increased by 11.6% when cultivated in a 5 L bioreactor. The transcription levels of the mSLDH subunit sldA and sldB in G. oxydans H-8 increased by 1.4- and 2.0-fold, respectively. Meanwhile, the intracellular PQQ concentration in G. oxydans H-8 was 16.1% higher than that of the parent strain, and qRT-PCR analysis showed that the genes pqqB and pqqC played an important role in PQQ biosynthesis (transcription levels increased by 2.4- and 1.8-fold, respectively). Relying on the advantages of the above, G. oxydans H-8 could produce 64.3 ± 2.2 g/L 6NSL after 36 h of bioconversion with resting cells, showing a 33.7% increase in the product yield.

Excessive Promoters as Silencers of Genes Horizontally Acquired by Escherichia coli.

Horizontally acquired genes are usually transcriptionally inactive, although most of them are associated with genomic loci enriched with promoter-like sequences forming “promoter islands.” We hypothesized that lateral DNA transfer induces local mutagenesis, accumulating AT base pairs and creating promoter-like sequences, whose occupancy with RNA polymerase and a specific silencer H-NS suppresses the transcription of foreign genes. Error-prone mutagenesis was implemented for the “promoter island” of a foreign gene appY and the promoter region of an inherent gene dps. Derivatives with changed transcriptional activity were selected using a reporter plasmid pET28_eGFP. Only one cycle of mutagenesis with negative selection suppressed the activity of the main dps promoter to the background level due to a single substitution in its -10 element, while positive selection gave a sequence with improved -35 element, thus testifying feasibility of the approach. The same suppression for appY was achieved by three cycles, while eightfold transcription activation required nine iterations of mutagenesis. In both cases, the number of potential start points decreased resulting in an ordinary regulatory region with only one dominant promoter in the case of positive selection. Efficiency of H-NS binding remained virtually unchanged in all mutant constructs. Based on these findings we conclude that excessive promoters can adversely affect transcription by providing a platform for interference between several RNA polymerase molecules, which can act as a silencer at promoter-dense regions.

An Efficient Approach for Two Distal Point Site-Directed Mutagenesis from Randomly Ligated PCR Products.

Site-directed mutagenesis is one of the most important tools in molecular biology. The majority of the mutagenesis methods have been developed to mutate one region of target DNA in each cycle of mutagenesis, while in some cases there is a need to mutate several distal points. We used a new method to simultaneously mutate two distal points in the target DNA. Different regions of the target DNA were amplified in three separate PCR reactions. The PCR products were back-to-back and together they made the complete length of the template DNA. Mutations were introduced to PCR products by middle mutagenic primers. PCR products were mixed and ligated with random blunt ligation, and then the desired mutated DNA fragments were selected in two steps by flanking restriction enzyme digestion and size selection. Selected fragments were amplified in another PCR reaction using flanking primers and finally cloned into the plasmid vector. This mutagenesis process is simple, there is no need to use modified primers and long or difficult PCR reactions.

Thermostabilization of firefly luciferase by in vivo directed evolution

Firefly luciferase is widely used in a number of areas of biotechnology and molecular biology. However, rapid inactivation of wild-type (WT) luciferases at elevated temperatures often hampers their application. A simple non-lethal in vivo screening scheme was used to identify thermostable mutants of luciferase in Escherichia coli colonies. This scheme allowed carrying out each cycle of mutagenesis in a rapid and efficient manner. Four rounds of directed evolution were conducted on a part of the gene coding for amino acid residues 130-390 of Luciola mingrelica luciferase. The resultant mutant designated 4TS had a half-life of 10 h at 42°C, which is 65-fold higher compared with the WT luciferase. Moreover, the mutant 4TS showed a 1.9-fold increase in specific activity, 5.7-fold reduction of K(m) for ATP and a higher-temperature optimum compared with the WT enzyme. 4TS contains eight mutations, fourof which are suggested to be mainly responsible for the enhancement of thermostability: R211L, A217V, E356K and S364C. Thus, directed evolution with non-lethal colony screening for in vivo bioluminescence activity proved to be an effective and efficient approach for increasing thermal stability of luciferase while retaining high catalytic activity.

Stabilization of Luciola mingrelica firefly luciferase by genetic engineering methods

The results of Luciola mingrelica firefly luciferase stabilization by genetic engineering methods are reviewed. The Cys62, Cys146, and Cys164 to Ser mutant enzymes with an enhanced thermostability and lower sensitivity to dithiothreitol were obtained by site-directed mutagenesis. The double mutant G216N, A217L was obtained, which displayed a higher thermostability and resistance to DMSO in comparison with WT luciferase. Random mutagenesis of the gene region encoding residues 1–225 and subsequent screening of the mutants resulted in the production of the mutant MT8 with a higher thermostability, as well as mutants MT3 and MT4 with higher resistance to dimethyl sulfoxide. The mutant 4TS was obtained by the method of directed evolution of the gene site encoding residues 130–390, which was shown to contain eight replacements after four cycles of mutagenesis and had two-fold higher specific activity, eight-fold lower K m value for ATP, and stability at 42°C, which was 65-fold higher that of WT luciferase. The stabilization mechanism of this mutant is discussed.

Screening and selection of lovastatin hyper-producing mutants ofAspergillus terreususing cyclic mutagenesis

134 fungal cultures isolated from different soil samples were screened for lovastatin production. Of these, 38 isolates produced different levels of lovastatin. An Aspergillus terreus strain GD13, producing 190 mg/l of lovastatin was selected and subjected to a rational mutation-selection programme based on the resistance to lovastatin and fatty acid synthase (FAS) inhibitors, viz., iodoacetamide and N-ethylmaleimide. After three cycles of mutagenesis, a hyper-producing mutant (EM19) exhibiting 7.5-fold (1424 mg/l) higher levels of lovastatin when compared to wild type parent strain was obtained.

Directed Evolution of the α-l-Fucosidase from Thermotoga maritima into an α-l-Transfucosidase

The alpha-L-fucosidase from Thermotoga maritima (Tm alpha fuc) was converted into alpha-L-transfucosidase variants by directed evolution. The wild-type enzyme catalyzes oligosaccharide synthesis by transfer of a fucosyl residue from a pNP-fucoside donor to pNP-fucoside (self-condensation) with alpha-(1-->3) regioselectivity or pNP-galactoside (transglycosylation) with alpha-(1-->2) regioselectivity at low yields (7%). The wild-type enzyme was submitted to one cycle of mutagenesis, followed by rational recombination of the selected mutations, which allowed identification of variants with improved transferase activity. The transferase and hydrolytic kinetics of all the mutants were assessed by NMR methods and capillary electrophoresis. It was shown that the best mutant exhibited a dramatic 32-fold increase in the transferase/hydrolytic kinetic ratio, while keeping 60% of the overall wild-type enzyme activity. Accordingly, the maximum yield of a specific transglycosylation product [pNP-Gal-alpha-(1-->2)-Fuc] reached more than 60% compared to 7% with WT enzyme at equimolar and low concentrations of donor and acceptor (10 mM). Such an improvement was obtained with only three mutations (T264A, Y267F, L322P), which were all located in the second amino acid shell of the fucosidase active site. Molecular modeling suggested that some of these mutations (T264A, Y267F) cause a reorientation of the amino acids that are in direct contact with the substrates, resulting in a better docking energy. Such mutants with high transglycosidase activity may constitute novel enzymatic tools for the synthesis of fucooligosaccharides.

Amylase hyperproduction by deregulated mutants of the thermophilic fungus Thermomyces lanuginosus

was subjected to three cycles of mutagenesis (UV/NTG) and a selection procedure to develop amylase-hyperproducing, catabolite-repression-resistant and partially constitutive strains. One of the selected derepressed mutant strain III51, produced ∼7- and 3-fold higher specific activity of α-amylase (190 U/mg protein) and glucoamylase (105 U/mg protein), respectively, compared to a wild-type parental strain. Further, the effect of production parameters on mutant strain III51 was studied using a Box–Behnken design. The regression models computed showed significantly high R2 values of 96 and 97% for α-amylase and glucoamylase activities, respectively, indicating that they are appropriate for predicting relationships between corn flour, soybean meal and pH with α-amylase and glucoamylase production. Journal of Industrial Microbiology & Biotechnology (2002) 29, 70–74 doi:10.1038/sj.jim.7000270

Amylase hyperproduction by deregulated mutants of the thermophilic fungus Thermomyces lanuginosus.

Thermomyces lanuginosus was subjected to three cycles of mutagenesis (UV/NTG) and a selection procedure to develop amylase-hyperproducing, catabolite-repression-resistant and partially constitutive strains. One of the selected derepressed mutant strain III(51), produced approximately 7- and 3-fold higher specific activity of alpha-amylase (190 U/mg protein) and glucoamylase (105 U/mg protein), respectively, compared to a wild-type parental strain. Further, the effect of production parameters on mutant strain III(51) was studied using a Box-Behnken design. The regression models computed showed significantly high R(2) values of 96 and 97% for alpha-amylase and glucoamylase activities, respectively, indicating that they are appropriate for predicting relationships between corn flour, soybean meal and pH with alpha-amylase and glucoamylase production.

Reciprocal domain evolution within a transactivator in a restricted sequence space.

offhough the concept of domain merging and shuffling as a major force in protein evolution is well established, it has been difficult to demonstrate how domains coadapt. Here we show evidence of coevolution of the Sinorhizobium meliloti NifA (SmNifA) domains. We found that, because of the lack of a conserved glycine in its DNA-binding domain, this transactivator protein interacts weakly with the enhancers. This defect, however, was compensated by evolving a highly efficient activation domain that, contrasting to Bradyrhizobium japonicum NifA (BjNifA), can activate in trans. To explore paths that lead to this enhanced activity, we mutagenized BjNifA. After three cycles of mutagenesis and selection, a highly active derivative was obtained. Strikingly, all mutations changed to amino acids already present in SmNifA. Our artificial process thus recreated the natural evolution followed by this protein and suggests that NifA is trapped in a restricted sequence space with very limited solutions for higher activity by point mutation.

Reciprocal domain evolution within a transactivator in a restricted sequence space

offhough the concept of domain merging and shuffling as a major force in protein evolution is well established, it has been difficult to demonstrate how domains coadapt. Here we show evidence of coevolution of the Sinorhizobium meliloti NifA (SmNifA) domains. We found that, because of the lack of a conserved glycine in its DNA-binding domain, this transactivator protein interacts weakly with the enhancers. This defect, however, was compensated by evolving a highly efficient activation domain that, contrasting to Bradyrhizobium japonicum NifA (BjNifA), can activate in trans. To explore paths that lead to this enhanced activity, we mutagenized BjNifA. After three cycles of mutagenesis and selection, a highly active derivative was obtained. Strikingly, all mutations changed to amino acids already present in SmNifA. Our artificial process thus recreated the natural evolution followed by this protein and suggests that NifA is trapped in a restricted sequence space with very limited solutions for higher activity by point mutation.

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.

In vitro evolution of horse heart myoglobin to increase peroxidase activity.

Random mutagenesis and screening for enzymatic activity has been used to engineer horse heart myoglobin to enhance its intrinsic peroxidase activity. A chemically synthesized gene encoding horse heart myoglobin was subjected to successive cycles of PCR random mutagenesis. The mutated myoglobin gene was expressed in Escherichia coli LE392, and the variants were screened for peroxidase activity with a plate assay. Four cycles of mutagenesis and screening produced a series of single, double, triple, and quadruple variants with enhanced peroxidase activity. Steady-state kinetics analysis demonstrated that the quadruple variant T39I/K45D/F46L/I107F exhibits peroxidase activity significantly greater than that of the wild-type protein with k1 (for H2O2 oxidation of metmyoglobin) of 1. 34 x 10(4) M-1 s-1 ( approximately 25-fold that of wild-type myoglobin) and k3 [for reducing the substrate (2, 2'-azino-di-(3-ethyl)benzthiazoline-6-sulfonic acid] of 1.4 x 10(6) M-1 s-1 (1.6-fold that of wild-type myoglobin). Thermal stability of these variants as measured with circular dichroism spectroscopy demonstrated that the Tm of the quadruple variant is decreased only slightly compared with wild-type (74.1 degreesC vs. 76.5 degreesC). The rate constants for binding of dioxygen exhibited by the quadruple variant are identical to the those observed for wild-type myoglobin (kon, 22.2 x 10(-6) M-1 s-1 vs. 22.3 x 10(-6) M-1 s-1; koff, 24.3 s-1 vs. 24.2 s-1; KO2, 0.91 x 10(-6) M-1 vs. 0.92 x 10(-6) M-1). The affinity of the quadruple variant for CO is increased slightly (kon, 0.90 x 10(-6) M-1s-1 vs. 0.51 x 10(-6) M-1s-1; koff, 5.08 s-1 vs. 3.51 s-1; KCO, 1.77 x 10(-7) M-1 vs. 1.45 x 10(-7) M-1). All four substitutions are in the heme pocket and within 5 A of the heme group.

Overexpression, immobilization and biotechnological application of Pseudomonas lipases

Pseudomonas lipases play an important role in biotechnology both as hydrolases for detergent additives and as synthases catalyzing the kinetic resolution of racemic compounds. Large-scale production of Pseudomonas lipases requires correct folding and secretion through the bacterial membranes. Controllable expression of the gene lipH encoding a lipase-specific foldase proves to be important for overexpression in the homologous host Escherichia coli. Construction of appropriate His-tagged fusion proteins permitted overexpression, secretion and one-step purification of lipase from culture supernatants of the homologous host Pseudomonas aeruginosa. The immobilization of lipases in hydrophobic sol–gel materials derived from alkylsilane precursors of the type RSi(OCH 3) 3 or mixtures of RSi(OCH 3) 3 and Si(OCH 3) 4 provides highly active and chemically and thermally stable heterogeneous biocatalysts. The entrapped lipases are excellent catalysts in a variety of synthetic organic transformations. Using directed evolution based on error prone PCR, the enantioselectivity of the hydrolysis of a chiral ester, catalyzed by the lipase from P. aeruginosa, can be increased from ee 2 to ee 81% in just four mutagenesis cycles.

In vitro evolution used to define a protein recognition site within a large RNA domain

A minimum of 460 nucleotides of 16S ribosomal RNA are needed to fold the target site for E. coli ribosomal protein S4, although a much smaller region within this large domain is protected from chemical reagents by the protein. Starting with a 531-nucleotide rRNA fragment, cycles of mutagenesis, selection with S4, and amplification (‘in vitro evolution’) were used to obtain a pool of 30 RNA sequences selected for S4 recognition but ∼30% different from wild type. Numerous compensatory base pair changes have largely preserved the same secondary structure among these RNAs as found in wild-type sequences. A 20-base deletion and a single nucleotide insertion are among several unusual features found in most of the selected sequences and also prevalent among other prokaryotic rRNAs. Most of the compensatory base changes and selected features are located outside of the region protected by S4 from chemical reagents. It was unexpected that S4 would select for RNA structures throughout such a large domain; the selected features are probably contributing indirectly to S4 recognition by promoting correct tertiary folding of the region actually contacted by S4. The role of S4 may be to stabilize this domain (nearly one-third of the 16S rRNA) in its proper conformation for ribosome function.

Identification of mutations contributing to the reduced virulence of a modified strain of respiratory syncytial virus

The nucleotide sequences of the genome of the RSS-2 wild type strain of respiratory syncytial (RS) virus, which is known to induce upper respiratory tract infection in adults, and that of the attenuated ts1C candidate vaccine derived from it by three cycles of mutagenesis and selection of temperature-sensitive (ts) mutants, have been determined. Comparison of the sequences has located the genetic changes which contribute to the reduced pathogenicity in adults of the candidate vaccine. Thirty-seven nucleotide changes distinguish the wild type and ts1C, 13 of which confer amino acid substitutions; no mutations are present in extragenic regions. Partial nucleotide sequencing of the genomes of the first stage ts mutant ( ts1A) and the second stage ts mutant ( ts1B), which were intermediates in the derivation of the third stage mutant ts1C, established that five mutations resulting in amino acid substitutions had been induced in the first cycle of mutagenesis, one in the second cycle, and seven in the third cycle. The unique mutation differentiating ts1B from ts1A substitutes an alanine for a threonine at residue 736 in the polymerase (L) protein. The occurrence of a mutation in ts1C inducing substitution of a phenylalanine for a serine residue at an adjacent site (731) suggests that mutations in this region of the polymerase can have significant attenuating effects. The data suggest also that a mutation in the F gene may contribute to the attenuated phenotype.

Somatic variegation and germinal mutability reflect the position of transposable element Dissociation within the maize R gene.

The R gene regulates the timing and tissue-specificity of anthocyanin deposition during maize development. The Ac/Ds system of transposable elements was used to induce insertional mutants of the R-sc:124 allele during two cycles of mutagenesis. Of 43 unstable, spotted-aleurone mutants generated, 42 contain inserts of the Ds6 transposable element differing only in the position and orientation of the element. The remaining mutant, r-sc:m1, contained an insert of a Ds element of the approximate size of the Ds1 transposable element. The patterns of somatic variegation of these mutants, resulting from excision of Ds, define a spectrum of phenotypes ranging from sparse to dense variegation. The sparsely variegated mutants produce few germinal revertants but relatively many stable null derivative alleles; densely variegated mutants produce many germinal revertants and few stable null derivatives. Molecular analysis shows that the sparsely variegated alleles are caused by Ds6 insertions in protein coding regions of R-sc:124 whereas the densely variegated mutants result from insertions in introns or in flanking regions of the gene. The excision rate of Ds6 from R, estimated as the proportion of R genomic DNA restriction fragments lacking the element, was uniform regardless of position, orientation or whether the element was inserted in R-sc:124 or another R allele. The excision rate was greater, however, for the mutable alleles involving the Ds element from r-sc:m1. These data indicate that, although the excision rates are uniform for a given Ds element, the somatic and germinal mutability patterns of alleles associated with that element vary widely and depend primarily on the position of the transposable element within coding or noncoding regions of the gene.

Response surface methods for optimizingSaccharopolyspora spinosa, a novel macrolide producer

Strain A83543, recently identified asSaccharopolyspora spinosa, was cultured in a variety of media to optimize macrolide titer. Response surface methodology (RSM) was used to improve the fermentation medium and to characterize the microorganism's response to systematic variations in medium composition. Three sequential RSM studies on wild-type A83543 and two high macrolide-producing mutants showed that each strain produced maximum titers in nearly identical fermentation media. No obvious differences in nutrient requirements were evident in the three strains indicating little interaction between mutational change and medium composition through at least two cycles of mutagenesis. The overall increase in macrolide titer starting from the wild-type organism in the original fermentation medium to the second-generation mutant in the optimized medium was over 25-fold.