Random Mutagenesis Method Research Articles

Creation of Single Cell Protein-Producing Mutants of Phaffia Rhodozyma

Recently, there has been an increasing demand for sustainable protein sources for aquaculture. In aquaculture, sustainable protein includes sources that can be produced with a minimal environmental footprint, such as single-cell proteins, proteins from algae, insects, and by-products from the agricultural industry. Single-cell proteins that are derived from microorganisms show a sustainable alternative to traditional protein sources due to their high protein content and rapid growth. This research focuses on creating mutants of the yeast Phaffia rhodozyma to increase single-cell protein production. This yeast has significant industrial potential and biological features. The yeast Phaffia rhodozyma is already known for its unique ability to synthesize astaxanthin, so this case will focus on its ability to produce protein. In the study, we used wild strain yeast and previously obtained white mutants on inorganic nitrogen media for further random mutagenesis methods to introduce specific mutations into the genome aimed at improving their ability to biosynthesize protein. Amino acid inhibitor was used to select potential protein-producing mutants. The protein content of the biomass obtained from the experiments was analyzed in detail using BCA (Protein Assay Reagent) analysis, which confirmed their potential as a source of nutrients for aquaculture. This method is based on the reaction between bicinchoninic acid and the peptide bonds of proteins, which results in the formation of a color whose intensity is proportional to the amount of protein in the sample. The results obtained allow further research into the development of cost-effective and environmentally friendly alternatives for feed in aquaculture.

Regional random mutagenesis driven by multiple sgRNAs and diverse on-target genome editing events to identify functionally important elements in non-coding regions.

Functional regions that regulate biological phenomena are interspersed throughout eukaryotic genomes. The most definitive approach for identifying such regions is to confirm the phenotype of cells or organisms in which specific regions have been mutated or removed from the genome. This approach is invaluable for the functional analysis of genes with a defined functional element, the protein-coding sequence. By contrast, no functional analysis platforms have been established for the study of cis-elements or microRNA cluster regions consisting of multiple microRNAs with functional overlap. Whole-genome mutagenesis approaches, such as via N-ethyl-N-nitrosourea and gene trapping, have greatly contributed to elucidating the function of coding genes. These methods almost never induce deletions of genomic regions or multiple mutations within a narrow region. In other words, cis-elements and microRNA clusters cannot be effectively targeted in such a manner. Herein, we established a novel region-specific random mutagenesis method named CRISPR- and transposase-based regional mutagenesis (CTRL-mutagenesis). We demonstrate that CTRL-mutagenesis randomly induces diverse mutations within target regions in murine embryonic stem cells. Comparative analysis of mutants harbouring subtly different mutations within the same region would facilitate the further study of cis-element and microRNA clusters.

Comparative Proteomics Reveals Evidence of Enhanced EPA Trafficking in a Mutant Strain of Nannochloropsis oculata.

The marine microalga Nannochloropsis oculata is a bioproducer of eicosapentaenoic acid (EPA), a fatty acid. EPA is incorporated into monogalactosyldiacylglycerol within N. oculata thylakoid membranes, and there is a biotechnological need to remodel EPA synthesis to maximize production and simplify downstream processing. In this study, random mutagenesis and chemical inhibitor-based selection method were devised to increase EPA production and accessibility for improved extraction. Ethyl methanesulfonate was used as the mutagen with selective pressure achieved by using two enzyme inhibitors of lipid metabolism: cerulenin and galvestine-1. Fatty acid methyl ester analysis of a selected fast-growing mutant strain had a higher percentage of EPA (37.5% of total fatty acids) than the wild-type strain (22.2% total fatty acids), with the highest EPA quantity recorded at 68.5 mg/g dry cell weight, while wild-type cells had 48.6 mg/g dry cell weight. Label-free quantitative proteomics for differential protein expression analysis revealed that the wild-type and mutant strains might have alternative channeling pathways for EPA synthesis. The mutant strain showed potentially improved photosynthetic efficiency, thus synthesizing a higher quantity of membrane lipids and EPA. The EPA synthesis pathways could also have deviated in the mutant, where fatty acid desaturase type 2 (13.7-fold upregulated) and lipid droplet surface protein (LDSP) (34.8-fold upregulated) were expressed significantly higher than in the wild-type strain. This study increases the understanding of EPA trafficking in N. oculata, leading to further strategies that can be implemented to enhance EPA synthesis in marine microalgae.

Characterizing the portability of phage-encoded homologous recombination proteins.

Efficient genome editing methods are essential for biotechnology and fundamental research. Homologous recombination (HR) is the most versatile method of genome editing, but techniques that rely on host RecA-mediated pathways are inefficient and laborious. Phage-encoded ssDNA annealing proteins (SSAPs) improve HR 1000-fold above endogenous levels; however, they are not broadly functional. Using Escherichia coli, Lactococcus lactis, Mycobacterium smegmatis, Lactobacillus rhamnosus, and Caulobacter crescentus we investigated the limited portability of SSAPs. We find that these proteins specifically recognize the C-terminal tail of the host’s single-stranded DNA-binding protein (SSB), and are portable between species if compatibility with this host domain is maintained. Furthermore, we find that co-expressing SSAPs with a paired SSB can significantly improve activity, in some species enabling SSAP functionality even without host-compatibility. Finally, we find that high-efficiency HR far surpasses the mutational capacity of commonly used random mutagenesis methods, generating exceptional phenotypes inaccessible through sequential nucleotide conversions.

Site-Directed and Random Mutagenesis in Porphyromonas gingivalis: Construction of Fimbriae-Related-Gene Mutant.

Porphyromonas gingivalis, an etiological agent of chronic periodontitis, is an asaccharolytic anaerobic gram-negative coccobacillus. Genetic approaches greatly facilitate research on organisms at the molecular level. Although with some challenges, the use of genetic techniques (such as constructing knockout mutants) in P. gingivalis are feasible. In this chapter, we describe detailed methods for site-directed and random mutagenesis through the construction of fimbriae-related gene mutants of P. gingivalis.

Extended structure of pleiotropic DNA repair-promoting protein PprA from Deinococcus radiodurans.

Pleiotropic protein promoting DNA repair A (PprA) is a key protein facilitating the extreme radiation resistance of Deinococcus radiodurans. PprA is a unique protein to the genus Deinococcus and exists as an oligomer ranging from a tetramer to an ∼100-mer depending on protein concentrations. Here, the X-ray crystal structure of PprA was determined to clarify how PprA confers radiation resistance. The tertiary structure of dimeric PprA was elucidated by using mutants obtained with random and site-directed mutagenesis methods (W183R and A139R); these mutants have disabled DNA binding and polymerization functions. Because the mutant A139R and W183R proteins have dimeric assemblies with 2 different interfaces (Interfaces 1 and 2), the linear and oligomerized PprA model was constructed as a left-handed face-to-face periodic screw structure. In addition, the linear structure in solution was confirmed by small-angle scattering experiments. The site-directed mutational analysis identified essential basic amino acids for DNA binding. These analytical data support the hypothesis that a complex assembly of PprA molecules, which are extended and have a screw structure, surrounds and stretches the DNA strand, acting as a novel guide to colocalize the DNA strands for efficient DNA repairs.-Adachi, M., Shimizu, R., Shibazaki, C., Satoh, K., Fujiwara, S., Arai, S., Narumi, I., Kuroki, R. Extended structure of pleiotropic DNA repair-promoting protein PprA from Deinococcus radiodurans.

Improvement of carbonyl reductase activity for the bioproduction of tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate

tert-Butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate ((3R,5S)-CDHH) is a key chiral intermediate for the side chain synthesis of rosuvastatin. In this study, random mutagenesis, site-saturation mutagenesis and combinatorial mutagenesis methods were applied to improve the activity of a synthesized stereoselective short chain carbonyl reductase (SCR) to prepare (3R,5S)-CDHH. After screened by high-throughput screening method and high-performance liquid chromatography, mut-Phe145Met/Thr152Ser and mut-Phe145Tyr/Thr152Ser, were obtained, and the enzyme activities of mutants were improved by 1.60- and 1.91-fold compared with parent enzyme, respectively. The catalytically efficiencies (kcat/Km) of mut-Phe145Met/Thr152Ser and mut-Phe145Tyr/Thr152Ser exhibited 5.11- and 8.07-fold improvements in initial activity toward (S)-6-chloro-5-hydroxy-3-oxohexanoate ((S)-CHOH), respectively. In the asymmetric reduction, mut-Phe145Tyr/Thr152Ser catalyzed 500 g L−1 of (S)-CHOH to produce (3R,5S)-CDHH with >99% yield and >99% e.e., and the highest space-time yield achieved at 752.76 mmol L−1 h−1 g−1 wet cell weight within 8 h bioconversion. This study provides a foundation for the preparation of (3R,5S)-CDHH by carbonyl reductase.

Are Directed Evolution Approaches Efficient in Exploring Nature’s Potential to Stabilize a Lipase in Organic Cosolvents?

Despite the significant advances in the field of protein engineering, general design principles to improve organic cosolvent resistance of enzymes still remain undiscovered. Previous studies drew conclusions to engineer enzymes for their use in water-miscible organic solvents based on few amino acid substitutions. In this study, we conduct a comparison of a Bacillus subtilis lipase A (BSLA) library—covering the full natural diversity of single amino acid substitutions at all 181 positions of BSLA—with three state of the art random mutagenesis methods: error-prone PCR (epPCR) with low and high mutagenesis frequency (epPCR-low and high) as well as a transversion-enriched Sequence Saturation Mutagenesis (SeSaM-Tv P/P) method. Libraries were searched for amino acid substitutions that increase the enzyme’s resistance to the water-miscible organic cosolvents 1,4-dioxane (DOX), 2,2,2-trifluoroethanol (TFE), and dimethyl sulfoxide (DMSO). Our analysis revealed that 5%–11% of all possible single substitutions (BSLA site-saturation mutagenesis (SSM) library) contribute to improved cosolvent resistance. However, only a fraction of these substitutions (7%–12%) could be detected in the three random mutagenesis libraries. To our knowledge, this is the first study that quantifies the capability of these diversity generation methods generally employed in directed evolution campaigns and compares them to the entire natural diversity with a single substitution. Additionally, the investigation of the BSLA SSM library revealed only few common beneficial substitutions for all three cosolvents as well as the importance of introducing surface charges for organic cosolvent resistance—most likely due to a stronger attraction of water molecules.

P-27 セルラーゼの機能改変を目指したランダム変異導入法とスクリーニング手法の開発(糖化・発酵,ポスター発表)

P-27 セルラーゼの機能改変を目指したランダム変異導入法とスクリーニング手法の開発(糖化・発酵,ポスター発表)

Comparative genomics analysis of a series of Yarrowia lipolytica WSH-Z06 mutants with varied capacity for α-ketoglutarate production

Yarrowia lipolytica is one of the most intensively investigated α-ketoglutaric acid (α-KG) producers, and metabolic engineering has proven effective for enhancing production. However, regulation of α-KG metabolism remains poorly understood. Genetic engineering of new strains is accompanied by potential safety concerns in some countries and regions. A series of mutants with varied capacity for α-KG production were obtained using random mutagenesis of Y. lipolytica WSH-Z06. Comparative genomics analysis was implemented to identify genes candidates associated with α-KG production. Manipulation of genes regulating mitochondrial biogenesis and energy metabolism could improve α-KG production, while genes involved in regulating transformation between keto acids and amino acids may decrease production. One gene associated with cell cycle control well represented in all mutants, whereas this gene involved in cell concentration do not appear to influence α-KG production. The results shed light on α-KG production in eukaryotic cells, and pave the way for a high-throughput screening and random mutagenesis method for enhancing α-KG production.

Use of Random and Site-Directed Mutagenesis to Probe Protein Structure-Function Relationships: Applied Techniques in the Study of Helicobacter pylori.

Mutagenesis is a valuable tool to examine the structure-function relationships of bacterial proteins. As such, a wide variety of mutagenesis techniques and strategies have been developed. This chapter details a selection of random mutagenesis methods and site-directed mutagenesis procedures that can be applied to an array of bacterial species. Additionally, the direct application of the techniques to study the Helicobacter pylori Ferric Uptake Regulator (Fur) protein is described. The varied approaches illustrated herein allow the robust investigation of the structural-functional relationships within a protein of interest.

Novel Random Mutagenesis Method for Directed Evolution.

Directed evolution is a powerful strategy for gene mutagenesis, and has been used for protein engineering both in scientific research and in the biotechnology industry. The routine method for directed evolution was developed by Stemmer in 1994 (Stemmer, Proc Natl Acad Sci USA 91, 10747-10751, 1994; Stemmer, Nature 370, 389-391, 1994). Since then, various methods have been introduced, each of which has advantages and limitations depending upon the targeted genes and procedure. In this chapter, a novel alternative directed evolution method which combines mutagenesis PCR with dITP and fragmentation by endonuclease V is described. The kanamycin resistance gene is used as a reporter gene to verify the novel method for directed evolution. This method for directed evolution has been demonstrated to be efficient, reproducible, and easy to manipulate in practice.

Development of simple random mutagenesis protocol for the protein expression system in Pichia pastoris.

BackgroundRandom mutagenesis is a powerful technique to obtain mutant proteins with different properties from the wild-type molecule. Error-prone PCR is often employed for random mutagenesis in bacterial protein expression systems, but has rarely been used in the methylotrophic yeast Pichia pastoris system, despite its significant advantages, mainly because large (μg-level) amounts of plasmids are required for transformation.ResultsWe developed a quick and easy technique for random mutagenesis in P. pastoris by sequential Phi29 DNA polymerase-based amplification methods, error-prone rolling circle amplification (RCA) and multiple displacement amplification (MDA). The methodology was validated by applying it for random mutation of the gene encoding cellulase from the basidiomycete Phanerochaete chrysosporium (PcCel6A), a key enzyme in degradation of cellulosic biomass. In the error-prone RCA step, the concentrations of manganese ion (Mn2+) and cellulase gene-containing plasmid were varied, and the products obtained under each condition were subjected to the second MDA step in the absence of Mn2+. The maximum error rate was 2.6 mutations/kb, as evaluated from the results of large-scale sequencing. Several μg of MDA products was transformed by electroporation into Pichia cells, and the activities of extracellularly expressed PcCel6A mutants towards crystalline and amorphous celluloses were compared with those of wild-type enzyme to identify key amino acid residues affecting degradation of crystalline cellulose.ConclusionsWe present a rapid and convenient random mutagenesis method that does not require laborious steps such as ligation, cloning, and synthesis of specific primers. This method was successfully applied to the protein expression system in P. pastoris.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0613-z) contains supplementary material, which is available to authorized users.

Random Mutagenesis Methods for Hyper-Production of Laccase and its Effect on Azo Dye Decolourization

Random Mutagenesis Methods for Hyper-Production of Laccase and its Effect on Azo Dye Decolourization

Approaches to Inactivate Genes in Zebrafish.

Animal models of tumor initiation and tumor progression are essential components toward understanding cancer and designing/validating future therapies. Zebrafish is a powerful model for studying tumorigenesis and has been successfully exploited in drug discovery. According to the zebrafish reference genome, 82 % of disease-associated genes in the Online Mendelian Inheritance in Man (OMIM) database have clear zebrafish orthologues. Using a variety of large-scale random mutagenesis methods developed to date, zebrafish can provide a unique opportunity to identify gene mutations that may be associated with cancer predisposition. On the other hand, newer technologies enabling targeted mutagenesis can facilitate reverse cancer genetic studies and open the door for complex genetic analysis of tumorigenesis. In this chapter, we will describe the various technologies for conducting genome editing in zebrafish with special emphasis on the approaches to inactivate genes.

A Branch Point of Streptomyces Sulfur Amino Acid Metabolism Controls the Production of Albomycin

Albomycin (ABM), also known as grisein, is a sulfur-containing metabolite produced by Streptomyces griseus ATCC 700974. Genes predicted to be involved in the biosynthesis of ABM and ABM-like molecules are found in the genomes of other actinomycetes. ABM has potent antibacterial activity, and as a result, many attempts have been made to develop ABM into a drug since the last century. Although the productivity of S. griseus can be increased with random mutagenesis methods, understanding of Streptomyces sulfur amino acid (SAA) metabolism, which supplies a precursor for ABM biosynthesis, could lead to improved and stable production. We previously characterized the gene cluster (abm) in the genome-sequenced S. griseus strain and proposed that the sulfur atom of ABM is derived from either cysteine (Cys) or homocysteine (Hcy). The gene product, AbmD, appears to be an important link between primary and secondary sulfur metabolic pathways. Here, we show that propargylglycine or iron supplementation in growth media increased ABM production by significantly changing the relative concentrations of intracellular Cys and Hcy. An SAA metabolic network of S. griseus was constructed. Pathways toward increasing Hcy were shown to positively impact ABM production. The abmD gene and five genes that increased the Hcy/Cys ratio were assembled downstream of hrdBp promoter sequences and integrated into the chromosome for overexpression. The ABM titer of one engineered strain, SCAK3, in a chemically defined medium was consistently improved to levels ∼400% of the wild type. Finally, we analyzed the production and growth of SCAK3 in shake flasks for further process development.

GWT1 encoding an inositol acyltransferase homolog is required for laccase repression and stress resistance in the basidiomycete Cryptococcus neoformans.

The transcriptional expression of laccase, which has been confirmed to contribute to the virulence of Cryptococcus neoformans, is often repressed by a high concentration of glucose in many fungi, including C. neoformans. The underlying mechanism of the repression remains largely unknown. In this study, we found that a GWT1 gene that encodes a glycosylphosphatidylinositol (GPI) anchor biosynthesis-related protein is required for laccase repression by glucose in the basidiomycete C. neoformans. Disruption of GWT1 with the Agrobacterium tumefaciens-mediated T-DNA random insertional mutagenesis (ATMT) method resulted in constitutive expression of the laccase gene LAC1 and constant melanin formation. The loss of GWT1 also dramatically affected the cell membrane integrity and stress resistance. Our results revealed a GPI-dependent glucose repression mechanism in C. neoformans, and it may be helpful for understanding the virulence of C. neoformans.

Massively parallel single-amino-acid mutagenesis.

Random mutagenesis methods only partially cover the mutational space, and are constrained by DNA synthesis length limitations. Here, we demonstrate PALS, a single-volume, site-directed mutagenesis approach using microarray-programmed oligonucleotides. We created libraries including nearly every missense mutation as singleton events for the yeast transcription factor Gal4 (99.9% coverage) and human tumor suppressor p53 (93.5%). PALS-based comprehensive missense mutational scans may aid structure-function studies, protein engineering, and the interpretation of variants identified by clinical sequencing.

Identification of genes essential for leptospirosis.

The development of methods for the construction of defined mutants of pathogenic Leptospira has been a breakthrough in the study of leptospiral virulence. These methods have allowed the identification of genes essential for infection in animal models. This chapter describes methods for random transposon mutagenesis of pathogenic leptospires, identification of transposon insertion sites using direct sequencing from genomic DNA and a nested PCR utilizing degenerate oligonucleotides, and methods for testing mutant attenuation in the hamster model of infection.

Influenza A viruses with different amino acid residues at PB2-627 display distinct replication properties in vitro and in vivo: Revealing the sequence plasticity of PB2-627 position

Sequence analyses of influenza PB2 sequences indicate that the 627 position almost exclusively contains either lysine (K) or glutamic acid (E), suggesting a high sequence constraint at this genetic marker. Here, we used a site-directed random mutagenesis method to demonstrate that PB2-627 position has a high sequence plasticity. Recombinant viruses carrying various amino acid residues at this position are viable in cell cultures. These PB2-627 mutants showed various polymerase activities and replication kinetics in mammalian and avian cells as well as pathogenicity in mice. Serially passaging these mutants in MDCK cells generated some compensatory PB2 mutations that can restore polymerase activities of the PB2-627 mutants. Of these, PB2-D309N was identified as a novel one. Besides showing that influenza virus can tolerate a wide range of amino acid residues at the PB2-627 position, this study also demonstrates a potential strategy to identify novel mutations that can enhance viral polymerase.