Efficient library construction by in vivo recombination with a telomere-originated autonomously replicating sequence of Hansenula polymorpha

A high frequency of transformation and an equal gene dosage between transformants are generally required for activity-based selection of mutants from a library obtained by directed evolution. An efficient library construction method was developed by using in vivo recombination in Hansenula polymorpha. Various linear sets of vectors and insert fragments were transformed and analyzed to optimize the in vivo recombination system. A telomere-originated autonomously replicating sequence (ARS) of H. polymorpha, reported as a recombination hot spot, facilitates in vivo recombination between the linear transforming DNA and chromosomes. In vivo recombination of two linear DNA fragments containing the telomeric ARS drastically increases the transforming frequency, up to 10-fold, compared to the frequency of circular plasmids. Direct integration of the one-end-recombined linear fragment into chromosomes produced transformants with single-copy gene integration, resulting in the same expression level for the reporter protein between transformants. This newly developed in vivo recombination system of H. polymorpha provides a suitable library for activity-based selection of mutants after directed evolution.     

Efficient Library Construction by In Vivo Recombination with a Telomere-Originated Autonomously Replicating Sequence of Hansenula polymorpha

A high frequency of transformation and an equal gene dosage between transformants are generally required for activity-based selection of mutants from a library obtained by directed evolution. An efficient library construction method was developed by using in vivo recombination in Hansenula polymorpha. Various linear sets of vectors and insert fragments were transformed and analyzed to optimize the in vivo recombination system. A telomere-originated autonomously replicating sequence (ARS) of H. polymorpha, reported as a recombination hot spot, facilitates in vivo recombination between the linear transforming DNA and chromosomes. In vivo recombination of two linear DNA fragments containing the telomeric ARS drastically increases the transforming frequency, up to 10-fold, compared to the frequency of circular plasmids. Direct integration of the one-end-recombined linear fragment into chromosomes produced transformants with single-copy gene integration, resulting in the same expression level for the reporter protein between transformants. This newly developed in vivo recombination system of H. polymorpha provides a suitable library for activity-based selection of mutants after directed evolution.     

Directed evolution strategies for improved enzymatic performance

The engineering of enzymes with altered activity, specificity and stability, using directed evolution techniques that mimic evolution on a laboratory timescale, is now well established. However, the general acceptance of these methods as a route to new biocatalysts for organic synthesis requires further improvement of the methods for both ease-of-use and also for obtaining more significant changes in enzyme properties than is currently possible. Recent advances in library design, and methods of random mutagenesis, combined with new screening and selection tools, continue to push forward the potential of directed evolution. For example, protein engineers are now beginning to apply the vast body of knowledge and understanding of protein structure and function, to the design of focussed directed evolution libraries, with striking results compared to the previously favoured random mutagenesis and recombination of entire genes. Significant progress in computational design techniques which mimic the experimental process of library screening is also now enabling searches of much greater regions of sequence-space for those catalytic reactions that are broadly understood and, therefore, possible to model.     

Recombination and chimeragenesis by in vitro heteroduplex formation and in vivo repair.

We describe a simple method for creating libraries of chimeric DNA sequences derived from homologous parental sequences. A heteroduplex formed in vitro is used to transform bacterial cells where repair of regions of non-identity in the heteroduplex creates a library of new, recombined sequences composed of elements from each parent. Heteroduplex recombination provides a convenient addition to existing DNA recombination methods ('DNA shuffling') and should be particularly useful for recombining large genes or entire operons. This method can be used to create libraries of chimeric polynucleotides and proteins for directed evolution to improve their properties or to study structure-function relationships. We also describe a simple test system for evaluating the performance of DNA recombination methods in which recombination of genes encoding truncated green fluorescent protein (GFP) reconstructs the full-length gene and restores its characteristic fluorescence. Comprising seven truncated GFP constructs, this system can be used to evaluate the efficiency of recombination between mismatches separated by as few as 24 bp and as many as 463 bp. The optimized heteroduplex recombination protocol is quite efficient, generating nearly 30% fluorescent colonies for recombination between two genes containing stop codons 463 bp apart (compared to a theoretical limit of 50%).     

Random mutagenesis and recombination of sam1 gene by integrating error-prone PCR with staggered extension process

An efficient method for creating a DNA library is presented in which gene mutagenesis and recombination can be introduced by integrating error-prone PCR with a staggered extension process in one test tube. In this process, less than 15 cycles of error-prone PCR are used to introduce random mutations. After precipitated and washed with ethanol solution, the error-prone PCR product is directly used both as template and primers in the following staggered extension process to introduce DNA recombination. The method was validated by using adenosyl-methionine (AdoMet) synthetase gene, sam1, as a model. The full-length target DNA fragment was available after a single round. After being selected with a competitive inhibitor, ethionine, a mutated gene was obtained that increased AdoMet accumulation in vivo by 56%.     

Effective Optimization of Antibody Affinity by Phage Display Integrated with High-Throughput DNA Synthesis and Sequencing Technologies

Phage display technology has been widely used for antibody affinity maturation for decades. The limited library sequence diversity together with excessive redundancy and labour-consuming procedure for candidate identification are two major obstacles to widespread adoption of this technology. We hereby describe a novel library generation and screening approach to address the problems. The approach started with the targeted diversification of multiple complementarity determining regions (CDRs) of a humanized anti-ErbB2 antibody, HuA21, with a small perturbation mutagenesis strategy. A combination of three degenerate codons, NWG, NWC, and NSG, were chosen for amino acid saturation mutagenesis without introducing cysteine and stop residues. In total, 7,749 degenerate oligonucleotides were synthesized on two microchips and released to construct five single-chain antibody fragment (scFv) gene libraries with 4 x 10⁶ DNA sequences. Deep sequencing of the unselected and selected phage libraries using the Illumina platform allowed for an in-depth evaluation of the enrichment landscapes in CDR sequences and amino acid substitutions. Potent candidates were identified according to their high frequencies using NGS analysis, by-passing the need for the primary screening of target-binding clones. Furthermore, a subsequent library by recombination of the 10 most abundant variants from four CDRs was constructed and screened, and a mutant with 158-fold increased affinity (K_d = 25.5 pM) was obtained. These results suggest the potential application of the developed methodology for optimizing the binding properties of other antibodies and biomolecules.     

Current approaches to the synthesis and reconstruction of genetic material

Advanced approaches to the synthesis and reconstruction of genetic material developed in the Institutes of Molecular Biology and Genetics during the past years are summarized. The evolution of methods for oligonucleotide synthesis and scopes for their use in gene production are discussed. The principles of localised mutagenesis methods developed in the Institute are described, such as: a) mutagenesis directed to the regulatory gene regions; b) segment-localized mutagenesis; c) mutagenesis directed by phosphotriester analogues of oligonucleotides. Examples of employing these methods for induction of regulatory mutants of phage lambda, production of fused genes, mutant interferon genes, construction of new DNA vectors, construction of hybrid H1-H3 subtype haemagglutinine gene of influenza virus etc. are presented. The approach to in vivo site-directed mutagenesis is experimentally substantiated.     

Selectable in-vivo recombination to increase antibody library size — an improved phage display vector system

Phage display technology permits the display of libraries of random combinations of light (LC) and heavy chain (HC) antibody genes. Maximizing the size of these libraries would enable the isolation of antibodies with high affinity and specificity. In this study, the loxP/Cre system of in-vivo recombination has been employed to construct an improved vector system for the display of antibodies. In this system, the chloramphenicol acetyl transferase (CAT) gene is linked to a HC library in a donor plasmid, pUX. This CAT gene is `silent' before recombination but active after recombination. A second acceptor phagemid, pMOX, is used for cloning the LC repertoire. Following infection with a Cre producing phage, pMOX accepts the CAT/HC library from pUX via site-specific recombination at the loxP sites. Recombinants can then be selected via chloramphenicol resistance. Using this vector system, we have generated libraries of 4×10⁹ recombinants. Restriction analysis and Fab expression confirmed that 100% of the colonies in the library were recombinants. This system provides a stable selectable mechanism for the generation of large libraries and avoids the isolation of non-recombinants encountered with earlier in-vivo recombination systems.     

Transplacement mutagenesis: a novel in situ mutagenesis system using phage-plasmid recombination

Site-specific mutagenesis provides the ability to alter DNA with precision so that the function of any given gene can be more fully understood. Several methods of in vitro mutagenesis are time-consuming and imprecise, requiring the subcloning and sequencing of products. Here we describe a rapid, high fidelity method of in situ mutagenesis in bacteriophage lambda using transplacement. Using this method, mutations are transferred from oligonucleotides to target phages using a plasmid interface. A small (50 bp) homology region bearing a centred point mutation is generated from oligonucleotides and subcloned into a transplacement plasmid bearing positive and negative phage selectable markers. Following a positive/negative selection cycle of integrative recombination and excision, the point mutation is transferred precisely from plasmid to phage in a subset ( approximately 25-50%) of recombinants. As the fidelity of both oligonucleotide synthesis and phage-plasmid recombination is great, this approach is extremely reliable. Using transplacement, point mutations can be accurately deposited within large phage clones and we demonstrate the utility of this technique in the construction of gene targeting vectors in bacteriophages.     

Temperature-sensitive variants of Saccharomyces cerevisiae iso-1-cytochrome c produced by random mutagenesis of codons 43 to 54.

In vitro random mutagenesis within the CYC1 gene from the yeast Saccharomyces cerevisiae was used to produce a library of mutants encompassing codons 43 to 54 of iso-1-cytochrome c. This region consists of an evolutionarily conserved structure within an evolutionarily diverse sequence. The library, on a low-copy-number yeast shuttle phagemid, was introduced into a yeast strain lacking cytochrome c. The ability of transformants harboring a functional cytochrome c to grow on the non-fermentable carbon source glycerol at 30 degrees C and 37 degrees C was used to determine the phenotype of nearly 1000 transformants. Approximately 90% of the missense mutants present in the library give rise to the wild-type phenotype, 7% result in the temperature-sensitive (Cycts) phenotype, and 3% give rise to the non-functional (Cyc-) phenotype. Phagemids from 20 Cycts and 30 Cyc- transformants were subjected to DNA sequence analysis. All the mutations occur within the targeted region. One-third of the mutants from Cyc- transformants and all the mutants from Cycts transformants are missense mutants. The remaining mutants from Cyc- transformants are nonsense or frame-shift mutants. Missense mutations within the codons for Gly45, Tyr46, Thr49, Asn52 or Ile53 alone are sufficient to produce temperature-sensitive behavior both in vivo and in the variant proteins. The deduced amino acid substitutions correlate remarkably well with side-chain dynamics, secondary structure and tertiary structure of the wild-type protein.     

Orthogonal combinatorial mutagenesis: a codon-level combinatorial mutagenesis method useful for low multiplicity and amino acid-scanning protocols

We describe here a method to generate combinatorial libraries of oligonucleotides mutated at the codon-level, with control of the mutagenesis rate so as to create predictable binomial distributions of mutants. The method allows enrichment of the libraries with single, double or larger multiplicity of amino acid replacements by appropriate choice of the mutagenesis rate, depending on the concentration of synthetic precursors. The method makes use of two sets of deoxynucleoside-phosphoramidites bearing orthogonal protecting groups [4,4′-dimethoxytrityl (DMT) and 9-fluorenylmethoxycarbonyl (Fmoc)] in the 5′ hydroxyl. These phosphoramidites are divergently combined during automated synthesis in such a way that wild-type codons are assembled with commercial DMT-deoxynucleoside-methyl-phosphoramidites while mutant codons are assembled with Fmoc-deoxynucleoside-methyl-phosphoramidites in an NNG/C fashion in a single synthesis column. This method is easily automated and suitable for low mutagenesis rates and large windows, such as those required for directed evolution and alanine scanning. Through the assembly of three oligonucleotide libraries at different mutagenesis rates, followed by cloning at the polylinker region of plasmid pUC18 and sequencing of 129 clones, we concluded that the method performs essentially as intended.     

Mutant library construction in directed molecular evolution

Directed molecular evolution imitates the natural selection process in the laboratory to find mutant proteins with improved properties in the expected aspects by exploring the encoding sequence space. The success of directed molecular evolution experiment depends on the quality of artificially prepared mutant libraries and the availability of convenient high-throughput screening methods. Well-prepared libraries promise the possibility of obtaining desired mutants by screening a library containing a relatively small number of mutants. This article summarizes and reviews the currently available methodologies widely used in directed evolution practices in the hope of providing a general reference for library construction. These methods include error-prone polymerase chain reaction (epPCR), oligonucleotide-based mutagenesis, and genetic recombination exemplified by DNA shuffling and its derivatives. Another designed method is also discussed, in which B-lymphocytes are fooled to mutate nonantibody foreign proteins through somatic hypermutation (SHM).     

Polishing the craft of genetic diversity creation in directed evolution

Genetic diversity creation is a core technology in directed evolution where a high quality mutant library is crucial to its success. Owing to its importance, the technology in genetic diversity creation has seen rapid development over the years and its application has diversified into other fields of scientific research. The advances in molecular cloning and mutagenesis since 2008 were reviewed. Specifically, new cloning techniques were classified based on their principles of complementary overhangs, homologous sequences, overlapping PCR and megaprimers and the advantages, drawbacks and performances of these methods were highlighted. New mutagenesis methods developed for random mutagenesis, focused mutagenesis and DNA recombination were surveyed. The technical requirements of these methods and the mutational spectra were compared and discussed with references to commonly used techniques. The trends of mutant library preparation were summarised. Challenges in genetic diversity creation were discussed with emphases on creating “smart” libraries, controlling the mutagenesis spectrum and specific challenges in each group of mutagenesis methods. An outline of the wider applications of genetic diversity creation includes genome engineering, viral evolution, metagenomics and a study of protein functions. The review ends with an outlook for genetic diversity creation and the prospective developments that can have future impact in this field.     

Molecular Mechanisms of Recombination Restriction in the Envelope Gene of the Human Immunodeficiency Virus

The ability of pathogens to escape the host''s immune response is crucial for the establishment of persistent infections and can influence virulence. Recombination has been observed to contribute to this process by generating novel genetic variants. Although distinctive recombination patterns have been described in many viral pathogens, little is known about the influence of biases in the recombination process itself relative to selective forces acting on newly formed recombinants. Understanding these influences is important for determining how recombination contributes to pathogen genome and proteome evolution. Most previous research on recombination-driven protein evolution has focused on relatively simple proteins, usually in the context of directed evolution experiments. Here, we study recombination in the envelope gene of HIV-1 between primary isolates belonging to subtypes that recombine naturally in the HIV/AIDS pandemic. By characterizing the early steps in the generation of recombinants, we provide novel insights into the evolutionary forces that shape recombination patterns within viral populations. Specifically, we show that the combined effects of mechanistic processes that determine the locations of recombination breakpoints across the HIV-1 envelope gene, and purifying selection acting against dysfunctional recombinants, can explain almost the entire distribution of breakpoints found within this gene in nature. These constraints account for the surprising paucity of recombination breakpoints found in infected individuals within this highly variable gene. Thus, the apparent randomness of HIV evolution via recombination may in fact be relatively more predictable than anticipated. In addition, the dominance of purifying selection in localized areas of the HIV genome defines regions where functional constraints on recombinants appear particularly strong, pointing to vulnerable aspects of HIV biology.     

In vitro recombinants of antibiotic-resistant Chlamydia trachomatis strains have statistically more breakpoints than clinical recombinants for the same sequenced loci and exhibit selection at unexpected loci

Lateral gene transfer (LGT) is essential for generating between-strain genomic recombinants of Chlamydia trachomatis to facilitate the organism's evolution. Because there is no reliable laboratory-based gene transfer system for C. trachomatis, in vitro generation of recombinants from antibiotic-resistant strains is being used to study LGT. However, selection pressures imposed on in vitro recombinants likely affect statistical properties of recombination relative to naturally occurring clinical recombinants, including prevalence at particular loci. We examined multiple loci for 16 in vitro-derived recombinants of ofloxacin- and rifampin-resistant L(1) and D strains, respectively, grown with both antibiotics, and compared these with the same sequenced loci among 11 clinical recombinants. Breakpoints and recombination frequency were examined using phylogenetics, bioinformatics, and statistics. In vitro and clinical isolates clustered perfectly into two groups, without misclassification, using Ward's minimum variance based on breakpoint data. As expected, gyrA (confers ofloxacin resistance) and rpoB (confers rifampin resistance) had significantly more breakpoints among in vitro recombinants than among clinical recombinants (P < 0.0001 and P = 0.02, respectively, using the Wilcoxon rank sum test). Unexpectedly, trpA also had significantly more breakpoints for in vitro recombinants (P < 0.0001). There was also significant selection at other loci. The strongest bias was for ompA in strain D (P = 3.3 × 10(-8)). Our results indicate that the in vitro model differs statistically from natural recombination events. Additional genomic studies are needed to determine the factors responsible for the observed selection biases at unexpected loci and whether these are important for LGT to inform approaches for genetically manipulating C. trachomatis.     

A new approach to directed gene evolution by recombined extension on truncated templates (RETT)

We describe a new approach to in vitro DNA recombination technique termed recombined extension on truncated templates (RETT). RETT generates random recombinant gene library by template-switching of unidirectionally growing polynucleotides from primers in the presence of unidirectional single-stranded DNA fragments used as templates. RETT was applied to the recombination of two homologous chitinase genes from S. marcescens ATCC 21074 and S. liquefaciens GM1403. When the shuffled genes were examined by restriction mapping and sequence analysis, it was found that chimeric genes were produced at a high frequency (more than 70%) between two chitinase genes with 83% of sequence identity. The number of crossovers within each chimeric gene ranged from one to four, and the recombination points were randomly distributed along entire DNA sequence. We also applied RETT to directed evolution of a chitinase variant for enhancing thermostability. Chimeric chitinases that were more thermostable than the parental enzyme were successfully obtained by RETT-based recombination.     

Genetic Code Expansion Through Quadruplet Codon Decoding

Noncanonical amino acid mutagenesis has emerged as a powerful tool for the study of protein structure and function. While triplet nonsense codons, especially the amber codon, have been widely employed, quadruplet codons have attracted attention for the potential of creating additional blank codons for noncanonical amino acids mutagenesis. In this review, we discuss methodologies and applications of quadruplet codon decoding in genetic code expansion both in vitro and in vivo.     

Construction and testing of a bacterial luciferase reporter gene system forin Vivo measurement of nonsense suppression inStreptomyces

A reporter gene system, based on luciferase genes from Vibrio harvei, was constructed for measurement of translation nonsense suppression in Streptomyces. Using the site-directed mutagenesis the TCA codon in position 13 of the luxB gene was replaced by all of the three stop codons individually. By cloning of luxA and luxB genes under the control of strong constitutive Streptomyces promoter ermE* in plasmid pUWL201 we created Wluxl with the wild-type sequence and pWlux2, pWlux3 and pWlux4 plasmids containing TGA-, TAG- and TAA-stop codons, respectively. Streptomyces lividans TK 24 was transformed with the plasmids and the reporter system was tested by growth of the strain in the presence of streptomycin as a translation accuracy modulator. Streptomycin increased nonsense suppression on UAA nearly 10-fold and more than 20-fold on UAG. On the other hand, UGA, the most frequent stop signal in Streptomyces, the effect was negligible.     

用基于"Neo/E"的技术构建重组质粒

根据重组工程原理,建立了一种用于构建重组质粒的“neo／E”(抗生素／单酶切位点)选择与反选择新方法。首先采用PCR方法扩增出线性打靶分子：然后进行两步体内同源重组,(1)neo／E基因敲入,重组子呈现neo抗性表型;(2)目的基因替换M／E基因。用限制酶E消化时,发生第二步重组的DNA分子不能被消化,能够转化大肠杆菌受体菌DH5α。应用该方法构建了重组质粒pGL3-Basic PC1900T。PCR及测序鉴定证明：外源片段重组率为20％,所建立的重组工程选择与反选择新技术为质粒构建提供了新的解决方案。     

寡聚核苷酸诱导突变法改造φ×174噬菌体A基因蛋白的DNA识别序列

 为了进一步研究φX174噬菌体A基因蛋白的复制功能与其所识别的30核苷酸保守序列的关系,我们采用寡聚核苷酸诱导的定点突变法成功地改造了这30核苷酸保守序列。将此保守序列重组到M_(13)mp9噬菌体后,以其单链为模板,在14或16寡聚核苷酸的诱导下,合成共价闭环DNA。经转化到E.coli JM103菌株,用点印迹(Dot blot)杂交法筛选,得到两种重组突变株。一种突变株其30核苷酸保守序列正链的第22碱基由A改为G。另一突变株为其第10碱基A改为C,第11碱基T改为A。突变效率约为5％。制备了此突变株单链及双链DNA,分别做了双脱氧末端终止法及Maxam和Gilbert法序列分析鉴定。