Dealing with complexity: evolutionary engineering and genome shuffling

Comparative analysis of the growing number of microbial genome sequences has shown a high plasticity of genomes and several mechanisms for the adaptation of microbial cells to changing environmental conditions have been discovered. By contrast, the underlying metabolic networks of microorganisms are under strict control and relatively rigid, which poses a significant challenge for rational metabolic engineering approaches. Recursive shuffling of whole genomes has recently been demonstrated as an effective new evolutionary whole-cell engineering approach for the rapid improvement of industrially important microbial phenotypes.     

An mRNA-protein fusion at N-terminus for evolutionary protein engineering

A novel method to link a nascent protein (phenotype) to its mRNA (genotype) covalently through the N-terminus was developed. The mRNA harboring amber stop codon at just downstream of initiation site was hybridized with hydrazide-modified ssDNA at upstream of coding region and was ligated to the DNA. This construct was then modified with 4-acetyl-phenylalanyl amber suppressor tRNA. This modified construct was fused with the nascent protein via the phenylalanine derivative when the mRNA uses the amber suppressor tRNA to decode the amber stop codon. The obtained fusion molecule was used successfully in selective enrichment experiments. It will be applicable for high-through-put screening in evolutionary protein engineering. In contrast to fusion molecules generated by other methods in which the protein is linked to genotype molecule through the C- terminus, our fusion molecule will serve to select a protein for which the C-terminus is essential to be active.     

Construction and characterization of band-specific DNA libraries

Summary A universally primed polymerase chain reaction was developed to amplify DNA dissected from GTG-banded human chromosomes. The amplification products are cloned into plasmid vectors, which allow the rapid characterization of recombinant clones. Starting from 20–40 chromosome fragments, several thousand independent clones detecting single-copy sequences can be obtained. Although these libraries comprise only a few percent of the dissected DNA, they provide narrowly spaced anchor clones for the molecular characterization of chromosome bands and the identification of gene sequences. Here we describe the construction and characterization of DNA libraries for the Langer-Giedion syndrome chromosome region (LGCR, 8q23–24.1), Wilms tumor chromosome region 1 (WT1, 11p13), Prader-Willi syndrome/Angelman syndrome chromosome region (PWCR/ANCR, 15q11.2–12), meningioma chromosome region (MGCR, 22q12–13), and fragile X chromosome region (FRAXA, Xq27.3).     

利用DNA Shuffling技术构建重组PRRSV ORF5基因

通过DNA shuffling技术对PRRSV ORF5基因进行改组,研究其对异源毒株交叉中和能力。将获得的嵌合基因△2ORF5与载体pET-32a连接,转化到大肠杆菌Escherichia coli BL21中,经诱导表达获得约42 kDa重组蛋白。将纯化的重组△2ORF5蛋白免疫BALB/c小鼠,制备多克隆抗体,Western blotting试验表明其与4株亲本毒株重组ORF5蛋白具有良好的反应性。病毒感染抑制试验结果表明,多克隆抗体对4株亲本毒株具有较高的抑制率(53%)。研究结果为研制新型PRRSV疫苗奠定了基础。     

Rapid generation of incremental truncation libraries for protein engineering using alpha-phosphothioate nucleotides

Incremental truncation for the creation of hybrid enzymes (ITCHY) is a novel tool for the generation of combinatorial libraries of hybrid proteins independent of DNA sequence homology. We herein report a fundamentally different methodology for creating incremental truncation libraries using nucleotide triphosphate analogs. Central to the method is the polymerase catalyzed, low frequency, random incorporation of alpha-phosphothioate dNTPs into the region of DNA targeted for truncation. The resulting phosphothioate internucleotide linkages are resistant to 3'-->5' exonuclease hydrolysis, rendering the target DNA resistant to degradation in a subsequent exonuclease III treatment. From an experimental perspective the protocol reported here to create incremental truncation libraries is simpler and less time consuming than previous approaches by combining the two gene fragments in a single vector and eliminating additional purification steps. As proof of principle, an incremental truncation library of fusions between the N-terminal fragment of Escherichia coli glycinamide ribonucleotide formyltransferase (PurN) and the C-terminal fragment of human glycinamide ribonucleotide formyltransferase (hGART) was prepared and successfully tested for functional hybrids in an auxotrophic E.coli host strain. Multiple active hybrid enzymes were identified, including ones fused in regions of low sequence homology.     

透明颤菌血红蛋白的DNA改组研究

为提高透明颤菌血红蛋白在限氧条件下促进宿主细胞生长的能力,首先通过易错PCR向透明颤菌血红蛋白基因中引入突变,再结合DNA改组对其进行改造。将改组基因置于透明颤菌血红蛋白天然启动子下游,转化大肠杆菌DH5α,构建改组文库。以限氧培养条件下菌体沉淀的颜色为指标进行试管初筛,再以限氧和极端限氧条件下菌体湿重为指标进行摇瓶复筛,最终得到一个高活性突变蛋白VHb'042506。该蛋白使宿主的菌体湿重在限氧和极端限氧条件下较原基因转化子分别提高了31.25%和58.75%。经测序和比对,该基因与原基因相比发生了11处碱基点突变,致氨基酸4处错义突变。CO差光谱实验显示该蛋白具有更强的特征吸收。     

MEGA: a biologist-centric software for evolutionary analysis of DNA and protein sequences

The Molecular Evolutionary Genetics Analysis (MEGA) software is a desktop application designed for comparative analysis of homologous gene sequences either from multigene families or from different species with a special emphasis on inferring evolutionary relationships and patterns of DNA and protein evolution. In addition to the tools for statistical analysis of data, MEGA provides many convenient facilities for the assembly of sequence data sets from files or web-based repositories, and it includes tools for visual presentation of the results obtained in the form of interactive phylogenetic trees and evolutionary distance matrices. Here we discuss the motivation, design principles and priorities that have shaped the development of MEGA. We also discuss how MEGA might evolve in the future to assist researchers in their growing need to analyze large data set using new computational methods.     

Genetic engineering of stimuli-sensitive silkelastin-like protein block copolymers

Differentially charged analogues of block copolymers containing repeating sequences from silk (GAGAGS) and elastin (GVGVP) were synthesized using genetic engineering techniques by replacing a valine residue with glutamic acid. The sensitivity to pH and temperature was examined at various polymer concentrations, ionic strengths, and polymer lengths. The polymers transitioned from soluble to precipitate state over narrow temperature ranges. The transition temperature T(t) (the temperature at which half-maximal spectrophotometric absorption was observed) increased with increasing pH up to pH 7.0 and leveled off above this value for the Glu-containing polymer (17E)(11). T(t) was independent of pH for the Val-containing polymer (17V)(11). It decreased with increasing ionic strength, polymer concentration, and polymer length for both polymers. These results suggest that by substituting charged amino acids for neutral amino acids at strategic locations in the polymer backbone and by control of the length of silkelastin-like block copolymers using genetic engineering techniques, it is possible to precisely control sensitivity to pH, temperature, and ionic strength.     

Computationally mapping sequence space to understand evolutionary protein engineering

Evolutionary protein engineering has been dramatically successful, producing a wide variety of new proteins with altered stability, binding affinity, and enzymatic activity. However, the success of such procedures is often unreliable, and the impact of the choice of protein, engineering goal, and evolutionary procedure is not well understood. We have created a framework for understanding aspects of the protein engineering process by computationally mapping regions of feasible sequence space for three small proteins using structure-based design protocols. We then tested the ability of different evolutionary search strategies to explore these sequence spaces. The results point to a non-intuitive relationship between the error-prone PCR mutation rate and the number of rounds of replication. The evolutionary relationships among feasible sequences reveal hub-like sequences that serve as particularly fruitful starting sequences for evolutionary search. Moreover, genetic recombination procedures were examined, and tradeoffs relating sequence diversity and search efficiency were identified. This framework allows us to consider the impact of protein structure on the allowed sequence space and therefore on the challenges that each protein presents to error-prone PCR and genetic recombination procedures.     

Random DNA fragmentation with endonuclease V: application to DNA shuffling

The enzyme endonuclease V nicks uracil-containing DNA at the second or third phosphodiester bond 3′ to uracil sites. I applied the enzyme to random fragmentation of DNA to revise the complex DNA shuffling protocol. The merit of using endonuclease V is that cleavage occurs at random sites and the length of the fragments can easily be adjusted by varying the concentration of dUTP in the polymerase chain reaction. Unlike the conventional method using DNase I, no partial digestion or gel separation of fragments is required. Therefore, labor is dramatically reduced and reproducibility ensured. I applied this method to recombine two truncated green fluorescent protein (GFP) genes and demonstrated successful DNA shuffling by the appearance of the fluorescent full-length GFP genes.     

Artificial protein block polymer libraries bearing two SADs: effects of elastin domain repeats

We have generated protein block polymer E(n)C and CE(n) libraries composed of two different self-assembling domains (SADs) derived from elastin (E) and the cartilage oligomeric matrix protein coiled-coil (C). As the E domain is shortened, the polymers exhibit an increase in inverse transition temperature (T(t)); however, the range of temperature change differs dramatically between the E(n)C and CE(n) library. Whereas all polymers assemble into nanoparticles, the bulk mechanical properties of the E(n)C are very different from CE(n). The E(n)C members demonstrate viscolelastic behavior under ambient conditions and assemble into elastic soft gels above their T(t) values. By contrast, the CE(n) members are predominantly viscous at all temperatures. All library members demonstrate binding to curcumin. The differential thermoresponsive behaviors of the E(n)C and CE(n) libraries in addition to their small molecule recognition abilities make them suitable for potential use in tissue engineering and drug delivery.     

Optimization of DNA shuffling for high fidelity recombination.

A convenient 'DNA shuffling' protocol for random recombination of homologous genes in vitro with a very low rate of associated point mutagenesis (0.05%) is described. In addition, the mutagenesis rate can be controlled over a wide range by the inclusion of Mn2+or Mg2+during DNase I digestion, by choice of DNA polymerase used during gene reassembly as well as how the genes are prepared for shuffling (PCR amplification versus restriction enzyme digestion of plasmid DNA). These protocols should be useful for in vitro protein evolution, for DNA based computing and for structure-function studies of evolutionarily related genes.     

DNA shuffling: Modifying the hand that nature dealt

Summary DNA shuffling is a technique being utilized for in vitro recombination of a single gene or pools of homologous genes. The genes are fragmented into randomly sized pieces, and polymerase chain reaction (PCR) reassembly of full-length genes from the fragments, via self-priming, yields recombination due to PCR template switching. After these PCR products are screened and the interesting products sequenced, improved clones are reshuffled to recombine useful mutations in additive or synergistic ways, in effect mimicking the process of natural sexual recombination. Proteins can be ‘bred’ with the appropriate individual properties and then their ‘progeny’ screened for the desired combination of traits. DNA shuffling is a powerful tool enabling rapid and directed evolution of new genes, operons and whole viral genomes.     

DNA shuffling and screening strategies for improving vaccine efficacy

The efficacy of vaccines can be improved by increasing their immunogenicity, broadening their crossprotective range, as well as by developing immunomodulators that can be coadministered with the vaccine antigen. One technology that can be applied to each of these aspects of vaccine development is MolecularBreeding directed molecular evolution. Essentially, this technology is used to evolve genes in vitro through an iterative process consisting of recombinant generation followed by selection of the desired recombinants. We have used DNA shuffling and screening strategies to develop and improve vaccine candidates against several infectious pathogens including Plasmodium falciparum (a common cause of severe and fatal human malaria), dengue virus, encephalitic alphaviruses such as Venezuelan, western and eastern equine encephalitis viruses (VEEV, WEEV, and EEEV, respectively), human immunodeficiency virus-1 (HIV-1), and hepatitis B virus (HBV). By recombining antigen-encoding genes from different serovar isolates, new chimeras are selected for crossreactivity; these vaccine candidates are expected to provide broader crossprotection than vaccines based on a single serovar. Furthermore, the vaccine candidates can be selected for improved immunogenicity, which would also improve their efficacy. In addition to vaccine candidates, we have applied the technology to evolve several immunomodulators that when coadministered with vaccines can improve vaccine efficacy by fine-tuning the T cell response. Thus, DNA shuffling and screening technology is a promising strategy to facilitate vaccine efficacy.     

Conservation of telomere protein complexes: shuffling through evolution

The rapid evolution of telomere proteins has hindered identification of orthologs from diverse species and created the impression that certain groups of eukaryotes have largely non-overlapping sets of telomere proteins. However, the recent identification of additional telomere proteins from various model organisms has dispelled this notion by expanding our understanding of the composition, architecture and range of telomere protein complexes present in individual species. It is now apparent that versions of the budding yeast CST complex and mammalian shelterin are present in multiple phyla. While the precise subunit composition and architecture of these complexes vary between species, the general function is often conserved. Despite the overall conservation of telomere protein complexes, there is still considerable species-specific variation, with some organisms having lost a particular subunit or even an entire complex. In some cases, complex components appear to have migrated between the telomere and the telomerase RNP. Finally, gene duplication has created telomere protein paralogs with novel functions. While one paralog may be part of a conserved telomere protein complex and have the expected function, the other paralog may serve in a completely different aspect of telomere biology.     

Cosmix-plexing: a novel recombinatorial approach for evolutionary selection from combinatorial libraries

The efficiency of existing combinatorial biological library methods has been moderate in terms of the success rates, the affinities of the ligands selected and the time and effort involved in trying to optimize the initial leads. Although mimicking natural evolution, existing strategies take little notice of the importance of recombination within a selected population to generate increased diversity. We present an overview of our recent progress which has resulted in the successful development of such a strategy, which we designate cosmix-plexing. We incorporate recombination as a central feature in obtaining high success rates and high affinities, even for short monomer peptides, in a very short time. The method uses type II restriction enzymes to re-assort small hypervariable DNA cassettes from an intermediate pre-selected population (e.g. from a phagemid display library), while maintaining the original open-reading frame. Since, in the naive library, each cassette contains all possible combinations of the polypeptide sequences it encodes, much longer regions can be optimized than was possible with methods which depend on a simple selection from the naive library. Short peptides can now be rapidly selected, which exhibit the same, or higher, specificity and affinity for a defined target molecule, than (say) an antibody or even the natural ligand.     

Genome shuffling and ribosome engineering of Streptomyces virginiae for improved virginiamycin production

Bioprocess and Biosystems Engineering - The production of virginiamycin (VGM) from Streptomyces virginiae was improved by genome shuffling and ribosome engineering companied with a high-throughput...