Evolutionary Molecular Engineering Research Articles

1M0930 lacプロモータの細胞型進化分子工学IV

1M0930 lacプロモータの細胞型進化分子工学IV

Evolutionary molecular engineering by random elongation mutagenesis.

We describe a new method of random mutagenesis that employs the addition of peptide tails with random sequences to the C-terminal of enzyme molecules. A mutant population of catalase I from Bacillus stearothermophilus prepared by this method has a diversity in thermostability and enzyme activity equal to that obtained after random point mutagenesis. When a triple mutant of catalase I (I108T/D130N/1222T)-the thermostability of which is much lower than that of the wild type-was subjected to random elongation mutagenesis, we generated a mutant population containing only mutants with higher thermostability than the triple mutant. Some had an even higher stability than the wild-type enzyme, whose thermostability is considered to be optimized. These results indicate that peptide addition expands the protein sequence space resulting in a new fitness landscape. The enzyme can then move along the routes of the new landscape until it reaches a new optimum. The combination of random elongation mutagenesis with random point mutagenesis should be a useful approach to the in vitro evolution of proteins with new properties.

3PA133 lacプロモーターの細胞型進化分子工学III

3PA133 lacプロモーターの細胞型進化分子工学III

Screening of conformationally constrained random polypeptide libraries displayed on a protein scaffold.

The selection of novel proteins or enzymes from random protein libraries has come to be a major objective in current biology, and these enzymes should prove useful in various biological and biomedical fields. New technologies such as in vitro selection of proteins in cell-free systems have high potential to realize evolutionary molecular engineering of proteins. This review highlights an application of insertional mutagenesis of proteins to evolutionary molecular engineering. Random sequence proteins are inserted into the surface of a host enzyme which serves as a scaffold to display random protein libraries. Constraints on random polypeptide conformations owing to the proximity of N- and C-termini on the scaffold would result in greater screening efficiency of libraries. The scaffold enzyme is also used as a probe for monitoring the hill climbing of random sequence proteins on a fitness landscape and navigating rapid protein folding in the sequence space.

Fitness Landscape of a Biopolymer Participating in a Multi-step Reaction

A biopolymer such as an enzyme participates in a multi-step reaction. When we take such a biopolymer as a target of evolutionary molecular engineering, the problem of frustration or interference among partial-fitness landscapes for elementary reaction steps arises. We examined the effect of the frustration on a fitness landscape for the whole reaction. From the results of a previous paper we assumed that each partial-fitness landscape is of the Mt. Fuji-type, based on the mutational additivity. Computer simulation for two-step or three-step reactions showed that the fitness landscape for a whole reaction was of the rough Mt. Fuji-type, in which optima were located near the top. However, there were fitter double-point mutants around each local optimum. This conclusion was supported by the result of the mathematical analysis of a simplified landscape for anL-step reaction: even if there are local optima on the fitness landscape, several fitter sequences exist at the Hamming distance ofLfrom every local optimum in almost all cases. Fitness distributions around the global optimum also showed that the fitness landscape can be approximated to a Mt. Fuji-type landscape.

思想として,技術としての進化分子工学

思想として,技術としての進化分子工学

Y-ligation: an efficient method for ligating single-stranded DNAs and RNAs with T4 RNA ligase.

Very efficient ligation of oligodeoxyribonucleotides was attained through a simple molecular construct, which is composed of one stem and two branches (Y-shape), with use of T4 RNA ligase. Single-stranded DNAs (naturally, RNAs also) of more than 100 nucleotides (even 800 nts) were considerably ligated, approximately as theoretically expected. Owing to the molecular construct adopted, such a tiny amount of ligation products could be amplified to a sufficient amount by PCR and then recovered as single-stranded DNAs. This advantage of being amplifiable is shown to be useful for both combinatorial chemistry and evolutionary molecular engineering, which deal with a pool of diversity molecules.

In vitro virus: Bonding of mRNA bearing puromycin at the 3′-terminal end to the C-terminal end of its encoded protein on the ribosome in vitro

Adequate means for genotype assignment to phenotype is essential in evolutionary molecular engineering. In this study, construction of ‘in vitro virus’ was carried out in which a genotype molecule (mRNA) covalently binds to the phenotype molecule (protein) through puromycin on the ribosome in a cell-free translation system. Bonding efficiency was ∼10%, thus indicating a population of the in vitro virus to have ∼10 12 protein variants, this number being 10 4 that in the phage display. The in vitro virus is useful for examining protein evolution in a test tube and the results may possibly serve as basis for a general method for selecting proteins possessing the most desirable functions.

Exhortation to Evolutionary Molecular Engineering : Towards An Adaptive Design of "Biopolymers" and An Approach to the Origin of Bioinformation

Exhortation to Evolutionary Molecular Engineering : Towards An Adaptive Design of "Biopolymers" and An Approach to the Origin of Bioinformation

A virus-like molecule in the early stage of encoded molecular evolution

The recent advances of the evolutionary molecular engineering revealed the effectiveness of bonding strategy for assignment of the phenotype to its genotype, which non-enveloped viruses such as simple bacteriophages adopt. On the other hand, cellular organisms adopt another kind of the strategy, namely the compartmentalzation of both genotype and phenotype molecules in a single compartment enclosed with a cell membrane. The simplest strategy is that adopted by ribozymes in the RNA world. A single molecule carries both genotype and its phenotype. Based on the definition of “virus”-type and “cell”-type of the assignment strategy, we propose a virus-early/cell-late model of the history of life.

Evolutionary molecular engineering in Japan.

Evolutionary molecular engineering in Japan.

Improvement of thermal stability of proteins by evolutionary molecular engineering.

Thermal stability of proteins can be improved by evolutionary molecular engineering, without the knowledge about 3D structures of the proteins nor the principle that governs the protein stability. The method will provide a way of improving the thermal stability of industrially important proteins. It can also be used as a tool to study protein stability. There are a substantial number of examples now. Our recent study suggested that it is easy to obtain thermo-stabilized proteins by single-base substitution. Although, it requires further improvement of the method to obtain mutants with double-base substitution within a codon.

A model of the virus-type strategy in the early stage of encoded molecular evolution

Recent advances in evolutionary molecular engineering have revealed that the essential nature of a “virus” in the evolutionary aspect is its bonding strategy for assignment of the phenotype to its genotype. Based on the definition of “virus”-type and “cell”-type of the assignment strategy, we propose a virus-early/cell-late model of the history of life. The first encoded protein is assumed to be a cofactor of replication ribozyme in the RNA world and to be bound to its genetic RNA. As such a virus-type strategy could introduce the Darwinian selection process into the hypercycle with translation, a hypercycle with virus-like members could make the replicase protein and the translation system gradually evolve together out of the RNA world without a proto-cell. Moreover, they could evolve much faster by this virus-type strategy than by a primitive cellular organism.

進化分子工学による高次分子システムと機能の創出

進化分子工学による高次分子システムと機能の創出

Period Dependent Selection in Continuous Culture of Viruses in a Periodic Environment

Selection in a cellstat culture of two mutant strains of a bacteriophage under periodically fluctuating temperature was analyzed mathematically and numerically. Each of the two viral strains (P1 and P2) was assumed to have a different Arrhenius activation energy for its reproduction reaction. A phase diagram of the final state in the continuous culture was drawn. The most noticeable was that there were both P1-only and P2-only phases of competitive exclusion depending on the period of oscillation and the dilution rate. The period-dependent selection was proved that it was based on the feedback effect via the host-population change. At a short period of oscillation, the strain with larger arithmetic average fitness ultimately dominated in the population; on the other hand, at a long period of oscillation, the strain with larger geometric average fitness ultimately dominated. There was a co-existence phase between the two exclusion phases. The results suggest the feasibility of a method to escape from trapping at local optima on a fitness landscape in evolutionary molecular engineering.

Stabilization of proteins by evolutionary molecular engineering techniques

Laboratory ‘evolution’ seems to be a promising route to improving and converting the properties of proteins and enzymes. As a model case, chimeric 3-isopropylmalate dehydrogenase encoded by a hybrid gene constructed from thermophile and mesophile IeuB genes, has been stabilized by evolutionary molecular engineering techniques. The results indicate that fine tuning of the protein conformation is crucial for its increased stability.

Evolutionary Molecular Engineering.

An emerging biotechnology, the evolutionary molecular engineering, provides a new design principle of functional RNAs or proteins based on the Darwinian mechanism of molecular evolution. There are three types of the evolution reactor corresponding to three types of assignment strategy between the genotype and the phenotype, namely, RNA-type, virus-type and cell-type.

Selection and evolution of bacteriophages in cellstat.

Objectives of this work were as follows: 1. to establish a laboratory experimental system utilizable in a biophysical approach to molecular evolution; and 2. to provide real world parameters to theories of molecular evolution, especially to Eigen's theory of quasi-species. Secretion type bacteriophage fd of E. coli, closely related phages and artificial chimera phages of fd, and a virulent phage Q beta of E. coli were cultured continuously in a specially designed fermenter called a "cellstat". A phage is cultured in a flow of host bacterial cells. Due to its high dilution rate, the mutant cell could not be selected in the cellstat. It was therefore recognized that the cellstat is suitable for study of the selection and evolution process of a bacteriophage under well-defined environmental conditions without interference from host cell mutations. Population dynamics of bacteriophages of various types in the cellstat were studied theoretically by computer simulation and experimentally. A genetically invariable pure population of phage behaves like an open non-linear chemical reaction system. An invariable mixed population shows a selection process, while a variable population generates an evolution process. Kinetic constants describing the dynamics were determined by curve fitting between the theoretical and the experimental curve obtained from competition experiments and from biological relaxation experiments. One of the most important kinetic parameters thus obtained was the selection coefficient, and its dependence on the base sequence of phage DNA. We drew a local landscape of the selection coefficient near the fd sequence on the base sequence space. From this landscape we were able to confirm the importance of slightly deleterious mutants in molecular evolution. We also confirmed the possibility of developing an evolutionary molecular engineering using a cellstat as an evolution reactor and fd phage as a working replicon. Novelties of this work were as follows: 1. the first stable continuous culture of a bacteriophage was achieved with a cellstat; 2. a local landscape of selection coefficient near the fd sequence on the sequence space was the first experimental drawing of such a map; 3. a biological relaxation method was realized to measure kinetic constants of a biological kinetic process, or molecular evolution; and 4. a practical engineering process of evolutionary molecular engineering was proposed.

Evolutionary molecular engineering based on RNA replication

Abstract