Directed Evolution Technique Research Articles

One-step combined focused epPCR and saturation mutagenesis for thermostability evolution of a new cold-active xylanase.

Enzymes active at low temperature are of great interest for industrial bioprocesses due to their high efficiency at a low energy cost. One of the particularities of naturally evolved cold-active enzymes is their increased enzymatic activity at low temperature, however the low thermostability presented in this type of enzymes is still a major drawback for their application in biocatalysis. Directed evolution of cold-adapted enzymes to a more thermostable version, appears as an attractive strategy to fulfill the stability and activity requirements for the industry. This paper describes the recombinant expression and characterization of a new and highly active cold-adapted xylanase from the GH-family 10 (Xyl-L), and the use of a novel one step combined directed evolution technique that comprises saturation mutagenesis and focused epPCR as a feasible semi-rational strategy to improve the thermostability. The Xyl-L enzyme was cloned from a marine-Antarctic bacterium, Psychrobacter sp. strain 2-17, recombinantly expressed in E. coli strain BL21(DE3) and characterized enzymatically. Molecular dynamic simulations using a homology model of the catalytic domain of Xyl-L were performed to detect flexible regions and residues, which are considered to be the possible structural elements that define the thermolability of this enzyme. Mutagenic libraries were designed in order to stabilize the protein introducing mutations in some of the flexible regions and residues identified. Twelve positive mutant clones were found to improve the T5015 value of the enzyme, in some cases without affecting the activity at 25°C. The best mutant showed a 4.3°C increase in its T5015. The efficiency of the directed evolution approach can also be expected to work in the protein engineering of stereoselectivity.

ITCHY: Incremental Truncation for the Creation of Hybrid enzYmes.

Incremental Truncation for the Creation of Hybrid enzYmes (ITCHY) is a directed evolution technique for randomly recombining two genes. The chief advantage of ITCHY is that there is no requirement for the two genes to share any sequence similarity. This distinguishes ITCHY from directed evolution methods that are based on homologous recombination, such as DNA shuffling. In ITCHY, Escherichia coli exonuclease III is used to incrementally truncate one of the parental genes from its 3' end and the other from its 5' end. Ligation of the randomly truncated gene fragments yields a combinatorial library of chimeras. In this chapter, we provide detailed protocols for constructing libraries using both the user-friendly thio-ITCHY method and also time-dependent incremental truncation. We illustrate the protocols with the data that we obtained when we recombined two alcohol dehydrogenase genes that only share 47 % sequence identity.

Fitness Loss and Library Size Determination in Saturation Mutagenesis

Saturation mutagenesis is a widely used directed evolution technique, in which a large number of protein variants, each having random amino acids in certain predetermined positions, are screened in order to discover high-fitness variants among them. Several metrics for determining the library size (the number of variants screened) have been suggested in the literature, but none of them incorporates the actual fitness of the variants discovered in the experiment. We present the results of an extensive simulation study, which is based on probabilistic models for protein fitness landscape, and which investigates how the result of a saturation mutagenesis experiment – the fitness of the best variant discovered – varies as a function of the library size. In particular, we study the loss of fitness in the experiment: the difference between the fitness of the best variant discovered, and the fitness of the best variant in variant space. Our results are that the existing criteria for determining the library size are conservative, so smaller libraries are often satisfactory. Reducing the library size can save labor, time, and expenses in the laboratory.

Directed Evolution of a Thermophilic β-glucosidase for Cellulosic Bioethanol Production

Characteristics that would make enzymes more desirable for industrial applications can be improved using directed evolution. We developed a directed evolution technique called random drift mutagenesis (RNDM). Mutant populations are screened and all functional mutants are collected and put forward into the next round of mutagenesis and screening. The goal of this technique is to evolve enzymes by rapidly accumulating mutations and exploring a greater sequence space by providing minimal selection pressure and high-throughput screening. The target enzyme was a beta-glucosidase isolated from the thermophilic bacterium, Caldicellulosiruptor saccharolyticus that cleaves cellobiose resulting from endoglucanase hydrolysis of cellulose. Our screening method was fluorescence-activated cell sorting (FACS), an attractive method for assaying mutant enzyme libraries because individual cells can be screened, sorted into distinct populations and collected very rapidly. However, FACS screening poses several challenges, in particular, maintaining the link between genotype and phenotype because most enzyme substrates do not remain associated with the cells. We employed a technique where whole cells were encapsulated in cell-like structures along with the enzyme substrate. We used RNDM, in combination with whole cell encapsulation, to create and screen mutant beta-glucosidase libraries. A mutant was isolated that, compared to the wild type, had higher specific and catalytic efficiencies (k(cat)/K(M)) with p-nitrophenol-glucopyranoside and -galactopyranoside, an increased catalytic turnover rate (k(cat)) with cellobiose, an improvement in catalytic efficiency with lactose and reduced inhibition (K(i)) with galactose and lactose. This mutant had three amino acid substitutions and one was located near the active site.

Expression in yeast of secreted lignin peroxidase with improved 2,4-dichlorophenol degradability by DNA shuffling

Lignin peroxidase (LiP) from Phanerochaete chrysosporium was shown to mineralize a variety of recalcitrant aromatic compounds and oxidize a number of polycyclic aromatic and phenolic compounds. The major problem of the wild type LiP is that it can be inactivated by excess H 2O 2 and high concentrations of aromatic compounds. We applied a directed evolution technique coupled with a rapid colorimetric screening method to obtain mutant genes with improved H 2O 2 stability and polychlorinated phenol degradability, and they were successfully expressed as the secretive LiPs in recombinant Saccharomyces cerevisiae. The resulting variants showed approximately 1.6-fold improved 2,4-dichlorophenol (2,4-DCP) degradation activity and stability against H 2O 2 compared with the parent strain. The kinetic properties of the variants toward 2,4-DCP and H 2O 2 were also increased compared with the wild type for all three mutants studied. Amino acid sequence analysis indicated that the greatest number of amino acid substitutions was located near the surface or Ca 2+ binding sites of the enzyme.

Directed evolution approach to a structural genomics project: Rv2002 from Mycobacterium tuberculosis.

One of the serious bottlenecks in structural genomics projects is overexpression of the target proteins in soluble form. We have applied the directed evolution technique and prepared soluble mutants of the Mycobacterium tuberculosis Rv2002 gene product, the wild type of which had been expressed as inclusion bodies in Escherichia coli. A triple mutant I6TV47MT69K (Rv2002-M3) was chosen for structural and functional characterizations. Enzymatic assays indicate that the Rv2002-M3 protein has a high catalytic activity as a NADH-dependent 3alpha, 20beta-hydroxysteroid dehydrogenase. We have determined the crystal structures of a binary complex with NAD(+) and a ternary complex with androsterone and NADH. The structure reveals that Asp-38 determines the cofactor specificity. The catalytic site includes the triad Ser-140Tyr-153Lys-157. Additionally, it has an unusual feature, Glu-142. Enzymatic assays of the E142A mutant of Rv2002-M3 indicate that Glu-142 reverses the effect of Lys-157 in influencing the pKa of Tyr-153. This study suggests that the Rv2002 gene product is a unique member of the SDR family and is likely to be involved in steroid metabolism in M. tuberculosis. Our work demonstrates the power of the directed evolution technique as a general way of overcoming the difficulties in overexpressing the target proteins in soluble form.

Thermostabilization of cellulosomal endoglucanase EngB from Clostridium cellulovorans by in vitro DNA recombination with non-cellulosomal endoglucanase EngD.

Enhancement of enzyme thermostability by protein engineering gives us information about the thermostabilization mechanism as well as advantages for industrial use of enzymes. In this study, we enhanced the thermostability of endoglucanase EngB, one component of the cellulase complex (cellulosome) from Clostridium cellulovorans, by the directed evolution technique. The library was constructed by in vitro recombination of the genes for EngB and non-cellulosomal cellulase EngD, based on the fact that the catalytic domains of both cellulases were highly homologous. To obtain thermostable clones without loss of activity, the library was screened by a combination of activity and thermostability screening. We obtained three mutants out of 8000 selected clones that showed significantly higher thermostability than those of EngB and EngD without compromising their endoglucanase activities. One of the mutants possessed a sevenfold higher thermostability than EngB. The possible mechanisms of thermostabilization are discussed.

Predicting the emergence of antibiotic resistance by directed evolution and structural analysis.

Directed evolution can be a powerful tool to predict antibiotic resistance. Resistance involves the accumulation of mutations beneficial to the pathogen while maintaining residue interactions and core packing that are critical for preserving function. The constraint of maintaining stability, while increasing activity, drastically reduces the number of possible mutational combination pathways. To test this theory, TEM-1 beta-lactamase was evolved using a hypermutator E. coli-based directed evolution technique with cefotaxime selection. The selected mutants were compared to two previous directed evolution studies and a database of clinical isolates. In all cases, evolution resulted in the generation of the E104K/M182T/G238S combination of mutations ( approximately 500-fold increased resistance), which is equivalent to clinical isolate TEM-52. The structure of TEM-52 was determined to 2.4 A. G238S widens access to the active site by 2.8 A whereas E104K stabilizes the reorganized topology. The M182T mutation is located 17 A from the active site and appears to be a global suppressor mutation that acts to stabilize the new enzyme structure. Our results demonstrate that directed evolution coupled with structural analysis can be used to predict future mutations that lead to increased antibiotic resistance.

Isolation of directed evolution mutants of chloroperoxidase resistant to suicide inactivation by primary olefins

AbstractWild‐type chloroperoxidase catalyzes the efficient chiral epoxidation of secondary olefins but is rapidly inactivated in a mechanism‐based suicide reaction when incubated with hydrogen peroxide and primary olefins. Directed evolution mutants of chloroperoxidase have now been isolated that are resistant to suicidal inactivation. Plasmid vectors containing error‐prone copies of the chloroperoxidase gene and a hygromycin B resistance marker gene have been used to transform Caldariomyces fumago spheroplasts and produce mutant libraries. The mutant library clones were screened for their ability to resist mechanism‐based inactivation by allylbenzene. Four generations of PCR‐based random mutagenesis and screening yielded mutants that were completely resistant to the suicide‐inactivation reaction. Rather surprisingly, the fourth generation mutant developed enhanced epoxidation activity in addition to resistance to allylbenzene inactivation. These initial results suggest that the directed evolution technique can be used to produce chloroperoxidase variants that can further exploit the potential of chloroperoxidase for the synthesis of chiral intermediates.