Directed Evolution Protocols Research Articles

DirectedCHO: A new miniaturized directed evolution process for phenotype stability trial test of CHO cells before bioreactor scale-up

Most of the biopharmaceuticals that are currently on the market are expressed using the Chinese Hamster Ovary (CHO) cell lines. However, the production yield of these biopharmaceuticals is affected due to CHO cellular heterogeneity and challenges in adaptability during the bioreactor scale-up stage. In this communication, we report the protocol for the miniaturized directed evolution process for CHO cells. The results of the directed evolution process would guide adapting the CHO cell line before bioreactor scale-up. With our approach, we have established the protocol that can be used to streamline superior CHO cell lines for biopharmaceutical production which would be the first of its kind in Africa. Our directed evolution protocol includes a method for a low-cost multiplex directed evolution process that can be used on CHO cells using 20 stressors in 8 concentrations and provides stable trial results for the scale-up process. Using our process, we can provide a simple consumable kit that manufacturers can use for the CHO cell phenotype stability test before the scale-up process. With our approach, we would further develop a platform that can streamline superior CHO cell lines for biopharmaceutical production. This approach would be the first of its kind in South Africa/ Africa.

A Directed Evolution Protocol for Engineering Minimal Transcription Factors, Based on CIS Display.

Directed evolution is an efficient strategy for obtaining desired biomolecules. Since the 1990s, the emergence of display techniques has enabled high-throughput screening of functional proteins. However, classical methods require library construction by plasmid cloning and are limited by transformation efficiencies, typically limiting library sizes to ~106-107 variants. More recently, in vitro techniques have emerged that avoid cloning, allowing library sizes of >1012 members. One of these, CIS display, is a DNA-based display technique which allows high-throughput selection of biomolecules in vitro. CIS display creates the genotype-phenotype link required for selection by a DNA replication initiator protein, RepA, that binds exclusively to the template from which it has been expressed. This method has been successfully used to evolve new protein-protein interactions but has not been used before to select DNA-binding proteins, which are major components in mammalian synthetic biology. In this chapter, we describe a directed evolution method using CIS display to efficiently select functional DNA-binding proteins from pools of nonbinding proteins. The method is illustrated by enriching the minimal transcription factor Cro from a low starting frequency (1 in 109). This protocol is also applicable to engineering other DNA-binding proteins or transcription factors from combinatorial libraries.

EnzyHTP Computational Directed Evolution with Adaptive Resource Allocation.

Directed evolution facilitates enzyme engineering via iterative rounds of mutagenesis. Despite the wide applications of high-throughput screening, building "smart libraries" to effectively identify beneficial variants remains a major challenge in the community. Here, we developed a new computational directed evolution protocol based on EnzyHTP, a software that we have previously reported to automate enzyme modeling. To enhance the throughput efficiency, we implemented an adaptive resource allocation strategy that dynamically allocates different types of computing resources (e.g., GPU/CPU) based on the specific need of an enzyme modeling subtask in the workflow. We implemented the strategy as a Python library and tested the library using fluoroacetate dehalogenase as a model enzyme. The results show that compared to fixed resource allocation where both CPU and GPU are on-call for use during the entire workflow, applying adaptive resource allocation can save 87% CPU hours and 14% GPU hours. Furthermore, we constructed a computational directed evolution protocol under the framework of adaptive resource allocation. The workflow was tested against two rounds of mutational screening in the directed evolution experiments of Kemp eliminase (KE07) with a total of 184 mutants. Using folding stability and electrostatic stabilization energy as computational readout, we identified all four experimentally observed target variants. Enabled by the workflow, the entire computation task (i.e., 18.4 μs MD and 18,400 QM single-point calculations) completes in 3 days of wall-clock time using ∼30 GPUs and ∼1000 CPUs.

Quantifying the Performance of Micro-Compartmentalized Directed Evolution Protocols.

High-throughput, in vitro approaches for the evolution of enzymes rely on a random micro-encapsulation to link phenotypes to genotypes, followed by screening or selection steps. In order to optimise these approaches, or compare one to another, one needs a measure of their performance at extracting the best variants of a library. Here, we introduce a new metric, the Selection Quality Index (SQI), which can be computed from a simple mock experiment, performed with a known initial fraction of active variants. In contrast to previous approaches, our index integrates the effect of random co-encapsulation, and comes with a straightforward experimental interpretation. We further show how this new metric can be used to extract general protocol efficiency trends or reveal hidden selection mechanisms such as a counterintuitive form of beneficial poisoning in the compartmentalized self-replication protocol.

Design and evolution of an enzyme with a non-canonical organocatalytic mechanism.

The combination of computational design and laboratory evolution is a powerful and potentially versatile strategy for the development of enzymes with new functions1-4. However, the limited functionality presented by the genetic code restricts the range of catalytic mechanisms that are accessible in designed active sites. Inspired by mechanistic strategies from small-molecule organocatalysis5, here we report the generation of a hydrolytic enzyme that uses Nδ-methylhistidine as a non-canonical catalytic nucleophile. Histidine methylation is essential for catalytic function because it prevents the formation of unreactive acyl-enzyme intermediates, which has been a long-standing challenge when using canonical nucleophiles in enzyme design6-10. Enzyme performance was optimized using directed evolution protocols adapted to an expanded genetic code, affording a biocatalyst capable of accelerating ester hydrolysis with greater than 9,000-fold increased efficiency over free Nδ-methylhistidine in solution. Crystallographic snapshots along the evolutionary trajectory highlight the catalytic devices that are responsible for this increase in efficiency. Nδ-methylhistidine can be considered to be a genetically encodable surrogate of the widely employed nucleophilic catalyst dimethylaminopyridine11, and its use will create opportunities to design and engineer enzymes for a wealth of valuable chemical transformations.

A robust protocol for directed aryl sulfotransferase evolution toward the carbohydrate building block GlcNAc.

Bacterial aryl sulfotransferases (AST) utilize p-nitrophenylsulfate (pNPS) as a phenolic donor to sulfurylate typically a phenolic acceptor. Interest in aryl sulfotransferases is growing because of their broad variety of acceptors and cost-effective sulfuryl-donors. For instance, aryl sulfotransferase A (ASTA) from Desulfitobacterium hafniense was recently reported to sulfurylate d-glucose. In this study, a directed evolution protocol was developed and validated for aryl sulfotransferase B (ASTB). Thereby the well-known pNPS quantification system was advanced to operate efficiently as a continuous screening system in 96-well MTP format with a true coefficient of variation of 14.3%. A random mutagenesis library (SeSaM library) of ASTB was screened (1,760 clones) to improve sulfurylation of the carbohydrate building block N-acetylglucosamine (GlcNAc). The beneficial variant ASTB-V1 (Val579Asp) showed an up to 3.4-fold increased specific activity toward GlcNAc when compared to ASTB-WT. HPLC- and MS-analysis confirmed ASTB-V1's increased GlcNAc monosulfurylation (2.4-fold increased product formation) representing the validation of the first successful directed evolution round of an AST for a saccharide substrate.

Directed evolution of polypropylene and polystyrene binding peptides.

Surface functionalization of biological inert polymers (e.g., polypropylene PP; polystyrene PS) with material binding peptides facilitates an efficient immobilization of enzymes, bioactive peptides or antigens at ambient temperature in water. The developed robust directed evolution protocol enables to tailor polymer binding anchor peptides (PBPs) for efficient binding under application conditions. Key for a successful directed evolution campaign was to develop an epPCR protocol with a very high mutation frequency (60 mutations/kb) to ensure sufficient diversity in PBPs (47 aas LCI: "liquid chromatography peak I"; 44 aas TA2: "Tachystatin A2"). LCI and TA2 were genetically fused to the reporter egfp to quantify peptide binding on PP and PS by fluorescence analysis. The Peptide-Polymer evolution protocol (PePevo protocol) was validated in two directed evolution campaigns for two PBPs and polymers (LCI: PP; TA2: PS). Surfactants were used as selection pressure for improved PBP binders (non-ionic surfactant Triton X-100; 1 mM for LCI-PP // anionic surfactant LAS; 0.5 mM for TA2-PS). PePevo yielded an up to three fold improved PP-binder (LCI-M1-PP: I24T, Y29H, E42 K and LCI-M2-PP: D31V, E42G) and an up to six fold stronger PS-binder (TA2-M1-PS: R3S, L6P, V12 K, S15P, C29R, R30L, F33S, Y44H and TA2-M2-PS: F9C, C24S, G26D, S31G, C41S, Y44Q).

Novel GP64 envelope variants for improved delivery to human airway epithelial cells.

Lentiviral vectors pseudotyped with the baculovirus envelope protein GP64 transduce primary cultures of human airway epithelia (HAE) at their apical surface. Our goal in this study was to harness a directed evolution approach to develop a novel envelope glycoprotein with increased transduction properties for HAE. Using error-prone PCR, a library of GP64 mutants was generated and used to prepare a diverse pool of lentiviral virions pseudotyped with GP64 variants. The library was serially passaged on HAE and three GP64 mutations were recovered. Single-, double- and the triple-combination mutant envelope glycoproteins were compared with wild-type GP64 for their ability to transduce HAE. Our results suggest that lentiviral vectors pseudotyped with evolved GP64 transduced HAE with greater efficiency than wild-type GP64. This effect was not observed in primary cultures of porcine airway epithelial cells, suggesting that the directed evolution protocol was species specific. In summary, our studies indicate that serial passage of a GP64 mutant library yielded specific variants with improved HAE cell tropism, yielding tools with the potential to improve the success of gene therapy for airway diseases.

Directed Evolution – A New Metaheuristc for Optimization

Abstract Recently, we have witnessed an infusion of calculating models based on models offered by nature, models with more or less fidelity to the original that have led to the development of various problem-solving computational procedures. Starting from the observation of natural processes at the macroscopic or microscopic level, various methods have been developed. Technological progress today allows the accelerated reproduction of natural phenomena in the laboratory, which is why a new niche has arisen in the landscape of nature-inspired methods. This niche is devoted to the emulation of artificial biological processes in computational problem-solving methods. This paper proposes a novel approach, which is to develop novel computational methods in the field of Natural Computing based on the semi-natural process, namely Directed Evolution. In the first step we explain Directed Evolution, defined as the artificial reproduction of the process of evolution in the laboratory in order to obtain performing biological entities. For computer scientists, this provide a strong source of inspiration in the search for efficient methods of optimization. The computational model that proposed here largely overlaps with the Directed Evolution protocol, and the results obtained in the numerical experiments confirm the viability of such techniques inspired by processes which are more artificial than natural. The paper describes a novel general algorithm, inspired by Directed Evolution, which is able to solve different optimization problems, such as single optimization, multiobjective optimization and combinatorial optimization problems.

Probabilistic methods in directed evolution: library size, mutation rate, and diversity.

Directed evolution has emerged as an important tool for engineering proteins with improved or novel properties. Because of their inherent reliance on randomness, directed evolution protocols are amenable to probabilistic modeling and analysis. This chapter summarizes and reviews in a nonmathematical way some of the probabilistic works related to directed evolution, with particular focus on three of the most widely used methods: saturation mutagenesis, error-prone PCR, and in vitro recombination. The ultimate aim is to provide the reader with practical information to guide the planning and design of directed evolution studies. Importantly, the applications and locations of freely available computational resources to assist with this process are described in detail.

Directed laccase evolution for improved ionic liquid resistance

In nature, the biodegradation of lignin is a challenging process since lignin is highly cross-linked and poorly water-soluble. Laccases (EC 1.10.3.2, benzenediol: oxygen oxidoreductase) play a key role in the enzymatic degradation of lignin and ionic liquids (ILs) have been used successfully to dissolve lignin. One limitation in lignin degradation using laccases is their low activity/resistance in the presence of ILs. In order to improve the resistance of laccase in IL, a directed evolution protocol based on the ABTS (2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid))-screening assay in 96-well microtiter plate format was developed. 1-Ethyl-3-methylimidazolium ethylsulfate ([EMIM] [EtSO4]) can dissolve lignin efficiently and its anion does not inhibit laccase. The stability of the ABTS radical cation was not affected in the presence of [EMIM] [EtSO4]. Therefore, ([EMIM] [EtSO4]) is a suitable cosolvent for directed laccase evolution. Four laccases (lcc1_2005, lcc1_1997, lcc2 and CVLG1) from T. versicolor (Trametes versicolor) were expressed in Saccharomyces cerevisiae and finally lcc2 was selected as the starting point due to its superior resistance and activity in presence of [EMIM] [EtSO4]. After two rounds of directed evolution, the lcc2 variant M3 (Phe265Ser/Ala318Val) displayed about 4.5-fold higher activity than the lcc2 wild type (WT) in the presence of 15% (v/v) [EMIM] [EtSO4] and a 3.5-fold higher activity than lcc2 WT in buffer. The IC50 value of [EMIM] [EtSO4] towards M3 increases from 392 mM (lcc2 WT) to 497 mM.

Directed evolution of Her2/neu-binding IgG1-Fc for improved stability and resistance to aggregation by using yeast surface display

An Fcab (Fc antigen binding) is a crystallizable fragment of IgG having C-terminal structural loops of CH3 domains engineered for antigen binding. Since introduction of novel binding sites might impair the immunoglobulin fold, repairing strategies are needed for improving the biophysical properties of promising binders without decreasing affinity to the antigen. Here, a directed evolution protocol was developed and applied for stabilization of a Her2/neu-binding Fcab. Distinct loop regions of the parental binder were softly randomized by parsimonious mutagenesis, followed by heat incubation of the yeast displayed protein library and selection for retained antigen binding. Selected Fcabs were expressed solubly in Pichia pastoris and human embryonic kidney 293 cells and characterized. Fcab clones that retained their affinity to Her2/neu but exhibited a significantly increased conformational stability and resistance to aggregation could be evolved. Moreover, we demonstrate that simultaneous selection for binding to the antigen and to structurally specific ligands (FcγRI and an antibody directed against the CH2 domain) yields even more stable Fcabs. To sum up, this study presents a very potent and generally applicable method for improving the fold and stability of antibodies, antibody fragments and alternative binding scaffolds.

Understanding radiation resistance using resistance‐promoting RecA mutants

Multiple bacterial species exist that can survive the extensive DNA damage caused by ionizing radiation, yet the exact mechanisms of this resistance are not entirely understood. Using a directed evolution protocol, our lab has generated several strains of E. coli that are extremely radiation‐resistant. The genomes of multiple isolates from the evolved populations have been sequenced to identify mutations responsible for this phenotype. Two alleles were identified which encode mutations in RecA (A289S and D276A), a central protein in DNA repair. Inserting these alleles into wildtype E. coli resulted in small increases in survival after exposure to radiation. Thus, understanding the altered activity of these mutants can provide insight into DNA repair mechanisms and how an organism can survive ionizing radiation.RecA D276A displays an increased ability to bind to DNA coated with the single stranded DNA binding protein (SSB). This suggests that faster activation of RecA may aid in cell survival after DNA damage. Conversely, the RecA A289S protein has a diminished capability to bind to SSB‐coated DNA, and thus, the RecF, RecO, and RecR loading proteins may have a greater role in facilitating RecA binding to SSB‐coated DNA with this mutant. Also, RecA A289S filaments disassemble faster when exposed to the inhibitory RecX protein. This data suggests that tighter regulation of RecA A289S activity may produce a filament form that enhances certain types of RecA‐mediated recombination events. This work was supported by a grant from the National Institutes of Health, GM32335.

Design of a Functional Nitric Oxide Reductase within a Myoglobin Scaffold

A review. In recent years, significant progress has been made in the computational design of functional artificial enzymes. When combined with directed evolution protocols, their efficiencies are comparable to those obtained with catalytic antibodies. In comparison, the in silico creation of artificial metalloenzymes remains challenging. This could be due to the difficulty in computing both the transition states for metal-catalyzed reactions and the corresponding entatic state for a metalloenzyme. In a recent study, structurally characterized a functional bacterial nitric oxide reductase within a myoglobin scaffold has been rationally designed. This ground-breaking study by Lu and coworkers has opened the way towards the rational design of artificial metalloenzymes for more challenging reactions. For the artificial nitric oxide reductase, several issues deserve further scrutiny: catalytic efficiency as well as detailed reaction mechanism. In a broader perspective, the use of more elaborate computational algorithms, combined with efficient directed-evolution protocols should enable the creation and optimization of highly versatile artificial metalloenzymes in a variety of protein folds. [on SciFinder(R)]

Computational design of protein–ligand binding: Modifying the specificity of asparaginyl‐tRNA synthetase

A method for computational design of protein-ligand interactions is implemented and tested on the asparaginyl- and aspartyl-tRNA synthetase enzymes (AsnRS, AspRS). The substrate specificity of these enzymes is crucial for the accurate translation of the genetic code. The method relies on a molecular mechanics energy function and a simple, continuum electrostatic, implicit solvent model. As test calculations, we first compute AspRS-substrate binding free energy changes due to nine point mutations, for which experimental data are available; we also perform large-scale redesign of the entire active site of each enzyme (40 amino acids) and compare to experimental sequences. We then apply the method to engineer an increased binding of aspartyl-adenylate (AspAMP) into AsnRS. Mutants are obtained using several directed evolution protocols, where four or five amino acid positions in the active site are randomized. Promising mutants are subjected to molecular dynamics simulations; Poisson-Boltzmann calculations provide an estimate of the corresponding, AspAMP, binding free energy changes, relative to the native AsnRS. Several of the mutants are predicted to have an inverted binding specificity, preferring to bind AspAMP rather than the natural substrate, AsnAMP. The computed binding affinities are significantly weaker than the native, AsnRS:AsnAMP affinity, and in most cases, the active site structure is significantly changed, compared to the native complex. This almost certainly precludes catalytic activity. One of the designed sequences has a higher affinity and more native-like structure and may represent a valid candidate for Asp activity.

Synonymous Genes Explore Different Evolutionary Landscapes

The evolutionary potential of a gene is constrained not only by the amino acid sequence of its product, but by its DNA sequence as well. The topology of the genetic code is such that half of the amino acids exhibit synonymous codons that can reach different subsets of amino acids from each other through single mutation. Thus, synonymous DNA sequences should access different regions of the protein sequence space through a limited number of mutations, and this may deeply influence the evolution of natural proteins. Here, we demonstrate that this feature can be of value for manipulating protein evolvability. We designed an algorithm that, starting from an input gene, constructs a synonymous sequence that systematically includes the codons with the most different evolutionary perspectives; i.e., codons that maximize accessibility to amino acids previously unreachable from the template by point mutation. A synonymous version of a bacterial antibiotic resistance gene was computed and synthesized. When concurrently submitted to identical directed evolution protocols, both the wild type and the recoded sequence led to the isolation of specific, advantageous phenotypic variants. Simulations based on a mutation isolated only from the synthetic gene libraries were conducted to assess the impact of sub-functional selective constraints, such as codon usage, on natural adaptation. Our data demonstrate that rational design of synonymous synthetic genes stands as an affordable improvement to any directed evolution protocol. We show that using two synonymous DNA sequences improves the overall yield of the procedure by increasing the diversity of mutants generated. These results provide conclusive evidence that synonymous coding sequences do experience different areas of the corresponding protein adaptive landscape, and that a sequence's codon usage effectively constrains the evolution of the encoded protein.

Directed evolution of a histone acetyltransferase – enhancing thermostability, whilst maintaining catalytic activity and substrate specificity

Histone acetylation plays an integral role in the epigenetic regulation of gene expression. Transcriptional activity reflects the recruitment of opposing classes of enzymes to promoter elements; histone acetyltransferases (EC 2.3.1.48) that deposit acetyl marks at a subset of histone residues and histone deacetylases that remove them. Many histone acetyltransferases are difficult to study in solution because of their limited stability once purified. We have developed a directed evolution protocol that allows the screening of hundreds of histone acetyltransferase mutants for histone acetylating activity, and used this to enhance the thermostability of the human P/CAF histone acetyltransferase. Two rounds of directed evolution significantly stabilized the enzyme without lowering the catalytic efficiency and substrate specificity of the enzyme. Twenty-four variants with higher thermostability were identified. Detailed analysis revealed twelve single amino acid mutants that were found to possess a higher thermostability. The residues affected are scattered over the entire protein structure, and are different from mutations predicted by sequence alignment approaches, suggesting that sequence comparison and directed evolution methods are complementary strategies in engineering increased protein thermostability. The stabilizing mutations are predominately located at surface of the enzyme, suggesting that the protein's surface is important for stability. The directed evolution approach described in the present study is easily adapted to other histone modifying enzymes, requiring only appropriate peptide substrates and antibodies, which are available from commercial suppliers.

Directed evolution of glucose oxidase from Aspergillus niger for ferrocenemethanol‐mediated electron transfer

A directed evolution protocol was developed for glucose oxidase (GOx) from Aspergillus niger that mimics applications conditions and employs a well-known mediator, oxidized ferrocenemethanol, in a medium throughput screen (96-well plate format). Upon reduction, oxidized ferrocenemethanol shows a color change from blue to pale yellow that can be recorded at 625 nm. Under optimized screening conditions, a CV of less than 20% was achieved in 96-well microtiter plates. For validating the screening system, two mutant libraries of GOx were generated by standard error-prone PCR conditions (0.04 mM MnCl(2)) and Saccharomyces cerevisiae was employed as host for secreted GOx expression. Two screening of approximately 2000 GOx mutants yielded a double mutant (T30S I94V) with improved pH and thermal resistance. Thermal resistance at a residual activity of 50% was increased from 58 degrees C (wild type, WT) to 62 degrees C (T30S I94V) and pH stability was improved at basic pH (pH 8-11). K(m) for glucose remained nearly unchanged (20.8 mM WT; 21.3 mM T30S I94V) and k(cat) increased (69.5/s WT; 137.7/s T30S I94V).

ECodonOpt: a systematic computational framework for optimizing codon usage in directed evolution experiments.

We present a systematic computational framework, eCodonOpt, for designing parental DNA sequences for directed evolution experiments through codon usage optimization. Given a set of homologous parental proteins to be recombined at the DNA level, the optimal DNA sequences encoding these proteins are sought for a given diversity objective. We find that the free energy of annealing between the recombining DNA sequences is a much better descriptor of the extent of crossover formation than sequence identity. Three different diversity targets are investigated for the DNA shuffling protocol to showcase the utility of the eCodonOpt framework: (i) maximizing the average number of crossovers per recombined sequence; (ii) minimizing bias in family DNA shuffling so that each of the parental sequence pair contributes a similar number of crossovers to the library; and (iii) maximizing the relative frequency of crossovers in specific structural regions. Each one of these design challenges is formulated as a constrained optimization problem that utilizes 0-1 binary variables as on/off switches to model the selection of different codon choices for each residue position. Computational results suggest that many-fold improvements in the crossover frequency, location and specificity are possible, providing valuable insights for the engineering of directed evolution protocols.