Rational and Combinatorial Engineering of the Glucan Synthesizing Enzyme Amylosucrase

Rational engineering of amylosucrase required detailed investigations of the molecular basis of catalysis. Biochemical characterization of the enzyme coupled to structural analyses enabled the polymerization mechanism to be elucidated. This provided key information for successfully changing amylosucrase to a hydrolase or an improved polymerase using site-directed mutagenesis. In parallel, a combinatorial approach was developed to further improve the catalyst. The method, based on random mutagenesis and recombination of parent sequences, has generated libraries of variants. These were searched for improved polymer formation using a first step of selection followed by a screening test including a double detection method. Several mutants with desired properties have been isolated, their sequences revealed that they could not have been designed following a rational approach.     

Combinatorial engineering to enhance amylosucrase performance: construction, selection, and screening of variant libraries for increased activity

Amylosucrase is a glucosyltransferase belonging to family 13 of glycoside hydrolases and catalyses the formation of an amylose-type polymer from sucrose. Its potential use as an industrial tool for the synthesis or the modification of polysaccharides, however, is limited by its low catalytic efficiency on sucrose alone, its low stability, and its side reactions resulting in sucrose isomer formation. Therefore, combinatorial engineering of the enzyme through random mutagenesis, gene shuffling, and selective screening (directed evolution) was started, in order to generate more efficient variants of the enzyme. A convenient zero background expression cloning strategy was developed. Mutant gene libraries were generated by error-prone polymerase chain reaction (PCR), using Taq polymerase with unbalanced dNTPs or Mutazyme™, followed by recombination of the PCR products by DNA shuffling. A selection method was developed to allow only the growth of amylosucrase active clones on solid mineral medium containing sucrose as the sole carbon source. Automated protocols were designed to screen amylosucrase activity from mini-cultures using dinitrosalicylic acid staining of reducing sugars and iodine staining of amylose-like polymer. A pilot experiment using the described mutagenesis, selection, and screening methods yielded two variants with significantly increased activity (five-fold under the screening conditions). Sequence analysis of these variants revealed mutations in amino acid residues which would not be considered for rational design of improved amylosucrase variants. A method for the characterisation of amylosucrase action on sucrose, consisting of accurate measurement of glucose and fructose concentrations, was introduced. This allows discrimination between hydrolysis and transglucosylation, enabling a more detailed comparison between wild-type and mutant enzymes.     

Combinatorial engineering to enhance thermostability of amylosucrase

Amylosucrase is a transglucosidase that catalyzes amylose-like polymer synthesis from sucrose substrate. About 60,000 amylosucrase variants from two libraries generated by the MutaGen random mutagenesis method were submitted to an in vivo selection procedure leading to the isolation of more than 7000 active variants. These clones were then screened for increased thermostability using an automated screening process. This experiment yielded three improved variants (two double mutants and one single mutant) showing 3.5- to 10-fold increased half-lives at 50 degrees C compared to the wild-type enzyme. Structural analysis revealed that the main differences between wild-type amylosucrase and the most improved variant (R20C/A451T) might reside in the reorganization of salt bridges involving the surface residue R20 and the introduction of a hydrogen-bonding interaction between T451 of the B' domain and D488 of flexible loop 8. This double mutant is the most thermostable amylosucrase known to date and the only one usable at 50 degrees C. At this temperature, amylose synthesis by this variant using high sucrose concentration (600 mM) led to the production of amylose chains twice as long as those obtained by the wild-type enzyme at 30 degrees C.     

Increased amylosucrase activity and specificity, and identification of regions important for activity, specificity and stability through molecular evolution

Amylosucrase is a transglycosidase which belongs to family 13 of the glycoside hydrolases and transglycosidases, and catalyses the formation of amylose from sucrose. Its potential use as an industrial tool for the synthesis or modification of polysaccharides is hampered by its low catalytic efficiency on sucrose alone, its low stability and the catalysis of side reactions resulting in sucrose isomer formation. Therefore, combinatorial engineering of the enzyme through random mutagenesis, gene shuffling and selective screening (directed evolution) was applied, in order to generate more efficient variants of the enzyme. This resulted in isolation of the most active amylosucrase (Asn387Asp) characterized to date, with a 60% increase in activity and a highly efficient polymerase (Glu227Gly) that produces a longer polymer than the wild-type enzyme. Furthermore, judged from the screening results, several variants are expected to be improved concerning activity and/or thermostability. Most of the amino acid substitutions observed in the totality of these improved variants are clustered around specific regions. The secondary sucrose-binding site and beta strand 7, connected to the important Asp393 residue, are found to be important for amylosucrase activity, whereas a specific loop in the B-domain is involved in amylosucrase specificity and stability.     

Improving the Efficiency of Transposon Mutagenesis in Salmonella Enteritidis by Overcoming Host-Restriction Barriers

Transposon mutagenesis using transposome complex is a powerful method for functional genomics analysis in diverse bacteria by creating a large number of random mutants to prepare a genome-saturating mutant library. However, strong host restriction barriers can lead to limitations with species- or strain-specific restriction-modification systems. The purpose of this study was to enhance the transposon mutagenesis efficiency of Salmonella Enteritidis to generate a larger number of random insertion mutants. Host-adapted Tn5 DNA was used to form a transposome complex, and this simple approach significantly and consistently improved the efficiency of transposon mutagenesis, resulting in a 46-fold increase in the efficiency as compared to non-adapted transposon DNA fragments. Random nature of Tn5 insertions was confirmed by high-throughput sequencing of the Tn5-junction sequences. The result based on S. Enteritidis in this study should find broad applications in preparing a comprehensive mutant library of other species using transposome complex.     

High level expression of a recombinant amylosucrase gene and selected properties of the enzyme

Two high-level heterologous expression systems for amylosucrase genes have been constructed. One depends on sigma-70 bacterial RNA polymerase, the other on phage T7 RNA polymerase. Translational fusions were formed between slightly truncated versions of the gene from Neisseria polysaccharea and sequences of expression vectors pQE-81L or pET33b(+), respectively. These constructs were introduced into different Escherichia coli strains. The resulting recombinants yielded up to 170 mg of dissolved enzyme per litre of culture at a moderate cell density of five OD₆₀₀. To our knowledge, this is the highest yield per cell described so far for amylosucrases. The recombinant enzymes could rapidly be purified through the use of histidine tags in the N-terminally attached sequences. These segments did not alter catalytic properties and therefore need not be removed for most applications. Investigations with glucose and malto-oligosaccharides of different lengths identified rate-limiting steps in the elongation (acceptor reaction) and truncation (donor reaction) of these substrates. The elongation of maltotriose and its reversal, the truncation of maltotetraose, were found to be particularly slow reactions. Potential reasons are discussed, based on the crystal structure of the enzyme. It is furthermore shown that amylosucrase is able to synthesise mixed disaccharides. All of the glucose epimers mannose, allose, and galactose served as acceptors, yielding between one and three main products. We also demonstrate that, as an alternative to the use of purified amylosucrase, cells of the constructed recombinant strains can be used to carry out glucosylations of acceptors.     

Expression of an amylosucrase gene in potato results in larger starch granules with novel properties

Main conclusion

Expression of amylosucrase in potato resulted in larger starch granules with rough surfaces and novel physico-chemical properties, including improved freeze–thaw stability, higher end viscosity, and better enzymatic digestibility. Starch is a very important carbohydrate in many food and non-food applications. In planta modification of starch by genetic engineering has significant economic and environmental benefits as it makes the chemical or physical post-harvest modification obsolete. An amylosucrase from Neisseria polysaccharea fused to a starch-binding domain (SBD) was introduced in two potato genetic backgrounds to synthesize starch granules with altered composition, and thereby to broaden starch applications. Expression of SBD–amylosucrase fusion protein in the amylose-containing potato resulted in starch granules with a rough surface, a twofold increase in median granule size, and altered physico-chemical properties including improved freeze–thaw stability, higher end viscosity, and better enzymatic digestibility. These effects are possibly a result of the physical interaction between amylosucrase and starch granules. The modified larger starches not only have great benefit to the potato starch industry by reducing losses during starch isolation, but also have an advantage in many food applications such as frozen food due to its extremely high freeze–thaw stability.     

insilico.mutagenesis: a primer selection tool designed for sequence scanning applications used in directed evolution experiments

Several primer prediction programs have been developed for a variety of applications. However none of these tools allows the prediction of a large set of primers for whole gene site-directed mutagenesis experiments using the megaprimer method. We report a novel primer prediction tool (insilico.mutagenesis), accessible at www.insilico.uni-duesseldorf.de, developed for the application to high-throughput mutagenesis used in directed evolution or structure-function dependency projects, which involve the subsequent mutagenesis of a large number of amino acid positions (e.g., in whole gene saturation or gene scanning mutagenesis experiments). Furthermore, the program is suitable for all site-directed (saturation) mutagenesis approaches, such as saturation mutagenesis of promoter sequences and other types of untranslated intergenic regions. In anticipation of downstream cloning steps, the primer design tool also includes a restriction site control feature alerting the user if unwanted restriction sites have been introduced within the mutagenesis primer. The use of our tool promises to speed up the process of site-directed mutagenesis, as it instantly allows predicting a large set of primers.     

Artificial protein sequences enable recognition of vicinal and distant protein functional relationships

High divergence in protein sequences makes the detection of distant protein relationships through homology-based approaches challenging. Grouping protein sequences into families, through similarities in either sequence or 3-D structure, facilitates in the improved recognition of protein relationships. In addition, strategically designed protein-like sequences have been shown to bridge distant structural domain families by serving as artificial linkers. In this study, we have augmented a search database of known protein domain families with such designed sequences, with the intention of providing functional clues to domain families of unknown structure. When assessed using representative query sequences from each family, we obtain a success rate of 94% in protein domain families of known structure. Further, we demonstrate that the augmented search space enabled fold recognition for 582 families with no structural information available a priori. Additionally, we were able to provide reliable functional relationships for 610 orphan families. We discuss the application of our method in predicting functional roles through select examples for DUF4922, DUF5131, and DUF5085. Our approach also detects new associations between families that were previously not known to be related, as demonstrated through new sub-groups of the RNA polymerase domain among three distinct RNA viruses. Taken together, designed sequences-augmented search databases direct the detection of meaningful relationships between distant protein families. In turn, they enable fold recognition and offer reliable pointers to potential functional sites that may be probed further through direct mutagenesis studies.     

Optimized and automated protocols for high-throughput screening of amylosucrase libraries

This article describes the design and validation of a general procedure for the high-throughput isolation of amylosucrase variants displaying higher thermostability or increased resistance to organic solvents. This procedure consists of 2 successive steps: an in vivo selection that eliminates inactive variants followed by automated screening of active variants to isolate mutants displaying enhanced features. The authors chose an Escherichia coli expression vector, allowing a high production rate of the recombinant enzyme in miniaturized culture conditions. The screening assay was validated by minimizing variability for various parameters of the protocol, especially bacterial growth and protein production in cultures in 96-well microplates. Recombinant amylosucrase production was normalized by decreasing the coefficient of variance from 27% to 12.5%. Selective screening conditions were defined to select variants displaying higher thermostability or increased resistance to organic solvents. A first-generation amylosucrase variant library, constructed by random mutagenesis, was subjected to this procedure, yielding a mutant displaying a 25-fold increased stability at 50 degrees C compared to the parental wild-type enzyme.     

Efficient Mutagenesis by Cas9 Protein-Mediated Oligonucleotide Insertion and Large-Scale Assessment of Single-Guide RNAs

The CRISPR/Cas9 system has been implemented in a variety of model organisms to mediate site-directed mutagenesis. A wide range of mutation rates has been reported, but at a limited number of genomic target sites. To uncover the rules that govern effective Cas9-mediated mutagenesis in zebrafish, we targeted over a hundred genomic loci for mutagenesis using a streamlined and cloning-free method. We generated mutations in 85% of target genes with mutation rates varying across several orders of magnitude, and identified sequence composition rules that influence mutagenesis. We increased rates of mutagenesis by implementing several novel approaches. The activities of poor or unsuccessful single-guide RNAs (sgRNAs) initiating with a 5′ adenine were improved by rescuing 5′ end homogeneity of the sgRNA. In some cases, direct injection of Cas9 protein/sgRNA complex further increased mutagenic activity. We also observed that low diversity of mutant alleles led to repeated failure to obtain frame-shift mutations. This limitation was overcome by knock-in of a stop codon cassette that ensured coding frame truncation. Our improved methods and detailed protocols make Cas9-mediated mutagenesis an attractive approach for labs of all sizes.     

An improved PCR-mutagenesis strategy for two-site mutagenesis or sequence swapping between related genes.

The QuikChangeTM protocol is one of the simplest and fastest methods for site-directed mutagenesis, but introduces mutations at only one site at a time, and requires two HPLC-purified complementary oligonucleotides. Here, we describe that this method can be used with non-overlapping oligonucleotides. By doing this, two separate sites can be mutagenised simultaneously, or money can be saved by using a second 'standard' oligonucleotide. By a further modification, we have also used the QuikChangeTM approach to exchange DNA sequences between closely related genes.     

Three-step PCR mutagenesis for 'linker scanning'.

'Linker scanning' has been used as an efficient method for systematically surveying a segment of DNA for functional elements by mutagenesis. A three-step PCR method was developed to simplify this process. In this method, a set of 'mutation primers' was made with 6 to 8 base substitutions in the center of the primers. In the first PCR reaction, these 'mutation primers' are paired with an 3' primer from the opposite end of the analyzed sequences to form a 'ladder' of fragments containing the base pair substitutions. These are used as templates in the second PCR with the 3' primer as the only primer to generate single stranded sequences, which are used as primers in the third PCR paired with an 5' primer to complete the mutagenesis. We have tested the method in a mutation screen of the steroid sulfatase promoter. Its application to general site specific mutagenesis is discussed.     

Rapid amplification and cloning of Tn5 flanking fragments by inverse PCR

A simple approach is described to efficiently amplify DNA sequences flanking transposon Tn5 insertions. The method involves: (i) digestion with a restriction enzyme that cuts within Tn5; (ii) self-ligation under conditions favouring the production of monomeric circles; (iii) four parallel PCR reactions using primers designed to amplify left or right flanking sequences, and to distinguish target amplicons from non-specific products. This reveals the number of Tn5 insertions and the size of flanking genomic restriction fragments, without Southern blot analysis. The amplified product contains restriction sites that facilitate cohesive-end cloning. This rapid method is demonstrated using Tn5 and Tn5-Mob tagged DNA sequences involved in albicidin biosynthesis in Xanthomonas albilineans. It is generally applicable for efficient recovery of DNA sequences flanking transposon Tn5 derivatives in insertional mutagenesis studies.     

In vitro evolution of single-chain antibodies using mRNA display

Here we describe the application of the in vitro virus mRNA display method, which involves covalent linkage of an in vitro-synthesized antibody (phenotype) to its encoding mRNA (genotype) through puromycin, for in vitro evolution of single-chain Fv (scFv) antibody fragments. To establish the validity of this approach to directed antibody evolution, we used random mutagenesis by error-prone DNA shuffling and off-rate selection to improve the affinity of an anti-fluorescein scFv as a model system. After four rounds of selection of the library of mRNA-displayed scFv mutants, we obtained six different sequences encoding affinity-matured mutants with five consensus mutations. Kinetic analysis of the mutant scFvs revealed that the off-rates have been decreased by more than one order of magnitude and the dissociation constants were improved ~30-fold. The antigen-specificity was not improved by affinity maturation, but remained similar to that of the wild type. Although the five consensus mutations of the high-affinity mutants were scattered over the scFv sequence, analysis by site-directed mutagenesis demonstrated that the critical mutations for improving affinity were the two that lay within the complementarity determining regions (CDRs). Thus, mRNA display is expected to be useful for rapid artificial evolution of high-affinity diagnostic and therapeutic antibodies by optimizing their CDRs.     

Unnatural amino acid mutagenesis: a precise tool for probing protein structure and function

The first general method for the biosynthetic incorporation of unnatural amino acids into proteins was reported in 1989. The ensuing years have seen the solid development and subsequent implementation of "unnatural amino acid mutagenesis" in a number of groundbreaking studies. Over 100 different amino acids have been incorporated into dozens of soluble and transmembrane proteins, using both cell-extract and cell-intact translation systems. The approach has provided insights into ligand-binding sites, conformational changes, and protein-protein interactions with a level of precision simply unparalleled by conventional mutagenesis. Here, the methodology is outlined, significant applications of the approach are summarized, and recent major improvements in the method are discussed. The future will likely see many more investigators utilizing this approach to manipulate proteins as it realizes its promise of becoming a tool with enormous potential.     

Tailored amino acid diversity for the evolution of antibody affinity

Antibodies are a unique class of proteins with the ability to adapt their binding sites for high affinity and high specificity to a multitude of antigens. Many analyses have been performed on antibody sequences and structures to elucidate which amino acids have a predominant role in antibody interactions with antigens. These studies have generally not distinguished between amino acids selected for broad antigen specificity in the primary immune response and those selected for high affinity in the secondary immune response. By studying a large data set of affinity matured antibodies derived from in vitro directed evolution experiments, we were able to specifically highlight a subset of amino acids associated with affinity improvements. In a comparison of affinity maturations using either tailored or full amino acid diversification, the tailored approach was found to be at least as effective at improving affinity while requiring fewer mutagenesis libraries than the traditional method. The resulting sequence data also highlight the potential for further reducing amino acid diversity for high affinity binding interactions.     

Tailored amino acid diversity for the evolution of antibody affinity

Antibodies are a unique class of proteins with the ability to adapt their binding sites for high affinity and high specificity to a multitude of antigens. Many analyses have been performed on antibody sequences and structures to elucidate which amino acids have a predominant role in antibody interactions with antigens. These studies have generally not distinguished between amino acids selected for broad antigen specificity in the primary immune response and those selected for high affinity in the secondary immune response. By studying a large data set of affinity matured antibodies derived from in vitro directed evolution experiments, we were able to specifically highlight a subset of amino acids associated with affinity improvements. In a comparison of affinity maturations using either tailored or full amino acid diversification, the tailored approach was found to be at least as effective at improving affinity while requiring fewer mutagenesis libraries than the traditional method. The resulting sequence data also highlight the potential for further reducing amino acid diversity for high affinity binding interactions.     

In vivo site-directed mutagenesis using oligonucleotides

Functional characterization of the genes of higher eukaryotes has been aided by their expression in model organisms and by analyzing site-specific changes in homologous genes in model systems such as the yeast Saccharomyces cerevisiae. Modifying sequences in yeast or other organisms such that no heterologous material is retained requires in vitro mutagenesis together with subcloning. PCR-based procedures that do not involve cloning are inefficient or require multistep reactions that increase the risk of additional mutations. An alternative approach, demonstrated in yeast, relies on transformation with an oligonucleotide, but the method is restricted to the generation of mutants with a selectable phenotype. Oligonucleotides, when combined with gap repair, have also been used to modify plasmids in yeast; however, this approach is limited by restriction-site availability. We have developed a mutagenesis approach in yeast based on transformation by unpurified oligonucleotides that allows the rapid creation of site-specific DNA mutations in vivo. A two-step, cloning-free process, referred to as delitto perfetto, generates products having only the desired mutation, such as a single or multiple base change, an insertion, a small or a large deletion, or even random mutations. The system provides for multiple rounds of mutation in a window up to 200 base pairs. The process is RAD52 dependent, is not constrained by the distribution of naturally occurring restriction sites, and requires minimal DNA sequencing. Because yeast is commonly used for random and selective cloning of genomic DNA from higher eukaryotes such as yeast artificial chromosomes, the delitto perfetto strategy also provides an efficient way to create precise changes in mammalian or other DNA sequences.