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PubMed2003

Cloning, over-expression, purification, and characterisation of N-acetylneuraminate synthase from Streptococcus agalactiae.

N-acetylneuraminate synthase (NeuAc-synthase; E.C. 4.1.3.19) is one of the two enzymes responsible for sialic acid (N-acetylneuraminic acid) synthesis in bacteria. Potential genes encoding NeuAc synthase in Streptococcus agalactiae and Bacillus subtilis were identified from a BLAST search of the EMBL/GenBank/DDBJ database using the E. coli neuB gene sequence as a probe and the genes cloned and expressed at high level in Escherichia coli. The neuB gene of S. agalactiae was shown to encode an active NeuAc synthase, whereas the spsE gene product from B. subtilis did not have this activity. Expression of the native S. agalactiae neuB gene product enzyme in E. coli resulted in a product that was prone to proteolysis during purification so the protein was tagged with a hexa-histidine tag at its N-terminus and the enzyme was rapidly purified to homogeneity by ammonium sulphate fractionation and Ni-chelating affinity chromatography in two steps. Measurement of the subunit molecular mass by electrospray ionisation mass spectrometry (M(r) = 38, 987 +/- 3) and of the native molecular mass by gel filtration chromatography (M(r) = 78,000) clearly demonstrated that the enzyme is dimeric. The effects of EDTA, temperature, and pH on the activity of the S. agalactiae NeuAc synthase were examined. Enzyme activity was maximal at pH 7 and was dependent on the presence of metal ions such as Mg(2+), Mn(2+) or Co(2+). The purified enzyme was inhibited by the reagent phenylglyoxal and the substrates N-acetyl mannosamine or phosphoenol pyruvate afforded protection against this inhibition, suggesting that one or more arginine residues are involved in substrate recognition and binding. The ease of expression and the properties of the enzyme should now permit a thorough study of the specificity of the enzyme and provide the prerequisites for attempts to alter this specificity by directed evolution for the production of novel sialic acid analogues.

Ammonium Sulfate↗

PubMed2001

Receptor-mediated gene transfer by phage-display vectors: applications in functional genomics and gene therapy.

Recent studies have demonstrated targeted gene-delivery to mammalian cells using modified phage-display vectors. Specificity is determined by the choice of the genetically displayed targeting ligand. Without targeting, phage particles have virtually no tropism for mammalian cells. Thus, novel ligands can be selected from phage libraries by their ability to deliver a reporter gene to targeted cells. Together with advances in cDNA display technologies, these findings offer new opportunities for the use of phage-display technology in functional genomics. In addition, targeted phage particles have potential as alternative gene therapy vectors that can be further improved using directed evolution.

Journal Article↗

PubMed2002

Incorporation of non-natural amino acids into proteins.

Chemical and biological diversity of protein structures and functions can be widely expanded by position-specific incorporation of non-natural amino acids carrying a variety of specialty side groups. After the pioneering works of Schultz's group and Chamberlin's group in 1989, noticeable progress has been made in expanding types of amino acids, in finding novel methods of tRNA aminoacylation and in extending genetic codes for directing the positions. Aminoacylation of tRNA with non-natural amino acids has been achieved by directed evolution of aminoacyl-tRNA synthetases or some ribozymes. Codons have been extended to include four-base codons or non-natural base pairs. Multiple incorporation of different non-natural amino acids has been achieved by the use of a different four-base codon for each tRNA. The combination of these novel techniques has opened the possibility of synthesising non-natural mutant proteins in living cells.

Acylation↗

PubMed1999

Extremozymes.

Extremozymes offer new opportunities for biocatalysis and biotransformations as a result of their extreme stability. From recent work, major approaches to extending the range of applications of extremozymes have emerged. Both the discovery of new extremophilic species and the determination of genome sequences provide a route to new enzymes, with the possibility that these will lead to novel applications. Of equal importance, protein engineering and directed evolution provide approaches to improve enzyme stability and modify specificity in ways that may not exist in the natural world.

Bacteria↗

PubMed2001

Metabolic engineering as an integrating platform for strain development.

Integration of the analytical framework and experimental tools of metabolic engineering with emerging technologies such as DNA microarrays and directed evolution stands to dramatically improve the approaches by which strain improvement and biocatalyst design are pursued in the future. Progress in genomics and applied molecular biology, together with increasing emphasis on renewable resource utilization for chemical production, has advanced metabolic engineering to the forefront of biotechnological interest.

Industrial Microbiology↗

PubMed1999

Commodity scale production of sugars from starches.

The production of sugars from starch sources is an industry that exists in its present form due to the application of industrial enzymology to solve process related problems. As the industry matures, the demand for more efficient enzymes leading to higher quality products and lower production costs for the starch processor has increased. Researchers are now finding or tailoring enzymes for specific operational needs of the processor using a combination of tools such as protein engineering, directed evolution and improved accessing of natural diversity.

Aspergillus↗

PubMed2002

Crystal structure of D-hydantoinase from Bacillus stearothermophilus: insight into the stereochemistry of enantioselectivity.

Industrial production of antibiotics, such as semisynthetic penicillins and cephalosporins, requires optically pure D-p-hydroxylphenylglycine and its derivatives as important side-chain precursors. To produce optically pure D-amino acids, microbial D-hydantoinase (E.C. 3.5.2.2) is used for stereospecific hydrolysis of chemically synthesized cyclic hydantoins. We report the apo-crystal structure of D-hydantoinase from B. stearothermophilus SD1 at 3.0 A resolution. The structure has a classic TIM barrel fold. Despite an undetectable similarity in sequence, D-hydantoinase shares a striking structural similarity with the recently solved structure of dihydroorotase. A structural comparison of hydantoinase with dihydroorotase revealed that the catalytic chemistry is conserved, while the substrate recognition is not. This structure provides insight into the stereochemistry of enantioselectivity in hydrolysis and illustrates how the enzyme recognizes stereospecific exocyclic substituents and hydrolyzes hydantoins. It should also provide a rationale for further directed evolution of this enzyme for hydrolysis of new hydantoins with novel exocyclic substituents.

Amidohydrolases↗

PubMedDate not supplied

Imaging polarimetry for high throughput chiral screening.

Imaging polarimetry was demonstrated as a highly parallel method of determining optical rotation of biochemical samples. The imaging polarimeter utilized a bright, uniform light source wavelength-filtered to near the sodium D line, a sample array flanked by inlet and analyzing polarizers, and a CCD camera fitted with an equal-perspective telecentric lens. The prototype apparatus was demonstrated to have an optical resolution better than 0.08 degrees. The potential for high throughput screening was demonstrated by imaging chiral solutions in 1536-well microtiter plates and by real-time monitoring of 30 simultaneous chiral enzymatic reactions. Improvements in polarizer and CCD technology may broadly expand the technique's applicability to fields such as directed evolution and combinatorial chemistry, where screening throughput is currently limiting for chiral applications.

Algorithms↗

PubMed2002

Structural chemistry of a green fluorescent protein Zn biosensor.

We designed a green fluorescent protein mutant (BFPms1) that preferentially binds Zn(II) (enhancing fluorescence intensity) and Cu(II) (quenching fluorescence) directly to a chromophore ligand that resembles a dipyrrole unit of a porphyrin. Crystallographic structure determination of apo, Zn(II)-bound, and Cu(II)-bound BFPms1 to better than 1.5 A resolution allowed us to refine metal centers without geometric restraints, to calculate experimental standard uncertainty errors for bond lengths and angles, and to model thermal displacement parameters anisotropically. The BFPms1 Zn(II) site (KD = 50 muM) displays distorted trigonal bipyrimidal geometry, with Zn(II) binding to Glu222, to a water molecule, and tridentate to the chromophore ligand. In contrast, the BFPms1 Cu(II) site (KD = 24 muM) exhibits square planar geometry similar to metalated porphyrins, with Cu(II) binding to the chromophore chelate and Glu222. The apo structure reveals a large electropositive region near the designed metal insertion channel, suggesting a basis for the measured metal cation binding kinetics. The preorganized tridentate ligand is accommodated in both coordination geometries by a 0.4 A difference between the Zn and Cu positions and by distinct rearrangements of Glu222. The highly accurate metal ligand bond lengths reveal different protonation states for the same oxygen bound to Zn vs Cu, with implications for the observed metal ion specificity. Crystallographic anisotropic thermal factor analysis validates metal ion rigidification of the chromophore in enhancement of fluorescence intensity upon Zn(II) binding. Thus, our high-resolution structures reveal how structure-based design has effectively linked selective metal binding to changes in fluorescent properties. Furthermore, this protein Zn(II) biosensor provides a prototype suitable for further optimization by directed evolution to generate metalloprotein variants with desirable physical or biochemical properties.

Biosensing Techniques↗

PubMed2016

Characterization of a novel adeno-associated viral vector with preferential oligodendrocyte tropism.

No adeno-associated virus (AAV) capsid has been described in the literature to exhibit a primary oligodendrocyte tropism when a constitutive promoter drives gene expression, which is a significant barrier for efficient in vivo oligodendrocyte gene transfer. The vast majority of AAV vectors, such as AAV1, 2, 5, 6, 8 or 9, exhibit a dominant neuronal tropism in the central nervous system. However, a novel AAV capsid (Olig001) generated using capsid shuffling and directed evolution was recovered after rat intravenous delivery and subsequent capsid clone rescue, which exhibited a >95% tropism for striatal oligodendrocytes after rat intracranial infusion where a constitutive promoter drove gene expression. Olig001 contains a chimeric mixture of AAV1, 2, 6, 8 and 9, but unlike these parental serotypes after intravenous administration Olig001 has very low affinity for peripheral organs, especially the liver. Furthermore, in mixed glial cell cultures, Olig001 exhibits a 9-fold greater binding when compared with AAV8. This novel oligodendrocyte-preferring AAV vector exhibits characteristics that are a marked departure from previously described AAV serotypes.

Animals↗

PubMed2002

Synthetic shuffling expands functional protein diversity by allowing amino acids to recombine independently.

We describe synthetic shuffling, an evolutionary protein engineering technology in which every amino acid from a set of parents is allowed to recombine independently of every other amino acid. With the use of degenerate oligonucleotides, synthetic shuffling provides a direct route from database sequence information to functional libraries. Physical starting genes are unnecessary, and additional design criteria such as optimal codon usage or known beneficial mutations can also be incorporated. We performed synthetic shuffling of 15 subtilisin genes and obtained active and highly chimeric enzymes with desirable combinations of properties that we did not obtain by other directed-evolution methods.

Amino Acid Sequence↗

PubMed2026

Programmable enzymes for targeted gene insertion.

Genome editing technologies have advanced from nuclease-based reagents that generate programmed DNA double-strand breaks, which can cause deleterious effects, to next-generation reagents that perform controlled DNA modification through double-strand break-independent mechanisms, such as base editing and prime editing. Although these approaches enable precise small-scale sequence changes, methods for programmable insertion of large DNA cargos have been limited. The ability to write entire genes or large regions into the genome could transform the treatment of genetically heterogeneous disorders, for which numerous pathogenic variants underlie a common disease and mutation-specific editing strategies are impractical. Recent advances in computational genome mining have accelerated the discovery of naturally occurring enzymes with novel biochemical and functional properties, including recombinases and transposases capable of large-scale modifications. Moreover, directed evolution, rational engineering and expanded homologue discovery are enabling the repurposing and optimization of these systems for genome engineering. Here we review recent technology development efforts that harness diverse enzymes for kilobase-scale genome engineering, with a particular focus on CRISPR-associated transposase systems.

Journal Article↗

PubMed2024

Binary vector copy number engineering improves Agrobacterium-mediated transformation.

The copy number of a plasmid is linked to its functionality, yet there have been few attempts to optimize higher-copy-number mutants for use across diverse origins of replication in different hosts. We use a high-throughput growth-coupled selection assay and a directed evolution approach to rapidly identify origin of replication mutations that influence copy number and screen for mutants that improve Agrobacterium-mediated transformation (AMT) efficiency. By introducing these mutations into binary vectors within the plasmid backbone used for AMT, we observe improved transient transformation of Nicotiana benthamiana in four diverse tested origins (pVS1, RK2, pSa and BBR1). For the best-performing origin, pVS1, we isolate higher-copy-number variants that increase stable transformation efficiencies by 60-100% in Arabidopsis thaliana and 390% in the oleaginous yeast Rhodosporidium toruloides. Our work provides an easily deployable framework to generate plasmid copy number variants that will enable greater precision in prokaryotic genetic engineering, in addition to improving AMT efficiency.

Genetic Vectors↗

PubMed2001

Continuous affinity-based selection: rapid screening and simultaneous amplification of bacterial surface-display libraries.

A new method for continuous biopanning has been developed. We have combined the power of affinity chromatography with the fecundity of bacteria in a unique process that mimics clonal selection. Mixed populations of bacteria were applied to a fermenter containing the immobilized ligand of interest. Bacteria retained in this affinity fermenter were allowed to grow under continuous washout conditions, such that weakly bound organisms were selectively lost. Those initially rare founder bacteria expressing a receptor for the immobilized ligand (R+ve) were thus enriched and amplified simultaneously. From an initial culture containing 1 x 10(10) R-ve cells spiked with fewer than 30 R+ve bacteria (<1 in 10(8)), final ratios of R+ve/R-ve bacteria as high as 1 in 12 were observed, representing an enrichment factor of 55 million-fold. This technology has considerable potential for rapid screening of bacterial surface-display libraries and in facilitating directed-evolution studies.

Bacterial Outer Membrane Proteins↗

PubMed2003

Mutations of penicillin acylase residue B71 extend substrate specificity by decreasing steric constraints for substrate binding.

Two mutant forms of penicillin acylase from Escherichia coli strains, selected using directed evolution for the ability to use glutaryl-L-leucine for growth [Forney, Wong and Ferber (1989) Appl. Environ. Microbiol. 55, 2550-2555], are changed within one codon, replacing the B-chain residue Phe(B71) with either Cys or Leu. Increases of up to a factor of ten in k (cat)/ K (m) values for substrates possessing a phenylacetyl leaving group are consistent with a decrease in K (s). Values of k (cat)/ K (m) for glutaryl-L-leucine are increased at least 100-fold. A decrease in k (cat)/ K (m) for the Cys(B71) mutant with increased pH is consistent with binding of the uncharged glutaryl group. The mutant proteins are more resistant to urea denaturation monitored by protein fluorescence, to inactivation in the presence of substrate either in the presence of urea or at high pH, and to heat inactivation. The crystal structure of the Leu(B71) mutant protein, solved to 2 A resolution, shows a flip of the side chain of Phe(B256) into the periphery of the catalytic centre, associated with loss of the pi-stacking interactions between Phe(B256) and Phe(B71). Molecular modelling demonstrates that glutaryl-L-leucine may bind with the uncharged glutaryl group in the S(1) subsite of either the wild-type or the Leu(B71) mutant but with greater potential freedom of rotation of the substrate leucine moiety in the complex with the mutant protein. This implies a smaller decrease in the conformational entropy of the substrate on binding to the mutant proteins and consequently greater catalytic activity.

Binding Sites↗

PubMed2002

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.

Amino Acid Sequence↗

PubMed2003

Programming peptidomimetic syntheses by translating genetic codes designed de novo.

Although the universal genetic code exhibits only minor variations in nature, Francis Crick proposed in 1955 that "the adaptor hypothesis allows one to construct, in theory, codes of bewildering variety." The existing code has been expanded to enable incorporation of a variety of unnatural amino acids at one or two nonadjacent sites within a protein by using nonsense or frameshift suppressor aminoacyl-tRNAs (aa-tRNAs) as adaptors. However, the suppressor strategy is inherently limited by compatibility with only a small subset of codons, by the ways such codons can be combined, and by variation in the efficiency of incorporation. Here, by preventing competing reactions with aa-tRNA synthetases, aa-tRNAs, and release factors during translation and by using nonsuppressor aa-tRNA substrates, we realize a potentially generalizable approach for template-encoded polymer synthesis that unmasks the substantially broader versatility of the core translation apparatus as a catalyst. We show that several adjacent, arbitrarily chosen sense codons can be completely reassigned to various unnatural amino acids according to de novo genetic codes by translating mRNAs into specific peptide analog polymers (peptidomimetics). Unnatural aa-tRNA substrates do not uniformly function as well as natural substrates, revealing important recognition elements for the translation apparatus. Genetic programming of peptidomimetic synthesis should facilitate mechanistic studies of translation and may ultimately enable the directed evolution of small molecules with desirable catalytic or pharmacological properties.

Base Sequence↗

PubMed1997

RNA-peptide fusions for the in vitro selection of peptides and proteins.

Covalent fusions between an mRNA and the peptide or protein that it encodes can be generated by in vitro translation of synthetic mRNAs that carry puromycin, a peptidyl acceptor antibiotic, at their 3' end. The stable linkage between the informational (nucleic acid) and functional (peptide) domains of the resulting joint molecules allows a specific mRNA to be enriched from a complex mixture of mRNAs based on the properties of its encoded peptide. Fusions between a synthetic mRNA and its encoded myc epitope peptide have been enriched from a pool of random sequence mRNA-peptide fusions by immunoprecipitation. Covalent RNA-peptide fusions should provide an additional route to the in vitro selection and directed evolution of proteins.

Base Sequence↗