Engineering precision RNA molecular switches

Ligand-specific molecular switches composed of RNA were created by coupling preexisting catalytic and receptor domains via structural bridges. Binding of ligand to the receptor triggers a conformational change within the bridge, and this structural reorganization dictates the activity of the adjoining ribozyme. The modular nature of these tripartite constructs makes possible the rapid construction of precision RNA molecular switches that trigger only in the presence of their corresponding ligand. By using similar enzyme engineering strategies, new RNA switches can be made to operate as designer molecular sensors or as a new class of genetic control elements.

Ethyl-substituted erythromycin derivatives produced by directed metabolic engineering

A previously unknown chemical structure, 6-desmethyl-6-ethylerythromycin A (6-ethylErA), was produced through directed genetic manipulation of the erythromycin (Er)-producing organism Saccharopolyspora erythraea. In an attempt to replace the methyl side chain at the C-6 position of the Er polyketide backbone with an ethyl moiety, the methylmalonate-specific acyltransferase (AT) domain of the Er polyketide synthase was replaced with an ethylmalonate-specific AT domain from the polyketide synthase involved in the synthesis of the 16-member macrolide niddamycin. The genetically altered strain was found to produce ErA, however, and not the ethyl-substituted derivative. When the strain was provided with precursors of ethylmalonate, a small quantity of a macrolide with the mass of 6-ethylErA was produced in addition to ErA. Because substrate for the heterologous AT seemed to be limiting, crotonyl-CoA reductase, a primary metabolic enzyme involved in butyryl-CoA production in streptomycetes, was expressed in the strain. The primary macrolide produced by the reengineered strain was 6-ethylErA.

Expressed protein ligation: A general method for protein engineering

A protein semisynthesis method—expressed protein ligation—is described that involves the chemoselective addition of a peptide to a recombinant protein. This method was used to ligate a phosphotyrosine peptide to the C terminus of the protein tyrosine kinase C-terminal Src kinase (Csk). By intercepting a thioester generated in the recombinant protein with an N-terminal cysteine containing synthetic peptide, near quantitative chemical ligation of the peptide to the protein was achieved. The semisynthetic tail-phosphorylated Csk showed evidence of an intramolecular phosphotyrosine-Src homology 2 interaction and an unexpected increase in catalytic phosphoryl transfer efficiency toward a physiologically relevant substrate compared with the non-tail-phosphorylated control. This work illustrates that expressed protein ligation is a simple and powerful new method in protein engineering to introduce sequences of unnatural amino acids, posttranslational modifications, and biophysical probes into proteins of any size.

Engineering a tRNA and aminoacyl-tRNA synthetase for the site-specific incorporation of unnatural amino acids into proteins in vivo

In an effort to expand the scope of protein mutagenesis, we have completed the first steps toward a general method to allow the site-specific incorporation of unnatural amino acids into proteins in vivo. Our approach involves the generation of an “orthogonal” suppressor tRNA that is uniquely acylated in Escherichia coli by an engineered aminoacyl-tRNA synthetase with the desired unnatural amino acid. To this end, eight mutations were introduced into tRNA2Gln based on an analysis of the x-ray crystal structure of the glutaminyl-tRNA aminoacyl synthetase (GlnRS)–tRNA2Gln complex and on previous biochemical data. The resulting tRNA satisfies the minimal requirements for the delivery of an unnatural amino acid: it is not acylated by any endogenous E. coli aminoacyl-tRNA synthetase including GlnRS, and it functions efficiently in protein translation. Repeated rounds of DNA shuffling and oligonucleotide-directed mutagenesis followed by genetic selection resulted in mutant GlnRS enzymes that efficiently acylate the engineered tRNA with glutamine in vitro. The mutant GlnRS and engineered tRNA also constitute a functional synthetase–tRNA pair in vivo. The nature of the GlnRS mutations, which occur both at the protein–tRNA interface and at sites further away, is discussed.

Rational engineering of a miniprotein that reproduces the core of the CD4 site interacting with HIV-1 envelope glycoprotein

Protein–protein interacting surfaces are usually large and intricate, making the rational design of small mimetics of these interfaces a daunting problem. On the basis of a structural similarity between the CDR2-like loop of CD4 and the β-hairpin region of a short scorpion toxin, scyllatoxin, we transferred the side chains of nine residues of CD4, central in the binding to HIV-1 envelope glycoprotein (gp120), to a structurally homologous region of the scorpion toxin scaffold. In competition experiments, the resulting 27-amino acid miniprotein inhibited binding of CD4 to gp120 with a 40 μM IC50. Structural analysis by NMR showed that both the backbone of the chimeric β-hairpin and the introduced side chains adopted conformations similar to those of the parent CD4. Systematic single mutations suggested that most CD4 residues from the CDR2-like loop were reproduced in the miniprotein, including the critical Phe-43. The structural and functional analysis performed suggested five additional mutations that, once incorporated in the miniprotein, increased its affinity for gp120 by 100-fold to an IC50 of 0.1–1.0 μM, depending on viral strains. The resulting mini-CD4 inhibited infection of CD4+ cells by different virus isolates. Thus, core regions of large protein–protein interfaces can be reproduced in miniprotein scaffolds, offering possibilities for the development of inhibitors of protein–protein interactions that may represent useful tools in biology and in drug discovery.

Metabolic pathway engineering in cotton: Biosynthesis of polyhydroxybutyrate in fiber cells

Alcaligenes eutrophus genes encoding the enzymes, β-ketothiolase (phaA), acetoacetyl-CoA reductase (phaB), and polyhydroxyalkanoate synthase (phaC) catalyze the production of aliphatic polyester poly-d-(−)-3-hydroxybutyrate (PHB) from acetyl-CoA. PHB is a thermoplastic polymer that may modify fiber properties when synthesized in cotton. Endogenous β-ketothiolase activity is present in cotton fibers. Hence cotton was transformed with engineered phaB and phaC genes by particle bombardment, and transgenic plants were selected based on marker gene, β-glucuronidase (GUS), expression. Fibers of 10 transgenic plants expressed phaB gene, while eight plants expressed both phaB and phaC genes. Electron microscopy examination of fibers expressing both genes indicated the presence of electron-lucent granules in the cytoplasm. High pressure liquid chromatography, gas chromatography, and mass spectrometry evidence suggested that the new polymer produced in transgenic fibers is PHB. Sixty-six percent of the PHB in fibers is in the molecular mass range of 0.6 × 106 to 1.8 × 106 Da. The presence of PHB granules in transgenic fibers resulted in measurable changes of thermal properties. The fibers exhibited better insulating characteristics. The rate of heat uptake and cooling was slower in transgenic fibers, resulting in higher heat capacity. These data show that metabolic pathway engineering in cotton may enhance fiber properties by incorporating new traits from other genetic sources. This is an important step toward producing new generation fibers for the textile industry.

Engineering the largest RNA virus genome as an infectious bacterial artificial chromosome

The construction of cDNA clones encoding large-size RNA molecules of biological interest, like coronavirus genomes, which are among the largest mature RNA molecules known to biology, has been hampered by the instability of those cDNAs in bacteria. Herein, we show that the application of two strategies, cloning of the cDNAs into a bacterial artificial chromosome and nuclear expression of RNAs that are typically produced within the cytoplasm, is useful for the engineering of large RNA molecules. A cDNA encoding an infectious coronavirus RNA genome has been cloned as a bacterial artificial chromosome. The rescued coronavirus conserved all of the genetic markers introduced throughout the sequence and showed a standard mRNA pattern and the antigenic characteristics expected for the synthetic virus. The cDNA was transcribed within the nucleus, and the RNA translocated to the cytoplasm. Interestingly, the recovered virus had essentially the same sequence as the original one, and no splicing was observed. The cDNA was derived from an attenuated isolate that replicates exclusively in the respiratory tract of swine. During the engineering of the infectious cDNA, the spike gene of the virus was replaced by the spike gene of an enteric isolate. The synthetic virus replicated abundantly in the enteric tract and was fully virulent, demonstrating that the tropism and virulence of the recovered coronavirus can be modified. This demonstration opens up the possibility of employing this infectious cDNA as a vector for vaccine development in human, porcine, canine, and feline species susceptible to group 1 coronaviruses.

Protein engineering of Bacillus thuringiensis δ-endotoxin: Mutations at domain II of CryIAb enhance receptor affinity and toxicity toward gypsy moth larvae

Substitutions or deletions of domain II loop residues of Bacillus thuringiensis δ-endotoxin CryIAb were constructed using site-directed mutagenesis techniques to investigate their functional roles in receptor binding and toxicity toward gypsy moth (Lymantria dispar). Substitution of loop 2 residue N372 with Ala or Gly (N372A, N372G) increased the toxicity against gypsy moth larvae 8-fold and enhanced binding affinity to gypsy moth midgut brush border membrane vesicles (BBMV) ≈4-fold. Deletion of N372 (D3), however, substantially reduced toxicity (>21 times) as well as binding affinity, suggesting that residue N372 is involved in receptor binding. Interestingly, a triple mutant, DF-1 (N372A, A282G and L283S), has a 36-fold increase in toxicity to gypsy moth neonates compared with wild-type toxin. The enhanced activity of DF-1 was correlated with higher binding affinity (18-fold) and binding site concentrations. Dissociation binding assays suggested that the off-rate of the BBMV-bound mutant toxins was similar to that of the wild type. However, DF-1 toxin bound 4 times more than the wild-type and N372A toxins, and it was directly correlated with binding affinity and potency. Protein blots of gypsy moth BBMV probed with labeled N372A, DF-1, and CryIAb toxins recognized a common 210-kDa protein, indicating that the increased activity of the mutants was not caused by binding to additional receptor(s). The improved binding affinity of N372A and DF-1 suggest that a shorter side chain at these loops may fit the toxin more efficiently to the binding pockets. These results offer an excellent model system for engineering δ-endotoxins with higher potency and wider spectra of target pests by improving receptor binding interactions.

Tissue engineering of cartilage in space

Tissue engineering of cartilage, i.e., the in vitro cultivation of cartilage cells on synthetic polymer scaffolds, was studied on the Mir Space Station and on Earth. Specifically, three-dimensional cell-polymer constructs consisting of bovine articular chondrocytes and polyglycolic acid scaffolds were grown in rotating bioreactors, first for 3 months on Earth and then for an additional 4 months on either Mir (10−4–10−6 g) or Earth (1 g). This mission provided a unique opportunity to study the feasibility of long-term cell culture flight experiments and to assess the effects of spaceflight on the growth and function of a model musculoskeletal tissue. Both environments yielded cartilaginous constructs, each weighing between 0.3 and 0.4 g and consisting of viable, differentiated cells that synthesized proteoglycan and type II collagen. Compared with the Earth group, Mir-grown constructs were more spherical, smaller, and mechanically inferior. The same bioreactor system can be used for a variety of controlled microgravity studies of cartilage and other tissues. These results may have implications for human spaceflight, e.g., a Mars mission, and clinical medicine, e.g., improved understanding of the effects of pseudo-weightlessness in prolonged immobilization, hydrotherapy, and intrauterine development.

Reverse engineering the (β/α)8 barrel fold

The (β/α)8 barrel is the most commonly occurring fold among protein catalysts. To lay a groundwork for engineering novel barrel proteins, we investigated the amino acid sequence restrictions at 182 structural positions of the prototypical (β/α)8 barrel enzyme triosephosphate isomerase. Using combinatorial mutagenesis and functional selection, we find that turn sequences, α-helix capping and stop motifs, and residues that pack the interface between β-strands and α-helices are highly mutable. Conversely, any mutation of residues in the central core of the β-barrel, β-strand stop motifs, and a single buried salt bridge between amino acids R189 and D227 substantially reduces catalytic activity. Four positions are effectively immutable: conservative single substitutions at these four positions prevent the mutant protein from complementing a triosephosphate isomerase knockout in Escherichia coli. At 142 of the 182 positions, mutation to at least one amino acid of a seven-letter amino acid alphabet produces a triosephosphate isomerase with wild-type activity. Consequently, it seems likely that (β/α)8 barrel structures can be encoded with a subset of the 20 amino acids. Such simplification would greatly decrease the computational burden of (β/α)8 barrel design.

Induction of male sterility in plants by metabolic engineering of the carbohydrate supply

Extracellular invertase mediates phloem unloading via an apoplastic pathway. The gene encoding isoenzyme Nin88 from tobacco was cloned and shown to be characterized by a specific spatial and temporal expression pattern. Tissue-specific antisense repression of Nin88 under control of the corresponding promoter in tobacco results in a block during early stages of pollen development, thus, causing male sterility. This result demonstrates a critical role of extracellular invertase in pollen development and strongly supports the essential function of extracellular sucrose cleavage for supplying carbohydrates to sink tissues via the apoplast. The specific interference with phloem unloading, the sugar status, and metabolic signaling during pollen formation will be a potentially valuable approach to induce male sterility in various crop species for hybrid seed production.

High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides

Homologous DNA recombination is a fundamental, regenerative process within living organisms. However, in most organisms, homologous recombination is a rare event, requiring a complex set of reactions and extensive homology. We demonstrate in this paper that Beta protein of phage λ generates recombinants in chromosomal DNA by using synthetic single-stranded DNAs (ssDNA) as short as 30 bases long. This ssDNA recombination can be used to mutagenize or repair the chromosome with efficiencies that generate up to 6% recombinants among treated cells. Mechanistically, it appears that Beta protein, a Rad52-like protein, binds and anneals the ssDNA donor to a complementary single-strand near the DNA replication fork to generate the recombinant. This type of homologous recombination with ssDNA provides new avenues for studying and modifying genomes ranging from bacterial pathogens to eukaryotes. Beta protein and ssDNA may prove generally applicable for repairing DNA in many organisms.

Metabolic engineering of essential oil yield and composition in mint by altering expression of deoxyxylulose phosphate reductoisomerase and menthofuran synthase

Peppermint (Mentha × piperita L.) was independently transformed with a homologous sense version of the 1-deoxy-d-xylulose-5-phosphate reductoisomerase cDNA and with a homologous antisense version of the menthofuran synthase cDNA, both driven by the CaMV 35S promoter. Two groups of transgenic plants were regenerated in the reductoisomerase experiments, one of which remained normal in appearance and development; another was deficient in chlorophyll production and grew slowly. Transgenic plants of normal appearance and growth habit expressed the reductoisomerase transgene strongly and constitutively, as determined by RNA blot analysis and direct enzyme assay, and these plants accumulated substantially more essential oil (about 50% yield increase) without change in monoterpene composition compared with wild-type. Chlorophyll-deficient plants did not afford detectable reductoisomerase mRNA or enzyme activity and yielded less essential oil than did wild-type plants, indicating cosuppression of the reductoisomerase gene. Plants transformed with the antisense version of the menthofuran synthase cDNA were normal in appearance but produced less than half of this undesirable monoterpene oil component than did wild-type mint grown under unstressed or stressed conditions. These experiments demonstrate that essential oil quantity and quality can be regulated by metabolic engineering. Thus, alteration of the committed step of the mevalonate-independent pathway for supply of terpenoid precursors improves flux through the pathway that leads to increased monoterpene production, and antisense manipulation of a selected downstream monoterpene biosynthetic step leads to improved oil composition.