Directed evolution

The epidermal growth factor receptor 1 (EGFR) is overexpressed in various malignancies and is associated with a poor patient prognosis. A small, receptor-specific, high-affinity imaging agent would be a useful tool in diagnosing malignant tumors and in deciding upon treatment and assessing the response to treatment. We describe here the affinity maturation procedure for the generation of Affibody molecules binding

The use of natural microorganisms in biotransformations is frequently constrained by their limited tolerance to the high concentrations of metabolites and solvents required for effective industrial production. In many cases, more robust strains have to be generated by random mutagenesis and selection. This process of directed evolution can be accelerated in mutator strains, which carry defects in one or more of their DNA repair genes. However, in order to use mutator strains, it is essential to restore the normal low mutation rate of the selected organisms immediately after selection to prevent the accumulation of undesirable spontaneous mutations. To enable this process, we constructed temperature-sensitive plasmids that temporarily increase the mutation frequency of their hosts by 20-to 4,000-fold. Under appropriate selection pressure, microorganisms transformed with mutator plasmids can be quickly evolved to exhibit new, complex traits. By using this approach, we were able to increase the tolerance of three bacterial strains to dimethylformamide by 10 to 20 g/liter during only two subsequent transfers. Subsequently, the evolved strains were returned to their normal low mutation rate by curing the cells of the mutator plasmids. Our results demonstrate a new and efficient method for rapid strain improvement based on in vivo mutagenesis.

We recently reported conversion of cytochrome P450 BM-3, a medium-chain (C 12 -C 18 ) fatty acid monooxygenase, into a highly efficient alkane hydroxylase by directed evolution [Nat. Biotechnol. 2002, 20, 1135. P450 BM-3 mutant 139-3 exhibited high activity towards a variety of fatty acid and alkane substrates, including C 3 -C 8 alkanes. We report here that mutant 139-3 is also active on benzene, styrene, cyclohexene, 1-hexene, and propylene. Benzene is converted to phenol, while styrene is converted to styrene oxide. Propylene oxidation generates only propylene oxide, but cyclohexene oxidation produces a mixture of cyclohexene oxide (85%) and 2-cyclohexene-1ol (15%), and 1-hexene is converted to the allylic hydroxylation product, 1-hexene-3-ol. Initial rates of NADPH oxidation for 139-3 in the presence of the substrates greatly (17-to .100-fold) surpass the wild-type in all cases. However, NADPH consumption is only partially coupled to product formation (14-79%). This cytochrome P450 epoxidation catalyst is a suitable starting point for further evolution to improve coupling and activity. q

In places previously regarded as incapable of sustaining life, such as Antarctic ice fields and volcanic pools, organisms are now being identified for whom these extreme environments are home, sweet home. In most cases, adaptation to such extreme environments has not required completely new molecular machinery; in fact, many 'extremophilic' enzymes are similar to their counterparts from the 'mesophilic' environments we find more hospitable. For example, enzymes that function at very different temperatures can have nearly superimposable three-dimensional structures . Sequence comparisons indicate that these enzymes are derived from a common ancestral enzyme and have accumulated mutations that allow them to adapt over millions of years.

BCF2, a monoclonal antibody raised against scorpion toxin Cn2, is capable of neutralizing both, the toxin and the whole venom of the Mexican scorpion Centruroides noxius Hoffmann. The single chain antibody fragment (scFv) of BCF2 was constructed and expressed in Escherichia coli. Although its affinity for the Cn2 toxin was shown to be in the nanomolar range, it was non-neutralizing in vivo due to a low stability. In order to recover the neutralizing capacity, the scFv of BCF2 was evolved by error-prone PCR and the variants were panned by phage display. Seven improved mutants were isolated from three different libraries. One of these mutants, called G5 with one mutation at CDR1 and another at CDR2 of the light chain, showed an increased affinity to Cn2, as compared to the parental scFv. A second mutant, called B7 with a single change at framework 2 of heavy chain, also had a higher affinity. Mutants G5 and B7 were also improved in their stability but they were unable to neutralize the toxin. Finally, we constructed a variant containing the changes present in G5 and B7. The purpose of this construction was to combine the increments in affinity and stability borne by these mutants. The result was a triple mutant capable of neutralizing the Cn2 toxin. This variant showed the best affinity constant (K D Z7.5!10 K11 M), as determined by surface plasmon resonance (BIAcore). The k on and k off were improved threefold and fivefold, respectively, leading to 15-fold affinity improvement. Functional stability determinations by ELISA in the presence of different concentrations of guanidinium hydrochloride (Gdn-HCl) revealed that the triple mutant is significantly more stable than the parental scFv. These results suggest that not only improving the affinity but also the stability of our scFv were important for recovering its neutralization capacity. These findings pave the way for the generation of recombinant neutralizing antisera against scorpion stings based on scFvs.

Development of artificial enzymes designed for industrial plants that could convert carbon dioxide into carbonates, with the ultimate aim of reducing CO2 emissions. Enzymes are biological catalysts that accelerate chemical reactions, such as the conversion of gaseous carbon dioxide (CO2) into carbonates. Carbonates are the basic component of coral reefs, mollusc shells, geological platforms and kidney stones. Although naturally occurring enzymes would be ideal for converting human-generated CO2 emissions into carbonates, they are generally incapable of coping with the extreme conditions of industrial plants. Ernesto and colleagues are now developing artificial enzymes that can withstand the harsh environments of industrial plants while accelerating chemical reactions. His team ultimately aims to create a clean, cheap, practical and socially responsible solution for global warming by reducing CO2 emissions. "We believe that our novel artificial enzymes will be the first tailor-made enzymes for industrial plants to produce carbonates," says Dr Hernandez. So far, Dr Hernandez and his colleagues have built an artificial environment composed of chimney-like equipment, measuring 1.5 metres in height and 15 centimetres in diameter, that mimics the smoke released by power plants. Using the artificial environment, the researchers will ensure that their artificial enzymes can function properly under extreme conditions consisting of hot, corrosive, poisonous and sticky smoke as well as soot and other gases produced by power plants. The team is basing the development of its artificial enzyme on naturally occurring carbonic anhydrase (CA), which accelerates the conversion of CO2 into carbonates. Carbonic anhydrase is capable of turning CO2 molecules into carbonates at a rate of one million molecules per second. However, "the enzyme's CO2 conversion rate slows down dramatically under industrial conditions," Dr Hernandez points out. He and his colleagues are now engineering artificial enzymes based on natural CA, using directed evolution techniques. Their first step involves the creation of a library of diverse genes that encode for carbonic anhydrases. "This library includes sequences of unique forms of carbonic anhydrases recently found near deep-ocean chimneys (hydrothermal vents)," says Dr Hernandez. The team plans to modify and multiply the genes encoding for carbonic anhydrases using a molecular technique called random mutagenesis. The researchers will then place the mutated genes in the artificial environment to see which ones are most effective at converting carbon dioxide into carbonates. The best mutations will then be put through the modification and multiplication processes again. The researchers will repeat the whole process until they have isolated a mutated gene encoding for recombinant carbonic anhydrase that can convert CO2 into carbonates under industrial conditions. With the help of artificial enzymes, CO2-converted carbonates could be used in everything from baking soda and chalk to Portland cement and lime manufacturing.

Advances in Biochemical Engineering/Biotechnology reviews actual trends in modern biotechnology. Its aim is to cover all aspects of this interdisciplinary technology where knowledge, methods and expertise are required for chemistry, biochemistry, micro-biology, genetics, chemical engineering and computer science. Special volumes are dedicated to selected topics which focus on new biotechnological products and new processes for their synthesis and purification. They give the state-of-the-art of a topic in a comprehensive way thus being a valuable source for the next 3-5 years.

Many people underestimate the effect that unifying principles have on the study of biology. Unifying principles are used to provide simplifying assumptions to complex problems, which allow them to be effectively tackled by the tools at hand. However, erroneous unifying principles will generate simplifying assumptions that lead towards mischaracterizations of problems which inevitably lead to invalid conclusions. The unifying principles of the current Modern Synthesis of evolution are presently being challenged by the Extended Evolutionary Synthesis. However, the Extended Evolutionary Synthesis has so far failed to provide unifying principles of its own, which has caused many to question whether or not the Extended Evolutionary Synthesis is indeed a unique synthesis of evolutionary biology. Here, the concept of evolutionary teleonomy (Ernst Mayr's concept of teleonomy applied to evolutionary processes themselves) is identified as a unifying principle of the Extended Evolutionary Synthesis. Additionally, specific examples are provided where modern research has been led astray by the unifying principles of the Modern Synthesis which would have been corrected by applying the Extended Evolutionary Synthesis with the unifying principle of evolutionary teleonomy.

By compartmentalizing reactions in aqueous microdroplets of water-in-oil emulsions, reaction volumes can be reduced by factors of up to 10 9 compared to conventional microtitre-plate based systems. This allows massively parallel processing of as many as 10 10 reactions in a total volume of only 1 ml of emulsion. This review describes the use of emulsions for directed evolution of proteins and RNAs, and for performing polymerase chain reactions (PCRs). To illustrate these applications we describe certain specific experiments, each of which exemplifies a different facet of the technique, in some detail. These examples include directed evolution of Diels-Alderase and RNA ligase ribozymes and several classes of protein enzymes, including DNA polymerases, phosphotriesterases, b-galactosidases and thiolactonases. We also describe the application of emulsion PCR to screen for rare mutations and for new ultra-high throughput sequencing technologies. Finally, we discuss the recent development of microfluidic tools for making and manipulating microdroplets and their likely impact on the future development of the field. Andrew Griffiths received a BSc in Biochemistry from the University of Sheffield, UK, in 1985. He completed a PhD, studying messenger RNA splicing, under the supervision of Prof. Ian Eperon, at the University of Leicester, UK, in 1988. He then worked for 15 years at the MRC Laboratory of Molecular Biology, Cambridge, UK, first as a post-doc with Dr Greg Winter, where he worked on selection of human antibodies for therapy using phage display, and later as a group leader, where he became interested in developing systems based on compartmentalisation of reactions in emulsions to select for catalysis. He is currently a Professor in the Institut de Science et d'Ingénierie Supramoléculaires (ISIS), a new multidisciplinary research centre founded by Prof. Jean-Marie Lehn in Strasbourg.

Recent studies on microbial cytochrome P450 enzymes have covered several new areas. Advances have been made in structure–function analysis and new non-enzymatic/electrochemical systems for the replacement of NAD (P) H in biocatalysis have been ...

Viruses can be engineered to efficiently deliver exogenous genes, but their natural gene delivery properties often fail to meet human therapeutic needs. Therefore, engineering viral vectors with new properties, including enhanced targeting abilities and resistance to immune responses, is a growing area of research. This review discusses protein engineering approaches to generate viral vectors with novel gene delivery capabilities. Rational design of viral vectors has yielded successful advances in vitro, and to an extent in vivo. However, there is often insufficient knowledge of viral structure-function relationships to reengineer existing functions or create new capabilities, such as virus-cell interactions, whose molecular basis is distributed throughout the primary sequence of the viral proteins. Therefore, high-throughput library and directed evolution methods offer alternative approaches to engineer viral vectors with desired properties. Parallel and integrated efforts in rational and library-based design promise to aid the translation of engineered viral vectors toward the clinic. 169 Annu. Rev. Biomed. Eng. 2008.10:169-194. Downloaded from arjournals.annualreviews.org by University of California -Berkeley on 07/22/08. For personal use only.

Laccases are multi-copper containing oxidases (EC 1.10.3.2), widely distributed in fungi, higher plants and bacteria. Laccase catalyses the oxidation of phenols, polyphenols and anilines by oneelectron abstraction, with the concomitant reduction of oxygen to water in a four-electron transfer process. In the presence of small redox mediators, laccase offers a broader repertory of oxidations including non-phenolic substrates. Hence, fungal laccases are considered as ideal green catalysts of great biotechnological impact due to their few requirements (they only require air, and they produce water as the only by-product) and their broad substrate specificity, including direct bioelectrocatalysis.

Expression of proteins on the surface of yeasts has a wide range of applications in biotechnology, such as directed evolution of proteins for increased affinity and thermal stability, screening of antibody libraries, epitope mapping, and use as whole-cell biocatalysts. However, hyperglycosylation can interfere with overall protein accessibility on the surface. Therefore, the less elaborate hyperglycosylation in wild type Pichia pastoris and the availability of glycoengineered strains make this yeast an excellent alternative for surface display of glycoproteins. Here, we report the implementation of the well-established a-agglutinin-based yeast surface display technology in P. pastoris. Four heterologous proteins were expressed on the surface of a wild type and a glycoengineered strain. Surface display levels were monitored by Western blot, immunofluorescence microscopy, and FACS analysis. The availability of glycoengineered strains makes P. pastoris an excellent alternative for surface display of glycoproteins and paves the way for new applications.

We report a DNA shuffling-based approach for developing cell type-specific vectors through directed evolution. Capsid genomes of adeno-associated virus (AAV) serotypes 1-9 were randomly fragmented and reassembled using PCR to generate a chimeric capsid library. A single infectious clone (chimeric-1829) containing genome fragments from AAV1, 2, 8, and 9 was isolated from an integrin minus hamster melanoma cell line previously shown to have low permissiveness to AAV. Molecular modeling studies suggest that AAV2 contributes to surface loops at the icosahedral threefold axis of symmetry, while AAV1 and 9 contribute to two-and fivefold symmetry interactions, respectively. The C-terminal domain (AAV9) was identified as a critical structural determinant of melanoma tropism through rational mutagenesis. Chimeric-1829 utilizes heparan sulfate as a primary receptor and transduces melanoma cells more efficiently than all serotypes. Further, chimeric-1829 demonstrates altered tropism in rodent skeletal muscle, liver, and brain including nonhuman primates. We determined a unique immunological profile based on neutralizing antibody (NAb) titer and crossreactivity studies strongly supporting isolation of a synthetic laboratory-derived capsid variant. Application of this technology to alternative cell/tissue types using AAV or other viral capsid sequences is likely to yield a new class of biological nanoparticles as vectors for human gene transfer.

On the basis of a bioinformatic approach, we suggest that in invertebrates many ligands interact with a single, ancestral and generalized receptor driving ligand evolution. In vertebrates, on the other hand, the occurrence of gene/genome duplications induced the shift to a ligand-directed evolution of receptors.

Body size variation across the Metazoa is immense, encompassing 17 orders of magnitude in biovolume. Factors driving this extreme diversification in size and the consequences of size variation for biological processes remain poorly resolved. Species diversity is invoked as both a predictor and a result of size variation, and theory predicts a strong correlation between the two. However, evidence has been presented both supporting and contradicting such a relationship. Here, we use a new comprehensive dataset for maximum and minimum body sizes across all metazoan phyla to show that species diversity is strongly correlated with minimum size, maximum size and consequently intra-phylum variation. Similar patterns are also observed within birds and mammals. The observations point to several fundamental linkages between species diversification and body size variation through the evolution of animal life.

Sequence data from a portion of the external transcribed spacer (ETS) and from the internal transcribed spacers (ITS1 and ITS2) of 18S-26S nuclear ribosomal DNA were used together with chloroplast DNA PCR-RFLP data to unravel patterns of allotetraploid speciation within the Western European Dactylorhiza polyploid complex. A maximum likelihood tree based on combined ETS and ITS sequences suggests that the Western European Dactylorhiza allotetraploids have evolved by hybridization between four main diploid lineages. Cloned sequences and the topology of the ITS plus ETS tree indicate that the allotetraploid species D. elata, D. brennensis, and D. sphagnicola have originated from the autotetraploid D. maculata together with the diploid D. incarnata, while D. majalis, D. traunsteineri, and D. angustata seem to have evolved by hybridization between the D. fuchsii s.str and D. incarnata lineages. Finally, the diploid D. saccifera lineage seems to have been involved together with the D. incarnata lineage in the formation of the allotetraploid D. praetermissa. The observed congruence between the chloroplast tree and the ITS/ETS tree suggests a directional evolution of the nrDNA after polyploidization in favor of the maternal genome. Considered together with morphological, biogeographical, and ecological evidence, the molecular analysis leads us to recognize four species within the investigated allotetraploid complex, namely D. majalis, D. praetermissa, D. elata, and D. sphagnicola.

The bright ultraluminous X-ray source (ULX), M33 X-8, has been observed several times by XMM-Newton, providing us with a rare opportunity to 'flux bin' the spectral data and search for changes in the average X-ray spectrum with flux level. The aggregated X-ray spectra appear unlike standard sub-Eddington accretion state spectra which, alongside the lack of discernible variability at any energy, argues strongly against conventional two-component, sub-Eddington models. Although the lack of variability could be consistent with disc-dominated spectra, sub-Eddington disc models are not sufficiently broad to explain the observed spectra. Fits with a ˜ Eddington accretion rate slim disc model are acceptable, but the fits show that the temperature decreases with flux, contrary to expectations, and this is accompanied by the appearance of a harder tail to the spectrum. Applying a suitable two-component model reveals that the disc becomes cooler and less advection dominated as the X-ray flux increases, and this is allied to the emergence of an optically thick Comptonization medium. We present a scenario in which this is explained by the onset of a radiatively driven wind from the innermost regions of the accretion disc, as M33 X-8 exceeds the Eddington limit. Furthermore, we argue that the direct evolution of this spectrum with increasing luminosity (and hence radiation pressure) leads naturally to the two-component spectra seen in more luminous ULXs.

Pitch control is an important aspect in pulp and paper manufacture, and the first example where microbial biotechnology provided successful solutions in this industrial sector. Triglycerides cause deposits in softwood mechanical pulping, and both microbial and enzymatic products have been commercialized to be applied on wood and pulp, respectively. The former are based on colorless strains of sapstain fungi. The latter are improved lipases, including thermostable variants from directed evolution. These enzymes are among the additives of choice in pulping of high-resin-content softwoods. However, lipases are not useful when pitch originates from other lipids, such as steroids and terpenes, and the sapstain inocula are also only partially effective. In the search for stronger biocatalysts to degrade recalcitrant lipids, the potential of white-rot fungi and their enzymes has been demonstrated. When inocula of these fungi are used, wood treatment must be controlled to avoid cellulose degradation. However, the efficiency and selectivity of the laccase-mediator system permits its integration as an additional bleaching stage. A double benefit can be obtained from these treatments since pitch is controlled at the same time that residual lignin is removed facilitating the implementation of totally chlorine free pulp bleaching.

Serum paraoxonases (PONs) exhibit a wide range of physiologically important hydrolytic activities, including drug metabolism and detoxification of nerve gases. PON1 and PON3 reside on high-density lipoprotein (HDL) (the "good cholesterol"), and are involved in the alleviation of atherosclerosis. Members of the PON family have been identified not only in mammals and other vertebrates, but also in invertebrates. We earlier described the first crystal structure of a PON family member, a directly-evolved variant of PON1, at 2.2 Å resolution. PON1 is a 6-bladed beta-propeller with a unique active-site lid which is also involved in binding to HDL. The 3-D structure, taken together with directed evolution studies, permitted analysis of mutations which enhanced the stability, solubility and crystallizability of this PON1 variant. The structure permits a detailed description of PON1's active site and suggests possible mechanisms for its catalytic activity on certain substrates.

A current challenge in synthetic organic chemistry is the development of methods that allow the regio-and stereoselective oxidative C-H activation of natural or synthetic compounds with formation of the corresponding alcohols. Cytochrome P450 enzymes enable C-H activation at non-activated positions, but the simultaneous control of both regio-and stereoselectivity is problematic. Here, we demonstrate that directed evolution using iterative saturation mutagenesis provides a means to solve synthetic problems of this kind. Using P450 BM3(F87A) as the starting enzyme and testosterone as the substrate, which results in a 1:1 mixture of the 2b-and 15b-alcohols, mutants were obtained that are 96-97% selective for either of the two regioisomers, each with complete diastereoselectivity. The mutants can be used for selective oxidative hydroxylation of other steroids without performing additional mutagenesis experiments. Molecular dynamics simulations and docking experiments shed light on the origin of regio-and stereoselectivity.

Synthetic genetic circuits are artificial networks of transcriptional control elements inserted into living cells in order to 'program' cellular behavior. We can extend this application to programming population behavior by incorporating cell-cell communications capabilities. By designing and building such networks, cellular circuit engineers expect to gain insight into how natural genetic networks function with remarkable robustness, stability, and adaptability to changing environments. Programmed cells also have promising applications in biotechnology and medicine. A major challenge that biological circuit engineers face is the difficulty of predicting circuit performance at the design stage, with the consequence that actual construction requires significant experimental effort, even for very simple circuits. To address this fundamental obstacle we propose the use of laboratory evolution methods to create new circuit components and optimize circuit performance inside living cells.

Organophosphate-degrading enzyme from Agrobacterium radiobacter P230 (OPDA) is a recently discovered enzyme that degrades a broad range of organophosphates. It is very similar to OPH first isolated from Pseudomonas diminuta MG. Despite a high level of sequence identity, OPH and OPDA exhibit different substrate specificities. We report here the structure of OPDA and identify regions of the protein that are likely to give it a preference for substrates that have shorter alkyl substituents. Directed evolution was used to evolve a series of OPH mutants that had activities similar to those of OPDA. Mutants were selected for on the basis of their ability to degrade a number of substrates. The mutations tended to cluster in particular regions of the protein and in most cases, these regions were where OPH and OPDA had significant differences in their sequences.

Peroxidases are widely distributed in nature. Reduction of peroxides at the expense of electron donating substrates, make peroxidases useful in a number of biotechnological applications. Enzymes such as lignin peroxidase and manganese peroxidase, both associated with lignin degradation, may be successfully used for biopulping and biobleaching in the paper industry, and can produce oxidative breakdown of synthetic azo dyes. Oxidative polymerization of phenols and aromatic amines conducted by horseradish peroxidase (HRP) in water and watermiscible organic solvents, may lead to new types of aromatic polymers. Site directed mutagenesis of HRP has been used to improve the enantioselectivity of arylmethylsulfide oxidations. Peroxidase has a potential for soil detoxification, while HRP as well as soybean and turnip peroxidases have been applied for the bioremediation of wastewater contaminated with phenols, cresols, and chlorinated phenols. Peroxidase based biosensors have found use in analytical systems for determination of hydrogen peroxide and organic hydroperoxides, while coimmobilized with a hydrogen peroxide producing enzyme, they can be used for determination of glucose, alcohols, glutamate and choline. Peroxidase has also been used for practical analytical applications in diagnostic kits, such as quantitation of uric acid, glucose, cholesterol, lactose, and so on. Enzyme linked immunorbent assay (ELISA) tests on which peroxidase is probably the most common enzyme used for labeling an antibody, are a simple and reliable way of detecting toxins, pathogens, cancer risk in bladder and prostate, and many other analytes. Directed evolution methods, appear to be a valuable alternative to engineer new catalyst forms of plant peroxidases from different sources to overcome problems of stability and to increase thermal resistance. Abbreviations: A-electron donor; A •-oxidized donor; AH-reduced electron donor; CDH-cellobiose dehydrogenase; CoI-compound I; CoII-compound II; EIA-enzyme immunoassay; ELISA-enzyme linked immunosorbent assay; EPR-electron paramagnetic resonance; ESR-electron spin resonance; ET-electron transfer; GC-MS-gas chromatography-Mass spectrometry; HPLC-high performance liquid chromatography; HRP-horseradish peroxidase; hTNF-alpha-Human tumor necrosis factor-alpha; IAA-indole-3-acetic acid; LiP-lignin peroxidase; MnP-manganese peroxidase; PEG-polyethylene glycol; TLC-thin layer chromatography; UKP-hard wood kraft pulp.

Chemostat enrichment is a classical microbiological method that is well suited for use in directed-evolution strategies. We used a two-phase sulfur-limited chemostat to select for gain-of-function mutants with mutations in the biodesulfurization (Dsz) system of Rhodococcus erythropolis IGTS8, enriching for growth in the presence of organosulfur compounds that could not support growth of the wild-type strain. Mutations arose that allowed growth with octyl sulfide and 5-methylbenzothiophene as sole sulfur sources. An isolate from the evolved chemostat population was genetically characterized and found to contain mutations in two genes, dszA and dszC. A transversion (G to T) in dszC codon 261 resulted in a V261F mutation that was determined to be responsible for the 5-methylbenzothiophene gain-of-function phenotype. By using a modified RACHITT (random chimeragenesis on transient templates) method, mutant DszC proteins containing all possible amino acids at that position were generated, and this mutant set was assayed for the ability to metabolize 5-methylbenzothiophene, alkyl thiophenes, and dibenzothiophene. No mutant with further improvements in these catalytic activities was identified, but several clones lost all activity, confirming the importance of codon 261 for enzyme activity.

An image analysis-based method for high throughput screening of an α-amylase mutant library using chromogenic assays was developed. Assays were performed in microplates and high resolution images of the assay plates were read using the Virtual Microplate Reader (VMR) script to quantify the concentration of the chromogen. This method is fast and sensitive in quantifying 0.025–0.3 mg starch/ml as well as 0.05–0.75 mg glucose/ml. It was also an effective screening method for improved α-amylase activity with a coefficient of variance of 18%.

Respiratory viruses evolve to maintain infectivity levels that permit spread yet prevent host and virus extinction, resulting in surprisingly low infection rates. Respiratory viruses harnessed as gene therapy vectors have illustrated this limitation. We used directed evolution in an organotypic human airway model to generate a highly infectious adeno-associated virus. This virus mediated gene transfer more than 100-fold better than parental strains and corrected the cystic fibrosis epithelial Cl ؊ transport defect. Thus, under appropriate selective pressures, viruses can evolve to be more infectious than observed in nature, a finding that holds significant implications for designing vectors for gene therapy and for understanding emerging pathogens.

The breadth of genomic diversity found among organisms in nature allows populations to adapt to diverse environments 1,2 . However, genomic diversity is difficult to generate in the laboratory and new phenotypes do not easily arise on practical timescales 3 . Although in vitro and directed evolution methods 4-9 have created genetic variants with usefully altered phenotypes, these methods are limited to laborious and serial manipulation of single genes and are not used for parallel and continuous directed evolution of gene networks or genomes. Here, we describe multiplex automated genome engineering (MAGE) for large-scale programming and evolution of cells. MAGE simultaneously targets many locations on the chromosome for modification in a single cell or across a population of cells, thus producing combinatorial genomic diversity. Because the process is cyclical and scalable, we constructed prototype devices that automate the MAGE technology to facilitate rapid and continuous generation of a diverse set of genetic changes (mismatches, insertions, deletions). We applied MAGE to optimize the 1-deoxy-Dxylulose-5-phosphate (DXP) biosynthesis pathway in Escherichia coli to overproduce the industrially important isoprenoid lycopene. Twenty-four genetic components in the DXP pathway were modified simultaneously using a complex pool of synthetic DNA, creating over 4.3 billion combinatorial genomic variants per day. We isolated variants with more than fivefold increase in lycopene production within 3 days, a significant improvement over existing metabolic engineering techniques. Our multiplex approach embraces engineering in the context of evolution by expediting the design and evolution of organisms with new and improved properties.