For millennia, selective breeding, on the basis of biparental mating, has led to the successful improvement of plants and animals to meet societal needs. At a molecular level, DNA shuffling mimics, yet accelerates, evolutionary processes, and allows the breeding and improvement of individual genes and subgenomic DNA fragments. We describe here whole-genome shuffling; a process that combines the advantage of multi-parental crossing allowed by DNA shuffling with the recombination of entire genomes normally associated with conventional breeding. We show that recursive genomic recombination within a population of bacteria can efficiently generate combinatorial libraries of new strains. When applied to a population of phenotypically selected bacteria, many of these new strains show marked improvements in the selected phenotype. We demonstrate the use of this approach through the rapid improvement of tylosin production from Streptomyces fradiae. This approach has the potential to facilitate cell and metabolic engineering and provide a non-recombinant alternative to the rapid production of improved organisms.
Fungicides continue to be essential for the effective control of plant diseases. New classes of fungicides with novel modes of action are being developed in the 1990s. These include the strobilurins, phenylpyrroles, anilinopyrimidines, phenoxyquinolines, and compounds that trigger defense mechanisms in the plant. For the foreseeable future, new toxophores will be identified through a process of random screening, with natural products representing a rich source of fungicide leads. Progress is being made in the development of high-throughput screens comprised of target enzyme sites or cell-based assays; these techniques will improve the probability of discovery. Following the identification of suitable leads, biorational design is used to optimize specific properties. In vivo glasshouse screens and field trials are expected to remain the dominant methods for characterizing new compounds. Low toxicity to humans and wildlife, low environmental impact, low residues in food, and compatibility with integrated pest management (IPM) programs are increasingly important considerations in the selection of fungicides for development.
This review describes the current state of biocatalysis in the chemical industry. Although we recognize the advantages of chemical approaches, we suggest that the use of biological catalysis is about to expand dramatically because of the recent developments in the artificial evolution of genes that code for enzymes. For the first time it is possible to consider the rapid development of an enzyme that is designed for a specific chemical reaction. This technology offers the opportunity to adapt the enzyme to the needs of the process. We describe herein the development of enzyme evolution technology and particularly DNA shuffling. We also consider several classes of enzymes, their current applications, and the limitations that should be addressed. In a review of this length it is impossible to describe all the enzymes with potential for industrial exploitation; there are other classes, which given appropriate activity, selectivity, and robustness, could become useful tools for the industrial chemist. This is an exciting era for biocatalysis and we expect great progress in the future.
Exploitation of potential new targets for drug and vaccine development has an absolute requirement for multimilligram quantities of soluble protein. While recombinant expression of full-length proteins is frequently problematic, high-yield soluble expression of functional subconstructs is an effective alternative, so long as appropriate termini can be identified. Bioinformatics localizes domains, but doesn't predict boundaries with sufficient accuracy, so that subconstructs are typically found by trial and error. Combinatorial Domain Hunting (CDH) is a technology for discovering soluble, highly expressed constructs of target proteins. CDH combines unbiased, finely sampled gene-fragment libraries, with a screening protocol that provides ''holistic'' readout of solubility and yield for thousands of protein fragments. CDH is free of the ''passenger solubilization'' and out-of-frame translational start artifacts of fusion-protein systems, and hits are ready for scale-up expression. As a proof of principle, we applied CDH to p85a, successfully identifying soluble and highly expressed constructs encapsulating all the known globular domains, and immediately suitable for downstream applications.
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