A Darzins

DNA shuffling method for generating highly recombined genes and evolved enzymes

We introduce a method of in vitro recombination or “DNA shuffling” to generate libraries of evolved enzymes. The approach relies on the ordering, trimming, and joining of randomly cleaved parental DNA fragments annealed to a transient polynucleotide scaffold. We generated chimeric libraries averaging 14.0 crossovers per gene, a several-fold higher level of recombination than observed for other methods. We also observed an unprecedented four crossovers per gene in regions of 10 or fewer bases of sequence identity. These properties allow generation of chimeras unavailable by other methods. We detected no unshuffled parental clones or duplicated “sibling” chimeras, and relatively few inactive clones. We demonstrated the method by molecular breeding of a monooxygenase for increased rate and extent of biodesulfurization on complex substrates, as well as for 20-fold faster conversion of a nonnatural substrate. This method represents a conceptually distinct and improved alternative to sexual PCR for gene family shuffling.

Characterization of the Pseudomonas aeruginosa alginate (alg) gene region II.

Pseudomonas aeruginosa region II alginate genes are involved in the biosynthesis of the uronic acid containing exopolysaccharide, alginic acid. We have subcloned and overexpressed various DNA fragments contained within region II in an attempt to further characterize and more precisely localize the genes involved in alginate production. Overexpression of the genes controlling alginate biosynthesis within region II was accomplished by placing various cloned restriction fragments under the transcriptional control of the hybrid trp-lac (tac) promoter, and plasmid encoded proteins were examined in a maxicell expression system. We correlated various region II plasmid constructions with the ability to complement specific alginate negative (alg) mutants and code for polypeptides. Several proteins suspected of being involved in alginate production were encoded by sequences within region II. The results of this study further reveal that the transcriptional orientation of the alg loci within region II appears to be in the direction from argF to pmi. The specific activities of phosphomannomutase (PMM) and GDP-mannose pyrophosphorylase (GMP), two enzymes involved in the formation of the alginate precursor GDP-mannuronic acid, were measured in region II alg mutants and in cells overexpressing cloned segments from region II. Based on these enzyme measurements, we conclude that the remaining region II alg genes do not encode either PMM or GMP. These results support the suggestion that the remaining alg genes in region II are likely to be involved in post GDP-mannuronic acid processing events such as mannuronic acid transport, polymerization, secretion, epimerization and acetylation.