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.

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There is great interest in engineering human growth factors as potential therapeutic agonists and antagonists. We approached this goal with a synthetic DNA recombination method. We aligned a pool of "top-strand" oligonucleotides incorporating polymorphisms from mammalian genes encoding epidermal growth factor (EGF) using multiple polymorphic "scaffold" oligonucleotides. Top strands were then linked by gap filling and ligation. This approach avoided heteroduplex annealing in the linkage of highly degenerate oligonucleotides and thus achieved completely random recombination. Cloned genes from a human-mouse chimeric library captured every possible permutation of the parental polymorphisms, creating an apparently complete recombined gene-family library, which has not been previously described. This library yielded a chimeric protein whose agonist activity was enhanced 123-fold. A second library from five mammalian EGF homologs possessed the highest reported recombination density (1 crossover per 12.4 bp). The five-homolog library yielded the strongest-binding hEGF variant yet reported. In addition, it contained strongly binding EGF variants with antagonist properties. Our less biased approach to DNA shuffling should be useful for the engineering of a wide variety of proteins.

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The objective of this study was to develop scaleup bioprocesses for producing 10‐hydroxystearic acid (10‐HSA) and 10‐ketostearic acid (10‐KSA) as well as their primary amides for potential new uses. A reactor process was examined to obtain the mono‐oxygenated FA using Sphingobacterium thalpophilum (NRRL B‐14797) and Bacillus sphaericus (NRRL NRS‐732), which solely produce 10‐HSA and 10‐KSA, respectively, from technical‐grade oleic acid. By using an 8‐h‐old B‐14797 culture grown in a manganese‐containing WF6 medium, pH 7.3, at 28°C under 350 rpm agitation and 0–50% dissolved oxygen concentrations provided by a controlled sparger aeration, the production of 10‐HSA reached 7 g/L with a 40% yield in 4 d. In using a 12‐h‐old NRS‐732 culture grown in a pyruvate‐containing PF6 medium, pH 6.5, at 30°C under 750 rpm agitation without any sparger aeration during the conversion reaction, 10‐KSA production reached 7.9 g/L with a yield of more than 54% in 72 h. The scaleup reactor process provided crystalline 10‐HSA and 10‐KSA for producting new primary amides via a lipase‐catalyzed amidation reaction with yields of 94 and 92%, respectively. The primary amides of 10‐HSA and 10‐KSA displayed m.p of 115 and 120°C, respectively.