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.

Oxygen inactivation and recovery of nitrogenase activity in cyanobacteria.

Exposure of nitrogen-fixing cultures of Anabaena spp. to 100% oxygen resulted in the rapid decline of nitrogenase activity. When oxygen-treated cells were transferred to 100% argon, nitrogenase activity was quickly restored in a process that required protein synthesis. Anaerobiosis was not essential for the recovery process; in fact, cells of Anabaena sp. strains CA and 1F will recover nitrogenase activity after prolonged incubation in 100% oxygen. Oxygen treatment acted directly on the intracellular nitrogenase and did not affect other metabolic processes. Examination of crude extracts of oxygen-treated Anabaena sp. strain CA indicated that both components of nitrogenase are inactivated. However, several lines of evidence suggest that oxygen treatment does not result in irreversible denaturation of nitrogenase, but rather results in a reversible inactivation which may serve as a protection mechanism. Nitrogenase present in crude extracts from cells of Anabaena sp. strain 1F which had been incubated for a prolonged period in 100% oxygen was less sensitive to oxygen in vitro than was nitrogenase of a crude extract of untreated cells.

Molybdenum accumulation and storage in Klebsiella pneumoniae and Azotobacter vinelandii.

In Klebsiella pneumoniae, Mo accumulation appeared to be coregulated with nitrogenase synthesis. O2 and NH+4, which repressed nitrogenase synthesis, also prevented Mo accumulation. In Azotobacter vinelandii, Mo accumulation did not appear to be regulated Mo was accumulated to levels much higher than those seen in K. pneumoniae even when nitrogenase synthesis was repressed. Accumulated Mo was bound mainly to a Mo storage protein, and it could act as a supply for the Mo needed in component I synthesis when extracellular Mo had been exhausted. When A. vinelandii was grown in the presence of WO2-(4) rather than MoO2-(4), it synthesized a W-containing analog of the Mo storage protein. The Mo storage protein was purified from both NH+4 and N2-grown cells of A. vinelandii and found to be a tetramer of two pairs of different subunits binding a minimum of 15 atoms of Mo per tetramer.

In vitro activation of inactive nitrogenase component I with molybdate.

When Azotobacter vinelandii was derepressed for nitrogenase synthesis in the presence of WO42- rather than MoO42-, it synthesized active component II and inactive component I of nitrogenase. This inactive component I could be activated in vitro with the iron-molybdenum cofactor or with MoO42-. The latter reaction required adenosine 5'-triphosphate and was inhibited by adenosine 5'-diphosphate. FeMo cofactor and MoO42- produced different levels of activation, but there was no evidence that they acted upon different species of demolybdo component I. Rather, it may be that an additional factor necessary for MoO42-mediated activation but not for FeMo cofactor-mediated activation was limiting. Mo was inserted into component I during both FeMo cofactor- and MoO42- mediated activations.

Molybdenum cofactors from molybdoenzymes and in vitro reconstitution of nitrogenase and nitrate reductase.

A molybdenum cofactor (Mo-co) from xanthine oxidase (xanthine:oxygen oxidoreductase, EC 1.2.3.2) can be isolated from the enzyme by a technique that has been used to isolate an iron-molybdenum cofactor (FeMo-co) from component I of nitrogenase. N-Methylformamide is used for the extraction of these molybdenum cofactors. Mo-co from xanthine oxidase activates nitrate reductase (NADPH:nitrate oxidoreductase, EC 1.6.6.2) in an extract from Neurospora crassa mutant strain Nit-1; however, FeMo-co is unable to activate nitrate reductase in strain Nit-1. Mo-co from xanthine oxidase is unable to activate nitrogenase in an extract of Azotobacter vinelandii mutant strain UW45. Inactive component I in this extract can be activated by FeMo-co. These results indicate that nitrate reductase and xanthine oxidase share a common molybdenum cofactor, but this cofactor is different from the molybdenum cofactor in nitrogenase.A. vinelandii synthesizes both Mo-co and FeMo-co. Mo-co is produced when the cells fix N(2) and also when they are repressed for nitrogenase synthesis by growth in a medium containing excess ammonium. However, FeMo-co is not produced when cells are grown in an ammonium-containing medium. Partially purified preparations of component I from A. vinelandii and Klebsiella pneumoniae contain both FeMo-co and Mo-co. The presence of both FeMo-co and Mo-co activities in partially purified preparations of component I explains previous reports of activation of inactive nitrate reductase in strain Nit-1 by acid-treated component I of nitrogenase. The Mo-co can be separated from FeMo-co in these preparations by chromatography on Sephadex G-100 in N-methylformamide. Both FeMo-co and Mo-co are sensitive to oxygen.

An unusual mutant affecting the frequency of organelle mutation.

A mutant of Euglena gracilis Z is described that spontaneously produces irreversibly bleached mutants at a high frequency. Bleaching only occurs during exponential growth under highly aerobic conditions. The kinetics for bleaching and growth of bleached and nonbleached cells suggest that mutation is induced by segregation of a cellular component that becomes limiting after several culture generations. The properties of spontaneous bleaching closely resemble the properties of induced organelle mutagenesis in E. gracilis and yeast.