We have shown that whole Escherichia coli cells overexpressing NADPH-dependent cyclohexanone monooxygenase carry out a model Baeyer-Villiger oxidation with high volumetric productivity (0.79 g epsilon-caprolactone/L.h ) under nongrowing conditions (Walton, A. Z.; Stewart, J. D. Biotechnol. Prog. 2002, 18, 262-268). This is approximately 20-fold higher than the space-time yield for reactions that used growing cells of the same strain. Here, we show that the intracellular stability of cyclohexanone monooxygenase and the rate of substrate transport across the cell membrane were the key limitations on the overall reaction duration and rate, respectively. Directly measuring the levels of intracellular nicotinamide cofactors under bioprocess conditions suggested that E. coli cells could support even more efficient NADPH-dependent bioconversions if a more suitable enzyme-substrate pair were identified. This was demonstrated by reducing ethyl acetoacetate with whole cells of an E. coli strain that overexpressed an NADPH-dependent, short-chain dehydrogenase from baker's yeast (Saccharomyces cerevisiae). Under glucose-fed, nongrowing conditions, this reduction proceeded with a space-time yield of 2.0 g/L.h and a final product titer of 15.8 g/L using a biocatalyst:substrate ratio (g/g) of only 0.37. These values are significantly higher than those obtained previously. Moreover, the stoichiometry linking ketone reduction and glucose consumption (2.3 +/- 0.1) suggested that the citric acid cycle supplied the bulk of the intracellular NADPH under our process conditions. This information can be used to improve the efficiency of glucose utilization even further by metabolic engineering strategies that increase carbon flux through the pentose phosphate pathway.

Previous studies (Mertens et al., Virology 157, 375-386, 1987) have shown that removal of the outer capsid layer from bluetongue virus (BTV) significantly reduces (approximately x 10(-4)) the infectivity of the resultant core particle for mammalian cells (BHK 21 cells). In contrast, the studies reported here, using a cell line (KC cells) derived from a species of Culicoides that can act as a vector for BTV (Culicoides variipennis), demonstrated a much higher infectivity of core particles than that in mammalian cells (approximately x 10(3)). This increase resulted in a specific infectivity for cores that was only 20-fold less than that of purified disaggregated virus particles (stored in the presence of 0.1% sodium-N-lauroylsarcosine (NLS)). Removal of this detergent caused intact virus particle aggregation and (as previously reported) resulted in an approximately 1 log10 drop in the specific infectivity of those virus particles which remained in suspension. In consequence the specific infectivity of core particles for the KC cells was directly comparable to that of the intact but aggregated virus. These data are compared with the results from oral infectivity studies using two vector species (C. variipennis and Culicoides nubeculosus), which showed similar infection rates at comparable concentrations of purified cores, or of the intact but aggregated virus particles (NLS was toxic to adult flies). The role of the outer core proteins (VP7) in cell attachment and penetration, as an alternative route of initiation of infection, is discussed. Previous studies (Mertens et al., Virology 157, 375-386, 1987) also showed that the outer capsid layer of BTV can be modified by proteases (including trypsin or chymotrypsin), thereby generating infectious subviral particles (ISVP). The specific infectivity of ISVP for mammalian cells (BHK21 cells) was shown to be similar to that of disaggregated virus particles. In contrast, we report a significantly higher specific infectivity of ISVP but not of the intact virus (approximately x 100) for two insect cell lines (KC cells and C6/36 mosquito cells (derived from Aedes albopictus)). In oral infection studies with adults of the two vector species, ISVP produced the same infection rate at approximately 100-fold lower concentrations than either core particles or the intact but aggregated virus particles. The importance of mammalian host serum proteases, or insect gut proteases, in modification of the intact virus particle to form ISVP and their role in initiation of infection and the vector status of the insect is discussed.

Economical methods of supplying NADPH must be developed before biotransformations involving this cofactor can be considered for large-scale applications. We have studied the enzymatic Baeyer-Villiger oxidation of cyclohexanone as a model for this class of reactions and developed a simple approach that uses whole, non-growing Escherichia coli cells to provide high productivity (0.79 g epsilon-caprolactone/L/h = 18 micromol epsilon-caprolactone/min/g dcw) and an 88% yield. Glucose supplied the reducing equivalents for this process, and no exogenous cofactor was required. The volumetric productivity of non-growing cells was an order of magnitude greater than that achieved with growing cells of the same strain. Cells of an engineered E. coli strain that overexpresses Acinetobacter sp. cyclohexanone monooxygenase were grown under inducing conditions in rich medium until the entry to stationary phase; the subsequent cyclohexanone oxidation was carried out in minimal salts medium lacking a nitrogen source. After the biotransformation was complete, the lactone product was adsorbed to a solid support and recovered by washing with an organic solvent.

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Methods were developed for the purification, at high yield, of four different particle types of African horsesickness virus serotype 9 (AHSV-9). These products included virus particles purified on CsC1 gradients which contain proteins apparently directly comparable to those ofbluetongue virus (VP1 to VP7); virus particles purified on sucrose gradients which also contain, as a variable component, protein NS2; infectious subviral particles (ISVPs), containing chymotrypsin cleavage products of VP2; and cores, obtained by treating purified ISVPs with 1 M-MgCIz to remove the components of the outer capsid layer (VP5 and VP2 cleavage products). Additional protein bands migrating with apparent Mrs lower than that of VP5 were detected during SDS-PAGE analysis of virus particles. These appear to be conform-