DNA mediated transformation of Aspergillus ficuum

Summary Two DNA-mediated transformation systems were successfully adapted to Aspergillus ficuum. Both the Escherichia coli hygromycin B resistance gene and the A. nidulans amdS gene transformation systems produced stable A. ficuum NRRL 3135 transformants. Cotransformation with the E. coli lacZ gene was also achieved with the hygromycin B system. In cotransformation a second unselected gene, in this case the lacZ gene which codes for -galactosidase, was also integrated and expressed in hygromycin B transformants. Since both of these transformation systems utilized dominant selection markers, they are potentially useful in other genetically uncharacterized filamentous fungi.     

Species‐specific ontogenetic diet shifts attenuate trophic cascades and lengthen food chains in exploited ecosystems

Ontogenetic diet shifts are pervasive in food websbut rules governing their emergence and the implications for trophic cascades are only partly understood. Recent theoretical advances in multispecies size spectrum models (MSSMs) predict that the emergence of ontogenetic diet shifts are driven primarily by size‐selective predation and changes in the relative abundances of suitably sized prey. Howeverthese assumptions have not yet been tested with data. Herewe developed alternative MSSMs based on different assumptions about the nature of species and size‐based preferences and tested them using an extensive dietary database for the Eastern Bering Sea (EBS). MSSMs with both size and species‐specific prey preferences correctly predicted approximately three‐fold more of the diet links than those that assumed fixed species preferences. Importantlythese model assumptions also had a profound effect on the strength of fishing‐induced trophic cascades and the emergent trophic structure of the community with and without fishing. The diet‐informed models exhibited lower predation mortality ratesparticularly for small individuals (less than 1 g) whichin turnreduced the intensity and reach of fishing‐induced trophic cascades up the size spectrum. If the level and size dependency of piscivory observed in EBS predators is typical of other systemsthe potential for fishing‐induced trophic cascades may be over‐stated in MSSMs as they are currently formulated and parameterized. Representation of species‐specific ontogenetic shifts in diet can strongly influence system responses to perturbationsand the extensions we propose should accelerate adoption of MSSMs as frameworks for exploring size‐based food web theory and developing modeling tools to support strategic management decisions.     

Expression of the Caldariomyces fumago chloroperoxidase in Aspergillus niger and characterization of the recombinant enzyme

The Caldariomyces fumago chloroperoxidase was successfully expressed in Aspergillus niger. The recombinant enzyme was produced in the culture medium as an active protein and could be purified by a three-step purification procedure. The catalytic behavior of recombinant chloroperoxidase (rCPO) was studied and compared with that of native CPO. The specific chlorination activity (47 units/nmol) of rCPO and its pH optimum (pH 2.75) were very similar to those of native CPO. rCPO catalyzes the oxidation of various substrates in comparable yields and selectivities to native CPO. Indole was oxidized to 2-oxindole with 99% selectivity and thioanisole to the corresponding R-sulfoxide (enantiomeric excess >98%). Incorporation of (18)O from labeled H(2)18O(2) into the oxidized products was 100% in both cases.     

Calcium measurement in living filamentous fungi expressing codon-optimized aequorin

Calcium signalling is little understood in filamentous fungi largely because easy and routine methods for calcium measurement in living hyphae have previously been unavailable. We have developed the recombinant aequorin method for this purpose. High levels of aequorin expression were obtained in Neurospora crassa, Aspergillus niger and Aspergillus awamori by codon optimization of the aequorin gene. Three external stimuli (mechanical perturbation, hypo-osmotic shock and high external calcium) were found transiently to increase [Ca(2+)](c). Each of the calcium signatures associated with these physico-chemical treatments was unique, suggesting the involvement of three distinct calcium-mediated signal transduction pathways. The fungal calcium channel blocker KP4 inhibited the [Ca(2+)](c) responses to hypo-osmotic shock and high external calcium, but not to mechanical perturbation. The divalent cation chelator BAPTA inhibited [Ca(2+)](c) responses to mechanical perturbation and hypo-osmotic shock. The calcium agonists A23187 and cyclopiazonic acid increased [Ca(2+)](c) levels.     

The production of antibody fragments and antibody fusion proteins by yeasts and filamentous fungi

In this review we will focus on the current status and views concerning the production of antibody fragments and antibody fusion proteins by yeasts and filamentous fungi. We will focus on single-chain antibody fragment production (scFv and V_HH) by these lower eukaryotes and the possible applications of these proteins. Also the coupling of fragments to relevant enzymes or other components will be discussed. As an example of the fusion protein strategy, the 'magic bullet' approach for industrial applications, will be highlighted.