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Thaumatin, a 22-kDa protein containing eight disulfide bonds, is secreted by the filamentous fungus Aspergillus awamori at levels which are dependent upon the extent of overexpression of protein disulfide isomerase (PDIA). Additional copies of the PDIA-encoding gene pdiA were introduced into a strain of A. awamori that expresses a cassette encoding thaumatin. Transformants with different levels of pdiA mRNA and measured PDIA levels were chosen for examination of the impact that PDIA levels had on thaumatin secretion. The secretion of two native proteins, alpha-amylase and acid phosphatase, was also examined in relation to varying levels of PDIA. Over a range of PDIA levels of 1-8, relative to the native level in strains with just one copy of the pdiA gene, the fraction of alpha-amylase and acid phosphatase in the total secreted protein was unaffected. In contrast, a peak level of thaumatin, about 5-fold higher than in the strain with one copy of pdiA, was found in strains with a relative PDIA level of between two and four. Improved thaumatin production was confirmed in 5-1 fermenters using a strain of A. awamori with six pdiA gene copies, containing 3.2-fold higher levels of PDIA than wild-type strains.

We examine the expression of constitutive or repressible, monocistronic genes from Pseudomonas aeruginosa and Escherichia coli after their transfer to the heterologous host. To this end, chromosomal DNA from P. aeruginosa was cloned into the mobilizable broad-host-range vector pKT240; recombinant plasmids carrying the argA, argF, or proC genes were identified by complementation of the corresponding auxotrophic mutations. The isofunctional E. coli genes and the E. coli proB gene were subcloned into pKT240 from existing recombinant plasmids. The enzyme expression specified by the Pseudomonas genes in E. coli, calculated per gene copy, ranged from 0.3%-5% of the levels observed in Pseudomonas. Fusion of the P. aeruginosa proC gene to the E. coli consensus tac promoter resulted in very high proC enzyme production in E. coli, indicating that, at least in this case, the expression barrier is essentially at the level of transcriptional initiation. The E. coli argA and argF enzymes, which are controlled by repression in their native host, were synthesized constitutively in P. aeruginosa at 5% of the levels measured in E. coli under derepressed conditions. The constitutive E. coli proB and proC genes were expressed at high levels (ca. 50%) in the heterologous host. These results support the idea that P. aeruginosa may be a more permissive host than E. coli for the heterologous expression of genes from gram-negative bacteria.

The Pseudomonas aeruginosa lysA gene encoding diaminopimelate decarboxylase (DAP-decarboxylase) was cloned into a broad host range vector. This gene complemented a lys mutation at the lys-12 locus of P. aeruginosa and a lysA defect in Escherichia coli. The P. aeruginosa DAP-decarboxylase was synthesized constitutively in P. aeruginosa as well as in E. coli, where the Pseudomonas lysA gene was poorly expressed. By contrast, the E. coli lysA gene was expressed well in P. aeruginosa and subject to lysine regulation when the E. coli LysR activator protein was provided. This indicates that the mechanism of transcriptional activation for the E. coli lysA gene is effective in the heterologous host.