The secretion pathway in filamentous fungi: a biotechnological view.

The high capacity of the secretion machinery of filamentous fungi has been widely exploited for the production of homologous and heterologous proteins; however, our knowledge of the fungal secretion pathway is still at an early stage. Most of the knowledge comes from models developed in yeast and higher eukaryotes, which have served as reference for the studies on fungal species. In this review we compile the data accumulated in recent years on the molecular basis of fungal secretion, emphasizing the relevance of these data for the biotechnological use of the fungal cell and indicating how this information has been applied in attempts to create improved production strains. We also present recent emerging approaches that promise to provide answers to fundamental questions on the molecular genetics of the fungal secretory pathway.

Control of replication of the Lactobacillus pentosus plasmid p353-2: evidence for a mechanism involving transcriptional attenuation of the gene coding for the replication protein.

The synthesis of plasmid DNA and of RNA encoded by the replication protein gene (rep) of plasmid p353-2 of Lactobacillus pentosus was studied for the wild-type plasmid and for a mutant plasmid with a deletion in the 5' untranslated region of the rep gene. Plasmid p353-2 codes for two countertranscript RNAs (CT-RNA) of approximately 75 and 250 nucleotides transcribed from the 5' untranslated region of the rep gene, in opposite directions. In a mutant plasmid with a deletion of the promoter and part of the CT-RNA-encoding sequence which shows a 5- to 10-fold increase in copy number compared to the wild-type plasmid, no CT-RNA could be detected. In the wild-type plasmid more than 90% of transcription initiated at a promoter upstream of the rep gene is prematurely terminated to form a 190 nucleotide truncated RNA, whereas in the mutant plasmid nearly all transcripts reach a size (1100 nucleotides) corresponding to that of the rep gene. A model is presented for the role of CT-RNA in control of plasmid replication, similar to that previously presented for the staphylococcal plasmid pT181, involving a mechanism of transcriptional attenuation of rep RNA at a site just upstream of the rep gene.

Structural and functional analysis of two cryptic plasmids from Lactobacillus pentosus MD353 and Lactobacillus plantarum ATCC 8014.

The DNA sequences of a 2.4 kb plasmid (p353-2) from Lactobacillus pentosus MD353 and a 1.9 kb plasmid (p8014-2) from Lactobacillus plantarum ATCC 8014 show 81.5% overall similarity. Both plasmids carry elements (replication protein gene, plus-origin and minus-origin of replication), which are typical of plasmids that replicate via a rolling-circle mechanism of replication (RCR). Direct evidence for an RCR mechanism was obtained by showing the accumulation of single-stranded plasmid intermediates in the presence of rifampicin. A minus-origin of replication was defined for plasmids p353-2 and p8014-2 based on DNA sequence analysis and on its ability to convert single-stranded into double-stranded plasmid DNA. Plasmids pLPE323, pLPE350 and pLPC37 that are derived from the p353-2 or p8014-2 replicon are structurally and segregationally stable in L. pentosus MD353, L. plantarum ATCC 8014 and in Lactobacillus casei ATCC 393. The presence of Escherichia coli or lambda DNA fragments in vectors derived from p353-2 or p8014-2 does not affect the structural stability but results in segregational instability of the vectors. The instability increases with increasing size of the inserted DNA fragment. Since vectors based on these replicons can be efficiently propagated in a wide variety of Lactobacillus species, they are highly suitable for cloning and expression of foreign DNA in Lactobacillus, provided that selective pressure is applied.

Incompatibility of Lactobacillus Vectors with Replicons Derived from Small Cryptic Lactobacillus Plasmids and Segregational Instability of the Introduced Vectors.

Three new Lactobacillus vectors based on cryptic Lactobacillus plasmids were constructed. The shuttle vector pLP3537 consists of a 2.3-kb plasmid from Lactobacillus pentosus MD353, an erythromycin resistance gene from Staphylococcus aureus plasmid pE194, and pUC19 as a replicon for Escherichia coli. The vectors pLPE317 and pLPE323, which do not contain E. coli sequences, were generated by introducing the erythromycin resistance gene of pE194 into a 1.7- and a 2.3-kb plasmid from L. pentosus MD353, respectively. These vectors and the shuttle vector pLP825 (M. Posno, R. J. Leer, J. M. M. van Rijn, B. C. Lokman, and P. H. Pouwels, p. 397-401, in A. T. Ganesan and J. A. Hoch, ed., Genetics and biotechnology of bacilli, vol. 2, 1988) could be introduced by electroporation into Lactobacillus casei, L. pentosus, L. plantarum, L. acidophilus, L. fermentum, and L. brevis strains with similar efficiencies. Transformation efficiencies were strain dependent and varied from 10 to 10 transformants per mug of DNA. Plasmid DNA analysis of L. pentosus MD353 transformants revealed that the introduction of pLP3537 or pLPE323 was invariably accompanied by loss of the endogenous 2.3-kb plasmid. Remarkably, pLPE317 could only be introduced into an L. pentosus MD353 strain that had been previously cured of its endogenous 1.7-kb plasmid. The curing phenomena are most likely to be explained by the incompatibility of the vectors and resident plasmids. Lactobacillus vectors are generally rapidly lost when cells are cultivated in the absence of selective pressure. However, pLPE323 is stable in three of four Lactobacillus strains tested so far.