TheniaDandniiAgenes ofAspergillus nidulans,which code, respectively, for nitrate and nitrite reductases, are divergently transcribed, and their ATGs are separated by 1,200 bp. The genes are under the control of the positivelyactingNirAtranscriptionfactor,whichmediatesnitrateinduction.TheDNAbindingdomainofNirA was expressed as a fusion protein with the glutathioneS-transferase ofSchistosoma japonicum.Gel shift and footprint experiments have shown that in the intergenic region there are four binding sites for the NirA transcription factor. These sites can be represented by the nonpalindromic consensus 5*CTCCGHGG3*. Making use of a bidirectional expression vector, we have analyzed the role of each of the sites inniaDandniiA expression. The sites were numbered from theniiAside. It appeared that site 1 is necessary for the inducibility of niiA only, while sites 2, 3, and to a lesser extent 4 (which is nearer to and strongly affects niaD) act bidirectionally. The results also suggest that of the 10 binding sites for the AreA protein, which mediates nitrogen metabolite repression, those which are centrally located are physiologically important. The insertion ofanunrelatedupstreamactivatingsequenceintotheintergenicregionstronglyaffectedtheexpressionofboth genes, irrespective of the orientation in which the element was inserted.

The intergenic region between the divergently transcribed niiA and niaD genes of Aspergillus nidulans contains multiple NirA binding sites which act bidirectionally.

To improve heterologous gene expression in Trichoderma reesei, a set of optimal artificial cellobiohydrolase I gene (cbh1) promoters was obtained. The region from -677 to -724 with three potential glucose repressor binding sites was deleted. Then the region from -620 to -820 of the modified cbh1 promoter, including the CCAAT box and the Ace2 binding site, was repeatedly inserted into the modified cbh1 promoter, obtaining promoters with copy numbers 2, 4, and 6. The results showed that the glucose repression effects were abolished and the expression level of the glucuronidase (gus) reporter gene regulated by these multi-copy promoters was markedly enhanced as the copy number increased simultaneously. The data showed the great promise of using the promoter artificial modification strategy to increase heterologous gene expression in filamentous fungi and provided a set of optional high-expression vectors for gene function investigation and strain modification.

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Improved heterologous gene expression in Trichoderma reesei by cellobiohydrolase I gene (cbh1) promoter optimization

Filamentous fungi are critical to production of many commercial enzymes and organic compounds. Fungal-based systems have several advantages over bacterial-based systems for protein production because high-level secretion of enzymes is a common trait of their decomposer lifestyle. Furthermore, in the large-scale production of recombinant proteins of eukaryotic origin, the filamentous fungi become the vehicle of choice due to critical processes shared in gene expression with other eukaryotic organisms. The complexity and relative dearth of understanding of the physiology of filamentous fungi, compared to bacteria, have hindered rapid development of these organisms as highly efficient factories for the production of heterologous proteins. In this review, we highlight several of the known benefits and challenges in using filamentous fungi (particularly Aspergillus spp., Trichoderma reesei, and Neurospora crassa) for the production of proteins, especially heterologous, nonfungal enzymes. We review various techniques commonly employed in recombinant protein production in the filamentous fungi, including transformation methods, selection of gene regulatory elements such as promoters, protein secretion factors such as the signal peptide, and optimization of coding sequence. We provide insights into current models of host genomic defenses such as repeat-induced point mutation and quelling. Furthermore, we examine the regulatory effects of transcript sequences, including introns and untranslated regions, pre-mRNA (messenger RNA) processing, transcript transport, and mRNA stability. We anticipate that this review will become a resource for researchers who aim at advancing the use of these fascinating organisms as protein production factories, for both academic and industrial purposes, and also for scientists with general interest in the biology of the filamentous fungi.

Heterologous Gene Expression in Filamentous Fungi

The role of the conserved sequence region III in the promoter regions of the amylase-encoding genes amyB, glaA and agdA of Aspergillus oryzae was examined. Introduction of multiple copies of the region III fragment into the agdA promoter resulted in a significant increase in promoter activity at the transcriptional level. This result suggests that the fragment comprising region III consists of one or more cis-acting sequence(s). Moreover, expression of the agdA gene under the control of the improved agdA promoter resulted in efficient overproduction of alpha-glucosidase, even in the presence of glucose. Thus, overexpression of genes controlled by the improved promoter incorporating region III is possible. Interestingly, expression of the amyB and glaA genes in the transformant was strongly repressed. This result suggests that the trans-acting regulatory protein(s) that interact with region III are common to these amylase genes and that the titration of regulatory protein(s) reduced the expression of the amyB and glaA genes.

Improvement of promoter activity by the introduction of multiple copies of the conserved region III sequence, involved in the efficient expression of Aspergillus oryzae amylase-encoding genes

Filamentous fungi, typically, are saprophytic organisms which, unlike yeasts, secrete a wide array of enzymes involved in the breakdown and recycling of complex polymers from both plant and animal tissues. This makes them attractive hosts for the production of secreted heterologous proteins (Jeenes et al., 1991; van den Hondel et al., 1991). In only a few examples, however, have the secreted yields of heterologous protein reached the gram per litre levels of many homologous fungal enzymes. In many cases, the problem does not appear to be at the level of transcription but, rather, occurs within the secretory pathway. Although the secretory process has barely been explored in filamentous fungi, we have attempted to identify areas upon which attention should be focused based on current knowledge gained from other systems. We also discuss recent developments in the dissection of transcriptional control in these organisms with particular reference to the interaction of regulatory proteins with fungal promoter regions and to the need for targeting expression cassettes to specific locations in the fungal genome. Understanding the detailed mechanisms of transcriptional control will help in designing modified promoter elements or regulatory factors optimized for a given set of growth conditions. Coupled with the proposed study of the secretory pathway, this should improve the yields of secreted heterologous proteins produced by filamentous fungi.

C. Kramer

Papers

An upstream activating sequence from the Aspergillus nidulans gpdA gene.