Michael F. Tuite

Heterologous gene expression in filamentous fungi

Abstract Filamentous fungi are central to a broad spectrum of activities in industrial microbiology. They have been used for decades in the elaboration of a wide range of compounds including antibiotics and organic acids. They are also prodigious producers of enzymes which are used, for example, in the production of soy sauce, in cheese ripening and in the biocatalytic conversion of starch to glucose. Two species of filamentous fungi, Aspergillus nidulans and Neurospora crassa , have been the subjects of sophisticated genetic and biochemical analyses for a number of years. In this article we discuss the recent progress made in the use of filamentous fungi as hosts for the expression of foreign genes, highlighting their established and potential advantages for the production of proteins of therapeutic and/or commercial significance.

Using Molecular Genetics to Improve the Production of Recombinant Proteins by the Yeast Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae has proven to be an excellent host for the production of a number of different recombinant proteins that have potential medical and commercial applications. The use of S. cerevisiae as a recombinant host has a number of advantages: (1) yeast cells are easily fermented to industrial scale using simple media; (2) yeast cells are free of endotoxin and nonpathogenic to man; (3) S. cerevisiae has well-developed genetics, which offers unparalleled possibilities for solving problems that may exist at various steps in the production of heterologous proteins through a combination of classical and molecular genetic approaches; and (4) yeast cells are capable of performing post-translational and cotranslational processing of proteins in a manner similar to higher eukaryotes. In addition, the secretion of heterologous proteins by yeast has several advantages: first, only low levels of native proteins are secreted into the culture medium, simplifying purification of a target protein; second, yeast is able in many cases to correctly fold proteins and form intramolecular disulfide bonds during secretion as demonstrated by the successful secretion of proteins containing multiple disulfide bonds in a biologically active f ~ r m . I ~ Two examples are echistatin4 and anti~tasin.~ Finally, S. cerevisiae has a number of strong promoters that are either inducible or constitutive. These promoters have been used in a variety of different yeast expression vectors that in turn can be used to readily transform yeast using several different selective markers (URA3, LEU2, T W I , etc.). As many of the above features of heterologous protein expression in yeast have been discussed in several recent they will not be discussed further here. This paper will highlight the well-defined genetics of S. cerevisiae, which enable one to engineer yeast host strains with desired genetic characteristics such that

Host-Plasmid Interactions in Saccharomyces cerevisiae: Effect of Host Ploidy on Plasmid Stability and Copy Number

The segregational stability of two chimaeric plasmids has been examined in an isogenic series of haploid, diploid and tetraploid strains of Saccharomyces cerevisiae, constructed by transformation-associated spheroplast fusion. For the highly unstable, ARS-based plasmid YRp7M, a significant increase in its segregational stability was observed with increasing ploidy, while the relatively stable, 2 microns-based plasmid pMA3a showed only a small increase in stability in strains of higher ploidy. The copy number of both pMA3a and the endogenous 2 microns plasmid increased in proportion with the host cell ploidy, while the copy number of TRp7M was increased in the higher ploidy strains but did not correlate with ploidy. These results suggest that the copy numbers of both the 2 microns plasmid and a plasmid derived from it are controlled by a nuclear gene and that, in addition, there are 2 microns sequences, other than those required for the FLP-mediated recombination system, that play a role in maintaining copy number.

Genetic control of translational fidelity in yeast: molecular cloning and analysis of the allosuppressor gene SAL3

SummaryThe fidelity of translation in the yeast Saccharomyces cerevisiae is controlled by a number of gene products. We have begun a molecular analysis of such genes and here describe the cloning and analysis of one of these genes, SAL3. Mutations at this locus, and at least four other unlinked loci (designated SAL1-SAL5), increase the efficiency of the tRNA ochre suppressor SUQ5, and are thus termed allosuppressors. We have cloned the SAL3 gene from a yeast genomic library by complementation of a sal3 mutation. Integration of the cloned sequence into the yeast chromosome was used to confirm that the SAL3 gene had been cloned. SAL3 gene is present in a single copy in the yeast genome, is transcribed into a 2.3-kb polyadenylated mRNA and encodes a protein of Mr 80 000. The size of the SAL3 gene product strongly suggests that it is not a ribosomal protein.

Expression of heterologous genes

With the advent of recombinant DNA technology in the mid-1970s a number of shrewd academics quickly realized the commercial potential of these tools in producing proteins of high commercial value, which, until that time, had proven both difficult and expensive to isolate in quantities sufficient for clinical trials. While Escherichia coli as a host organism was an obvious choice in the early stages of commercial genetic engineering (Harris, 1983), it soon became apparent that it was far from the ideal host. It did not make sense, for example, to expect a eukaryotic gene product to be expressed in a prokaryotic environment and be correctly folded and modified at the posttranslational level. So by 1980 the search was underway to find a more suitable host and the budding yeast Saccharomyces cerevisiae immediately presented itself as an attractive alternative. It is a host that is widely accepted for human consumption by both the consumer and the regulatory authorities, has no associated toxins, and, with the development of an effective plasmid-based gene cloning system (see Chapter 5), its exploitation as a host for producing high-value proteins has progressed rapidly.

A novel suppressor tRNA from the dimorphic fungus Candida albicans

Unfractionated tRNAs from a number of prokaryotes and eukaryotes were examined for their ability to promote termination codon readthrough in a cell-free system isolated from Saccharomyces cerevisiae. tRNA from the dimorphic fungus Candida albicans was found to have significant UGA and UAG readthrough activity and this activity was present in tRNA extracted from both the yeast and the hyphal phase of the fungus. Unusually the efficiency of readthrough activity in vitro was not affected by the [psi] determinant. C. albicans tRNA was fractionated by one-dimensional and two-dimensional gel electrophoresis and both readthrough activities appeared to be associated with a single species of tRNA.

Strategies for the Genetic Manipulation of Saccharomyces cerevisiae

The budding yeast Saccharomyces cerevisiae is now widely used as a model organism in the study of gene structure, function, and regulation in addition to its more traditional use as a workhorse of the brewing and baking industries. In this article the plethora of methods available for manipulating the genome of S. cerevisiae are reviewed. This will include a discussion of methods for manipulating individual genes and whole chromosomes, and will address both classic genetic and recombinant DNA-based methods. Furthermore, a critical evaluation of the various genetic strategies for genetically manipulating this simple eukaryote will be included, highlighting the requirements of both the new and the more traditional biotechnology industries.

Antifungal drug development: the identification of new targets

With the increasing prevalence of life-threatening systemic fungal infections in the human population, there is a need to develop new, more-effective antifungal agents. This, in turn, will depend upon the identification and exploitation of new antifungal targets--aspects of fungal cytology, metabolism and gene expression which are important for fungal pathogenesis, but which have no mammalian host counterpart. Such new targets have been identified through a combination of classical genetic, cytological and biochemical studies and are reviewed here, as is the potential for applying recombinant DNA techniques as a means of confirming the role of the identified gene products in pathogenesis.

Fungal cloning vectors

Despite the industrial utility and metabolic diversity of the fungi, efforts to develop cloning vectors have so far been largely confined to the Ascomycotina. This article reviews progress within this subdivision and considers current work and prospects for the application of recombinant DNA techniques to the other subdivisions of the fungi.

The influence of cell ploidy on the thermotolerance of Saccharomyces cerevisiae

SummaryThe resistance of Saccharomyces cerevisiae to inactivation by DNA damaging agents has long been known to be affected by cell ploidy. Resistance is greater for diploid than for haploid cells, but exhibits decreases for further increases in ploidy beyond diploid. In this study S. cerevisiae cells whose genomes differ only in their ploidy were employed to investigate how ploidy directly influences resistance to thermal killing. In virtually all species resistance to thermal killing is a cellular property that is elevated by heat shock and other agents that induce the heat shock response. We therefore investigated how ploidy affected the thermal killing of S. cerevisiae cells both before and after elevation of thermotolerance by means of a 40 min 25 °C to 38 °C heat shock. Without such induction of thermotolerance there was negligible effect of ploidy on thermal killing. In contrast in the heat shocked cultures there was an appreciable decrease in thermotolerance as ploidy increased. This difference indicates that the lethal thermal damage in the thermotolerance induced cultures is not totally equivalent to that in cells not given a prior heat shock, and that gene expression changes after heat shock result in a ploidy effect on heat tolerance which is absent from cells in which the heat shock response has not been induced.