Expression cloning of fungal enzyme genes; a novel approach for efficient isolation of enzyme genes of industrial relevance

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Abstract

Expression cloning is a relatively new method for fast and efficient cloning of enzyme genes from fungi that are known to make complex enzyme mixtures. In contrast to traditional cloning methods that are usually dependent on knowledge of at least a partial amino acid sequence in order to synthezise appropriate DNA probes or primers, the expression cloning method solely relies on access to reliable and sensitive enzyme assays. A representative expression cDNA library is made in Saccharomyces cerevisiae from the donor strain and relevant cDNA clones are detected directly based on the encoded enzyme activity. Thus, time-consuming enzyme purification and characterization steps are avoided. The method has been applied on the characterization of extracellular enzyme genes from the filamentous fungus Aspergillus aculeatus and has resulted in the isolation of 20 different enzyme genes such as endo-glucanases, xylanases, pectinases, proteases, hemicellulases and rhamnogalacturonan-degrading enzymes. All enzymes have been expressed in Aspergillus oryzae, purified and characterized. In the present review a description of the expression cloning technique will be given as well as examples of how the technique has been used in the exploration and characterization of a commercial enzyme product that is known to consist of a complex mixture of more than 25 different enzyme activities.

Introduction

Filamentous fungi have been used for more than 50 years in the production of industrial enzymes. However, most fungi are known to produce several enzymes simultaneously and the classic product is therefore most often a mixture of different enzymes that `contaminate' the one enzyme of interest (Fig. 1). Although the enzyme mixture can be used in certain applications and can give a significant effect, the complex nature of the enzyme mixture has several disadvantages. First, it is impossible to obtain any detailed knowledge of the action of the enzyme mixture: an effect is seen, but which enzyme is responsible or necessary for what cannot be elucidated. Second, a reasonable production economy can be difficult to obtain, as it may be impossible to optimize the production of a specific enzyme without knowing the target gene. Third, without understanding the effect and action of the enzyme/enzymes it is difficult to develop new and improved enzymes.

As a consequence, more and more enzymes are produced by recombinant technology. The recombinant enzymes can be produced in high yields thus providing new tools for functional studies through careful selection of the expression system in substantially higher purity. However, although the number of recombinant enzyme products on the market is increasing the introduction of cloned products is hampered by the relatively time-consuming standard cloning process, based on enzyme purification, amino acid sequence determination and subsequent probing of libraries with DNA probes. Recently a new method for fast and efficient isolation of enzyme genes from filamentous fungi was described [1]. The method combines the ability of Saccharomyces cerevisiae to express heterologous genes with the utilization of sensitive and reliable enzyme assays. A cDNA library is constructed in a S. cerevisiae/E. coli shuttle vector in E. coli from the fungi of interest. Plasmid DNA is isolated from sub-pools of the libraries and transformed into S. cerevisiae. Next the yeast transformants are replicated onto sets of agar plates containing appropriate enzyme substrates that allow detection of enzyme activity. After a subsequent characterization of the clones by DNA sequence analysis a representative cDNA for each enzyme is sub-cloned in an Aspergillus vector and expressed in high levels in A. oryzae.

Section snippets

A classic enzyme product

One of the first industries to benefit from the use of enzymes was the juice industry where the enzyme technology was first introduced in the 1930's [2]. An extensive use of enzymes, especially pectinases has since followed. A classic pectinase enzyme, or rather enzyme mixture as it consists of many different enzymes (Fig. 1), used in the juice processing, is derived from the filamentous fungus Aspergillus. The pectinases reduce the viscosity of the juice and cause precipitation of the pectic

The principles of expression cloning

The method is based on isolation of enzyme genes from fungi by combining the ability of S. cerevisiae to express heterologous genes with the utilization of sensitive and reliable enzyme assays. The overall principles of expression cloning are outlined in Fig. 3Fig. 4. The donor strain is propagated in a complex media which can induce as many as possible of the enzyme activities of interest. On the precise day of maximal enzyme production the mycelium is harvested and used for mRNA isolation. A

The three key factors of importance for efficient expression cloning

The three main factors to consider in order to establish an efficient expression cloning system are the synthesis of cDNA, the enzyme assays and the expression of the enzymes in yeast.

Expression cloning from Aspergillus aculeatus

From A. aculeatus 20 enzymes have been cloned by expression cloning (see Table 4). All the enzymes have been expressed in A. oryzae, purified and characterized with respect to molecular mass, pI, pH, temperature optimum and stability, substrate specificity, Km and Vmax and specific activity. Below a more detailed description of the applied assays and the isolated genes will be given.

Pectin methyl esterase; a new enzyme for industrial applications.

The smooth regions in pectin can be methyl-esterified to a varying degree and the degree of methylation has a significant impact on the physico-chemical and thus gel-forming properties of the pectin. Un-methylated pectin is able to form gels in the presence of Ca2+ ions and is being extensively used in the food industry as a gelling or conditioning agent. As pectin methyl esterases hydrolyze the ester linkage between methanol and galacturonic acid in esterified pectin, an improvement of the

Conclusion

The expression cloning method has contributed significantly to an increase in the speed with which new enzymes can be cloned and thus be produced in a pure form in large quantities. During the last few years expression cloning has been applied on several fungi and has proven very efficient in the cloning of enzyme genes 1, 8, 9, 12, 13and more than 150 different fungal enzyme genes such as arabinanases, endo-glucanases, galactanases, mannanases, polygalacturonases, pectin lyases, pectin methyl

Acknowledgements

I would like to thank all my colleagues from GeneExpress and Enzyme Development and Applications at Novo Nordisk for their significant scientific contributions and excellent collaboration, without which this work would not have been possible. Furthermore I wish to thank Janice B.S. Kongsbak for proofreading of this manuscript.

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