Cloning for new products
Filamentous fungi have been used for over 50 years in the production of industrial enzymes. Most fungi, however, produce several enzymes simultaneously and the classical enzyme products are thus often mixtures of different enzymes in addition to the enzyme of interest. Although these mixtures can be used in many applications, monocomponent enzymes produced by recombinant DNA technology are now being introduced to the market.
For the past years a considerable effort has been made within Novozymes to build up a microbial screening and cloning platform focusing on the utilization of nature?s biodiversity in the discovery of new industrial enzymes. This has triggered the research scientists within Novozymes to undertake the cloning of a large number of microbial enzymes, in attempts to create new products with potential industrial interest. Recent developments in molecular techniques, especially expression cloning and molecular screening by PCR, have significantly contributed to the efficiency and accuracy by which the new enzymes have been discovered.
Expression cloning of fungal enzyme cDNAs combines the ability of baker?s yeast (Saccharomyces cerevisiae) to express heterologous (fungal) enzyme genes with the utilization of sensitive, simple and reliable enzyme assays. The fungus of interest is fermented under conditions that give high-level production of the desired enzyme(s), poly(A)+RNA is subsequently isolated from fungal mycelium and used to construct a cDNA expression library in a yeast or a fungal expression vector. Plasmid DNA isolated from subpools of the library is transformed into the yeast and the relevant cDNA clones are detected and isolated directly based on the encoded enzyme activity. After characterization and classification of the cDNAs by sequencing, a representative cDNA
for each enzyme is subcloned into e.g. an Aspergillus expression vector for high-level production of the monocomponent enzyme in e.g. Aspergillus oryzae.
Key factors for success
The key factors for establishing a successful expression cloning system are: (1) synthesis of full-length cDNA; (2) development of selective and reliable enzyme assays, and (3) functional expression of the enzymes in yeast. The technique has several advantages compared to standard cloning methods. First, it is totally independent of any knowledge of the enzymes to be cloned. The time consuming enzyme purification, characterization and amino acid sequence determination steps can thus be omitted. Second, the method allows simultaneous screening of the cDNA library for many different enzymes simply by making several replicas of the yeast-containing agar plates and applying different screening assays. Within the last few years, expression cloning in yeast, and recently also in Aspergillus, has been extensively used at Novozymes, resulting in the cloning of more than 300 enzyme genes from a number of different fungi.
An expanding inventory of cloned enzyme genes along with the complete genetic blueprints for several organisms, spanning all three domains of life; prokaryotes, Archea, and eukaryotes, has rapidly increased the available sequence information for a large selection of enzymes and enzyme families.