Exploitation of the cellulase enzyme complex of neurospora

Details Exploitation of the cellulase enzyme complex of neurospora Exploitation of the cellulase enzyme complex of neurospora

1996-07-15

1999-09-21

Ketter, James

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

435 691, 4353201, 435197, 530350, 536 231, C12Q 168, C12N 1563, C07K 1300, C07H 2104

Patent

active

059552700

DESCRIPTION:

BRIEF SUMMARY
The invention relates to a method and recombinant means particularly, but not exclusively, expression cassettes and expression/export cassettes for the production of heterologous peptides and the enhanced production of cellulases especially cellobiohydrolase-1. The method and means have particular application in the production of such peptides and enzymes from the biotechnological exploitation of filamentous fungi and particularly Neurospora crassa.
The most abundant cell-wall and structural polysaccharide in the plant world is cellulose. Cellulose is a linear polymer of D-glucose arranged in a Beta 1-4 linkage. Cellulose is a major component of wood and thus of paper, it is also a major component of cotton and other plant materials.
On complete hydrolysis, cellulose is broken down to D-glucose, but partial hydrolysis yields a reducing disaccharide cellobiose in which the linkage between the D-glucose units is a glycosidic Beta 1-4 arrangement. Enzymes capable of hydrolysing cellulose are not secreted in the digestive tract of most mammals and therefore cellulose is not a source of food. However, ruminants can use celluloses as food because in the rumen of their stomachs they house bacteria which produce the enzyme cellulase.
As fossil fuel reserves become depleted, a renewable feed-stock for the chemical industry becomes more significant The obvious renewable resource is cellulose, which is already in embarrassingly large supply and largely wasted. However, the conversion of cellulose to a more readily utilisable substance such as sugars and alcohols is problematical.
Neurospora crassa grows well on cellulosic substrates. In doing so, it secretes enzymes of the cellulase complex, hydrolysing the substrate outside the cell. The resulting soluble sugars may be recovered before they are taken up by the cell and further metabolised. The amount of cellulose/cellobiose typically required to activate gene expression is represented by 1-2% by weight of cellulose/cellobiose.
The cellobiohydrolase-1 enzyme of Neurospora crassa is the major enzyme in the cellulase complex, and one of the major exported proteins of the organism when induced by cellulose or cellobiose (the product of partial hydrolysis of cellulose). Furthermore, Neurospora crassa is a very efficient cellulolytic species, able to hydrolyse cellulose efficiently, and grow on it as the sole carbon source. We have grown it on a range of cellulosic substrates, including pressed-sugar-beet pulp, cereal straw and spent malted grains from breweries. Indeed, Neurospora has been isolated in the wild from burnt sugar cane, and so is likely to grow well on bagasse from sugar cane processing. In addition, it grows very well on starch and a wide range of soluble sugars. Its nitrogen requirement for growth is readily satisfied by the supply of any one of a wide range of nitrogen sources, including protein, amino acids, ammonium ions, nitrate, nitrite, and urea. Its only complex biochemical requirement is for trace amounts of the vitamin biotin.
It follows from the above that genetic manipulation of the gene encoding the promoter and associated enzyme sequence structure for the enzyme cellobiohydrolase-1 will enable us to do a number of things, namely: the copy number of the cbh-1 gene or increasing the strength of the promoter of the cbh-1 gene. Both of these ways could be achieved by transforming in either additional copies or an altered copy of the gene, or possibly both. Thus for example, one could produce by transformation multiple copies of the gene encoding the cellobiohydrolase-1 enzyme so as to increase the level of cellobiohydrolase-1 production. of the cellobiohydrolase promoter thus increasing the level of cellobiohydrolase-1 enzyme. thus ensuring that such gene is transcribed by cellulose- or cellobiose-induction and at the high rate that the enzyme cellobiohydrolase would normally be produced, resulting in the production of high levels of the heterologous gene product
The expression constructs are of the following types; sequences upstream from a multi

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