We have developed the yeast Kluyveromyces lactis as a host organism for the production of the milk-clotting enzyme chymosin. In contrast to Saccharomyces cerevisiae, we found that this yeast is capable of the synthesis and secretion of fully active prochymosin. Various signal sequences could be used to efficiently direct the secretion of prochymosin in Kluyveromyces, but not in S. cerevisiae. We conclude that the efficient synthetic and secretory capacity of this heterologous protein is a property of the yeast Kluyveromyces. These results have led to the development of a large scale production process for chymosin.

We describe a novel fungal expression system which utilizes the Quorn myco-protein fungus Fusarium graminearum A 3/5. A transformation system was developed for F. graminearum and was used to introduce the coding and regulatory regions of a trypsin gene from Fusarium oxysporum. The protein was efficiently expressed, processed and secreted by the recombinant host strain. In addition, the promoter and terminator of the F. oxysporum trypsin gene have been successfully utilized to drive the expression of a cellulase gene from Scytalidium thermophilum and a lipase gene from Thermomyces lanuginosus in F. graminearum.

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The fructose-l,6-bisphosphatase [Fru(l ,6)P2ase] gene of the budding yeast, Kluyveromyces luctis, was cloned and sequenced. The gene encodes one open reading frame predicting a 354-amino-acid polypeptide. The polypeptide is different from other Fru(l,6)P2ases in that it contains a short amino-acid-insert region close to a basic residue located at the binding site for the allosteric inhibitor AMP. Comparison of the biochemical properties of the K. Zuctis enzyme with its closest homolog, the Succhuromyces cerevisiue Fru(1 ,6)P2ase (74% amino acid identity), reveals that the K. luctis enzyme is significantly less sensitive to AMP (Kl = 540 pM) than the S. cerevisiue enzyme (K,= 190 pM). However, studies with a K. luctis Fru(l,6)Pzase mutant, in which the insert region (amino acids 152-160) was deleted by site-directed mutagenesis [(des-152-160)Fru(l,6)Pzase], showed that the mutant enzyme had higher sensitivity to AMP inhibition (Kl = 280 pM) than the control K. luctis enzyme. Thus, the nine-amino-acid insert region appears to be responsible for the decreased AMP inhibition shown by the K. luctis wild-type enzyme. Catabolite-repression and catabolite-inactivation studies show that, unlike the complete repression of FBPI mRNA and inactivation of enzyme activity by glucose seen in S. cerevisiue, mRNA levels and enzyme activity of K. Zuctis Fru(16)P2ase decreased only about 2-4-fold due to the presence of glucose in the cell-culture medium.