Construction of a flocculent brewer's yeast strain secreting Aspergillus niger beta-galactosidase.

One way of improving heterologous protein production is to use high cell density systems, one of the most attractive being the flocculating yeast production system. Also, lactose is available in large amounts as a waste product from cheese production processes. The construction of flocculent and non-flocculent brewer's yeast strains secreting beta-galactosidase and growing on lactose is presented. A plasmid was constructed coding for an extracellular beta-galactosidase of Aspergillus niger and having, as selective marker, the yeast CUP1 gene conferring resistance to copper. This selective marker allows for the transformation of wild-type yeasts. This work represents an important step towards the study of heterologous protein secretion by flocculent cells.

Regulation of cellulase gene expression in the filamentous fungus Trichoderma reesei.

Basic features of regulation of expression of the genes encoding the cellulases of the filamentous fungus Trichoderma reesei QM9414, the genes cbh1 and cbh2 encoding cellobiohydrolases and the genes egl1, egl2 and egl5 encoding endoglucanases, were studied at the mRNA level. The cellulase genes were coordinately expressed under all conditions studied, with the steady-state mRNA levels of cbh1 being the highest. Solka floc cellulose and the disaccharide sophorose induced expression to almost the same level. Moderate expression was observed when cellobiose or lactose was used as the carbon source. It was found that glycerol and sorbitol do not promote expression but, unlike glucose, do not inhibit it either, because the addition of 1 to 2 mM sophorose to glycerol or sorbitol cultures provokes high cellulase expression levels. These carbon sources thus provide a useful means to study cellulase regulation without significantly affecting the growth of the fungus. RNA slot blot experiments showed that no expression could be observed on glucose-containing medium and that high glucose levels abolish the inducing effect of sophorose. The results clearly show that distinct and clear-cut mechanisms of induction and glucose repression regulate cellulase expression in an actively growing fungus. However, derepression of cellulase expression occurs without apparent addition of an inducer once glucose has been depleted from the medium. This expression seems not to arise simply from starvation, since the lack of carbon or nitrogen as such is not sufficient to trigger significant expression.

Functional analysis of the cellobiohydrolase I promoter of the filamentous fungus Trichoderma reesei.

Functional analysis of the cellulase promoter cbh1 of the filamentous fungus Trichoderma reesei was carried out using the Escherichia coli lacZ gene as a reporter. An assay based on cultivation on solid medium in microtiter plates was developed that allows rapid and reliable semiquantitative analysis of beta-galactosidase expression of a large number of transformants. A series of deletions and specifically designed alterations were made covering 2.2 kb of the cbh1 promoter. Removal of sequences upstream of nucleotide -500 in relation to the initiator ATG abolished glucose repression. Mutation of a single hexanucleotide sequence 5'GTGGGG at nucleotide -720 was sufficient for derepression. This site is similar to the binding sites of the glucose repressors MIG1 of Saccharomyces cerevisiae and CREA/CREI of filamentous fungi. Removal of the glucose repressor site did not affect sophorose induction. Sophorose induction of the promoter was retained even in deletion derivatives lacking sequences upstream of position -161, which retained about 70 bp upstream of the transcription start point and only 30 bp upstream of the TATA box.

Use of a modified alcohol dehydrogenase, ADH1, promoter in construction of diacetyl non-producing brewer's yeast.

The bacterial gene, encoding alpha-acetolactate decarboxylase (alpha-ALDC), was expressed in a bottom-fermenting brewer's yeast under the control of a modified Saccharomyces cerevisiae alcohol dehydrogenase (ADH1) promoter which lacks the upstream regions from -800 bp to -1500 bp. In pilot scale brewing conditions, the level of alpha-ALDC produced was high enough to reduce the concentration of diacetyl so that lagering was not required. alpha-ALDC active brewer's yeast strains were also shown to be suitable for high gravity brewing.

Molecular cloning and analysis of the yeast flocculation gene FLO1.

The DNA sequence of the flocculation gene FLO1 of Saccharomyces cerevisiae, which is located on chromosome I (Watari et al., 1989) was determined. The sequence contains a large open reading frame (ORF) of 2586 bp and codes for a protein of 862 amino acids. However, further study (genomic Southern and polymerase chain reaction analyses) indicated that the gene we cloned was not the intact FLO1 gene but a form with an approximately 2 kb deletion in the ORF region. The intact FLO1 gene was then cloned and its nucleotide sequence determined. The sequence revealed that the ORF of the intact gene is composed of 4611 bp which code for a protein of 1537 amino acids. A remarkable feature of the putative Flo1 protein is that it contains four families of repeated sequences composed of 18, 2, 3 and 3 repeats and that it has a large number of serines and threonines. In the deleted FLO1 form, a large part of these repeated sequences was missing. The N- and C-terminal regions are hydrophobic and both contain a potential membrane-spanning region, suggesting that the Flo1 protein is an integral membrane protein and a cell wall component.

Characterization of an intronless collagen gene family in the marine sponge Microciona prolifera.

Two independent clones from the genomic DNA of a marine sponge Microciona prolifera were isolated by hybridization to the Caenorhabditis elegans Col-1 gene and one clone was obtained from genomic DNA by PCR. They contain open reading frames (MpCol1, MpCol2, MpCol3, MpCol4) capable of coding for a family of collagens different from those previously found in sponges. Southern blotting of genomic DNA suggested the presence of several other homologous genes. cDNA clones covering most of the triple-helical coding domain and the 3' untranslated region of MpCol1 were isolated by specific primers and reverse PCR. Two cDNA clones end in the middle of an AATAAA sequence 170 bp downstream from the translation stop codon of MpCol1. The putative NH2-terminal noncollagenous peptide is composed of only seven amino acid residues. The 1074-bp triple-helical coding region is not interrupted by intervening sequences. It codes for a polypeptide of 120 Gly-Xaa-Yaa triplets with only one short interruption near the COOH terminus. A putative N-glycosylation sequence (Asn-Gly-Ser), three Arg-Gly-Asp triplets known as cell recognition peptides, frequent Lys residues in the Yaa position (which are templates for hydroxylation), several Lys-Gly-Asn/Xaa-Arg peptides known as the lysyl oxidase recognition site, and long stretches without imino acids could be found within the triple-helical domain. The short COOH-terminal noncollagenous domain closely resembles that of nematode cuticular collagens and vertebrate nonfibrillar collagens. Our results strongly support the idea that the diversity of collagen genes and gene families found in higher organisms already existed in sponge.