Expression of an alpha-galactosidase gene under control of the homologous inulinase promoter in Kluyveromyces marxianus.

For expression of the alpha-galactosidase gene from Cyamopsis tetragonoloba in Kluyveromyces marxianus CBS 6556 we have used the promoter of the homologous inulinase-encoding gene (INU1). The INU1 gene has been cloned and sequenced and the coding region shows an identity of 59% with the Saccharomyces cerevisiae invertase gene (SUC2). In the 5'-flanking region of INU1 we found a sequence (TAAATCCGGGG) that perfectly matches to the MIG1 binding consensus sequence (WWWWTSYGGGG) of the S. cerevisiae GAL1, GAL4 and SUC2 genes. Using the K. marxianus INU1 promoter and prepro-signal sequence, we obtained a high alpha-galactosidase production level (153 mg/l) and a secretion efficiency of 99%. Both the production level and the secretion efficiency were significantly reduced when the INU1 pro-peptide was deleted. With either the S. cerevisiae PGK or GAL7 promoter we could obtain only low alpha-galactosidase production levels (2 mg/l).

Cloning and sequencing of the URA3 gene of Kluyveromyces marxianus CBS 6556.

The URA3 gene, coding for orotidine-5'-phosphate decarboxylase, from Kluyveromyces marxianus CBS 6556, was isolated from a genomic DNA library. The K. marxianus URA3 gene encodes a protein of 267 amino acids with a calculated molecular weight of 29.3 kDa. Comparison of the K. marxianus protein with the corresponding enzymes of Saccharomyces cerevisiae and Kluyveromyces lactis showed amino acid sequence identities of 81% and 88%, respectively. Using contour-clamped homogenous electric field gel electrophoresis, the genomic copy was found to be located on chromosome VI. We have used the cloned gene for the construction of a K. marxianus leu2 mutant. This mutant contains no heterologous sequences, which is essential to make it acceptable for application in the food industry.

Effect of a pmr 1 disruption and different signal sequences on the intracellular processing and secretion of Cyamopsis tetragonoloba alpha-galactosidase by Saccharomyces cerevisiae.

We fused the yeast-derived sequences encoding the invertase, acid phosphatase and alpha-factor pre- and prepro-signal peptides (SP) to the Cyamopsis tetragonoloba (guar plant) alpha-galactosidase(alpha Gal)-encoding gene and expressed these gene fusions in yeast. Whereas the amount of fusion protein produced by each of the constructs did not vary significantly, the secretion efficiency of the fusion protein that carried the SP of the prepro-alpha-factor (MF alpha 1) was consistently found to be about 10% higher than that of the other fusions (99% vs. 90%). Furthermore, when the secretion of alpha Gal was directed by the invertase (SUC2) SP, the intracellular enzyme localized to the endoplasmic reticulum (ER), whereas use of the MF alpha 1 SP caused the intracellular enzyme to be outer-chain-glycosylated and processed by the KEX2 endoproteinase, implying that it had passed the ER. These results suggest that the pro-peptide of MF alpha 1 stimulates the efflux of the heterologous protein from the ER. Null mutants of PMR1 (encoding a Ca(2+)-dependent ATPase) are known to give higher secretion efficiencies for a number of different heterologous proteins. Therefore, we also studied the secretion of alpha Gal in a pmr 1 disruption mutant. Structural analysis of the enzyme secreted by the mutant cells showed that it was completely processed by KEX2 and outer-chain-glycosylated, although the length of the outer-chain carbohydrate moiety was reduced when compared with the enzyme secreted by wild-type cells. These results contradict the hypothesis advanced by Rudolph et al. [Cell 58 (1989) 133-145] that disruption of PMR1 causes the secretory pathway to bypass the Golgi apparatus.

Multiple-copy integration of the alpha-galactosidase gene from Cyamopsis tetragonoloba into the ribosomal DNA of Kluyveromyces lactis.

We have developed a vector system for high-copy-number integration into the ribosomal DNA of the yeast Kluyveromyces lactis. This system is analogous to the pMIRY-system developed for Saccharomyces cerevisiae. Plasmids containing a portion of K. lactis rDNA for targeted homologous recombination, as well as the S. cerevisiae TRP1 gene with various promoter deletions, were constructed and, after transformation to K. lactis, analyzed for both copy number and stability. These plasmids were found to be present in about 60 copies per cell and were stably maintained during growth under non-selective conditions. Using this vector system, we expressed a fusion construct containing the S. cerevisiae GAL7 promoter, the SUC2 (invertase) signal sequence and the gene coding for alpha-galactosidase from the plant Cyamopsis tetragonoloba. Although the maximum copy number of these integrated plasmids was only about 15, we nevertheless obtained a high level of alpha-galactosidase production (250 mg/l) with a secretion efficiency of about 95%. When compared to extrachromosomal K. lactis vectors containing the same fusion construct, the multicopy integrants showed a much higher alpha-galactosidase production level and a considerably higher stability under non-selective conditions.

Cloning and nucleotide sequence of the Escherichia coli K-12 ppsA gene, encoding PEP synthase.

We have cloned and sequenced the Escherichia coli K-12 ppsA gene. The ppsA gene codes for PEP synthase, which converts pyruvate into phosphoenolpyruvate (PEP), an essential step in gluconeogenesis when pyruvate or lactate are used as a carbon source. The open reading frame consists of 792 amino acids and shows homology with other phosphohistidine-containing enzymes that catalyze the conversion between pyruvate and PEP. These enzymes include pyruvate, orthophosphate dikinases from plants and Bacteroides symbiosus and Enzyme I of the bacterial PEP:carbohydrate phosphotransferase system.

Cloning and disruption of the LEU2 gene of Kluyveromyces marxianus CBS 6556.

The LEU2 gene, coding for beta-isopropylmalate dehydrogenase, of the yeast Kluyveromyces marxianus was isolated and sequenced. An open reading frame, coding for a protein with a molecular weight of 38 kDa was found. Comparison of the deduced amino acid sequence of the LEU2 gene with the corresponding enzymes of three other yeasts and two thermophilic bacteria, revealed extensive sequence similarities. The cloned gene could complement a leuB mutation of Escherichia coli and a leu2 mutation of Saccharomyces cerevisiae. Using orthogonal field alternation gel electrophoresis, the genomic copy of the gene was found to be located at chromosome VI or VII. Analysis of the 5'-untranslated region indicated the presence of a putative binding site for the LEU3 protein, which is involved in the leucine-specific regulation of transcription. We show that the cloned gene can be used for the construction of a non-reverting K. marxianus leu2 mutant.

The repressor of the PEP:fructose phosphotransferase system is required for the transcription of the pps gene of Escherichia coli.

We have cloned the pps gene, coding for PEP synthase, of Escherichia coli. PEP synthase catalyses the ATP-dependent conversion of pyruvate into phosphoenol-pyruvate and is required for gluconeogenesis. The pps gene was cloned by an in vivo cloning method using a mini-Mulac bacteriophage containing a plasmid replicon. Upon expression of the cloned pps gene in the maxicell system a protein with an apparent molecular weight of 84 kDa was synthesized. The position of the pps gene of the plasmid was localized by restriction analysis of isolated transposon insertions and the determination of the PEP synthase activities of the different clones. An operon fusion between the pps gene and the galK gene was constructed. Measurements of the galactokinase activity in Salmonella typhimurium galK and galK fruR mutants showed that the transcription of the pps gene requires the presence of FruR, the repressor of the PEP: fructose phosphotransferase system (PTS) in E. coli and S. typhimurium. To test whether the components of the Fructose PTS, in particular FPr, are involved in the expression of the pps gene, we investigated a S. typhimurium galK strain, containing the fusion plasmid, in which the chromosomal fru operon was inactivated by a transposon insertion. Measurements of the galactokinase activity showed that the absence of the Fructose PTS proteins has no significant influence on the regulation of the pps gene.

The PEP: fructose phosphotransferase system in Salmonella typhimurium: FPr combines enzyme IIIFru and pseudo-HPr activities.

We have cloned the fru operon of Salmonella typhimurium, coding for the enzymes of the phosphoenolpyruvate: fructose phosphotransferase system (Fructose PTS). The fruFKA operon consists of three genes: fruF coding for FPr, fruK for fructose 1-phosphate kinase and fruA for Enzyme IIFru. Insertions of Tn5 in the different genes were isolated and the activities of the gene products were measured. Expression of the plasmid-encoded fru operon in the maxicell system resulted in the synthesis of three proteins with molecular weights of 47 kDa (fruA), 39 kDa (fruF) and 32 kDa (fruK). We have sequenced the fruF gene and the regulatory region of the fru operon. In contrast to previously published results, we have found that the fruF gene codes for a 39 kDa protein, FPr, that combines Enzyme IIIFru and pseudo-HPr activities. The N-terminal part of FPr is homologous to the cytoplasmic domain of the Escherichia coli Enzyme IIMtl, as well as several Enzymes IIIMtl from gram-positive bacteria. The C-terminal domain shows homology to HPr of E. coli and several gram-positive organisms. The fru operon is regulated by a repressor, FruR. We have constructed an operon fusion between fru and the galK gene and shown that regulation of the fru operon by FruR takes place at the level of transcription.

Relationship between pseudo-HPr and the PEP: fructose phosphotransferase system in Salmonella typhimurium and Escherichia coli.

We have studied in Salmonella typhimurium and Escherichia coli the properties of pseudo-HPr suppressor mutations. These mutations suppressed the defects in a ptsH mutant which lacks HPr, one of the enzymes of the phosphoenolpyruvate: carbohydrate phosphotransferase system. The suppressor mutation was mapped in S. typhimurium at 3 min, closely linked to leu. The corresponding chromosomal fragment of 1.7 kb from S. typhimurium and E. coli (extending clockwise from ilvH) was cloned. In a maxicell system a protein with an approximate molecular weight of 36,000 was synthesized. Pseudo-HPr suppressor mutations (fruR) and a deletion extending clockwise from leu resulted in the constitutive expression of the fru operon containing the genes for IIFru (fruA), IIIFru (fruB), fructose 1-phosphate kinase (fruK) and pseudo-HPr (fruF). fruR probably codes for a repressor of the fru operon. Tn10 mutagenesis revealed the following order of genes in the fru operon: fruB-(fruK, fruF)-fruA. Pseudo-HPr activity could replace HPr in PEP-dependent phosphorylation of PTS carbohydrates. IIIFru could be phosphorylated both via HPr and pseudo-HPr, since mutants lacking pseudo-HPr activity were still able to phosphorylate fructose in the presence of added HPr. Both the pseudo-HPr suppressor mutations at 3 min and the deletion extending from leu had an additional phenotype. Introduction of these mutations or deletions was always accompanied by disappearance of PEP synthase activity. Complementation of such a mutant with the cloned fragments reversed both phenotypes at the same time. Possibly, the fruR gene product acts as an activator of the gene coding for PEP synthase.

Analysis of regulatory sequences upstream of the E. coli uvrB gene; involvement of the DnaA protein.

A region located upstream of the uvrB promoters P1 and P2 was found to cause high plasmid loss when cloned in multicopy vectors. Two sequence elements responsible for this phenomenon were identified by mapping of spontaneous mutations that restore plasmid maintenance: a sequence known to have in vitro promoter activity and a partially overlapping sequence that shows extensive homology to recognition sites for the DnaA protein. Accordingly alterations in the level of DnaA protein in vivo were found to affect the extent of plasmid loss. A possible role for interaction of the DnaA protein with the region of interest is discussed in relation to regulation of uvrB expression.

In vivo transcription of the E. coli uvrB gene: both promoters are inducible by UV.

The transcriptional activity of the tandem promoters of the Escherichia coli uvrB gene was measured in vivo. Both promoters are shown to be inducible by UV irradiation. P1, the most proximal promoter, is responsible for the main part of transcription both in uninduced and induced cells. Plasmids have been constructed carrying small deletions in the lexA binding site that overlaps with P2, the distal promoter. These deletions result in constitutive transcription from P1. This indicates that the DNA region which contains P2 functions mainly as a target site for regulation of P1 transcription in vivo.