A defined level of protein disulfide isomerase expression is required for optimal secretion of thaumatin by Aspergillus awamori

Thaumatin, a 22-kDa protein containing eight disulfide bonds, is secreted by the filamentous fungus Aspergillus awamori at levels which are dependent upon the extent of overexpression of protein disulfide isomerase (PDIA). Additional copies of the PDIA-encoding gene pdiA were introduced into a strain of A. awamori that expresses a cassette encoding thaumatin. Transformants with different levels of pdiA mRNA and measured PDIA levels were chosen for examination of the impact that PDIA levels had on thaumatin secretion. The secretion of two native proteins, alpha-amylase and acid phosphatase, was also examined in relation to varying levels of PDIA. Over a range of PDIA levels of 1-8, relative to the native level in strains with just one copy of the pdiA gene, the fraction of alpha-amylase and acid phosphatase in the total secreted protein was unaffected. In contrast, a peak level of thaumatin, about 5-fold higher than in the strain with one copy of pdiA, was found in strains with a relative PDIA level of between two and four. Improved thaumatin production was confirmed in 5-1 fermenters using a strain of A. awamori with six pdiA gene copies, containing 3.2-fold higher levels of PDIA than wild-type strains.     

Secretion of the sweet-tasting protein thaumatin by recombinant strains of Aspergillus niger var. awamori

A recombinant form of the sweet-tasting protein thaumatin has been produced in the filamentous fungus Aspergillus niger var. awamori. Expression cassettes containing a synthetic gene encoding thaumatin II were prepared and used to transform Aspergillus niger var. awamori strain NRRL312. Several fungal strains capable of synthesizing and secreting thaumatin into the culture medium were generated, and their production capabilities were determined, first in shake flasks and later in a laboratory fermentor. We report the expression and secretion of thaumatin in concentrations of 5–7 mg/l. This recombinant thaumatin is sweet. Received: 7 October 1997 / Received revision: 21 November 1997 / Accepted: 21 November 1997     

Production of Fusarium solani f. sp. pisi cutinase in Fusarium venenatum A3/5

Fusarium venenatum A3/5 was transformed using the Aspergillus niger expression plasmid, pIGF, in which the coding sequence for the F. solani f. sp. pisi cutinase gene had been inserted in frame, with a KEX2 cleavage site, with the truncated A. niger glucoamylase gene under control of the A. niger glucoamylase promoter. The transformant produced up to 21 U cutinase l⁻¹ in minimal medium containing glucose or starch as the primary carbon source. Glucoamylase (165 U l⁻¹ or 8 mg l⁻¹) was also produced. Both the transformant and the parent strain produced cutinase in medium containing cutin.     

Overexpression and lack of degradation of thaumatin in an aspergillopepsin A‐defective mutant of Aspergillus awamori containing an insertion in the pep A gene

A gene encoding the sweet-tasting protein thaumatin (tha) with optimized codon usage was expressed in Aspergillus awamori. Mutants of A. awamori with reduced proteolytic activity were isolated. One of these mutants, named lpr66, contained an insertion of about 200 bp in the pepA gene, resulting in an inactive aspergillopepsin A. In vitro thaumatin degradation tests confirmed that culture broths of mutant lpr66 showed only a small thaumatin-degrading activity. A. awamori lpr66 has been used as host strain for thaumatin expression cassettes containing the tha gene under the control of either the cahB (cephalosporin acetylhydrolase) promoter of Acremonium chrysogenum or the gdhA (glutamate dehydrogenase) promoter of Aspergillus awamori. Residual proteolytic activities were repressed by using a mixture of glucose and sucrose as carbon sources and l-asparagine as nitrogen source. Degradation of thaumatin by acidic proteases was prevented by maintaining the pH value at 6.2 in the fermentor. Expression of cassettes containing the gdhA promoter was optimal in ammonium sulfate as nitrogen source, whereas transformants expressing the tha gene from the cahB promoter yielded higher thaumatin levels using l-asparagine as nitrogen source. Under optimal fermentation conditions, yields of 105 mg thaumatin/l were obtained, thus making this fermentation a process of industrial interest. Received: 27 March 2000 / Received revision: 14 June 2000 / Accepted: 18 June 2000     

Efficient expression of a Phanerochaete chrysosporium manganese peroxidase gene in Aspergillus oryzae.

A manganese peroxidase gene (mnp1) from Phanerochaete chrysosporium was efficiently expressed in Aspergillus oryzae. Expression was achieved by fusing the mature cDNA of mnp1 with the A. oryzae Taka amylase promoter and secretion signal. The 3' untranslated region of the glucoamylase gene of Aspergillus awamori provided the terminator. The recombinant protein (rMnP) was secreted in an active form, permitting rapid detection and purification. Physical and kinetic properties of rMnP were similar to those of the native protein. The A. oryzae expression system is well suited for both mechanistic and site-directed mutagenesis studies.     

Analysis of heterologous protein production in defined recombinant Aspergillus awamori strains.

A study was carried out to obtain more insight into the parameters that determine the secretion of heterologous proteins from filamentous fungi. A strategy was chosen in which the mRNA levels and protein levels of a number of heterologous genes of different origins were compared. All genes were under control of the Aspergillus awamori 1,4-beta-endoxylanase A (exlA) expression signals and were integrated in a single copy at the A. awamori pyrG locus. A Northern (RNA) analysis showed that large differences occurred in the steady-state mRNA levels obtained with the various genes; those levels varied from high values for genes of fungal origin (A. awamori 1,4-beta-endoxylanase A, Aspergillus niger glucoamylase, and Thermomyces lanuginosa lipase) to low values for genes of nonfungal origin (human interleukin 6 and Cyamopsis tetragonoloba [guar] alpha-galactosidase). With the C. tetragonoloba alpha-galactosidase wild-type gene full-length mRNA was even undetectable. Surprisingly, small amounts of full-length mRNA could be detected when a C. tetragonoloba alpha-galactosidase gene with an optimized Saccharomyces cerevisiae codon preference was expressed. In all cases except human interleukin 6, the protein levels corresponded to the amounts expected on basis of the mRNA levels. For human interleukin 6, very low protein levels were observed, whereas relatively high steady-state mRNA levels were obtained. Our data suggest that intracellular protein degradation is the most likely explanation for the low levels of secreted human interleukin 6.     

Effects of pre- and pro-sequence of thaumatin on the secretion by Pichia pastoris

Thaumatin is a 22-kDa sweet-tasting protein containing eight disulfide bonds. When thaumatin is expressed in Pichia pastoris using the thaumatin cDNA fused with both the alpha-factor signal sequence and the Kex2 protease cleavage site from Saccharomyces cerevisiae, the N-terminal sequence of the secreted thaumatin molecule is not processed correctly. To examine the role of the thaumatin cDNA-encoded N-terminal pre-sequence and C-terminal pro-sequence on the processing of thaumatin and efficiency of thaumatin production in P. pastoris, four expression plasmids with different pre-sequence and pro-sequence were constructed and transformed into P. pastoris. The transformants containing pre-thaumatin gene that has the native plant signal, secreted thaumatin molecules in the medium. The N-terminal amino acid sequence of the secreted thaumatin molecule was processed correctly. The production yield of thaumatin was not affected by the C-terminal pro-sequence, and the pro-sequence was not processed in P. pastoris, indicating that pro-sequence is not necessary for thaumatin synthesis.     

Synthesis and processing of the plant protein thaumatin in yeast

Various maturation forms of the plant protein thaumatin were expressed in yeast, using a promoter fragment of the glyceraldehyde-3P-dehydrogenase (GAPDH) gene. Plasmids encoding preprothaumatin were shown to direct the synthesis of a processed form of the plant protein. The important role of signal sequences in the expression of the plant protein in yeast was indicated by the observation that plasmids encoding processed thaumatin forms were only poorly expressed, if at all. Nucleotide sequence analysis of the 843 nucleotide GAPDH promoter fragment revealed a characteristic structure with two regions of dyad symmetry containing translational starts of GAPDH and a putative 38 amino acid peptide. A promoter fragment from which the upstream region was deleted proved to be less efficient in thaumatin expression.     

Transgenic tomato plants as supersweet protein thaumatin II producers

Yalf tomato plants have been transformed with a gene for thaumatin II from Thaumatococcus daniellii Benth. The nucleotide sequence for thaumatin II cDNA was cloned in the pBI121 vector under the control of the CaMV 35S promoter of cauliflower mosaic virus. Expression of the thaumatin II gene was detected in all of the studied transgenic lines. A quantitative estimation of the thaumatin II accumulation in fruits was performed by ELISA. The highest content of thaumatin in transgenic tomato fruits (line 91) was 46.4 ± 10.5 μg/mg of total soluble protein (4.6%). In the other studied lines, the thaumatin content ranged from 17.6 ± 6.1 to 41.3 ± 12.3 μg/mg of total soluble protein (1.8–4.1%). The fruits of transgenic plants had a well-defined sweet taste with a long aftertaste typical of thaumatin II. Transgenic tomato lines with high expression levels can be potentially used as producers of thaumatin for the food and pharmaceutical industries.     

Novel secretion system of recombinant Saccharomyces cerevisiae using an N-terminus residue of human IL-1 beta as secretion enhancer.

An N-terminus sequence of human interleukin 1beta (hIL-1beta) was used as a fusion expression partner for the production of two recombinant therapeutic proteins, human granulocyte-colony stimulating factor (hG-CSF) and human growth hormone (hGH), using Saccharomyces cerevisiae as a host. The expression cassette comprised the leader sequence of killer toxin of Kluyveromyces lactis, the N-terminus 24 amino acids (Ser5-Ala28) of mature hIL-1beta, the KEX2 dibasic endopeptidase cleavage site, and the target protein (hG-CSF or hGH). The gene expression was controlled by the inducible UAS(gal)/MF-alpha1 promoter. With the expression vector above, both recombinant proteins were well secreted into culture medium with high secretion efficiencies, and especially, the recombinant hGH was accumulated up to around 1.3 g/L in the culture broth. This is due presumably to the significant role of fused hIL-1beta as secretion enhancer in the yeast secretory pathway. In our recent report, various immunoblotting analyses have shown that the presence of a core N-glycosylation resident in the hIL-1beta fragment is likely to be of crucial importance in the high-level secretion of hG-CSF from the recombinant S. cerevisiae. When the N-glycosylation was completely blocked with the addition of tunicamycin to the culture, the secretion of hG-CSF and hGH was decreased to a negligible level although the other host-derived proteins were well secreted to the culture broth regardless of the presence of tunicamycin. The N-terminal sequencing of the purified hG-CSF verified that the hIL-1beta fusion peptide was correctly removed by in vivo KEX2 protease upon the exit of fusion protein from Golgi complex. From the results presented in this article, it is strongly suggested that the N-terminus fusion of the hIL-1beta peptide could be utilized as a potent secretion enhancer in the expression systems designed for the secretory production of other heterologous proteins from S. cerevisiae.     

Creation of a heterologous gene expression system on the basis of Aspergillus awamori recombinant strain

A heterologous gene expression system was created in a domestic Aspergillus awamori Co-6804 strain, which is a producer of the glucoamylase gene. Vector pGa was prepared using promoter and terminator areas of the glucoamylase gene, and A. niger phytase, Trichoderma reesei endoglucanase, and Penicillium canescens xylanase genes were then cloned into pGa vector. Separation of enzyme samples using FPLC showed the amount of the recombinant proteins to be within the 0.6-14% range of total protein.     

Kinetics of mRNA and protein synthesis of genes controlled by the 1,4-beta-endoxylanase A promoter in controlled fermentations of Aspergillus awamori.

In this study, induction and repression kinetics of the expression of the Aspergillus awamori 1,4-beta-endoxylanase A (exlA) gene under defined physiological conditions was analyzed at the mRNA and the protein levels. Induction was analyzed by pulsing D-xylose to a sucrose-limited continuous culture of an A. awamori 1,4-beta-endoxylanase A (EXLA)-overproducing strain. Directly after the D-xylose pulse, exIA mRNA was synthesized, and it reached a constant maximal level after 45 to 60 min. This level was maintained as long as D-xylose was present. The kinetics of mRNA synthesis of the genes encoding Thermomyces lanuginosa lipase (lplA) and Escherichia coli beta-glucuronidase (uidA), which were also under the control of the exlA promoter, were similar to those observed for exlA mRNA. The repression of exlA expression was analyzed by pulsing D-glucose to a D-xylose-limited continuous culture. Immediately after the glucose pulse, the exlA mRNA level declined rapidly, with a half-life of approximately 20 to 30 min, and it reached a minimal level after 60 to 90 min. The time span between mRNA synthesis and the secretion of proteins was determined for EXLA and lipase. In both cases, mRNA became visible after approximately 7.5 min. After 1 h, both proteins became detectable in the medium but the rate of secretion of EXLA was faster than that of lipase.     

Construction of yeast secretion vectors designed for production of mature proteins using the signal sequence of yeast invertase

Summary Two kinds of yeast secretion vectors were constructed by site-directed mutagenesis of the invertase signal sequence and ligation of synthetic oligonucleotides coding appropriate signals. Each has a cloning site for a foreign gene preceded by a sequence encoding either the signal peptide cleavage site or a Lys-Arg sequence which is a cleavage site for the product of the KEX2 gene. Both vectors were able to direct the expression and secretion of mouse amylase. One of them has a SalI site within the signal sequence, and an attempt to clone sequences enhancing secretion of amylase with this vector is reported.