Analysis of the role of the gene bipA, encoding the major endoplasmic reticulum chaperone protein in the secretion of homologous and heterologous proteins in black Aspergilli

The function of the endoplasmic-reticulum-localized chaperone binding protein (BiP) in relation to protein secretion in filamentous fungi was studied. It was shown that the overproduction of several homologous and heterologous recombinant proteins by Aspergillus strains induces the expression of bipA, the BiP-encoding gene from Aspergillus niger and Aspergillus awamori. As this result could imply that BiP plays a role in protein overproduction, the effect of modulation of bipA gene expression on protein secretion was studied in several recombinant strains expressing glucoamylase (glaA) fusion genes. For overproduction of BiPA in these strains, extra copies of the bipA gene under the control of an inducible promoter were introduced. To allow analysis of the effect of a decreased bipA expression level on protein secretion, replacement of the wild-type gene for a bipA gene driven by the glaA promoter was attempted. However, this endeavour failed because of the lethality of this replacement. Although the final amount of secreted recombinant protein did not change significantly in strains with increased BiPA levels, increased levels of unprocessed fusion protein were detected in the total protein extracts of these strains. Received: 9 February 1998 / Received last revision: 26 May 1998 / Accepted: 14 June 1998     

Enhancing granulocyte colony-stimulating factor expression in Pichia pastoris through fusion with human serum albumin

Protein fusion technology has emerged as one of the important strategies to increase the level of expression and half-life of therapeutic proteins in heterologous expression systems. Granulocyte colony-stimulating factor (G-CSF) is a hematopoietic growth factor and is clinically used against neutropenia. Enhanced expression and stability of G-CSF were achieved in Pichia pastoris by the way of constructing a fusion protein with human serum albumin (HSA). The strategy involved polymerase chain reaction (PCR) amplification of fragments corresponding to codon-optimized G-CSF and domain 3 of HSA. Overlapping PCR was used to obtain the full-length fused gene (1,184?bp) with a 15-bp linker sequence comprising of 4 Gly and 1 Ser residues. Extracellular expression was carried out downstream of α-factor secretion signal sequence under the control of alcohol oxidase 1 promoter using pPICZαB. Excreted protein in the range of 110–380?mg?L^?1 was observed among the transformants. Effect of aeration and temperature was investigated in one of the transformants (35) overexpressing fusion protein and levels of G-CSF enhanced by 1.8-fold and 2.3-fold, respectively. Assay of biological activity indicated the fusion protein to retain similar cell proliferation activity as the commercial G-CSF preparation.     

Overexpression of barley <Emphasis Type="Italic">hva1</Emphasis> gene in creeping bentgrass for improving drought tolerance

The objectives of this study were to test the feasibility of introducing barley hva1 gene, a LEA3 member, into perennial grass species using the Agrobacterium-mediated transformation technique and to determine whether heterologous expression of hva1 would alleviate water-deficit injury in grass species. Creeping bentgrass (Agrostis stolonifera var. palustris), a drought-intolerant grass species, was transformed transiently or stably using three different promoters in conjunction with the downstream report/target genes. Two abscisic acid (ABA)-inducible promoters, ABA1 and ABA2 derived from ABA-response complex (ABRC3) were used to examine stress-responsive expression of the green fluorescent protein (GFP). Transient expression of GFP demonstrated the inducibility of ABA1 and ABA2 promoters in response to exogenous ABA application. The ABA2 promoter was further studied for stress-responsive expression of hva1 and a maize Ubi-1 promoter was tested for constitutive expression of the gene. In the T₀ generation, the Ubi-1::hva1 transformants displayed variable expression levels of HVA1 protein under normal growth conditions. The hva1 gene in the ABA2::hva1 transformants maintained low expression under well-watered conditions, but was upregulated under water-deficit conditions. The tolerance to water deficit of T₀ transgenic lines was assessed by measuring leaf relative water content and visually rating the severity of leaf wilting during to water stress. Under water-stressed conditions, some transgenic lines maintained high water content in leaves and showed significantly less extent of leaf wilting compared with non-transgenic control plants. These results indicated that the introduction of barley hva1 gene using constitutive or stress-inducible promoters lessened water-deficit injury in creeping bentgrass, suggesting that heterologous expression of LEA3 protein genes may enhance the survival ability of creeping bentgrass in water limiting environments.     

Expression of Soluble Bovine Pancreatic Ribonuclease A in Pichia pastoris and its Purification and Characterization

A Pichia pastoris expression system for bovine pancreatic RNase A was constructed: the RNase A sequence was fused to the PHO1 signal and the AOX1 promoter was used for efficient secretion. Approximately 5 mg of soluble enzymes were secreted per liter of the culture, but one half of them were glycosylated. After a series of purifications by cation-exchange chromatography, the glycosylated enzyme was removed and the pure recombinant soluble unglycosylated RNase A was obtained in the final yield of 1 mg per liter of the culture. N-Terminal sequence, molecular weight, secondary structure, thermal stability, and activity were completely identical with those of commercial RNase A. Glycosylated RNase A had a decreased k _cat, 60-70% of the activity of wild-type RNase A, as in the case of RNase B. Its carbohydrate moiety seemed to destabilize the enzyme differently from RNase B since T _m of the glycosylated RNase A was decreased by 6°C. The carbohydrate moiety of the glycosylated enzyme contained no GlcNAc. The N34A mutant RNase A, in which the only potential N-glycosylation site, Asn34, is mutated to alanine, was also glycosylated, implying that glycosylation is not N-linked but O-linked.     

Calcineurin Subunits A and B Interact to Regulate Growth and Asexual and Sexual Development in Neurospora crassa

Calcineurin is a calcium/calmodulin dependent protein phosphatase in eukaryotes that consists of a catalytic subunit A and a regulatory subunit B. Previous studies in the filamentous fungus Neurospora crassa had suggested that the catalytic subunit of calcineurin might be an essential protein. We generated N. crassa strains expressing the A (cna-1) and B (cnb-1) subunit genes under the regulation of P_tcu-1, a copper-responsive promoter. In these strains, addition of bathocuproinedisulfonic acid (BCS), a copper chelator, results in induction of cna-1 and cnb-1, while excess Cu²⁺ represses gene expression. Through analysis of these strains under repressing and inducing conditions, we found that the calcineurin is required for normal growth, asexual development and female fertility in N. crassa. Moreover, we isolated and analyzed cnb-1 mutant alleles generated by repeat-induced point mutation (RIP), with the results further supporting roles for calcineurin in growth and fertility in N. crassa. We demonstrated a direct interaction between the CNA-1 and CNB-1 proteins using an assay system developed to study protein-protein interactions in N. crassa.     

A Novel Glyceraldehyde-3-Phosphate Dehydrogenase (<Emphasis Type="Italic">GAPDH</Emphasis>) Promoter for Expressing Transgenes in the Halotolerant Alga <Emphasis Type="Italic">Dunaliella salina</Emphasis>

A major challenge for efficient transgene expression in Dunaliella salina is to find strong endogenous promoters to drive the transgene expression. In the present study, a novel glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter was cloned and used to drive expressions of the bialaphos resistance (bar) gene and of the N-terminal fragment of human canstatin (Can-N). The results showed that the bar gene was transcribed by the GAPDH promoter and integrated into the genome of the transformants of D. salina. Furthermore, the PCR identification, Southern and western blots indicated that Can-N was expressed in transgenic D. salina, demonstrating that the promoter of the D. salina GAPDH gene is suitable for driving expression of heterologous genes in transgenic D. salina.     

Cloning and characterization of a tyrosinase gene from the white-rot fungus Pycnoporus sanguineus, and overproduction of the recombinant protein in Aspergillus niger

A new tyrosinase-encoding gene (2,204 bp) and the corresponding cDNA (1,857 nucleotides) from the white-rot fungus Pycnoporus sanguineus BRFM49 were cloned. This gene consisted of seven exons and six introns and encoded a predicted protein of 68 kDa, exceeding the mature tyrosinase by 23 kDa. P. sanguineus tyrosinase cDNA was over-expressed in Aspergillus niger, a particularly suitable fungus for heterologous expression of proteins of biotechnological interest, under the control of the glyceraldehyde-3-phosphate-dehydrogenase promoter as strong and constitutive promoter. The glucoamylase preprosequence of A. niger was used to target the secretion. This construction enabled the production of recombinant tyrosinase in the extracellular medium of A. niger. The identity of the purified recombinant protein was confirmed by N-terminal amino acid sequencing. The maturation process was shown to be effective in A. niger, and the recombinant enzyme was fully active, with a molecular mass of 45 kDa. The best transformant obtained, A. niger D15#26-e, produced extracellular tyrosinase activities of 534 and 1,668 U l⁻¹ for monophenolase and diphenolase, respectively, which corresponded to a protein yield of ca. 20 mg l⁻¹.     

Folding, quality control, and secretion of pancreatic ribonuclease in live cells

Although bovine pancreatic RNase is one of the best characterized proteins in respect to structure and in vitro refolding, little is known about its synthesis and maturation in the endoplasmic reticulum (ER) of live cells. We expressed the RNase in live cells and analyzed its folding, quality control, and secretion using pulse-chase analysis and other cell biological techniques. In contrast to the slow in vitro refolding, the protein folded almost instantly after translation and translocation into the ER lumen (t_½ < 3 min). Despite high stability of the native protein, only about half of the RNase reached a secretion competent, monomeric form and was rapidly transported from the rough ER via the Golgi complex (t_½ = 16 min) to the extracellular space (t_½ = 35 min). The rest remained in the ER mainly in the form of dimers and was slowly degraded. The dimers were most likely formed by C-terminal domain swapping since mutation of Asn¹¹³, a residue that stabilizes such dimers, to Ser increased the efficiency of secretion from 59 to 75%. Consistent with stringent ER quality control in vivo, the secreted RNase in the bovine pancreas was mainly monomeric, whereas the enzyme present in the cells also contained 20% dimers. These results suggest that the efficiency of secretion is not only determined by the stability of the native protein but by multiple factors including the stability of secretion-incompetent side products of folding. The presence of N-glycans had little effect on the folding and secretion process.     

Improvement of ligninolytic properties by recombinant expression of glyoxal oxidase gene in hyper lignin-degrading fungus Phanerochaete sordida YK-624

Glyoxal oxidase (GLOX) is a source of the extracellular H₂O₂ required for the oxidation reactions catalyzed by the ligninolytic peroxidases. In the present study, the GLOX-encoding gene (glx) of Phanerochaete chrysosporium was cloned, and bee2 promoter of P. sordida YK-624 was used to drive the expression of glx. The expression plasmid was transformed into a P. sordida YK-624 uracil auxotrophic mutant (strain UV-64), and 16 clones were obtained as GLOX-introducing transformants. These transformants showed higher GLOX activities than wild-type P. sordida YK-624 and control transformants harboring marker plasmid. RT-PCR analysis indicated that the increased GLOX activity was associated with elevated recombinant glx expression. Moreover, these transformants showed higher ligninolytic activity than control transformants. These results suggest that the ligninolytic properties of white-rot fungi can be improved by recombinant expression of glx.     

Effective expression of the gene encoding an extracellular ribonuclease of <Emphasis Type="Italic">Zinnia elegans</Emphasis> in the SR1 <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> plants

Complementary DNA for the extracellular RNase of Zinnia elegans was cloned under control of the cauliflower mosaic virus 35S RNA constitutive promoter and transferred into the Nicotiana tabacum SR1 plants. Primary tobacco transformants were characterized by a high level of RNase activity.     

A food-grade delivery system for<Emphasis Type="Italic"> Lactococcus lactis</Emphasis> and evaluation of inducible gene expression

The genetic improvement of Lactococcus lactis is a matter of biotechnological interest in the food industry and in the pharmaceutical and medical fields. However, to construct a food-grade delivery system, both the presence of antibiotic markers or plasmid sequences should be avoided and the maintenance and expression of the cloned gene should be guaranteed. The objective of this work was to produce crossover mutants of L. lactis with a reporter gene under the control of an inducible promoter in order to evaluate the level of gene expression. We utilized a nuclease gene of Staphylococcus aureus as a reporter gene, P _nisA as the nisin-inducible promoter, a non-essential gene involved in histidine biosynthesis of L. lactis as the site for homologous recombination, and pRV300 as a suicide vector for the genomic integration in L. lactis NZ9000. Single- and double-crossover mutants were identified by genotype and phenotype. Relative to episomal transformants of L. lactis, the level of expression of the heterologous protein after nisin induction was similar in the crossover mutants, suggesting that a single copy of the heterologous gene can be used to produce the protein of interest.     

A novel expression system for intracellular production and purification of recombinant affinity-tagged proteins in Aspergillus niger

A set of different integrative expression vectors for the intracellular production of recombinant proteins with or without affinity tag in Aspergillus niger was developed. Target genes can be expressed under the control of the highly efficient, constitutive pkiA promoter or the novel sucrose-inducible promoter of the β-fructofuranosidase (sucA) gene of A. niger in the presence or absence of alternative carbon sources. All expression plasmids contain an identical multiple cloning sequence that allows parallel construction of N- or C-terminally His₆- and StrepII-tagged versions of the target proteins. Production of two heterologous model proteins, the green fluorescence protein and the Thermobifida fusca hydrolase, proved the functionality of the vector system. Efficient production and easy detection of the target proteins as well as their fast purification by a one-step affinity chromatography, using the His₆- or StrepII-tag sequence, was demonstrated.     

Expression of laccase gene lcc1 in Coprinopsis cinerea under control of various basidiomycetous promoters

Coprinopsis cinerea laccase gene lcc1 was expressed in this basidiomycete under naturally non-inductive conditions using various homologous and heterologous promoters. Laccase expression was achieved in solid and liquid media with promoter sequences from the C. cinerea tub1 gene, the Agaricus bisporus gpdII gene, the Lentinus edodes priA gene and the Schizophyllum commune Sc3 gene. As measured by enzyme activity in liquid cultures, a 277-bp gpdII promoter fragment, followed by a 423-bp priA fragment, was most efficient. A shorter priA sequence of 372 bp was inactive. tub1 promoter fragments were reasonably active, whereas the S. commune Sc3 promoter sequence was less active, in comparison. Irrespective of the promoter used, addition of copper to the medium increased enzymatic activities for highly active transformants by 10- to 50-fold and for less active transformants for 2- to 7-fold. The highest enzymatic activities (3 U/ml) were reached with the gpdII promoter in the presence of 0.1 mM CuSO₄.     

Hydrophobin signal sequence mediates efficient secretion of recombinant proteins in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Pichia pastoris is an important eukaryotic organism for the expression, processing, and secretion of recombinant proteins. Here, the secretion of enhanced green fluorescent protein (EGFP) in P. pastoris by using three novel secretion signals originating from the HFBI and HFBII class 2 hydrophobins of Trichoderma reesei was investigated. EGFP was fused to the carboxyl terminus of hydrophobin secretion signals and expressed under the control of the constitutive GAP promoter. In every case, recombinant EGFP entered the secretory pathway of P. pastoris. SDS-polyacrylamide gel electrophoresis, Western blot analysis of the cells' supernatant, and fluorescence measurements on single-cell level via flow cytometry confirmed the efficient secretion of EGFP mediated by the novel secretion sequences. In conclusion, the data clearly show that the secretion sequences derived from HFBI and HFBII of T. reesei have the potential to achieve an efficient secretion of heterologous proteins in P. pastoris. Due to the small size of the hydrophobin-derived secretion signals, their coding sequence can be easily introduced to the gene of interest by PCR.     

A heterologous production of the <Emphasis Type="Italic">Trametes hirsuta</Emphasis> laccase in the fungus <Emphasis Type="Italic">Penicillium canescens</Emphasis>

A heterologous protein expression in the fungus Penicillium canescens is described for the first time. The fungal strains producing Trametes hirsuta 072 accase under control of a highly efficient promoter of the P. canescens gene bgaS has been constructed. These strains efficiently transcribe the T. hirsuta 072 laccase gene with a correct intron splicing. Activity of the secreted heterologous laccase in the culture liquid reaches 3 U/ml, accounting for 98% of the total laccase activity, which demonstrates a high efficiency of heterologous secretion. The synthesized P. canescens laccase has the same molecular weight as the enzyme produced by T. hirsuta 072.