Gene expression systems for filamentous fungi.

The extraordinary capacity of filamentous fungi to produce large quantities of extracellular protein, together with the advent of DNA-mediated fungal transformation, has resulted in rapid advances in the development of gene expression systems for filamentous fungi. This review focuses on recent developments in the expression of both fungal and non-fungal genes and improvements to the host.

Isolation and characterization of the Aspergillus oryzae gene encoding aspergillopepsin O.

We have cloned and determined the nucleotide sequence of a genomic DNA segment from Aspergillus oryzae which contains pepO, the gene encoding the aspartic proteinase, aspergillopepsin O (PEPO). The organization of pepO is strikingly similar to that of pepA from A. niger var. awamori (previously called A. awamori) in that both are composed of four exons and three introns with virtually identical lengths, and the positions of the introns are exactly conserved. From the deduced amino acid (aa) sequence, it appears that PEPO, like other fungal aspartic proteinases, is synthesized as a zymogen containing a putative N-terminal prepro-region of 77 aa followed by a mature protein of 327 aa. Southern blotting experiments suggest that a single copy of pepO exists in the A. oryzae genome.

The promoter of the glucoamylase-encoding gene of Aspergillus niger functions in Ustilago maydis.

Promoter sequences from the Aspergillus niger glucoamylase-encoding gene (glaA) were linked to the bacterial hygromycin (Hy) phosphotransferase-encoding gene (hph) and this chimeric marker was used to select Hy-resistant (HyR) Ustilago maydis transformants. This is an example of an Ascomycete promoter functioning in a Basidiomycete. HyR transformants varied with respect to copy number of integrated vector, mitotic stability, and tolerance to Hy. Only 216 bp of glaA promoter sequence is required for expression in U. maydis but this promoter is not induced by starch as it is in Aspergillus spp. The transcriptional start points are the same in U. maydis and A. niger.

Molecular cloning and deletion of the gene encoding aspergillopepsin A from Aspergillus awamori.

We have cloned genomic pepA sequences encoding the aspartic proteinase aspergillopepsin A (PEPA) from Aspergillus awamori using a synthetic oligodeoxyribonucleotide probe. Nucleotide sequence data from the pepA gene revealed that it is composed of four exons of 320, 278, 249, and 338 bp. Three introns which interrupt the coding sequence are 51, 52, and 59 bp in length. Directly downstream from the putative start codon lies a sequence encoding 69 amino acids (aa) which are not present in mature PEPA. Based on similarities to other aspartic proteinases, this region may represent a 20-aa signal peptide followed by a 49-aa propeptide that is rich in basic aa residues. Northern blots of total cellular RNA extracted from A. awamori cells indicate that pepA is transcribed as a single 1.4-kb mRNA. Mutants of A. awamori lacking the pepA structural gene were derived by the following gene replacement strategy. First, we constructed a plasmid in which a 2.4-kb SalI fragment containing the entire pepA coding region was deleted from a 9-kb Eco RI genomic DNA clone and replaced by a synthetic DNA polylinker. Second, a selectable argB gene was inserted into the polylinker. Third, the EcoRI fragment which contained the argB marker flanked by pepA sequences was excised from the plasmid and used to transform an argB auxotroph of A. awamori. From 16-40% of the resulting prototrophic transformants were found to have a PEPA-deficient phenotype when screened with an immunoassay using antibodies specific for PEPA. Southern hybridization experiments confirmed that these mutants resulted from a gene replacement event at the pepA locus.

The development of gene expression systems for filamentous fungi.

Filamentous fungi have been used for decades in the commercial production of enzymes, antibiotics, and specialty chemicals. Traditionally, improving the yields of these products has involved either mutagenesis and screening or modification of fermentation conditions. Generally, selective breeding of strains has not been successful, because most of the commercially important fungal species lack a sexual cycle. For a few species, strain improvements have been made possible by employing the parasexual cycle for genetic crosses (30). The recent development of DNA-mediated transformation systems for several industrially important fungal species has spawned a flurry of research activity directed toward the development of gene expression systems for these microorganisms. This technology is now a viable means for novel and more directed approaches to improving existing fungal strains which produce enzymes or antibiotics. In addition, fungal expression systems are now being tested for the production of heterologous gene products such as mammalian pharmaceutical proteins. The goal of this review is to present a summary of the gene expression systems which have recently been developed for some filamentous fungi of commercial importance. To insure that the most recent developments are presented we have included data from not only scientific papers, but also from personal communications, abstracts, symposia, and our own laboratory.

Cloning and amplification of the gene encoding an extracellular beta-glucosidase from Trichoderma reesei: evidence for improved rates of saccharification of cellulosic substrates.

We have cloned and determined the nucleotide sequence of the gene encoding an extracellular beta-glucosidase (bgl1) from the cellulolytic fungus Trichoderma reesei. The predicted open reading frame of the bgl1 gene is interrupted by two putative introns of 70 and 64 bp and encodes a protein with a calculated molecular weight of 75,341. The genomic segment encoding bgl1 was cloned into a vector that contained the selectable marker gene, amdS. Transformation of T. reesei with this vector resulted in several stable transformant strains all possessing an increased copy number of the bgl1 gene integrated into the genome together with elevated rates of glucose production from avicel. One transformant produced an extracellular cellulase with a five-fold increase in the rate of production of glucose from cellobiose, a 33% rate increase from avicel, and a 17% increase from phosphoric acid swollen cellulose. These data suggest that the cellulolytic activity of T. reesei strains may be specifically improved by transformation with cloned cellulase genes.

Improved production of chymosin in Aspergillus by expression as a glucoamylase-chymosin fusion.

We have extended the work on chymosin production in Aspergillus by constructing an expression vector in which the cDNA encoding bovine prochymosin B was fused in frame immediately following the codon for the last amino acid of the A. awamori glucoamylase (glaA) gene. Transformation of A. awamori with this plasmid led to the secretion of considerably higher amounts of chymosin than obtained with previous chymosin expression vectors. We present evidence that mature chymosin is autocatalytically released from the glucoamylase-chymosin fusion protein after secretion.

Commercial levels of chymosin production by Aspergillus.

We have increased the production of bovine chymosin in Aspergillus niger var. awamori to more than one gram per liter of secreted authentic enzyme by combining a mutagenesis protocol with a novel robotic screening program. Analysis of the superior chymosin producing strains indicated that they have enhanced capabilities to secrete extracellular proteins.

Phospholipase C (heat-labile hemolysin) of Pseudomonas aeruginosa: purification and preliminary characterization.

Phospholipase C (heat-labile hemolysin) was purified from Pseudomonas aeruginosa culture supernatants to near homogeneity by ammonium sulfate precipitation followed by a novel application of DEAE-Sephacel chromatography. Enzymatic activity remained associated with DEAE-Sephacel even in the presence of 1 M NaCl, but was eluted with a linear gradient of 0 to 5% tetradecyltrimethylammonium bromide. Elution from DEAE-Sephacel was also obtained with 2% lysophosphatidylcholine, and to a lesser extent with 2% phosphorylcholine, but not at all with choline. The enzyme was highly active toward phospholipids possessing substituted ammonium groups (e.g., phosphatidycholine, lysophosphatidylcholine, and sphingomyelin); however, it had little if any activity toward phospholipids lacking substituted ammonium groups (e.g., phosphatidylethanolamine, phosphatidylserine, and phosphaditylglycerol). Collectively, these data suggest that phospholipase C from P. aeruginosa exhibits high affinity for substituted ammonium groups, but requires an additional hydrophobic moiety for optimum binding. The specific activity of the purified enzyme preparation increased 1,900-fold compared with that of culture supernatants. The molecular weight of the phospholipase C was estimated to be 78,000 by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Sephacryl S-200 column chromatography and was 76,000 by high-performance size exclusion chromatography. The isoelectric point was 5.5. Amino acid analysis showed that phospholipase C was rich in glycine, serine, threonine, aspartyl, glutamyl, and aromatic amino acids, but was cystine free.

Mutations in the small subunit of cyanobacterial ribulose-bisphosphate carboxylase/oxygenase that modulate interactions with large subunits.

In the cyanobacterium Anacystis nidulans (Synechococcus PCC6301), ribulose 1,5-bisphosphate carboxylase/oxygenase (Rbu-P2 carboxylase) is composed of eight large subunits and eight small subunits. There are three regions of the small subunit that contain amino acids that are conserved throughout evolution, from bacteria to higher plants. Since the function of the small subunit is not fully understood, site-directed mutagenesis was performed on highly conserved residues in the first and second conserved regions. Ser-16, Pro-19, Leu-21, and Tyr-54 were replaced by Asp-16, His-19, Glu-21, and Ser-54, respectively. Crude extracts containing the recombinant His-19 mutant enzyme indicated that there was little effect on either Rbu-P2 carboxylase activity or interactions between large and small subunits. However, the Asp-16, Glu-21, and Ser-54 mutations showed effects on Rbu-P2 carboxylase activity and the interaction between large and small subunits. The large and small subunits of the Asp-16, Glu-21, and Ser-54 enzymes were found to dissociate during nondenaturing gel electrophoresis or sucrose density gradient centrifugation. However, the dissociated small subunits remained functional and were capable of reconstituting Rbu-P2 carboxylase activity when added to large subunits. These results indicated that Ser-16, Leu-21, and Tyr-54 might play an important role in interactions between large and small subunits of the A. nidulans enzyme.

Phospholipase C regulatory mutation of Pseudomonas aeruginosa that results in constitutive synthesis of several phosphate-repressible proteins.

We describe here a new mutant of Pseudomonas aeruginosa PAO, strain D10C (genotype plcB), which produces phospholipase C and alkaline phosphatase constitutively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the extracellular proteins produced by this mutant in high- and low-Pi media revealed that the mutation resulted in a marked deficiency of one major Pi-regulated protein of 41,000 molecular weight and constitutive synthesis of all other major extracellular Pi-regulated proteins. Furthermore, the plcB mutant was deficient in phosphate transport. A plcA mutation, which also led to a loss of the 41,000-molecular-weight protein, was similarly deficient in Pi transport. The genetic loci, plcA and plcB, located at 22 to 23 min on the PAO chromosome, were indistinguishable by conjugational and transductional mapping, and may therefore be in the same gene or in a cluster of genes which regulate the synthesis of Pi-repressible proteins.

A Pseudomonas aeruginosa mutant non-derepressible for orthophosphate-regulated proteins.

Using a rapid screening assay based on the hydrolysis of p-nitrophenylphosphorylcholine, we isolated several mutants of Pseudomonas aeruginosa deficient in the production of phospholipase C. One, designated strain A50N, was also markedly deficient in the synthesis of alkaline phosphatase and several unidentified extracellular proteins. Because strain A50N produces these proteins under conditions of derepression at levels equal to those produced by the parental strain PAO1 grown in medium containing excess phosphate, it appears to have a mutation in a genetic element involved in the derepression of phosphate-repressible proteins.

Studies of phospholipase C (heat-labile hemolysin) in Pseudomonas aeruginosa.

The chromogenic substrate p-nitrophenylphosphorylcholine was used to measure phospholipase C (heat-labile hemolysin) production by clinical strains of Pseudomonas aeruginosa. Analysis of strains from various types of infection showed that phospholipase C production by urinary tract isolates was significantly greater than that by lung, blood, or other isolates (e.g., wounds, etc.). The above method was also found to be effective for the isolation of several classes of phospholipase C mutants.

Regulation of the glaA gene of Aspergillus niger.

The glucoamylase gene of Aspergillus niger, glaA, is expressed at high levels in the presence of starch. We have determined the nucleotide sequence of 1966 bp of the 5' flanking region of the glaA gene and have begun to identify sequences important for the control of glaA expression by deletion analysis. Constructs containing deletions extending into the glaA gene promoter were introduced into an A. niger host whose own glaA gene had been disrupted by a gene replacement event. Secreted levels of glucoamylase, expressed from each of the recombinant glaA genes, were measured by enzyme immunoassay. The effect of each deletion on the expression of glaA, when grown on differing carbon sources, was used to determine the limits of sequences upstream of glaA responsible for gene regulation. A region between -562 and -318 appears to direct high-level expression, whereas only 214 bp of 5' flanking sequence is required to initiate the start to transcription.

Cloning of the Aspergillus oryzae 5-aminolevulinate synthase gene and its use as a selectable marker.

The hemA gene encoding 5-aminolevulinate synthase, the first enzyme in heme biosynthesis, was cloned from Aspergillus oryzae and evaluated as a selectable marker for the transformation of filamentous fungi. Deletion of the hemA gene resulted in a lethal phenotype that could be rescued either by the supplementation of culture media with 5-aminolevulinic acid (ALA) or by transformation with the wild-type hemA gene, but not by growth on rich media, nor by the addition of exogenous heme. Transformation of a hemA deletion strain with the hemA gene linked to a lipase expression cassette yielded ALA prototrophs expressing lipase. The hemA gene can therefore be used as a selectable marker for the transformation of A. oryzae.

The catR gene encoding a catalase from Aspergillus niger: primary structure and elevated expression through increased gene copy number and use of a strong promoter.

Synthetic oligonucleotide probes based on amino acid sequence data were used to identify and clone cDNA sequences encoding a catalase (catalase-R) of Aspergillus niger. One cDNA clone was subsequently used to isolate the corresponding genomic DNA sequences (designated catR). Nucleotide sequence analysis of both genomic and cDNA clones suggested that the catR coding region consists of five exons interrupted by four small introns. The deduced amino acid sequence of catalase-R spans 730 residues which show significant homology to both prokaryotic and eukaryotic catalases, particularly in regions involved in catalytic activity and binding of the haem prosthetic group. Increased expression of the catR gene was obtained by transformation of an A. niger host strain with an integrative vector carrying the cloned genomic DNA segment. Several of these transformants produced three- to fivefold higher levels of catalase than the untransformed parent strain. Hybridization analyses indicated that these strains contained multiple copies of catR integrated into the genome. A second expression vector was constructed in which the catR coding region was functionally joined to the promoter and terminator elements of the A. niger glucoamylase (glaA) gene. A. niger transformants containing this vector produced from three- to 10-fold higher levels of catalase-R than the untransformed parent strain.

Molecular characterization and expression of a phytase gene from the thermophilic fungus Thermomyces lanuginosus.

The phyA gene encoding an extracellular phytase from the thermophilic fungus Thermomyces lanuginosus was cloned and heterologously expressed, and the recombinant gene product was biochemically characterized. The phyA gene encodes a primary translation product (PhyA) of 475 amino acids (aa) which includes a putative signal peptide (23 aa) and propeptide (10 aa). The deduced amino acid sequence of PhyA has limited sequence identity (ca. 47%) with Aspergillus niger phytase. The phyA gene was inserted into an expression vector under transcriptional control of the Fusarium oxysporum trypsin gene promoter and used to transform a Fusarium venenatum recipient strain. The secreted recombinant phytase protein was enzymatically active between pHs 3 and 7.5, with a specific activity of 110 micromol of inorganic phosphate released per min per mg of protein at pH 6 and 37 degrees C. The Thermomyces phytase retained activity at assay temperatures up to 75 degrees C and demonstrated superior catalytic efficiency to any known fungal phytase at 65 degrees C (the temperature optimum). Comparison of this new Thermomyces catalyst with the well-known Aspergillus niger phytase reveals other favorable properties for the enzyme derived from the thermophilic gene donor, including catalytic activity over an expanded pH range.

Cloning of a phosphate-regulated hemolysin gene (phospholipase C) from Pseudomonas aeruginosa.

Phospholipase C (heat-labile hemolysin) of Pseudomonas aeruginosa is a phosphate (P(i))-regulated extracellular protein which may be a significant virulence factor of this organism. The gene for this hemolytic enzyme was cloned on a 4.1-megadalton (Mdal) fragment from a BamHI digest of P. aeruginosa PAO1 genomic DNA and was inserted into the BamHI sites of the multicopy Escherichia coli(pBR322) and P. aeruginosa(pMW79) vectors. The E. coli and P. aeruginosa recombinant plasmids were designated pGV26 and pVB81, respectively. A restriction map of the 4.1-Mdal fragment from pGV26 was constructed, using double and single digestions with BamHI and EcoRI and several different restriction enzymes. Based on information from this map, a 2.4-Mdal BamHI/BglII fragment containing the gene for phospholipase C was subcloned to pBR322. The hybrid plasmids pGV26 and pVB81 direct the synthesis of enzymatically active phospholipase C, which is also hemolytic. The plasmid-directed synthesis of phospholipase C in E. coli or P. aeruginosa is not repressible by P(i) as is the chromosomally directed synthesis in P. aeruginosa. Data are presented which suggest that the synthesis of phospholipase C from pGV26 and pVB81 is directed from the tetracycline resistance gene promoter. The level of enzyme activity produced by E. coli(pGV26) is slightly higher than the levels produced by P. aeruginosa(pMW79) under repressed conditions. In contrast, the levels produced by P. aeruginosa(pVB81) are at least 600-fold higher than the levels produced by P. aeruginosa(pMW79) under repressed conditions and approximately 20-fold higher than those produced by P. aeruginosa(pMW79) under derepressed conditions. The majority (85%) of the enzyme produced by E. coli(pGV26) remained cell associated, whereas >95% of the enzyme produced by P. aeruginosa(pVB81) was extracellular. Analysis of extracellular proteins from cultures of P. aeruginosa(pMW79) and P. aeruginosa(pVB81) by high-performance liquid chromotography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the phospholipase C gene was cloned intact, and it is likely that several additional genes were cloned on the 4.1-Mdal fragment of DNA. It was also found that some of these genes encode proteins which are the same molecular weight as some previously described P(i)-repressible proteins of P. aeruginosa. The existence of a P(i) regulon of P. aeruginosa is proposed. It is likely that one of these genes also regulates the level of pyocyanin production by P. aeruginosa and that one or more play a role in transport or binding of P(i). The availability of the hybrid plasmids described herein will be useful in further studies on the role of this hemolysin in the virulence of P. aeruginosa and in the study of the genetics and physiology of P(i)-regulated proteins.

Identification and characterization of Campylobacter jejuni outer membrane proteins.

Outer membrane proteins from isolates of Campylobacter jejuni were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Sarcosinate-insoluble membrane preparations were outer membrane enriched based on increased ketodeoxyoctonate concentrations, the presence of surface-exposed 125I-labeled proteins that were hydrophobic, and similarity to membrane vesicle (bleb) sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles. Most isolates contained a single major band with molecular weight of 41,000 to 45,000. Profiles of C. jejuni and Campylobacter coli isolates were indistinguishable, but either could be easily differentiated from Campylobacter fetus and Campylobacter faecalis. The profiles were stable for strains under a variety of growth, incubation and passage conditions. We classified 110 isolates from patients with sporadic campylobacter enteritis into nine subtypes based on differences in outer membrane sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles. Two categories accounted for 76% of the isolates. Complete concordance was observed in subtypes of strains obtained from epidemiologically related cases. Thus, comparison of the major outer membrane proteins of C. jejuni is a useful technique for investigating the transmission of this organism and may provide a basis for immunological characterization of the outer membrane proteins.

Polygalacturonase gene expression in ripe melon fruit supports a role for polygalacturonase in ripening-associated pectin disassembly.

Ripening-associated pectin disassembly in melon is characterized by a decrease in molecular mass and an increase in the solubilization of polyuronide, modifications that in other fruit have been attributed to the activity of polygalacturonase (PG). Although it has been reported that PG activity is absent during melon fruit ripening, a mechanism for PG-independent pectin disassembly has not been positively identified. Here we provide evidence that pectin disassembly in melon (Cucumis melo) may be PG mediated. Three melon cDNA clones with significant homology to other cloned PGs were isolated from the rapidly ripening cultivar Charentais (C. melo cv Reticulatus F1 Alpha) and were expressed at high levels during fruit ripening. The expression pattern correlated temporally with an increase in pectin-degrading activity and a decrease in the molecular mass of cell wall pectins, suggesting that these genes encode functional PGs. MPG1 and MPG2 were closely related to peach fruit and tomato abscission zone PGs, and MPG3 was closely related to tomato fruit PG. MPG1, the most abundant melon PG mRNA, was expressed in Aspergillus oryzae. The culture filtrate exponentially decreased the viscosity of a pectin solution and catalyzed the linear release of reducing groups, suggesting that MPG1 encodes an endo-PG with the potential to depolymerize melon fruit cell wall pectin. Because MPG1 belongs to a group of PGs divergent from the well-characterized tomato fruit PG, this supports the involvement of a second class of PGs in fruit ripening-associated pectin disassembly.