Heterologous gene expression in Aspergillus niger: a glucoamylase-porcine pancreatic prophospholipase A2 fusion protein is secreted and processed to yield mature enzyme

Roberts, Ian N.; Jeenes, David J.; MacKenzie, Donald; Wilkinson, A. W.; Sumner, Ian G.; Archer, David B.

doi:10.1016/0378-1119(92)90043-o

Cited by 67 publications

(44 citation statements)

References 24 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Previous work in this lab showed that protease degradation contributed the most to the GFP loss [10]. Other researchers have also shown that proteases are responsible for the degradation of heterologous proteins such as human interleukin-6 [5], bovine chymosin [18] and porcine pancreatic phospholipase A [14]. Since protease activity is strongly affected by pH values, the recombinant protein production can increase if broth pH is properly controlled at a value at which protease activity is low, e.g., pH 5 or 6.…”

Section: Effect Of Ph On Protease Activitymentioning

confidence: 99%

Enhanced heterologous protein production in Aspergillus niger through pH control of extracellular protease activity

O’Donnell

Wang

et al. 2001

Biochemical Engineering Journal

View full text Add to dashboard Cite

Section: Effect Of Ph On Protease Activitymentioning

confidence: 99%

Enhanced heterologous protein production in Aspergillus niger through pH control of extracellular protease activity

O’Donnell

Wang

et al. 2001

Biochemical Engineering Journal

View full text Add to dashboard Cite

“…Large-scale fermentation technology for filamentous fungi is well established and several species have U.S. Food and Drug Administration approval for use in the food industry ("Generally Regarded as Safe" status). There is a great interest in the production of heterologous proteins in filamentous fungi (Fowler and Berka, 1991;Gouka et al, 1996;Jeenes et al, 1994;Roberts et al, 1992;van den Hondel et al, 1991). In general, the secreted yields of most heterologous proteins are low when compared with the homologous proteins, and reach levels that in most cases do not exceed a few milligrams per liter of culture medium.…”

Section: Introductionmentioning

confidence: 98%

Expression of a synthetic copy of the bovine chymosin gene in Aspergillus awamori from constitutive and pH‐regulated promoters and secretion using two different pre‐pro sequences

Cardoza

Gutiérrez

Ortega

et al. 2003

Biotech & Bioengineering

View full text Add to dashboard Cite

A copy of the bovine chymosin gene (chy) with a codon usage optimized for its expression in Aspergillus awamori was constructed starting from synthetic oligonucleotides. To study the ability of this filamentous fungus to secrete bovine prochymosin, two plasmids were constructed in which the transcriptional, translational, and secretory control regions of the A. nidulans gpdA gene and pepB genes were coupled to either preprochymosin or prochymosin genes. Secretion of a protein enzymatically and immunologically indistinguishable from bovine chymosin was achieved in A. awamori transformants with each of these constructions. In all cases, the primary translation product (40.5 kDa) was self-processed to a mature chymosin polypeptide having a molecular weight of 35.6 kDa. Immunological assays indicated that most of the chymosin was secreted to the extracellular medium. Hybridization analysis of genomic DNA from chymosin transformants showed chromosomal integration of prochymosin sequences and, in some transformants, multiple copies of the expression cassettes were observed. Expression from the gpdA promoter was constitutive, whereas expression from the pepB promoter was strongly influenced by pH. A very high expression from the pepB promoter was observed during the growth phase. The A. awamori pepB gene terminator was more favorable for chymosin production than the S. cerevisiae CYC1 terminator.

show abstract

“…This improvement factor is less than that obtained for homologous production of FAEA in A. niger (34). Apparently, as seen for other fusion proteins, the production yield was negatively affected by the presence of the bacterial domain (21,31,35,36). In addition, we observed that FAEADoc protein was produced as a truncated form.…”

Section: Discussionmentioning

confidence: 52%

“…In earlier studies, methods were developed to alleviate limitations for protein production in fungal hosts. An efficient strategy is based on the use of translational fusions in which the targeted protein is fused to an endogenous secreted carrier protein at the N-terminal end of the heterologous protein (17,35). The N-terminal carrier protein is believed to improve the secretion efficiency of the heterologous protein by facilitating translocation and folding in the endoplasmic reticulum.…”

Section: Discussionmentioning

confidence: 99%

“…Moreover, the esterase activity was even 4-and 20-fold higher, respectively, than those obtained for the FAEA-Doc transformant and the wild-type strain under optimal induction conditions, confirming that glucoamylase is the carrier protein of choice for such a construct. Glucoamylase was used in previous work to successfully produce recombinant proteins such as porcine pancreatic prophospholipase A2, human interleukin-6, or hen egg white lysozyme (6,17,20,21,35). Based on the specific activity of purified Doc-FAEA, we estimated a production yield of 100 mg per liter.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Design and Production in Aspergillus niger of a Chimeric Protein Associating a Fungal Feruloyl Esterase and a Clostridial Dockerin Domain

Levasseur¹,

Pagès

Fierobe

et al. 2004

Appl Environ Microbiol

View full text Add to dashboard Cite

A chimeric enzyme associating feruloyl esterase A (FAEA) from Aspergillus niger and dockerin from Clostridium thermocellum was produced in A. niger. A completely truncated form was produced when the dockerin domain was located downstream of the FAEA (FAEA-Doc), whereas no chimeric protein was produced when the bacterial dockerin domain was located upstream of the FAEA (Doc-FAEA). Northern blot analysis showed similar transcript levels for the two constructs, indicating a posttranscriptional bottleneck for Doc-FAEA production. The sequence encoding the first 514 amino acids from A. niger glucoamylase and a dibasic proteolytic processing site (kex-2) were fused upstream of the Doc-FAEA sequence. By using this fusion strategy, the esterase activity found in the extracellular medium was 20-fold-higher than that of the wild-type reference strain, and the production yield was estimated to be about 100 mg of chimeric protein/liter. Intracellular and extracellular production was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, dockerin-cohesin interaction assays, and Western blotting. Labeled cohesins detected an intact extracellular Doc-FAEA of about 43 kDa and a cleaved-off dockerin domain of about 8 kDa. In addition, an intracellular 120-kDa protein was recognized by using labeled cohesins and antibodies raised against FAEA. This protein corresponded to the unprocessed Doc-FAEA form fused to glucoamylase. In conclusion, these results indicated that translational fusion to glucoamylase improved the secretion efficiency of a chimeric Doc-FAEA protein and allowed production of the first functional fungal enzyme joined to a bacterial dockerin.Plant cell walls are composed of various polysaccharides and lignin, forming a rigid and complex matrix recalcitrant to microbial degradation. This structure is strengthened by crosslinkages such as diferulic acid bridges between adjacent hemicellulose chains (28) or between lignin and hemicellulose (24), increasing its resistance to microbial invasion. Aerobic and anaerobic microorganisms have developed two main efficient mechanisms of plant cell wall degradation. In the first system, microorganisms secrete free extracellular enzymes in contact with a substrate. The products of degradation constitute both nutrients for growth and regulators of the production of lignocellulolytic enzymes (9, 42, 43). Among these microorganisms, filamentous fungi such as Trichoderma spp. and Aspergillus spp. are especially good secretors of lignocellulolytic enzymes (1). Moreover, Aspergillus niger produces enzymes named feruloyl esterases (EC 3.1.1.73), a subclass of the carboxylic ester hydrolases, that are able to hydrolyze diferulate cross-links in plant cell walls, facilitating the access of main-chain-degrading enzymes to the polysaccharide backbone (8,46). Furthermore, feruloyl esterases release cross-linked aromatic acids, such as ferulic acid, which is an attractive industrial compound by virtue of its antioxidant, photoprotectant properties (18) and its potential biotr...

show abstract

Heterologous gene expression in Aspergillus niger: a glucoamylase-porcine pancreatic prophospholipase A2 fusion protein is secreted and processed to yield mature enzyme

Cited by 67 publications

References 24 publications

Enhanced heterologous protein production in Aspergillus niger through pH control of extracellular protease activity

Enhanced heterologous protein production in Aspergillus niger through pH control of extracellular protease activity

Expression of a synthetic copy of the bovine chymosin gene in Aspergillus awamori from constitutive and pH‐regulated promoters and secretion using two different pre‐pro sequences

Design and Production in Aspergillus niger of a Chimeric Protein Associating a Fungal Feruloyl Esterase and a Clostridial Dockerin Domain

Contact Info

Product

Resources

About