Ann Westerholm-Parvinen

Genome sequencing and analysis of the biomass-degrading fungus Trichoderma reesei (syn. Hypocrea jecorina).

Trichoderma reesei is the main industrial source of cellulases and hemicellulases used to depolymerize biomass to simple sugars that are converted to chemical intermediates and biofuels, such as ethanol. We assembled 89 scaffolds (sets of ordered and oriented contigs) to generate 34 Mbp of nearly contiguous T. reesei genome sequence comprising 9,129 predicted gene models. Unexpectedly, considering the industrial utility and effectiveness of the carbohydrate-active enzymes of T. reesei, its genome encodes fewer cellulases and hemicellulases than any other sequenced fungus able to hydrolyze plant cell wall polysaccharides. Many T. reesei genes encoding carbohydrate-active enzymes are distributed nonrandomly in clusters that lie between regions of synteny with other Sordariomycetes. Numerous genes encoding biosynthetic pathways for secondary metabolites may promote survival of T. reesei in its competitive soil habitat, but genome analysis provided little mechanistic insight into its extraordinary capacity for protein secretion. Our analysis, coupled with the genome sequence data, provides a roadmap for constructing enhanced T. reesei strains for industrial applications such as biofuel production.

Eukaryotic protein production in designed storage organelles

BackgroundProtein bodies (PBs) are natural endoplasmic reticulum (ER) or vacuole plant-derived organelles that stably accumulate large amounts of storage proteins in seeds. The proline-rich N-terminal domain derived from the maize storage protein γ zein (Zera) is sufficient to induce PBs in non-seed tissues of Arabidopsis and tobacco. This Zera property opens up new routes for high-level accumulation of recombinant proteins by fusion of Zera with proteins of interest. In this work we extend the advantageous properties of plant seed PBs to recombinant protein production in useful non-plant eukaryotic hosts including cultured fungal, mammalian and insect cells.ResultsVarious Zera fusions with fluorescent and therapeutic proteins accumulate in induced PB-like organelles in all eukaryotic systems tested: tobacco leaves, Trichoderma reesei, several mammalian cultured cells and Sf9 insect cells. This accumulation in membranous organelles insulates both recombinant protein and host from undesirable activities of either. Recombinant protein encapsulation in these PBs facilitates stable accumulation of proteins in a protected sub-cellular compartment which results in an enhancement of protein production without affecting the viability and development of stably transformed hosts. The induced PBs also retain the high-density properties of native seed PBs which facilitate the recovery and purification of the recombinant proteins they contain.ConclusionThe Zera sequence provides an efficient and universal means to produce recombinant proteins by accumulation in ER-derived organelles. The remarkable cross-kingdom conservation of PB formation and their biophysical properties should have broad application in the manufacture of non-secreted recombinant proteins and suggests the existence of universal ER pathways for protein insulation.

Production and characterization of a secreted, C‐terminally processed tyrosinase from the filamentous fungus Trichoderma reesei

A homology search of the genome database of the filamentous fungus Trichoderma reesei identified a new T. reesei tyrosinase gene tyr2, encoding a protein with a putative signal sequence. The gene was overexpressed in the native host under the strong cbh1 promoter, and the tyrosinase enzyme was secreted into the culture supernatant. This is the first report on a secreted fungal tyrosinase. Expression of TYR2 in T. reesei resulted in good yields, corresponding to approximately 0.3 and 1 g·L−1 tyrosinase in shake flask cultures and laboratory‐scale batch fermentation, respectively. T. reesei TYR2 was purified with a three‐step purification procedure, consisting of desalting by gel filtration, cation exchange chromatography and size exclusion chromatography. The purified TYR2 protein had a significantly lower molecular mass (43.2 kDa) than that calculated from the putative amino acid sequence (61.151 kDa). According to N‐terminal and C‐terminal structural analyses by fragmentation, chromatography, MS and peptide sequencing, the mature protein is processed from the C‐terminus by a cleavage of a peptide fragment of about 20 kDa. The T. reesei TYR2 polypeptide chain was found to be glycosylated at its only potential N‐glycosylation site, with a glycan consisting of two N‐acetylglucosamines and five mannoses. Also, low amounts of shorter glycan forms were detected at this site. T. reesei TYR2 showed the highest activity and stability within a neutral and alkaline pH range, having an optimum at pH 9. T. reesei tyrosinase retained its activity well at 30 °C, whereas at higher temperatures the enzyme started to lose its activity relatively quickly. T. reesei TYR2 was active on both l‐tyrosine and l‐dopa, and it showed broad substrate specificity.

Screening of candidate regulators for cellulase and hemicellulase production in Trichoderma reesei and identification of a factor essential for cellulase production

BackgroundThe soft rot ascomycetal fungus Trichoderma reesei is utilized for industrial production of secreted enzymes, especially lignocellulose degrading enzymes. T. reesei uses several different enzymes for the degradation of plant cell wall-derived material, including 9 characterized cellulases, 15 characterized hemicellulases and at least 42 genes predicted to encode cellulolytic or hemicellulolytic activities. Production of cellulases and hemicellulases is modulated by environmental and physiological conditions. Several regulators affecting the expression of cellulase and hemicellulase genes have been identified but more factors still unknown are believed to be present in the genome of T. reesei.ResultsWe have used transcriptional profiling data from T. reesei cultures in which cellulase/hemicellulase production was induced by the addition of different lignocellulose-derived materials to identify putative novel regulators for cellulase and hemicellulase genes. Based on this induction data, supplemented with other published genome-wide data on different protein production conditions, 28 candidate regulatory genes were selected for further studies and they were overexpressed in T. reesei. Overexpression of seven genes led to at least 1.5-fold increased production of cellulase and/or xylanase activity in the modified strains as compared to the parental strain. Deletion of gene 77513, here designated as ace3, was found to be detrimental for cellulase production and for the expression of several cellulase genes studied. This deletion also significantly reduced xylanase activity and expression of xylan-degrading enzyme genes. Furthermore, our data revealed the presence of co-regulated chromosomal regions containing carbohydrate-active enzyme genes and candidate regulatory genes.ConclusionsTranscriptional profiling results from glycoside hydrolase induction experiments combined with a previous study of specific protein production conditions was shown to be an effective method for finding novel candidate regulatory genes affecting the production of cellulases and hemicellulases. Recombinant strains with improved cellulase and/or xylanase production properties were constructed, and a gene essential for cellulase gene expression was found. In addition, more evidence was gained on the chromatin level regional regulation of carbohydrate-active enzyme gene expression.

Novel Coprinopsis cinerea Polyesterase That Hydrolyzes Cutin and Suberin

ABSTRACT Three cutinase gene-like genes from the basidiomycete Coprinopsis cinerea (Coprinus cinereus) found with a similarity search were cloned and expressed in Trichoderma reesei under the control of an inducible cbh1 promoter. The selected transformants of all three polyesterase constructs showed activity with p-nitrophenylbutyrate, used as a model substrate. The most promising transformant of the cutinase CC1G_09668.1 gene construct was cultivated in a laboratory fermentor, with a production yield of 1.4 g liter−l purified protein. The expressed cutinase (CcCUT1) was purified to homogeneity by immobilized metal affinity chromatography exploiting a C-terminal His tag. The N terminus of the enzyme was found to be blocked. The molecular mass of the purified enzyme was determined to be around 18.8 kDa by mass spectrometry. CcCUT1 had higher activity on shorter (C2 to C10) fatty acid esters of p-nitrophenol than on longer ones, and it also exhibited lipase activity. CcCUT1 had optimal activity between pH 7 and 8 but retained activity over a wide pH range. The enzyme retained 80% of its activity after 20 h of incubation at 50°C, but residual activity decreased sharply at 60°C. Microscopic analyses and determination of released hydrolysis products showed that the enzyme was able to depolymerize apple cutin and birch outer bark suberin.

A Cutinase from Trichoderma reesei with a Lid-Covered Active Site and Kinetic Properties of True Lipases.

Cutinases belong to the α/β-hydrolase fold family of enzymes and degrade cutin and various esters, including triglycerides, phospholipids and galactolipids. Cutinases are able to degrade aggregated and soluble substrates because, in contrast with true lipases, they do not have a lid covering their catalytic machinery. We report here the structure of a cutinase from the fungus Trichoderma reesei (Tr) in native and inhibitor-bound conformations, along with its enzymatic characterization. A rare characteristic of Tr cutinase is its optimal activity at acidic pH. Furthermore, Tr cutinase, in contrast with classical cutinases, possesses a lid covering its active site and requires the presence of detergents for activity. In addition to the presence of the lid, the core of the Tr enzyme is very similar to other cutinase cores, with a central five-stranded β-sheet covered by helices on either side. The catalytic residues form a catalytic triad involving Ser164, His229 and Asp216 that is covered by the two N-terminal helices, which form the lid. This lid opens in the presence of surfactants, such as β-octylglucoside, and uncovers the catalytic crevice, allowing a C11Y4 phosphonate inhibitor to bind to the catalytic serine. Taken together, these results reveal Tr cutinase to be a member of a new group of lipolytic enzymes resembling cutinases but with kinetic and structural features of true lipases and a heightened specificity for long-chain triglycerides.

Enabling Low Cost Biopharmaceuticals: A Systematic Approach to Delete Proteases from a Well-Known Protein Production Host Trichoderma reesei

The filamentous fungus Trichoderma reesei has tremendous capability to secrete proteins. Therefore, it would be an excellent host for producing high levels of therapeutic proteins at low cost. Developing a filamentous fungus to produce sensitive therapeutic proteins requires that protease secretion is drastically reduced. We have identified 13 major secreted proteases that are related to degradation of therapeutic antibodies, interferon alpha 2b, and insulin like growth factor. The major proteases observed were aspartic, glutamic, subtilisin-like, and trypsin-like proteases. The seven most problematic proteases were sequentially removed from a strain to develop it for producing therapeutic proteins. After this the protease activity in the supernatant was dramatically reduced down to 4% of the original level based upon a casein substrate. When antibody was incubated in the six protease deletion strain supernatant, the heavy chain remained fully intact and no degradation products were observed. Interferon alpha 2b and insulin like growth factor were less stable in the same supernatant, but full length proteins remained when incubated overnight, in contrast to the original strain. As additional benefits, the multiple protease deletions have led to faster strain growth and higher levels of total protein in the culture supernatant.

Methods for identifying lipoxygenase producing microorganisms on agar plates

Plate assays for lipoxygenase producing microorganisms on agar plates have been developed. Both potassium iodide-starch and indamine dye formation methods were effective for detecting soybean lipoxygenase activity on agar plates. A positive result was also achieved using the β-carotene bleaching method, but the sensitivity of this method was lower than the other two methods. The potassium iodide-starch and indamine dye formation methods were also applied for detecting lipoxygenase production by Trichoderma reesei and Pichia pastoris transformants expressing the lipoxygenase gene of the fungus Gaeumannomyces graminis. In both cases lipoxygenase production in the transformants could be identified. For detection of the G. graminis lipoxygenase produced by Aspergillus nidulans the potassium iodide-starch method was successful. When Escherichia coli was grown on agar and soybean lipoxygenase was applied on the culture lipoxygenase activity could clearly be detected by the indamine dye formation method. This suggests that the method has potential for screening of metagenomic libraries in E. coli for lipoxygenase activity.

Elastic and pH responsive hybrid interfaces created with engineered resilin and nanocellulose

We investigated how a genetically engineered resilin fusion protein modifies cellulose surfaces. We characterized the pH-responsive behavior of a resilin-like polypeptide (RLP) having terminal cellulose binding modules (CBM) and showed its binding to cellulose nanofibrils (CNF). Characterization of the resilin fusion protein at different pHs revealed substantial conformational changes of the protein, which were observed as swelling and contraction of the protein layer bound to the nanocellulose surface. In addition, we showed that employment of the modified resilin in cellulose hydrogel and nanopaper increased their modulus of stiffness through a cross-linking effect.

Coacervation of resilin fusion proteins containing terminal functionalities

Liquid-liquid phase transition known as coacervation of resilin-like-peptide fusion proteins containing different terminal domains were investigated. Two different modular proteins were designed and produced and their behavior were compared to a resilin-like-peptide without terminal domains. The size of the particle-like coacervates was modulated by the protein concentration, pH and temperature. The morphology and three-dimensional (3D) structural details of the coacervate particles were investigated by cryogenic transmission electron microscopy (cryo-TEM) and tomography (cryo-ET) reconstruction. Selective adhesion of the coacervates on cellulose and graphene surfaces was demonstrated.