Production of bovine beta-lactoglobulin and hen egg ovalbumin by Trichoderma reesei using precision fermentation technology and testing of their techno-functional properties.

The food protein ingredient market is dominated by dairy and egg proteins. Both milk whey and egg proteins are challenging proteins to replace, e.g. with plant proteins, due to the unique structural features of the animal proteins that render them highly functional. Thus, to provide a non-animal source of these important proteins the fungal host Trichoderma reesei was utilized for the biotechnical production of recombinant hen ovalbumin (TrOVA) and bovine beta lactoglobulin (TrBLG). These food proteins were investigated using two different promoter systems to test the concept of effectively expressing them in a fungal host. Both proteins were successfully produced in 24 well plate and bioreactor scale. The production level of TrBLG and TrOVA were 1 g/L and 2 g/L, respectively. Both proteins were further purified and characterized, and their functional properties were tested. TrBLG and TrOVA secondary structures determined by circular dichroism corresponded to the proteins of bovine and hen. The T. reesei produced proteins were found to be N-glycosylated, mostly with Man 5. TrBLG had emulsification properties matching to corresponding bovine protein. TrOVA showed excellent foaming characteristics and heat-induced gelation, although the strength of the gel was somewhat lower than with hen ovalbumin, possibly due to the partial degradation of TrOVA or presence of other host proteins. Biotechnical production of whey and egg proteins using precision fermentation technology offers an innovative way to increase the sustainability of the conventional food industry, without further reliance on animal farming. Industrial relevance: The food protein ingredient market is dominated by dairy (largely whey proteins) and egg proteins. Whey proteins are valuable and versatile food ingredients due to their functional and nutritional quality. They are largely used in meat and milk products, low fat products, bakery, confectionary, infant formulas and sports nutrition. Similarly, egg white protein ovalbumin is a highly functional protein ingredient that facilitates structure formation and high nutritional quality in most food products. Together they comprise 40-70% of the revenue in the animal protein ingredients market. Both whey and egg proteins are extremely challenging proteins to replace, e.g., by plant proteins due to their unique structural features that render them with high functionality. Biotechnical production of whey and egg proteins using precision fermentation technology offers an innovative way to increase the sustainability of the conventional food industry, without further reliance on animal farming.

Functional characterization of a highly specific L-arabinose transporter from Trichoderma reesei.

BACKGROUND: Lignocellulose biomass has been investigated as a feedstock for second generation biofuels and other value-added products. Some of the processes for biofuel production utilize cellulases and hemicellulases to convert the lignocellulosic biomass into a range of soluble sugars before fermentation with microorganisms such as yeast Saccharomyces cerevisiae. One of these sugars is L-arabinose, which cannot be utilized naturally by yeast. The first step in L-arabinose catabolism is its transport into the cells, and yeast lacks a specific transporter, which could perform this task. RESULTS: We identified Trire2_104072 of Trichoderma reesei as a potential L-arabinose transporter based on its expression profile. This transporter was described already in 2007 as D-xylose transporter XLT1. Electrophysiology experiments with Xenopus laevis oocytes and heterologous expression in yeast revealed that Trire2_104072 is a high-affinity L-arabinose symporter with a Km value in the range of [Formula: see text] 0.1-0.2 mM. It can also transport D-xylose but with low affinity (Km [Formula: see text] 9 mM). In yeast, L-arabinose transport was inhibited slightly by D-xylose but not by D-glucose in an assay with fivefold excess of the inhibiting sugar. Comparison with known L-arabinose transporters revealed that the expression of Trire2_104072 enabled yeast to uptake L-arabinose at the highest rate in conditions with low extracellular L-arabinose concentration. Despite the high specificity of Trire2_104072 for L-arabinose, the growth of its T. reesei deletion mutant was only affected at low L-arabinose concentrations. CONCLUSIONS: Due to its high affinity for L-arabinose and low inhibition by D-glucose or D-xylose, Trire2_104072 could serve as a good candidate for improving the existing pentose-utilizing yeast strains. The discovery of a highly specific L-arabinose transporter also adds to our knowledge of the primary metabolism of T. reesei. The phenotype of the deletion strain suggests the involvement of other transporters in L-arabinose transport in this species.

Electrophysiological characterization of a diverse group of sugar transporters from Trichoderma reesei.

Trichoderma reesei is an ascomycete fungus known for its capability to secrete high amounts of extracellular cellulose- and hemicellulose-degrading enzymes. These enzymes are utilized in the production of second-generation biofuels and T. reesei is a well-established host for their production. Although this species has gained considerable interest in the scientific literature, the sugar transportome of T. reesei remains poorly characterized. Better understanding of the proteins involved in the transport of different sugars could be utilized for engineering better enzyme production strains. In this study we aimed to shed light on this matter by characterizing multiple T. reesei transporters capable of transporting various types of sugars. We used phylogenetics to select transporters for expression in Xenopus laevis oocytes to screen for transport activities. Of the 18 tested transporters, 8 were found to be functional in oocytes. 10 transporters in total were investigated in oocytes and in yeast, and for 3 of them no transport function had been described in literature. This comprehensive analysis provides a large body of new knowledge about T. reesei sugar transporters, and further establishes X. laevis oocytes as a valuable tool for studying fungal sugar transporters.

Ovalbumin production using Trichoderma reesei culture and low-carbon energy could mitigate the environmental impacts of chicken-egg-derived ovalbumin.

Ovalbumin (OVA) produced using the fungus Trichoderma reesei (Tr-OVA) could become a sustainable replacement for chicken egg white protein powder-a widely used ingredient in the food industry. Although the approach can generate OVA at pilot scale, the environmental impacts of industrial-scale production have not been explored. Here, we conducted an anticipatory life cycle assessment using data from a pilot study to compare the impacts of Tr-OVA production with an equivalent functional unit of dried chicken egg white protein produced in Finland, Germany and Poland. Tr-OVA production reduced most agriculture-associated impacts, such as global warming and land use. Increased impacts were mostly related to industrial inputs, such as electricity production, but were also associated with glucose consumption. Switching to low-carbon energy sources could further reduce environmental impact, demonstrating the potential benefits of cellular agriculture over livestock agriculture for OVA production.

Studies on sugar transporter CRT1 reveal new characteristics that are critical for cellulase induction in Trichoderma reesei.

BACKGROUND: Trichoderma reesei is an ascomycete fungus that has a tremendous capability of secreting extracellular proteins, mostly lignocellulose-degrading enzymes. Although many aspects of the biology of this organism have been unfolded, the roles of the many sugar transporters coded in its genome are still a mystery with a few exceptions. One of the most interesting sugar transporters that has thus far been discovered is the cellulose response transporter 1 (CRT1), which has been suggested to be either a sugar transporter or a sensor due to its seemingly important role in cellulase induction. RESULTS: Here we show that CRT1 is a high-affinity cellobiose transporter, whose function can be complemented by the expression of other known cellobiose transporters. Expression of two sequence variants of the crt1 gene in Saccharomyces cerevisiae revealed that only the variant listed in the RUT-C30 genome annotation has the capability to transport cellobiose and lactose. When expressed in the Δ crt1 strain, the variant listed in the QM6a genome annotation offers partial complementation of the cellulase induction, while the expression of the RUT-C30 variant or cellobiose transporters from two other fungal species fully restore the cellulase induction. CONCLUSIONS: These results add to our knowledge about the fungal metabolism of cellulose-derived oligosaccharides, which have the capability of inducing the cellulase production in many species. They also help us to deepen our understanding of the T. reesei lactose metabolism, which can have important consequences as this sugar is used as the inducer of protein secretion in many industrial processes which employ this species.

Biomimetic composites with enhanced toughening using silk-inspired triblock proteins and aligned nanocellulose reinforcements.

Silk and cellulose are biopolymers that show strong potential as future sustainable materials. They also have complementary properties, suitable for combination in composite materials where cellulose would form the reinforcing component and silk the tough matrix. A major challenge concerns balancing structure and functional properties in the assembly process. We used recombinant proteins with triblock architecture, combining structurally modified spider silk with terminal cellulose affinity modules. Flow alignment of cellulose nanofibrils and triblock protein allowed continuous fiber production. Protein assembly involved phase separation into concentrated coacervates, with subsequent conformational switching from disordered structures into ß sheets. This process gave the matrix a tough adhesiveness, forming a new composite material with high strength and stiffness combined with increased toughness. We show that versatile design possibilities in protein engineering enable new fully biological materials and emphasize the key role of controlled assembly at multiple length scales for realization.

Novel genetic tools that enable highly pure protein production in Trichoderma reesei.

Trichoderma reesei is an established protein production host with high natural capacity to secrete enzymes. The lack of efficient genome engineering approaches and absence of robust constitutive gene expression systems limits exploitation of this organism in some protein production applications. Here we report engineering of T. reesei for high-level production of highly enriched lipase B of Candida antarctica (calB) using glucose as a carbon source. Multiplexed CRISPR/Cas9 in combination with the use of our recently established synthetic expression system (SES) enabled accelerated construction of strains, which produced high amounts of highly pure calB. Using SES, calB production levels in cellulase-inducing medium were comparable to the levels obtained by using the commonly employed inducible cbh1 promoter, where a wide spectrum of native enzymes were co-produced. Due to highly constitutive expression provided by the SES, it was possible to carry out the production in cellulase-repressing glucose medium leading to around 4 grams per liter of fully functional calB and simultaneous elimination of unwanted background enzymes.

Phase transitions as intermediate steps in the formation of molecularly engineered protein fibers.

A central concept in molecular bioscience is how structure formation at different length scales is achieved. Here we use spider silk protein as a model to design new recombinant proteins that assemble into fibers. We made proteins with a three-block architecture with folded globular domains at each terminus of a truncated repetitive silk sequence. Aqueous solutions of these engineered proteins undergo liquid-liquid phase separation as an essential pre-assembly step before fibers can form by drawing in air. We show that two different forms of phase separation occur depending on solution conditions, but only one form leads to fiber assembly. Structural variants with one-block or two-block architectures do not lead to fibers. Fibers show strong adhesion to surfaces and self-fusing properties when placed into contact with each other. Our results show a link between protein architecture and phase separation behavior suggesting a general approach for understanding protein assembly from dilute solutions into functional structures.

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.

A universal gene expression system for fungi.

Biotechnological production of fuels, chemicals and proteins is dependent on efficient production systems, typically genetically engineered microorganisms. New genome editing methods are making it increasingly easy to introduce new genes and functionalities in a broad range of organisms. However, engineering of all these organisms is hampered by the lack of suitable gene expression tools. Here, we describe a synthetic expression system (SES) that is functional in a broad spectrum of fungal species without the need for host-dependent optimization. The SES consists of two expression cassettes, the first providing a weak, but constitutive level of a synthetic transcription factor (sTF), and the second enabling strong, at will tunable expression of the target gene via an sTF-dependent promoter. We validated the SES functionality in six yeast and two filamentous fungi species in which high (levels beyond organism-specific promoters) as well as adjustable expression levels of heterologous and native genes was demonstrated. The SES is an unprecedentedly broadly functional gene expression regulation method that enables significantly improved engineering of fungi. Importantly, the SES system makes it possible to take in use novel eukaryotic microbes for basic research and various biotechnological applications.

Transient Silencing of DNA Repair Genes Improves Targeted Gene Integration in the Filamentous Fungus Trichoderma reesei.

Trichoderma reesei is a filamentous fungus that is used worldwide to produce industrial enzymes. Industrial strains have traditionally been created though systematic strain improvement using mutagenesis and screening approaches. It is also desirable to specifically manipulate the genes of the organism to further improve and to modify the strain. Targeted integration in filamentous fungi is typically hampered by very low frequencies of homologous recombination. To address this limitation, we have developed a simple transient method for silencing genes in T. reesei Using gene-specific small interfering RNAs (siRNAs) targeted to mus53, we could achieve up to 90% knockdown of mus53 mRNA. As a practical example, we demonstrated that transient silencing of DNA repair genes significantly improved homologous integration of DNA at a specific locus in a standard protoplast transformation. The best transient silencing of mus53 with siRNAs in protoplasts could achieve up to 59% marker gene integration.IMPORTANCE The previous solution for improving targeted integration efficiency has been deleting nonhomologous end joining (NHEJ) DNA repair genes. However, deleting these important repair genes may lead to unintended consequences for genomic stability and could lead to the accumulation of spontaneous mutations. Our method of transiently silencing NHEJ repair pathway genes allows recovery of their important repair functions. Here we report a silencing approach for improving targeted DNA integration in filamentous fungi. Furthermore, our transient silencing method is a truly flexible approach that is capable of knocking down the expression of a target gene in growing mycelial cultures, which could facilitate the broad study of gene functions in T. reesei.

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.

Single-Molecule Force Spectroscopy Study on Modular Resilin Fusion Protein.

The adhesive and mechanical properties of a modular fusion protein consisting of two different types of binding units linked together via a flexible resilin-like-polypeptide domain are quantified. The adhesive domains have been constructed from fungal cellulose-binding modules (CBMs) and an amphiphilic hydrophobin HFBI. This study is carried out by single-molecule force spectroscopy, which enables stretching of single molecules. The fusion proteins are designed to self-assemble on the cellulose surface, leading into the submonolayer of proteins having the HFBI pointing away from the surface. A hydrophobic atomic force microscopy (AFM) tip can be employed for contacting and lifting the single fusion protein from the HFBI-functionalized terminus by the hydrophobic interaction between the tip surface and the hydrophobic patch of the HFBI. The work of rupture, contour length at rupture and the adhesion forces of the amphiphilic end domains are evaluated under aqueous environment at different pHs.

Enabling low cost biopharmaceuticals: high level interferon alpha-2b production in Trichoderma reesei.

BACKGROUND: The filamentous fungus Trichoderma reesei has tremendous capability to secrete over 100 g/L of proteins and therefore it would make an excellent host system for production of high levels of therapeutic proteins at low cost. We have developed T. reesei strains suitable for production of therapeutic proteins by reducing the secreted protease activity. Protease activity has been the major hindrance to achieving high production levels. We have constructed a series of interferon alpha-2b (IFNα-2b) production strains with 9 protease deletions to gain knowledge for further strain development. RESULTS: We have identified two protease deletions that dramatically improved the production levels. Deletion of the subtilisin protease slp7 and the metalloprotease amp2 has enabled production levels of IFNα-2b up to 2.1 and 2.4 g/L, respectively. With addition of soybean trypsin protease inhibitor the level of production improved to 4.5 g/L, with an additional 1.8 g/L still bound to the secretion carrier protein. CONCLUSIONS: High levels of IFNα-2b were produced using T. reesei strains with reduced protease secretion. Further strain development can be done to improve the production system by reducing protease activity and improving carrier protein cleavage.

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.

Dipeptidyl peptidase IV as a potential target for selective prodrug activation and chemotherapeutic action in cancers.

The efficacy of chemotherapeutic drugs is often offset by severe side effects attributable to poor selectivity and toxicity to normal cells. Recently, the enzyme dipeptidyl peptidase IV (DPPIV) was considered as a potential target for the delivery of chemotherapeutic drugs. The purpose of this study was to investigate the feasibility of targeting chemotherapeutic drugs to DPPIV as a strategy to enhance their specificity. The expression profile of DPPIV was obtained for seven cancer cell lines using DNA microarray data from the DTP database, and was validated by RT-PCR. A prodrug was then synthesized by linking the cytotoxic drug melphalan to a proline-glycine dipeptide moiety, followed by hydrolysis studies in the seven cell lines with a standard substrate, as well as the glycyl-prolyl-melphalan (GP-Mel). Lastly, cell proliferation studies were carried out to demonstrate enzyme-dependent activation of the candidate prodrug. The relative RT-PCR expression levels of DPPIV in the cancer cell lines exhibited linear correlation with U95Av2 Affymetrix data (r(2) = 0.94), and with specific activity of a standard substrate, glycine-proline-p-nitroanilide (r(2) = 0.96). The significantly higher antiproliferative activity of GP-Mel in Caco-2 cells (GI50 = 261 µM) compared to that in SK-MEL-5 cells (GI50 = 807 µM) was consistent with the 9-fold higher specific activity of the prodrug in Caco-2 cells (5.14 pmol/min/µg protein) compared to SK-MEL-5 cells (0.68 pmol/min/µg protein) and with DPPIV expression levels in these cells. Our results demonstrate the great potential to exploit DPPIV as a prodrug activating enzyme for efficient chemotherapeutic drug targeting.

Chemical inhibitors of the calcium entry channel TRPV6.

PURPOSE: Calcium entry channels in the plasma membrane are thought to play a major role in maintaining cellular Ca(2+) levels, crucial for growth and survival of normal and cancer cells. The calcium-selective channel TRPV6 is expressed in prostate, breast, and other cancer cells. Its expression coincides with cancer progression, suggesting that it drives cancer cell growth. However, no specific inhibitors for TRPV6 have been identified thus far. METHODS: To develop specific TRPV6 inhibitors, we synthesized molecules based on the lead compound TH-1177, reported to inhibit calcium entry channels in prostate cancer cells in vitro and in vivo. RESULTS: We found that one of our compounds (#03) selectively inhibited TRPV6 over five times better than TRPV5, whereas TH-1177 and the other synthesized compounds preferentially inhibited TRPV5. The IC(50) value for growth inhibition by blocking endogenous Ca(2+) entry channels in the LNCaP human prostate cancer cell line was 0.44 ± 0.07 µM compared to TH-1177 (50 ± 0.4 µM). CONCLUSIONS: These results suggest that compound #03 is a relatively selective and potent inhibitor for TRPV6 and that it is an interesting lead compound for the treatment of prostate cancer and other cancers of epithelial origin.

Tamoxifen inhibits TRPV6 activity via estrogen receptor-independent pathways in TRPV6-expressing MCF-7 breast cancer cells.

The epithelial calcium channel TRPV6 is upregulated in breast carcinoma compared with normal mammary gland tissue. The selective estrogen receptor modulator tamoxifen is widely used in breast cancer therapy. Previously, we showed that tamoxifen inhibits calcium uptake in TRPV6-transfected Xenopus oocytes. In this study, we examined the effect of tamoxifen on TRPV6 function and intracellular calcium homeostasis in MCF-7 breast cancer cells transiently transfected with EYFP-C1-TRPV6. TRPV6 activity was measured with fluorescence microscopy using Fura-2. The basal calcium level was higher in transfected cells compared with nontransfected cells in calcium-containing solution but not in nominally calcium-free buffer. Basal influxes of calcium and barium were also increased. In transfected cells, 10 mumol/L tamoxifen reduced the basal intracellular calcium concentration to the basal calcium level of nontransfected cells. Tamoxifen decreased the transport rates of calcium and barium in transfected cells by 50%. This inhibitory effect was not blocked by the estrogen receptor antagonist, ICI 182,720. Similarly, a tamoxifen-induced inhibitory effect was also observed in MDA-MB-231 estrogen receptor-negative cells. The effect of tamoxifen was completely blocked by activation of protein kinase C. Inhibiting protein kinase C with calphostin C decreased TRPV6 activity but did not alter the effect of tamoxifen. These findings illustrate how tamoxifen might be effective in estrogen receptor-negative breast carcinomas and suggest that the therapeutic effect of tamoxifen and protein kinase C inhibitors used in breast cancer therapy might involve TRPV6-mediated calcium entry. This study highlights a possible role of TRPV6 as therapeutic target in breast cancer therapy.

Interferon-gamma increases expression of the di/tri-peptide transporter, h-PEPT1, and dipeptide transport in cultured human intestinal monolayers.

The di/tri-peptide transporter h-PEPT1 plays an important role in the oral absorption of di/tri-peptides and numerous drugs. Inflammatory conditions may influence intestinal xenobiotic transporter function; however, the effects of inflammation on h-PEPT1 have not been well described. This study was conducted to determine the effects of the inflammatory cytokine interferon-gamma (IFN-gamma) on h-PEPT1 mediated dipeptide absorption. Caco-2 monolayers were grown on permeable supports. The effective apical-to-basolateral permeability (P(eff)) of glycylsarcosine (Gly-Sar) was measured following incubation with IFN-gamma or control media. Additional experiments were conducted at 4 degrees C, and with escalating concentrations of Gly-Sar. h-PEPT1 expression was determined using semiquantitative RT-PCR. IFN-gamma 50 ng/ml increased Gly-Sar P(eff) 28.6% compared to controls (p=0.03). In experiments conducted at 4 degrees C, Gly-Sar P(eff) decreased 39.6% in IFN-gamma treated cells (p=0.003) and 28.4% in controls (p=0.006). In controls and IFN-gamma treated cells, concentration dependent transport was seen with escalating concentrations of Gly-Sar. Compared to controls, IFN-gamma 50 and 100 ng/ml increased h-PEPT1 mRNA expression by 14.2% and 11.5%, respectively (p=0.019). In summary, IFN-gamma increases h-PEPT1 expression and permeation of the dipeptide Gly-Sar in Caco-2 monolayers. These findings imply that intestinal absorption of peptides and peptidomimetic drugs may be increased in certain inflammatory conditions.

Enhanced absorption and growth inhibition with amino acid monoester prodrugs of floxuridine by targeting hPEPT1 transporters.

A series of amino acid monoester prodrugs of floxuridine was synthesized and evaluated for the improvement of oral bioavailability and the feasibility of target drug delivery via oligopeptide transporters. All floxuridine 5'-amino acid monoester prodrugs exhibited PEPT1 affinity, with inhibition coefficients of Gly-Sar uptake (IC50) ranging from 0.7 - 2.3 mM in Caco-2 and 2.0 - 4.8 mM in AsPC-1 cells, while that of floxuridine was 7.3 mM and 6.3 mM, respectively. Caco-2 membrane permeabilities of floxuridine prodrugs (1.01 - 5.31 x 10(-6 )cm/sec) and floxuridine (0.48 x 10(-6 )cm/sec) were much higher than that of 5-FU (0.038 x 10(-6) cm/sec). MDCK cells stably transfected with the human oligopeptide transporter PEPT1 (MDCK/hPEPT1) exhibited enhanced cell growth inhibition in the presence of the prodrugs. This prodrug strategy offers great potential, not only for increased drug absorption but also for improved tumor selectivity and drug efficacy.