Backbone and sidechain 1H, 13C and 15N chemical shift assignments of the hydrophobin DewA from Aspergillus nidulans

Hydrophobins are proteins secreted by filamentous fungi that are able to self-assemble into monolayers at hydrophobic:hydrophilic interfaces. The layers are amphipathic and can reverse the wettability of surfaces. Hydrophobins have several roles in fungal development, including the formation of coatings on fungal structures to render them hydrophobic. Here we report the backbone and sidechain assignments for the class I hydrophobin DewA from the fungus Aspergillus nidulans.     

Expression of an AT-rich xylanase gene from the anaerobic fungus <Emphasis Type="Italic">Orpinomyces</Emphasis> sp. strain PC-2 in and secretion of the heterologous enzyme by <Emphasis Type="Italic">Hypocrea jecorina</Emphasis>

The catalytic domain encoded by an adenine–thymine (AT)-rich xylanase gene (xynA) of the anaerobic fungus Orpinomyces was expressed in Hypocrea jecorina under the control of the cel7A promoter and terminator. No XynA protein was detected in H. jecorina culture supernatants when the original sequence was fused to the H. jecorina cel5A region coding for its signal peptide, carbohydrate-binding module, and hinge. Replacing the xynA (56% AT content) with a synthetic sequence containing lower AT content (39%) supported the extracellular production (150 mg l⁻¹) of the fusion xylanase by H. jecorina. Northern analysis revealed that successful production after the decrease in AT content was related to higher levels of the xylanase-specific mRNA. Another construct with an RDKR-coding sequence inserted between the cel5A linker and the xynA catalytic domain allowed production of the fully processed active xylanase catalytic domain. Both the fusion (40 kDa) and the fully processed (28 kDa) forms displayed enzymatic properties of family 11 xylanases. Both the R and the Kex2-like KR sites were recognized during secretion, resulting in a mixture of two amino termini for the 28-kDa xylanase. The work demonstrated for the first time that glycoside hydrolases derived from anaerobic fungi can be produced by H. jecorina. The mention of firm names or trade products does not imply that they are endorsed or recommended by the US Department of Agriculture over other firms or similar products not mentioned.     

Six Hydrophobins Are Involved in Hydrophobin Rodlet Formation in Aspergillus nidulans and Contribute to Hydrophobicity of the Spore Surface

Hydrophobins are amphiphilic proteins able to self-assemble at water-air interphases and are only found in filamentous fungi. In Aspergillus nidulans two hydrophobins, RodA and DewA, have been characterized, which both localize on the conidiospore surface and contribute to its hydrophobicity. RodA is the constituent protein of very regularly arranged rodlets, 10 nm in diameter. Here we analyzed four more hydrophobins, DewB-E, in A. nidulans and found that all six hydrophobins contribute to the hydrophobic surface of the conidiospores but only deletion of rodA caused loss of the rodlet structure. Analysis of the rodlets in the dewB-E deletion strains with atomic force microscopy revealed that the rodlets appeared less robust. Expression of DewA and DewB driven from the rodA promoter and secreted with the RodA secretion signal in a strain lacking RodA, restored partly the hydrophobicity. DewA and B were able to form rodlets to some extent but never reached the rodlet structure of RodA. The rodlet-lacking rodA-deletion strain opens the possibility to systematically study rodlet formation of other natural or synthetic hydrophobins.     

Asn64-glycosylation affects Hypocrea jecorina (syn. Trichoderma reesei) cellobiohydrolase Cel7A activity expressed in Pichia pastoris

In this study, four N-glycosylation sites, Asn45, Asn64, Asn270 and Asn384 of Hypocrea jecorina (syn. Trichoderma reesei) Cel7A (family 7 cellobiohydrolase I) were replaced by serines using site-directed mutagenesis. These four mutants and wild type H. jecorina Cel7A gene were transformed into P. pastoris, and the recombinant enzymes were purified and analyzed. The enzymatic activities of recombinant Cel7A (rCel7A), and mutants N45S, N270S and N384S were very low while mutant N64S displayed about seven times higher activity than that of rCel7A, and about 10% of the wild-type Cel7A activity from H. jecorina. The results indicate that N-glycosylation of Asn64 had an effect on the activity of the Cel7A enzyme expressed in P. pastoris, and that glycosylation at this site would be only a subordinate reason for the low activity of the recombinant enzyme.     

Secretion by Saccharomyces cerevisiae of rat apolipoprotein E as a fusion to Mucor rennin

As the first step for production of rat apolipoprotein E (rApoE) in Saccharomyces cerevisiae, the rApoE cDNA was cloned and its nucleotide sequence was determined. When the intact rApoE gene including the presequence-encoding region was expressed under the control of the yeast GAL7 promoter, no protein immunoreactive with anti-rApoE antibody was detected either in the culture medium or inside the cells. For the purpose of the extracellular production of rApoE, three fusion genes were constructed in which the mature rApoE-encoding sequence was connected after the pre, prepro, and whole regions of the gene encoding a fungal aspartic proteinase, Mucor pusillus rennin (MPP), since MPP is efficiently secreted from recombinant S. cerevisiae containing the MPP gene. When these three fusion genes were expressed under the control of the GAL7 promoter, only one, encoding the mature rApoE connected to the whole MPP sequence, directed efficient secretion of the fused protein. The maximum yield of the fused protein secreted into the medium reached 11.8 mg/l and the calculated rApoE part was 5.3 mg in the fused protein. The excreted fusion protein was glycosylated at the original two sites in the MPP part. The fused protein was gradually degraded in the medium probably by proteases of the host cell, because no such degradation occured in a yeast pep4mutant strain.     

Escherichia coli “TatExpress” strains super‐secrete human growth hormone into the bacterial periplasm by the Tat pathway

Numerous high‐value proteins are secreted into the Escherichia coli periplasm by the General Secretory (Sec) pathway, but Sec‐based production chassis cannot handle many potential target proteins. The Tat pathway offers a promising alternative because it transports fully folded proteins; however, yields have been too low for commercial use. To facilitate Tat export, we have engineered the TatExpress series of super‐secreting strains by introducing the strong inducible bacterial promoter, ptac , upstream of the chromosomal tatABCD operon, to drive its expression in E. coli strains commonly used by industry (e.g., W3110 and BL21). This modification significantly improves the Tat‐dependent secretion of human growth hormone (hGH) into the bacterial periplasm, to the extent that secreted hGH is the dominant periplasmic protein after only 1 hr induction. TatExpress strains accumulate in excess of 30 mg L^?1 periplasmic recombinant hGH, even in shake flask cultures. A second target protein, an scFv, is also shown to be exported at much higher rates in TatExpress strains.

     

Evolution and Ecophysiology of the Industrial Producer Hypocrea jecorina (Anamorph Trichoderma reesei) and a New Sympatric Agamospecies Related to It

Background

Trichoderma reesei, a mitosporic green mould, was recognized during the WW II based on a single isolate from the Solomon Islands and since then used in industry for production of cellulases. It is believed to be an anamorph (asexual stage) of the common pantropical ascomycete Hypocrea jecorina.

Methodology/Principal Findings

We combined molecular evolutionary analysis and multiple methods of phenotype profiling in order to reveal the genetic relationship of T. reesei to H. jecorina. The resulting data show that the isolates which were previously identified as H. jecorina by means of morphophysiology and ITS1 and 2 (rRNA gene cluster) barcode in fact comprise several species: i) H. jecorina/T. reesei sensu stricto which contains most of the teleomorphs (sexual stages) found on dead wood and the wild-type strain of T. reesei QM 6a; ii) T. parareesei nom. prov., which contains all strains isolated as anamorphs from soil; iii) and two other hypothetical new species for which only one or two isolates are available. In silico tests for recombination and in vitro mating experiments revealed a history of sexual reproduction for H. jecorina and confirmed clonality for T. parareesei nom. prov. Isolates of both species were consistently found worldwide in pantropical climatic zone. Ecophysiological comparison of H. jecorina and T. parareesei nom. prov. revealed striking differences in carbon source utilization, conidiation intensity, photosensitivity and mycoparasitism, thus suggesting adaptation to different ecological niches with the high opportunistic potential for T. parareesei nom. prov.

Conclusions

Our data prove that T. reesei belongs to a holomorph H. jecorina and displays a history of worldwide gene flow. We also show that its nearest genetic neighbour - T. parareesei nom. prov., is a cryptic phylogenetic agamospecies which inhabits the same biogeographic zone. These two species thus provide a so far rare example of sympatric speciation within saprotrophic fungi, with divergent ecophysiological adaptations and reproductive strategies.     

Sophorolipids-induced cellulase production in cocultures of Hypocrea jecorina Rut C30 and Candida bombicola

Sophorose is a potent but expensive inducer for cellulase production. In this study the feasibility of using sophorolipids, natural lipids that contain sophorose, for cellulase induction was investigated. Enhanced cellulase production by Hypocrea jecorina Rut C30 grown on glycerol, a substrate without cellulase-inducing ability, was first confirmed by addition of the crude sophorolipids collected from Candida bombicola fermentation. Cocultures of H. jecorina Rut C30 and C. bombicola were then employed to evaluate the effects of coculture conditions: the cell concentration ratio between the two cultures, the concentration of vegetable oil (as lipid precursor for sophorolipid synthesis, in addition to glycerol as the primary carbon source), the presence of nitrogen source for growth, and the substitution of glucose for glycerol as the primary carbon source. Specific cellulase productivity of H. jecorina Rut C30 was significantly higher under the conditions that promoted sophorolipid production by C. bombicola. The ability of H. jecorina Rut C30 to degrade sophorolipids was also confirmed. The results of the study indicated that the sophorolipids produced by C. bombicola can be degraded by H. jecorina Rut C30 and the sophorose generated from the degradation can effectively induce the fungal cellulase synthesis.     

PRODUCTION AND OPTIMIZATION OF L-GLUTAMINASE ENZYME FROM Hypocrea jecorina PURE CULTURE

L-Glutaminase (L-glutamine amidohydrolase, EC 3.5.1.2) is the important enzyme that catalyzes the deamination of L-glutamine to L-glutamic acid and ammonium ions. Recently, L-glutaminase has received much attention with respect to its therapeutic and industrial applications. It acts as a potent antileukemic agent and shows flavor-enhancing capacity in the production of fermented foods. Glutaminase activity is widely distributed in plants, animal tissues, and microorganisms, including bacteria, yeasts, and fungi. This study presents microbial production of glutaminase enzyme from Hypocrea jecorina pure culture and determination of optimum conditions and calculation of kinetic parameters of the produced enzyme. The optimum values were determined by using sa Nesslerization reaction for our produced glutaminase enzyme. The optimum pH value was determined as 8.0 and optimum temperature as 50°C for the glutaminase enzyme. The Km and Vmax values, the kinetic parameters, of enzyme produced from Hypocrea jecorina, pure culture were determined as 0.491 mM for Km and 13.86 U/L for Vmax by plotted Lineweaver–Burk graphing, respectively. The glutaminase enzyme from H. jecorina microorganism has very high thermal and storage stability.     

Evaluation of promoter sequences for the secretory production of a Clostridium thermocellum cellulase in Paenibacillus polymyxa

Due to their high secretion capacity, Gram-positive bacteria from the genus Bacillus are important expression hosts for the high-yield production of enzymes in industrial biotechnology; however, to date, strains from only few Bacillus species are used for enzyme production at industrial scale. Herein, we introduce Paenibacillus polymyxa DSM 292, a member of a different genus, as a novel host for secretory protein production. The model gene cel8A from Clostridium thermocellum was chosen as an easily detectable reporter gene with industrial relevance to demonstrate heterologous expression and secretion in P. polymyxa. The yield of the secreted cellulase Cel8A protein was increased by optimizing the expression medium and testing several promoter sequences in the expression plasmid pBACOV. Quantitative mass spectrometry was used to analyze the secretome in order to identify promising new promoter sequences from the P. polymyxa genome itself. The most abundantly secreted host proteins were identified, and the promoters regulating the expression of their corresponding genes were selected. Eleven promoter sequences were cloned and tested, including well-characterized promoters from Bacillus subtilis and Bacillus megaterium. The best result was achieved with the promoter for the hypothetical protein PPOLYM_03468 from P. polymyxa. In combination with the optimized expression medium, this promoter enabled the production of 5475 U/l of Cel8A, which represents a 6.2-fold increase compared to the reference promoter P_aprE. The set of promoters described in this work covers a broad range of promoter strengths useful for heterologous expression in the new host P. polymyxa.

     

Expression of an endoglucanase from Tribolium castaneum (TcEG1) in Saccharomyces cerevisiae

Insects are a largely unexploited resource in prospecting for novel cellulolytic enzymes to improve the production of ethanol fuel from lignocellulosic biomass. The cost of lignocellulosic ethanol production is expected to decrease by the combination of cellulose degradation (saccharification) and fermentation of the resulting glucose to ethanol in a single process, catalyzed by the yeast Saccharomyces cerevisiae transformed to express efficient cellulases. While S. cerevisiae is an established heterologous expression system, there are no available data on the functional expression of insect cellulolytic enzymes for this species. To address this knowledge gap, S. cerevisiae was transformed to express the full‐length cDNA encoding an endoglucanase from the red flour beetle, Tribolium castaneum (TcEG1), and evaluated the activity of the transgenic product (rTcEG1). Expression of the TcEG1 cDNA in S. cerevisiae was under control of the strong glyceraldehyde‐3 phosphate dehydrogenase promoter. Cultured transformed yeast secreted rTcEG1 protein as a functional β‐1,4‐endoglucanase, which allowed transformants to survive on selective media containing cellulose as the only available carbon source. Evaluation of substrate specificity for secreted rTcEG1 demonstrated endoglucanase activity, although some activity was also detected against complex cellulose substrates. Potentially relevant to uses in biofuel production rTcEG1 activity increased with pH conditions, with the highest activity detected at pH 12. Our results demonstrate the potential for functional production of an insect cellulase in S. cerevisiae and confirm the stability of rTcEG1 activity in strong alkaline environments.     

Biosynthesis of Inositol by an Enzyme System in the Rice Seeds

As the first step for production of human apolipoprotein E (hApoE) in Saccharomyces cerevisiae, the hApoE cDNA was cloned in Escherichia coli, on the basis of the nucleotide sequence reported previously. When the hApoE cDNA including its pre-sequence-encoding region was expressed under the control of the GAL7 promoter, no protein immunoreactive with anti-hApoE antibody was detected either in the culture medium or inside the cells. For efficient production and secretion of hApoE in S. cerevisiae, the mature hApoE-encoding region was fused to the prepro-sequence region of Rhizomucor rennin (MPR) and to the whole MPR gene including its prepro- and mature-MPR regions. When the fusion gene consisting of the prepro-sequence-encoding region and hApoE regions was expressed in S. cerevisiae, no protein reactive with the anti-hApoE antibody was detected in any fraction of the yeast cells, probably due to rapid degradation of the hApoE protein by yeast proteases. On the othe hand, when hApoE was expressed as a fusion to the whole MPR protein, a considerable amount of the fused protein was secreted into the medium. The preprosequence of MPR was correctly processed from the fused protein in the medium by autocatalytic activity of MPR and by a protease(s) of the host cell. Integration of the fusion gene into the chromosome at a copy number of eight led to secretion of the fused protein in a larger amount than the case when the fusion gene was carried on a 2-µm plasmid with its copy number of a few hundreds, because the 2-µm derived plasmid containing the fusion gene was very unstable in the yeast cells. The secretion level was also improved by changing g the culture conditions. A maximum yield of hApoE part in the secreted fused protein was estimated to be 23.7 mg per liter and the amount of the fused protein was calculated to be 53.0 mg per liter.     

Catabolite repression of the colonization factor antigen I (CFA/I) operon ofEscherichia coli

Experiments were performed to study whether the synthesis of the fimbrial colonization factor antigen I (CFA/I) of enterotoxigenicEscherichia coli is affected by glucose. The CFA/I-producing strain H-10407 (O78:H11:CFA/I) was grown in CFA medium containing various concentrations of glucose. Addition of 1% glucose into the medium resulted in a pronounced decrease in CFA/I production by H-10407 as assessed by ELISA, hemagglutination, and electron microscopy. The repressive effect of glucose was reversed by the addition of 10 mM cAMP to the medium. Examination of the promoter sequence of thecfaA gene of the CFA/I operon revealed a consensus binding site for the catabolite activator protein-cAMP complex. With a reporter plasmid containing a fusion of thecfaA promoter, a portion of thecfaA gene, and thelacZ gene, it was shown that the activity of this promoter was influenced by glucose. In a wild-typeE. coli strain, addition of 0.5% glucose to the growth medium diminished the promoter activity more than 70%. ThecfaA promoter also exhibited a lower level of activity incya (adenyl cyclase) andcrp (cAMP receptor protein) mutants than in the wild-type strain. The addition of 10 mM cAMP resulted in a marked increase in the expression from thecfaA promoter in thecya but not in thecrp mutant. These results suggest that the suppressive effect of glucose in the CFA/I system is mediated via the mechanism of catabolite repression through thecfaA promoter of the CFA/I operon.     

Expressed Peromyscus maniculatus (Pema) MHC class I genes: evolutionary implications and the identification of a gene encoding a Qa1-like antigen

To gain insight into the evolution of rodent major histocompatibility complex (MHC) class I genes and identify important (conserved) nonclassical class I (class Ib) gene products and residues in these proteins, sixPeromyscus maniculatus MHC (Pema) class I cDNA clones were isolated and sequenced. FivePema class I cDNAs appeared most similar to mouse and rat classical class I (class Ia) genes. One exhibited highest similarity to anH2 class Ib gene,H2-T23 (encoding the Qa1 antigen). Phylogenetic trees constructed withPema, RT1, andH2 class I sequences suggested that the lineages of some rodent class Ib genes (e.g.,T23 andT24) originated prior toMus andPeromyscus speciation [>50 million years (My) ago]. Sequences of four Qa1-like proteins from three species permitted the identification of ten Qa1-specific amino acids. On the basis of molecular modeling, three residues showed the potential to interact with T-cell receptors and three residues (all corresponding to polymorphic positions among H2 class Ia proteins) were predicted to influence antigen binding. The recognition of mouse Qa1 proteins by a subset of T-cells in influenced by a locus,Qdm, which encodes the H2-D leader peptide. One of thePema class I cDNA clones classified asH2-K, D/L-like (class Ia) is predicted to encode an identical peptide, implying that an antigen binding protein (Qa1) and the antigen to which it binds (the product ofQdm) has been conserved for over 50 My. The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers U12822 (Pm13), U12885 (Pm41), U12886 (Pm52), U12887 (Pm62), U16846 (Pm11), and U16847 (Pm53)