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.

The Gram-positive eubacterium Bacillus subtilis is well known for its high capacity to secrete proteins into the environment. Even though high-level secretion of proteins is an efficient process, it imposes stress on the cell. The present studies were aimed at the identification of systems required to combat this so-called secretion stress. A two-component regulatory system, named CssR-CssS, was identified, which bears resemblance to the CpxR-CpxA system of Escherichia coli. The results show that the CssR/S system is required for the cell to survive the severe secretion stress caused by a combination of high-level production of the alpha-amylase AmyQ and reduced levels of the extracytoplasmic folding factor PrsA. As shown with a prsA3 mutation, the Css system is required to degrade misfolded exported proteins at the membrane-cell wall interface. This view is supported by the observation that transcription of the htrA gene, encoding a predicted membrane-bound protease of B. subtilis, is strictly controlled by CssS. Notably, CssS represents the first identified sensor for extracytoplasmic protein misfolding in a Gram-positive eubacterium. In conclusion, the results show that quality control systems for extracytoplasmic protein folding are not exclusively present in the periplasm of Gram-negative eubacteria, but also in the Gram-positive cell envelope.

The PrsA protein of Bacillus subtilis is an essential membrane-bound lipoprotein that is assumed to assist post-translocational folding of exported proteins and stabilize them in the compartment between the cytoplasmic membrane and cell wall. This folding activity is consistent with the homology of a segment of PrsA with parvulin-type peptidyl-prolyl cis/trans isomerases (PPIase). In this study, molecular modeling showed that the parvulin-like region can adopt a parvulin-type fold with structurally conserved active site residues. PrsA exhibits PPIase activity in a manner dependent on the parvulin-like domain. We constructed deletion, peptide insertion, and amino acid substitution mutations and demonstrated that the parvulin-like domain as well as flanking N-and C-terminal domains are essential for in vivo PrsA function in protein secretion and growth. Surprisingly, none of the predicted active site residues of the parvulin-like domain was essential for growth and protein secretion, although several active site mutations reduced or abolished the PPIase activity or the ability of PrsA to catalyze proline-limited protein folding in vitro. Our results indicate that PrsA is a PPIase, but the essential role in vivo seems to depend on some non-PPIase activity of both the parvulin-like and flanking domains.

Hypocrea jecorina is an industrially important filamentous fungus due to its effective production of hydrolytic enzymes. It has received increasing interest because of its ability to convert lignocellulosic biomass to monomeric sugars, which can be converted into biofuels or platform chemicals. Genetic engineering of strains is a highly important means of meeting the requirements of tailor-made applications. Therefore, we report the development of a transformation system that allows highly efficient gene targeting by using a tmus53 (human LIG4 homolog) deletion strain. Moreover, it permits the unlimited reuse of the same marker by employing a Cre/loxP-based excision system. Both marker insertion and marker excision can be positively selected for by combining resistance to hygromycin B and loss of sensitivity to fluoroacetamide. Finally, the marker pyr4, also positively selectable for insertion and loss, can be used to remove the cre gene.

Regulated expression of AmyQ ␣-amylase of Bacillus amyloliquefaciens was used to examine the capacity of the protein secretion apparatus of B. subtilis. One B. subtilis cell was found to secrete maximally 10 fg of AmyQ per h. The signal peptidase SipT limits the rate of processing of the signal peptide. Another limit is set by PrsA lipoprotein. The wild-type level of PrsA was found to be 2 ؋ 10 4 molecules per cell. Decreasing the cellular level of PrsA did not decrease the capacity of the protein translocation or signal peptide processing steps but dramatically affected secretion in a posttranslocational step. There was a linear correlation between the number of cellular PrsA molecules and the number of secreted AmyQ molecules over a wide range of prsA and amyQ expression levels. Significantly, even when amyQ was expressed at low levels, overproduction of PrsA enhanced its secretion. The finding is consistent with a reversible interaction between PrsA and AmyQ. The high cellular level of PrsA suggests a chaperone-like function. PrsA was also found to be essential for the viability of B. subtilis. Drastic depletion of PrsA resulted in altered cellular morphology and ultimately in cell death.Proteins synthesized with a signal peptide are secreted from bacterial cells by the action of the protein secretion apparatus, which consists of several components involved in protein targeting, translocation, signal peptide processing, and posttranslocational folding (4, 7). Extensive studies of Escherichia coli and Bacillus subtilis have identified and characterized to a substantial extent the components of the apparatus that translocates secretory proteins across the cytoplasmic membrane. In B. subtilis, they include the SecY, SecE, and SecG proteins, which form the core of the translocation channel or translocator (30,47) and are associated in the membrane with the SecDF protein (3). Furthermore, there are several signal peptidases (43, 45). The role of SecA ATPase on the cis side of the membrane in targeting and coupling the energy required for translocation has been well established (15,48). Many components of the secretion apparatus are known to be under temporal control; their maximal level of expression parallels the onset of protein secretion in the early stationary growth phase (15, 43).The stages of protein secretion that take place outside the cytoplasmic membrane are less well understood. A central feature of secretion is posttranslocational folding. The correct folding of many secreted proteins is not spontaneous but dependent on assisting folding factors. In E. coli they include protein-specific chaperones, periplasmic peptidyl-prolyl cis/trans isomerases, and enzymes (Dsb proteins) involved in the formation and rearrangement of disulfide bonds (8,16,19,31,32). The depletion of foldases such as SurA and PpiD causes misfolding stress that activates the E -and cpx-dependent stress response (5,6,31). This results in the induction of expression of the periplasmic protease and foldases (6, 37), often resulting in t...

SummaryThe PrsA protein is a membrane-anchored peptidylprolyl cis-trans isomerase in Bacillus subtilis and most other Gram-positive bacteria. It catalyses the post-translocational folding of exported proteins and is essential for normal growth of B. subtilis. We studied the mechanism behind this indispensability. We could construct a viable prsA null mutant in the presence of a high concentration of magnesium. Various changes in cell morphology in the absence of PrsA suggested that PrsA is involved in the biosynthesis of the cylindrical lateral wall. Consistently, four penicillin-binding proteins (PBP2a, PBP2b, PBP3 and PBP4) were unstable in the absence of PrsA, while muropeptide analysis revealed a 2% decrease in the peptidoglycan cross-linkage index. Misfolded PBP2a was detected in PrsA-depleted cells, indicating that PrsA is required for the folding of this PBP either directly or indirectly. Furthermore, strongly increased uniform staining of cell wall with a fluorescent vancomycin was observed in the absence of PrsA. We also demonstrated that PrsA is a dimeric or oligomeric protein which is localized at distinct spots organized in a helical pattern along the cell membrane. These results suggest that PrsA is essential for normal growth most probably as PBP folding is dependent on this PPIase.

BackgroundTrichoderma reesei is the main industrial producer of cellulases and hemicellulases that are used to depolymerize biomass in a variety of biotechnical applications. Many of the production strains currently in use have been generated by classical mutagenesis. In this study we characterized genomic alterations in high-producing mutants of T. reesei by high-resolution array comparative genomic hybridization (aCGH). Our aim was to obtain genome-wide information which could be utilized for better understanding of the mechanisms underlying efficient cellulase production, and would enable targeted genetic engineering for improved production of proteins in general.ResultsWe carried out an aCGH analysis of four high-producing strains (QM9123, QM9414, NG14 and Rut-C30) using the natural isolate QM6a as a reference. In QM9123 and QM9414 we detected a total of 44 previously undocumented mutation sites including deletions, chromosomal translocation breakpoints and single nucleotide mutations. In NG14 and Rut-C30 we detected 126 mutations of which 17 were new mutations not documented previously. Among these new mutations are the first chromosomal translocation breakpoints identified in NG14 and Rut-C30. We studied the effects of two deletions identified in Rut-C30 (a deletion of 85 kb in the scaffold 15 and a deletion in a gene encoding a transcription factor) on cellulase production by constructing knock-out strains in the QM6a background. Neither the 85 kb deletion nor the deletion of the transcription factor affected cellulase production.ConclusionsaCGH analysis identified dozens of mutations in each strain analyzed. The resolution was at the level of single nucleotide mutation. High-density aCGH is a powerful tool for genome-wide analysis of organisms with small genomes e.g. fungi, especially in studies where a large set of interesting strains is analyzed.

Aims: To explore the potential to enhance secretion of heterologous proteins in Bacillus subtilis by engineering cell factors affecting extracytoplasmic protein folding and degradation. Methods and Results: Bottleneck components affecting the extracytoplasmic phase of protein secretion were genetically engineered and their effects on the secretion of 11 industrially interesting heterologous proteins were studied by Western blotting and enzymatic assays. Overproduction of PrsA lipoprotein enhanced the secretion of a-amylase of Bacillus stearothermophilus (fourfold) and pneumolysin (1AE5-fold). Increasing the net negative charge of the cell wall because of lack of the D D-alanine substitution of anionic cell wall polymers enhanced the secretion of pneumolysin c. 1AE5-fold. Decreasing the level of HtrA-type quality control proteases caused harmful effects on growth and did not enhance secretion. Pertussis toxin subunit, S1 was found to be a substrate for HtrA-type proteases and its secretion was dependent on these proteases. Conclusions: Secretion of heterologous proteins can be enhanced by engineering components involved in late stages of secretion in a protein-dependent manner. Significance and Impact of the Study: The study revealed both possibilities and limitations of modulating the post-translocational phase of secretion as a means to improve the yield of heterologous proteins.