Micheelsen, P.O., Østergaard, P.R., Lange, L., Skjøt, M.
"High-level expression of the native barley α-amylase/subtilisin inhibitor in Pichia pastoris"
Journal of Biotechnology, 133 (4), pp. 424-432. (2008)
An expression system for high-level expression of the native Hordeum vulgare α-amylase/subtilisin inhibitor (BASI) has been developed in Pichia pastoris, using the methanol inducible alcohol oxidase 1 (AOX1) promoter. To optimize expression, two codon-optimized coding regions have been designed and expressed alongside the wild-type coding region. To ensure secretion of the native mature protein, a truncated version of the alpha mating factor secretion signal from Saccharomyces cerevisiae was used. In order to be able to compare expression levels from different clones, single insertion transformants generated by gene replacement of the AOX1 gene was selected by PCR screening. Following methanol induction, expression levels reached 125 mg L-1 from the wild-type coding region while expression from the two codon-optimized variants reached 65 and 125 mg L-1, respectively. The protein was purified and characterized by Edman degradation, liquid chromatography mass spectrometry and insoluble blue starch assay, and was shown to posses the same characteristics as wild-type protein purified from barley grains. © 2007 Elsevier B.V. All rights reserved.
Hamann, T., Lange, L.
"Discovery, cloning and heterologous expression of secreted potato proteins reveal erroneous pre-mRNA splicing in Aspergillus oryzae"
Journal of Biotechnology, 126 (3), pp. 265-276. (2006)
A novel transposon assisted signal trapping (TAST) technology, developed to specifically select only the secreted proteins, was used to discover novel extracellular plant proteins from Solarium tuberosum infected with Phytophthora infestans. Analysis of 384 hits provided 191 P. infestans and S. tuberosum sequences of secreted proteins, with an approx. 2/3 of these originating from potato. Subsequent screening for interesting genes was carried out using bioinformatics. A selected variety of the discovered sequences are presented, including a novel S. tuberosum xyloglucan endotransglucosylase (StXTH), which was cloned and subjected to detailed heterologous expression studies in Aspergillus oryzae. RT-PCR analysis of mRNA from A. oryzae StXTH1 transformants revealed that parts of the mRNA pool had been incorrectly processed, and only weak and inconsistent indications of active protein could be detected. A high AT content of StXTH1 and the occurrence of A. oryzae intron donor, acceptor, and branch point recognition sites resulted in erroneous intron interpretation (cryptic introns) of parts of the mRNA coding sequence. This may explain the difficulties generally experienced in expressing plant genes in filamentous fungi. © 2006 Elsevier B.V. All rights reserved.
Herzberg, K., Bashkirov, V.I., Rolfsmeier, M., Haghnazari, E., McDonald, W.H., Anderson, S., Bashkirova, E.V., Yates III, J.R., Heyer, W.-D.
"Phosphorylation of Rad55 on serines 2, 8, and 14 is required for efficient homologous recombination in the recovery of stalled replication forks"
Molecular and Cellular Biology, 26 (22), pp. 8396-8409. (2006)
DNA damage checkpoints coordinate the cellular response to genotoxic stress and arrest the cell cycle in response to DNA damage and replication fork stalling. Homologous recombination is a ubiquitous pathway for the repair of DNA double-stranded breaks and other checkpoint-inducing lesions. Moreover, homologous recombination is involved in postreplicative tolerance of DNA damage and the recovery of DNA replication after replication fork stalling. Here, we show that the phosphorylation on serines 2, 8, and 14 (S2,8,14) of the Rad55 protein is specifically required for survival as well as for normal growth under genome-wide genotoxic stress. Rad55 is a Rad51 paralog in Saccharomyces cerevisiae and functions in the assembly of the Rad51 filament, a central intermediate in recombinational DNA repair. Phosphorylation-defective rad55-S2,8,14A mutants display a very slow traversal of S phase under DNA-damaging conditions, which is likely due to the slower recovery of stalled replication forks or the slower repair of replication-associated DNA damage. These results suggest that Rad55-S2,8,14 phosphorylation activates recombinational repair, allowing for faster recovery after genotoxic stress. Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Brinch-Pedersen, H., Hatzack, F., Stöger, E., Arcalis, E., Pontopidan, K., Holm, P.B.
"Heat-stable phytases in transgenic wheat (Triticum aestivum L.): Deposition pattern, thermostability, and phytate hydrolysis"
Journal of Agricultural and Food Chemistry, 54 (13), pp. 4624-4632. (2006)
The present paper addresses the question of thermotolerance of in planta synthesized heterologous enzymes using phytase as a model. Two individual transgenic wheat materials expressing an Aspergillus fumigatus phytase with a low denaturation temperature (62.5°C) but a high refolding capacity, and a rationally designed consensus phytase engineered to a high denaturation temperature (89.3°C), were evaluated. High levels of endosperm specific expression were ensured by the wheat high molecular weight glutenin 1DX5 promoter. Immunodetection at the light and electron microscopical level shows unequivocally that the heterologous phytase is deposited in the vacuole, albeit that the transformation constructs were designed for secretion to the apoplast. Evaluation of heat stability properties and kinetic properties unraveled that, under these deposition conditions, heat stability based on high unfolding temperature is superior to high refolding capacity and represents a realistic strategy for improving phosphate and mineral bioavailability in cereal-based feed and food. © 2006 American Chemical Society.
Widner, B., Behr, R., Von Dollen, S., Tang, M., Heu, T., Sloma, A., Sternberg, D., Deangelis, P.L., Weigel, P.H., Brown, S.
"Hyaluronic acid production in Bacillus subtilis"
Applied and Environmental Microbiology, 71 (7), pp. 3747-3752. (2005)
The hasA gene from Streptococcus equisimilis, which encodes the enzyme hyaluronan synthase, has been expressed in Bacillus subtilis, resulting in the production of hyaluronic acid (HA) in the 1-MDa range. Artificial operons were assembled and tested, all of which contain the hasA gene along with one or more genes encoding enzymes involved in the synthesis of the UDP-precursor sugars that are required for HA synthesis. It was determined that the production of UDP-glucuronic acid is limiting in B. subtilis and that overexpressing the hasA gene along with the endogenous tuaD gene is sufficient for high-level production of HA. In addition, the B. subtilis-derived material was shown to be secreted and of high quality, comparable to commercially available sources of HA. Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Widner, W., Behr, R., Tang, M., Hue, T., Sternberg, D., Von Dollen, S., Sloma, A., Weigel, P.H., Brown, S.H.
"Metabolic engineering of Bacillus subtilis for biosynthesis of hyaluronic acid - A superior system for industrial HA production"
Transactions - 7th World Biomaterials Congress, p. 40. (2004)
Biosynthesis of Hyaluronic acid (HA) by Bacillus Subtilis was discussed. It was found that the presence of tuaD in the expression cassette was required for HA production above trace levels. Overall, yields of HA from the recombinant B. subtilis strains were in the multi-gram per liter range, comparable to reported Streptocioccal yields. GPC-MALLS analysis of fermentation samples indicated a molecular weight in the 1-2 MDa range with a polydispersity index below 1.5.
C. Muller; C. M. Hjort; K. Hansen; J. Nielsen.
"Altering the expression of two chitin synthase genes differentially affects the growth and morphology of Aspergillus oryzae."
Microbiology (Reading), 148 (12), 4025-4033 (2002)
In Aspergillus oryzae, one full-length chitin synthase (chsB) and fragments of two other chitin synthases (csmA and chsC) were identified. The deduced amino acid sequence of chsB was similar (87% identity) to chsB from Aspergillus nidulans, which encodes a class III chitin synthase. The sequence obtained for csmA indicated that it had high similarity to class V chitin synthases. chsB and csmA disruption strains and a strain in which chsB transcription was controlled were constructed using the nitrite reductase (niiA) promoter. The strains were examined during hyphal growth by Northern analysis, analysis of the cell-wall composition and growth in the presence of Calcofluor white (CFW). The chsB disrupted strain and the uninduced p(niiA)-chsB strain exhibited hyperbranching, they had a lower level of conidiation than the wild-type and were sensitive to CFW at 50 mg l(-1). When chsB transcription was induced in the strain containing the p(niiA)-chsB construct, the strain displayed wild-type morphology on solid medium and at sub-maximum growth rates but the wild-type morphology was not fully restored during rapid growth in batch cultivation. The csmA disruption strain displayed morphological abnormalities, such as ballooning cells, intrahyphal hyphae and conidial scars. The growth was severely inhibited in the presence of 10 mg CFW l(-1). In none of the constructed strains did the cell-wall composition differ from the wild-type. Northern analysis indicated no change in the transcription of the chitin synthase genes csmA and chsC when chsB expression was altered, and there was no change in the transcription of chsB and chsC when csmA was disrupted.
H. Pedersen; B. Christensen; C. Hjort; J. Nielsen.
"Construction and characterization of an oxalic acid nonproducing strain of Aspergillus niger."
Metab Eng, 2(1), 34-41 (2000)
Aspergillus niger produces oxalic acid as a by-product which causes problems with downstream processing of industrial enzymes. To overcome this problem the oah gene encoding oxaloacetate hydrolase (EC 18.104.22.168) was disrupted in a glucoamylase-producing strain of A. niger and the resulting strain was incapable of producing oxalic acid. The strain with the disrupted gene was compared with the wild-type strain producing oxalic acid in batch cultivations. The specific growth rate of both strains was 0.20 h(-1). The citric acid yields were identical, but the glucoamylase yield was only 50% in the disruptant compared with the wild-type strain. Batch experiments with 13C-labeled glucose as substrate were carried out to determine the metabolic fluxes through the central metabolism. The two strains had almost identical metabolic fluxes, which suggested that it was possible to disrupt the oah gene without pleiotropic consequences. The flux through the pentose phosphate pathway was around 60% of the glucose uptake for both strains, which suggested that a sufficient supply of NADPH was available for biosynthesis.
P.L. Jørgense n; M. Tangney; P.E. Pedersen; S. Hastrup ; B. Diderichsen; S.T. Jørgensen.
"Cloning and sequencing of an alkaline protease gene from Bacillus lentus and amplification of the gene on the B. lentus chromosome by an improved technique."
Appl. Environ. Microbiol. 66, 825-827 (2000)
A gene encoding an alkaline proteinase (subtilisin), commercially known as Savinase®, was cloned from alkalophilic Bacillus lentus NCIB 10309 into B. subtilis DN497, and its nucleotide sequence was determined. The cloned gene was used to increase the copy number of the proteinase gene on the chromosome. From these studies, an improved genetic technique for integration and gene amplification was developed using the temp. sensitive plasmid pE194.
K. L. Petersen; J Lehmbeck; T. Christensen.
"A new transcriptional activator for amylase genes in Aspergillus."
Mol Gen Genet, 262, 668-676 (1999)
We have cloned a regulatory gene for amylase synthesis in Aspergillus oryzae. This gene, amyR, encodes a 604-amino acid transcriptional activator with a Cys6 zinc cluster, that shows extensive homology to the DNA binding domain of GAL4 from Saccharomyces cerevisiae. The DNA binding domain of amyR binds to two types of sequences found in a number of promoters from Aspergillus genes coding for starch-degrading enzymes. One type of binding site is characterized by two CGG triplets separated by eight nucleotides. The other type has only one CGG trip let, which is followed by the sequence AAATTTAA.