Herman Jan Pel

Genome sequencing and analysis of the versatile cell factory Aspergillus niger CBS 513.88

The filamentous fungus Aspergillus niger is widely exploited by the fermentation industry for the production of enzymes and organic acids, particularly citric acid. We sequenced the 33.9-megabase genome of A. niger CBS 513.88, the ancestor of currently used enzyme production strains. A high level of synteny was observed with other aspergilli sequenced. Strong function predictions were made for 6,506 of the 14,165 open reading frames identified. A detailed description of the components of the protein secretion pathway was made and striking differences in the hydrolytic enzyme spectra of aspergilli were observed. A reconstructed metabolic network comprising 1,069 unique reactions illustrates the versatile metabolism of A. niger. Noteworthy is the large number of major facilitator superfamily transporters and fungal zinc binuclear cluster transcription factors, and the presence of putative gene clusters for fumonisin and ochratoxin A synthesis.

Comparative genomics of citric-acid-producing Aspergillus niger ATCC 1015 versus enzyme-producing CBS 513.88

The filamentous fungus Aspergillus niger exhibits great diversity in its phenotype. It is found globally, both as marine and terrestrial strains, produces both organic acids and hydrolytic enzymes in high amounts, and some isolates exhibit pathogenicity. Although the genome of an industrial enzyme-producing A. niger strain (CBS 513.88) has already been sequenced, the versatility and diversity of this species compel additional exploration. We therefore undertook whole-genome sequencing of the acidogenic A. niger wild-type strain (ATCC 1015) and produced a genome sequence of very high quality. Only 15 gaps are present in the sequence, and half the telomeric regions have been elucidated. Moreover, sequence information from ATCC 1015 was used to improve the genome sequence of CBS 513.88. Chromosome-level comparisons uncovered several genome rearrangements, deletions, a clear case of strain-specific horizontal gene transfer, and identification of 0.8 Mb of novel sequence. Single nucleotide polymorphisms per kilobase (SNPs/kb) between the two strains were found to be exceptionally high (average: 7.8, maximum: 160 SNPs/kb). High variation within the species was confirmed with exo-metabolite profiling and phylogenetics. Detailed lists of alleles were generated, and genotypic differences were observed to accumulate in metabolic pathways essential to acid production and protein synthesis. A transcriptome analysis supported up-regulation of genes associated with biosynthesis of amino acids that are abundant in glucoamylase A, tRNA-synthases, and protein transporters in the protein producing CBS 513.88 strain. Our results and data sets from this integrative systems biology analysis resulted in a snapshot of fungal evolution and will support further optimization of cell factories based on filamentous fungi.

Effective lead selection for improved protein production in Aspergillus niger based on integrated genomics

The filamentous fungus Aspergillus niger is widely exploited for industrial production of enzymes and organic acids. An integrated genomics approach was developed to determine cellular responses of A. niger to protein production in well-controlled fermentations. Different protein extraction methods in combination with automated sample processing and protein identification allowed quantitative analysis of 898 proteins. Three different enzyme overproducing strains were compared to their isogenic fungal host strains. Clear differences in response to the amount and nature of the overproduced enzymes were observed. The corresponding genes of the differentially expressed proteins were studied using transcriptomics. Genes that were up-regulated both at the proteome and transcriptome level were selected as leads for generic strain improvement. Up-regulated proteins included proteins involved in carbon and nitrogen metabolism as well as (oxidative) stress response, and proteins involved in protein folding and endoplasmic reticulum-associated degradation (ERAD). Reduction of protein degradation through the removal of the ERAD factor doaA combined with overexpression of the oligosaccharyl transferase sttC in A. niger overproducing beta-glucuronidase (GUS) strains indeed resulted in a small increase in GUS expression.

Functional characterization of yeast mitochondrial release factor 1.

The yeast Saccharomyces cerevisiaemitochondrial release factor was expressed from the clonedMRF1 gene, purified from inclusion bodies, and refolded to give functional activity. The gene encoded a factor with release activity that recognized cognate stop codons in a termination assay with mitochondrial ribosomes and in an assay with Escherichia coli ribosomes. The noncognate stop codon, UGA, encoding tryptophan in mitochondria, was recognized weakly in the heterologous assay. The mitochondrial release factor 1 protein bound to bacterial ribosomes and formed a cross-link with the stop codon within a mRNA bound in a termination complex. The affinity was strongly dependent on the identity of stop signal. Two alleles of MRF1 that contained point mutations in a release factor 1 specific region of the primary structure and that in vivo compensated for mutations in the decoding site rRNA of mitochondrial ribosomes were cloned, and the expressed proteins were purified and refolded. The variant proteins showed impaired binding to the ribosome compared with mitochondrial release factor 1. This structural region in release factors is likely to be involved in codon-dependent specific ribosomal interactions.

Exploring Sequence Characteristics Related to High-Level Production of Secreted Proteins in Aspergillus niger

Protein sequence features are explored in relation to the production of over-expressed extracellular proteins by fungi. Knowledge on features influencing protein production and secretion could be employed to improve enzyme production levels in industrial bioprocesses via protein engineering. A large set, over 600 homologous and nearly 2,000 heterologous fungal genes, were overexpressed in Aspergillus niger using a standardized expression cassette and scored for high versus no production. Subsequently, sequence-based machine learning techniques were applied for identifying relevant DNA and protein sequence features. The amino-acid composition of the protein sequence was found to be most predictive and interpretation revealed that, for both homologous and heterologous gene expression, the same features are important: tyrosine and asparagine composition was found to have a positive correlation with high-level production, whereas for unsuccessful production, contributions were found for methionine and lysine composition. The predictor is available online at http://bioinformatics.tudelft.nl/hipsec. Subsequent work aims at validating these findings by protein engineering as a method for increasing expression levels per gene copy.

Efficient in vitro translational termination in Escherichia coli is constrained by the orientations of the release factor, stop signal and peptidyl-tRNA within the termination complex.

There have been contrasting reports of whether the positioning of a translational stop signal immediately after a start codon in a single oligonucleotide can act as a model template to support efficient in vitro termination. This paradox stimulated this study of what determines the constraints on the positioning of the components in the termination complex. The mini mRNA, AUGUGAA, was unable to support efficient in vitro termination in contrast to separate AUG/UGA(A) codons, unless the ribosomal interaction of the stop signal with the decoding factor, release factor 2, was stimulated with ethanol or with nucleotide-free release factor 3, or by using (L11-)-ribosomes which have a higher affinity for release factor 2, or unless the fMet-tRNA was first bound to 30S subunits independently of the mini mRNA. An additional triplet stop codon could restore activity of the mini mRNA, indicating that its recognition was not sterically restrained by the stop signal already within it. This suggests that in an initiation complex an adjoining start/stop signal is not positioned to support efficient decoding by release factor unless it is separated from the start codon. Site-directed crosslinking from mRNAs to components of the termination complex has shown that mRNA elements like the Shine-Dalgarno sequence and the codon preceding the stop signal can affect the crosslinking to release factor, and presumably the orientation of the signal to the factor.

THE STOP SIGNAL CONTROLS THE EFFICIENCY OF RELEASE FACTOR­ MEDIATED TRANSLATIONAL TERMINATION

There are three important steps in protein synthesis where signals in the mRNA are critical for a successful outcome, namely the production of a functional protein. First the information in the nucleic acid which is to be translated into an amino acid sequence is signalled by successive triplet sense codons, second the frame is set by one sense codon, the initiation codon, which acts as the start of translation of the encoded information, and third the end of the information frame also has to be marked by a specific signal. The use of a range of different signals to mark each of these steps allows for differences in the efficiency with which different proteins are produced. In this review the focus is on the signal that marks the end of the frame, the translational termination signal. For a long time it was thought that termination would be the least interesting phase of protein synthesis but it has subsequently been found to have unexpected dimensions, providing a substratum of cellular regulation. The translational stop signal should now be thought of as a full stop in the large majority of cases, but as a pause in a fundamentally important minority of cases where alternative genetic events can occur.

Identification of a mitotic recombination hotspot on chromosome III of the asexual fungus Aspergillus niger and its possible correlation with elevated basal transcription

Genetic recombination is an important tool in strain breeding in many organisms. We studied the possibilities of mitotic recombination in strain breeding of the asexual fungus Aspergillus niger. By identifying genes that complemented mapped auxotrophic mutations, the physical map was compared to the genetic map of chromosome III using the genome sequence. In a program to construct a chromosome III-specific marker strain by selecting mitotic crossing-over in diploids, a mitotic recombination hotspot was identified. Analysis of the mitotic recombination hotspot revealed some physical features, elevated basal transcription and a possible correlation with purine stretches.

Sequence-based prediction of protein secretion success in Aspergillus niger

The cell-factory A spergillus niger is widely used for industrial enzyme production. To select potential proteins for large-scale production, we developed a sequence-based classifier that predicts if an over-expressed homologous protein will successfully be produced and secreted. A dataset of 638 proteins was used to train and validate a classifier, using a 10-fold cross-validation protocol. Using a linear discriminant classifier, an average accuracy of 0.85 was achieved. Feature selection results indicate what features are mostly defining for successful protein production, which could be an interesting lead to couple sequence characteristics to biological processes involved in protein production and secretion.