C. Sagt

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.

Introduction of an N-Glycosylation Site Increases Secretion of Heterologous Proteins in Yeasts

ABSTRACT Saccharomyces cerevisiae is often used to produce heterologous proteins that are preferentially secreted to increase economic feasibility. We used N-glycosylation as a tool to enhance protein secretion. Secretion of cutinase, a lipase, and llama VHH antibody fragments by S. cerevisiae orPichia pastoris improved following the introduction of an N-glycosylation site. When we introduced an N-glycosylation consensus sequence in the N-terminal region of a hydrophobic cutinase, secretion increased fivefold. If an N-glycosylation site was introduced in the C-terminal region, however, secretion increased only 1.8-fold. These results indicate that the use of N glycosylation can significantly enhance heterologous protein secretion.

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.

Impaired Cutinase Secretion in Saccharomyces cerevisiae Induces Irregular Endoplasmic Reticulum (ER) Membrane Proliferation, Oxidative Stress, and ER-Associated Degradation

ABSTRACT Impaired secretion of the hydrophobic CY028 cutinase invokes an unfolded protein response (UPR) in Saccharomyces cerevisiae cells. Here we show that the UPR in CY028-expressing S. cerevisiae cells is manifested as an aberrant morphology of the endoplasmic reticulum (ER) and as extensive membrane proliferation compared to the ER morphology and membrane proliferation of wild-type CY000-producing S. cerevisiae cells. In addition, we observed oxidative stress, which resulted in a 21-fold increase in carbonylated proteins in the CY028-producing S. cerevisiae cells. Moreover, CY028-producing S. cerevisiae cells use proteasomal degradation to reduce the amount of accumulated CY028 cutinase, thereby attenuating the stress invoked by CY028 cutinase expression. This proteasomal degradation occurs within minutes and is characteristic of ER-associated degradation (ERAD). Our results clearly show that impaired secretion of the heterologous, hydrophobic CY028 cutinase in S. cerevisiae cells leads to protein aggregation in the ER, aberrant ER morphology and proliferation, and oxidative stress, as well as a UPR and ERAD.

Effects of a defective ERAD pathway on growth and heterologous protein production in Aspergillus niger

Endoplasmic reticulum associated degradation (ERAD) is a conserved mechanism to remove misfolded proteins from the ER by targeting them to the proteasome for degradation. To assess the role of ERAD in filamentous fungi, we have examined the consequences of disrupting putative ERAD components in the filamentous fungus Aspergillus niger. Deletion of derA, doaA, hrdC, mifA, or mnsA in A. niger yields viable strains, and with the exception of doaA, no significant growth phenotype is observed when compared to the parental strain. The gene deletion mutants were also made in A. niger strains containing single- or multicopies of a glucoamylase–glucuronidase (GlaGus) gene fusion. The induction of the unfolded protein response (UPR) target genes (bipA and pdiA) was dependent on the copy number of the heterologous gene and the ERAD gene deleted. The highest induction of UPR target genes was observed in ERAD mutants containing multiple copies of the GlaGus gene. Western blot analysis revealed that deletion of the derA gene in the multicopy GlaGus overexpressing strain resulted in a 6-fold increase in the intracellular amount of GlaGus protein detected. Our results suggest that impairing some components of the ERAD pathway in combination with high expression levels of the heterologous protein results in higher intracellular protein levels, indicating a delay in protein degradation.

Systems metabolic engineering in an industrial setting

Systems metabolic engineering is based on systems biology, synthetic biology, and evolutionary engineering and is now also applied in industry. Industrial use of systems metabolic engineering focuses on strain and process optimization. Since ambitious yields, titers, productivities, and low costs are key in an industrial setting, the use of effective and robust methods in systems metabolic engineering is becoming very important. Major improvements in the field of proteomics and metabolomics have been crucial in the development of genome-wide approaches in strain and process development. This is accompanied by a rapid increase in DNA sequencing and synthesis capacity. These developments enable the use of systems metabolic engineering in an industrial setting. Industrial systems metabolic engineering can be defined as the combined use of genome-wide genomics, transcriptomics, proteomics, and metabolomics to modify strains or processes. This approach has become very common since the technology for generating large data sets of all levels of the cellular processes has developed quite fast into robust, reliable, and affordable methods. The main challenge and scope of this mini review is how to translate these large data sets in relevant biological leads which can be tested for strain or process improvements. Experimental setup, heterogeneity of the culture, and sample pretreatment are important issues which are easily underrated. In addition, the process of structuring, filtering, and visualization of data is important, but also, the availability of a genetic toolbox and equipment for medium/high-throughput fermentation is a key success factor. For an efficient bioprocess, all the different components in this process have to work together. Therefore, mutual tuning of these components is an important strategy.

Identification of growth phenotype-related genes in Aspergillus oryzae by heterologous macroarray and suppression subtractive hybridization

Abstract Aspergillus oryzae requires polarized growth for colonization of solid substrates, and this growth phenotype differs from that seen in liquid medium. Various experimental approaches were used to identify genes that are differentially expressed when A. oryzae is grown on wheat kernels and in a wheat-based liquid medium. Hybridization of A. oryzae RNAs to a macroarray bearing cDNAs isolated from a library representing at least 16% of the total number of A. niger genes identified 14 differentially expressed cDNA clones, showing that heterologous macroarray analysis with an A. niger cDNA library can be used to identify regulated gene transcripts in the related species A. oryzae. Moreover, Northern analysis with a selection of eight probes for A. niger genes encoding proteins involved in morphological development and cell wall biosynthesis identified five more differentially expressed genes. A suppression subtractive hybridization procedure revealed another 12 differentially expressed genes. The results presented show that, of the 29 identified genes which are expressed at higher levels during growth on wheat kernels, six encode proteins that are functionally related to polarized growth, four encode products known to be involved in morphogenesis, three code for proteins related to cell wall composition, and nine of the cDNA clones encode novel proteins. These findings pinpoint genes associated with the changes in cellular morphogenesis seen in A. oryzae grown on wheat kernels as opposed to wheat-based liquid medium.

Peroxicretion: a novel secretion pathway in the eukaryotic cell

BackgroundEnzyme production in microbial cells has been limited to secreted enzymes or intracellular enzymes followed by expensive down stream processing. Extracellular enzymes consists mainly of hydrolases while intracellular enzymes exhibit a much broader diversity. If these intracellular enzymes could be secreted by the cell the potential of industrial applications of enzymes would be enlarged. Therefore a novel secretion pathway for intracellular proteins was developed, using peroxisomes as secretion vesicles.ResultsPeroxisomes were decorated with a Golgi derived v-SNARE using a peroxisomal membrane protein as an anchor. This allowed the peroxisomes to fuse with the plasma membrane. Intracellular proteins were transported into the peroxisomes by adding a peroxisomal import signal (SKL tag). The proteins which were imported in the peroxisomes, were released into the extra-cellular space through this artificial secretion pathway which was designated peroxicretion. This concept was supported by electron microscopy studies.ConclusionOur results demonstrate that it is possible to reroute the intracellular trafficking of vesicles by changing the localisation of SNARE molecules, this approach can be used in in vivo biological studies to clarify the different control mechanisms regulating intracellular membrane trafficking. In addition we demonstrate peroxicretion of a diverse set of intracellular proteins. Therefore, we anticipate that the concept of peroxicretion may revolutionize the production of intracellular proteins from fungi and other microbial cells, as well as from mammalian cells.

A novel screening system for secretion of heterologous proteins in Bacillus subtilis

High‐level production of secretory proteins in Bacillus subtilis leads to a stress response involving the two‐component system CssRS and its target genes htrA and htrB. Here, we used this sensing system in a reporter strain in which gfp is under control of PhtrA, the secretion stress responsive promoter of htrA. Overexpression of heterologous secretory proteins in this strain results in green fluorescent cells, which can be separated from non‐secreting, low fluorescent cells using a fluorescence‐activated cell sorter (FACS). Using this principle, genomic libraries of uncharacterized prokaryotic organisms, expressed in the reporter strain, can be screened for genes encoding secretory proteins.

Sample Preparation and Biostatistics for Integrated Genomics Approaches

Genomics is based on the ability to determine the transcriptome, proteome, and metabolome of a cell. These technologies only have added value when they are integrated and based on robust and reproducible workflows. This chapter describes the experimental design, sampling, sample pretreatment, data evaluation, integration, and interpretation. The actual generation of the data is not covered in this chapter since it is highly depended on available equipment and infrastructure. The enormous amount of data generated by these technologies are integrated and interpreted inorder to generate leads for strain and process improvement. Biostatistics are becoming very important for the whole work flow therefore, some general recommendations how to set up experimental design and how to use biostatistics in enhancing the quality of the data and the selection of biological relevant leads for strain engineering and target identification are described.