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

Jacobs, Denise I.; Olsthoorn, Maurien M. A.; Maillet, Isabelle; Akeroyd, Michiel; Breestraat, Stefaan; Donkers, Serge; Hoeven, R. A. M. van der; Hondel, Cees A. M. J. J. van den; Kooistra, Rolf; Lapointe, Thomas; Menke, Hildegard; Meulenberg, R.; Misset, Marijke; Müller, Wally H.; Peij, Noël N. M. E. van; Ram, Arthur F. J.; Rodriguez, Sabrina; Roelofs, Marc S.; Roubos, Johannes A.; Tilborg, Marcel W. E. M. van; Verkleij, Arie J.; Pel, Herman J.; Stam, H.; Sagt, Cees M. J.

doi:10.1016/j.fgb.2008.08.012

Cited by 68 publications

(44 citation statements)

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“…With the availability of the A. niger genome sequence (50), systems biology tools are being developed (4,5,33) which, together with new efficient methods for constructing gene knockout mutants (43), open new possibilities for further improvement of A. niger as a cell factory.…”

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confidence: 99%

Transcriptomic Insights into the Physiology of Aspergillus niger Approaching a Specific Growth Rate of Zero

Jørgensen

Nitsche

Lamers

et al. 2010

Appl Environ Microbiol

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The physiology of filamentous fungi at growth rates approaching zero has been subject to limited study and exploitation. With the aim of uncoupling product formation from growth, we have revisited and improved the retentostat cultivation method for Aspergillus niger. A new retention device was designed allowing reliable and nearly complete cell retention even at high flow rates. Transcriptomic analysis was used to explore the potential for product formation at very low specific growth rates. The carbon-and energy-limited retentostat cultures were highly reproducible. While the specific growth rate approached zero (<0.005 h ؊1 ), the growth yield stabilized at a minimum (0.20 g of dry weight per g of maltose). The severe limitation led to asexual differentiation, and the supplied substrate was used for spore formation and secondary metabolism. Three physiologically distinct phases of the retentostat cultures were subjected to genome-wide transcriptomic analysis. The severe substrate limitation and sporulation were clearly reflected in the transcriptome. The transition from vegetative to reproductive growth was characterized by downregulation of genes encoding secreted substrate hydrolases and cell cycle genes and upregulation of many genes encoding secreted small cysteine-rich proteins and secondary metabolism genes. Transcription of known secretory pathway genes suggests that A. niger becomes adapted to secretion of small cysteine-rich proteins. The perspective is that A. niger cultures as they approach a zero growth rate can be used as a cell factory for production of secondary metabolites and cysteine-rich proteins. We propose that the improved retentostat method can be used in fundamental studies of differentiation and is applicable to filamentous fungi in general.The filamentous fungus Aspergillus niger is one of the most important microorganisms in industrial biotechnology because of its ability to produce high levels of enzymes and organic acids (6,50,57). Apart from their biotechnological importance, filamentous fungi in the Aspergillus section Nigri (the black aspergilli) represent some of the most widespread storage molds which contaminate food and feedstocks with mycotoxins (26,37,48).Both metabolic engineering approaches and the search for optimal cultivation conditions have long been used to improve A. niger as a production host (e.g., 14, 22, 41). With the availability of the A. niger genome sequence (50), systems biology tools are being developed (4, 5, 33) which, together with new efficient methods for constructing gene knockout mutants (43), open new possibilities for further improvement of A. niger as a cell factory.A major ongoing challenge for microbial production processes is to minimize the amount of biomass formed while maintaining high productivity. Solutions to uncouple product formation from biomass accumulation or growth are therefore highly desirable. However, production at zero growth is difficult to achieve when nutrients are supplied to allow formation of a desired product. Carbon-a...

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confidence: 99%

Transcriptomic Insights into the Physiology of Aspergillus niger Approaching a Specific Growth Rate of Zero

Jørgensen

Nitsche

Lamers

et al. 2010

Appl Environ Microbiol

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“…These studies have addressed overall transcriptional and translational responses in A. niger by studying the impact of secretion stress-inducing chemicals, temperature shifts, protein overproduction, or growth on carbon sources that induce changes in secretory states (20,23,26).…”

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confidence: 99%

Shotgun Proteomics of Aspergillus niger Microsomes upon d -Xylose Induction

Oliveira

Passel

Schaap

et al. 2010

Appl Environ Microbiol

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Protein secretion plays an eminent role in cell maintenance and adaptation to the extracellular environment of microorganisms. Although protein secretion is an extremely efficient process in filamentous fungi, the mechanisms underlying protein secretion have remained largely uncharacterized in these organisms. In this study, we analyzed the effects of the D-xylose induction of cellulase and hemicellulase enzyme secretion on the protein composition of secretory organelles in Aspergillus niger. We aimed to systematically identify the components involved in the secretion of these enzymes via mass spectrometry of enriched subcellular microsomal fractions. Under each condition, fractions enriched for secretory organelles were processed for tandem mass spectrometry, resulting in the identification of peptides that originate from 1,081 proteins, 254 of which-many of them hypothetical proteins-were predicted to play direct roles in the secretory pathway. D-Xylose induction led to an increase in specific small GTPases known to be associated with polarized growth, exocytosis, and endocytosis. Moreover, the endoplasmic-reticulum-associated degradation (ERAD) components Cdc48 and all 14 of the 20S proteasomal subunits were recruited to the secretory organelles. In conclusion, induction of extracellular enzymes results in specific changes in the secretory subproteome of A. niger, and the most prominent change found in this study was the recruitment of the 20S proteasomal subunits to the secretory organelles.Aspergillus niger is a soil-dwelling filamentous fungus with a high capacity for decomposing plant materials. Many of the secreted depolymerizing enzymes have important biotechnological applications, e.g., to modify plant-derived food products. Homologous protein secretion in filamentous fungi may yield up to 20 g per liter of extracellular medium (14). For this reason, A. niger has been used intensively as a cell factory for enzyme production (3,14,32). In contrast to this, yields are much lower for heterologous protein secretion, with exceptions (11,18,35,59).The secretory potential of A. niger is not well understood, and only a limited number of functional studies have been performed to investigate the major components of the fungal secretion pathway. These studies have addressed overall transcriptional and translational responses in A. niger by studying the impact of secretion stress-inducing chemicals, temperature shifts, protein overproduction, or growth on carbon sources that induce changes in secretory states (20,23,26).In recent years, high-throughput shotgun proteomics has been used to study cell organelle makeup and function. Through the combined use of liquid chromatography and tandem mass spectrometry (LC-MS/MS), various studies have identified many organelle-specific proteins, including proteins related to protein secretion (18,24,29,51,51). In the case of aspergilli, such studies have focused mainly on the secretome and not on the actual components of the secretory pathway (34,36,55).In a previous study, we...

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“…The amount of secreted heterologous proteins was increased by deletion of the doaA gene, encoding an essential factor for ubiquitin-mediated proteolysis. This was further optimized by overexpression of the sttC gene, encoding a protein involved in the glycosylation of secreted products (Jacobs et al 2009). …”

Section: Strain Improvement Using Genomics and Functional Genomicsmentioning

confidence: 99%

Inducible promoters and functional genomic approaches for the genetic engineering of filamentous fungi

Kluge

Terfehr

Kück

2018

Appl Microbiol Biotechnol

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In industry, filamentous fungi have a prominent position as producers of economically relevant primary or secondary metabolites. Particularly, the advent of genetic engineering of filamentous fungi has led to a growing number of molecular tools to adopt filamentous fungi for biotechnical applications. Here, we summarize recent developments in fungal biology, where fungal host systems were genetically manipulated for optimal industrial applications. Firstly, available inducible promoter systems depending on carbon sources are mentioned together with various adaptations of the Tet-Off and Tet-On systems for use in different industrial fungal host systems. Subsequently, we summarize representative examples, where diverse expression systems were used for the production of heterologous products, including proteins from mammalian systems. In addition, the progressing usage of genomics and functional genomics data for strain improvement strategies are addressed, for the identification of biosynthesis genes and their related metabolic pathways. Functional genomic data are further used to decipher genomic differences between wild-type and high-production strains, in order to optimize endogenous metabolic pathways that lead to the synthesis of pharmaceutically relevant end products. Lastly, we discuss how molecular data sets can be used to modify products for optimized applications.

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

Cited by 68 publications

References 50 publications

Transcriptomic Insights into the Physiology of Aspergillus niger Approaching a Specific Growth Rate of Zero

Transcriptomic Insights into the Physiology of Aspergillus niger Approaching a Specific Growth Rate of Zero

Shotgun Proteomics of Aspergillus niger Microsomes upon d -Xylose Induction

Inducible promoters and functional genomic approaches for the genetic engineering of filamentous fungi

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