Metabolic Engineering of the Morphology of Aspergillus

McIntyre, Mhairi; Müller, Christian; Dynesen, Jens; Nielsen, Jens

doi:10.1007/3-540-45300-8_6

Cited by 53 publications

(57 citation statements)

References 127 publications

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“…The control of fungal morphology would improve the growth properties of filamentous fungi in a fermenter and contribute to increased biomass levels and product yields. Efforts to modulate hyphal branching, improve secretion and reduce the problems associated with high viscosity, oxygen and mass transfer are being pursued (McIntyre et al, 2001).…”

Section: Introductionmentioning

confidence: 99%

Protein kinase A is involved in the control of morphology and branching during aerobic growth of Mucor circinelloides

et al. 2004

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The cAMP signal transduction pathway controls many processes in fungi. The Mucor circinelloides pkaR and pkaC genes, encoding the regulatory (PKAR) and catalytic (PKAC) subunit of the cAMP-dependent protein kinase A (PKA), have been cloned recently. Expression analysis during the dimorphic shift and colony morphology suggested a role for PKAR in the control of morphology and branching. Here strain KFA121, which overexpresses the M. circinelloides pkaR gene, was used to quantify growth and branching under different aerobic growth conditions in a flow-through cell by computerized image analysis. An inverse relationship between the pkaR expression level in KFA121 and the hyphal growth unit length was observed in KFA121, suggesting a central role for PKAR in branching. A biochemical analysis of PKAR using antibodies and enzyme assay demonstrated that the level of PKAR is higher in KFA121 under inducing conditions, i.e. in the presence of high glucose, than in the vector control strain KFA89. Measurement of cAMP binding demonstrated a significant increase (two-to threefold) in PKAR level for KFA121 at the time of germ-tube emission in medium containing 10 g glucose l "1 . The level of PKA activity was determined using kemptide in the same crude cell extracts used to determine cAMP binding. Strain KFA121 showed a twofold increase in PKA activity. An excess of free PKAR subunit over PKA holoenzyme was determined using sucrose gradient centrifugation of extracts from KFA89 and KFA121. The data indicate that cAMP-dependent PKA in M. circinelloides might be down-regulated during hyphal-tube emergence and that an increase in PKAR levels results in increased branching.

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Section: Introductionmentioning

confidence: 99%

Protein kinase A is involved in the control of morphology and branching during aerobic growth of Mucor circinelloides

et al. 2004

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“…This results in poor mixing and poor mass transfer of the substrate, and it makes the morphologies of filamentous fungi an obvious, yet unanswered, challenge in process optimization. Recently, however, there have been major advances in the tools available for metabolic engineering of aspergilli (reviewed by McIntyre et al [17]), such as developments in genomics, the increased number of suitable transformation vectors, and powerful image analysis tools that enable rapid quantification of the fungal morphology. This has made metabolic engineering of the morphology for process optimization possible.…”

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confidence: 99%

Metabolic Engineering of the Morphology of Aspergillus oryzae by Altering Chitin Synthesis

Müller

McIntyre

Hansen

et al. 2002

Appl Environ Microbiol

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Morphology and ␣-amylase production during submerged cultivation were examined in a wild-type strain (A1560) and in strains of Aspergillus oryzae in which chitin synthase B (chsB) and chitin synthesis myosin A (csmA) have been disrupted (ChsB/G and CM101). In a flowthrough cell, the growth of submerged hyphal elements was studied online, making it possible to examine the growth kinetics of the three strains. The average tip extension rates of the CM101 and ChsB/G strains were 25 and 88% lower, respectively, than that of the wild type. The branching intensity in the CM101 strain was 25% lower than that in the wild type, whereas that in the ChsB/G strain was 188% higher. During batch cultivation, inseparable clumps were formed in the wild-type strain, while no or fewer large inseparable clumps existed in the cultivations of the ChsB/G and CM101 strains. The ␣-amylase productivity was not significantly different in the three strains. A strain in which the transcription of chsB could be controlled by the nitrogen source-regulated promoter niiA (NiiA1) was examined during chemostat cultivation, and it was found that the branching intensity could be regulated by regulating the promoter, signifying an important role for chsB in branching. However, the pattern of branching responded very slowly to the change in transcription, and increased branching did not affect ␣-amylase productivity. ␣-Amylase residing in the cell wall was stained by immunofluorescence, and the relationship between tip number and enzyme secretion is discussed.Filamentous fungi, such as Aspergillus oryzae, are widely used for industrial enzyme production, since they are able to secrete large amounts of proteins (e.g., amylases, proteases, phytases, and lipases) into the medium. Filamentous fungi grow by hyphal extension and branching through processes that are regulated in a way that is still not completely understood. During submerged cultivation, the growing hyphal elements tend to entangle, and this affects the rheology of the cultivation medium and the mixing characteristics in an undesirable fashion. This results in poor mixing and poor mass transfer of the substrate, and it makes the morphologies of filamentous fungi an obvious, yet unanswered, challenge in process optimization. Recently, however, there have been major advances in the tools available for metabolic engineering of aspergilli (reviewed by McIntyre et al. [17]), such as developments in genomics, the increased number of suitable transformation vectors, and powerful image analysis tools that enable rapid quantification of the fungal morphology. This has made metabolic engineering of the morphology for process optimization possible.Metabolic engineering of morphology requires detailed knowledge of the relationships among productivity, growth, and morphology. The morphologies of filamentous organisms can be classified into macroscopic and microscopic morphologies (21), and techniques for analyzing morphology on both of these levels have advanced rapidly during the last decade (reviewed...

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“…31 Morphology also poses a challenge due to the filamentous nature of the growth and the differentiation of cellular compartments as hyphae extend. 32,33 This leads to heterogeneity in terms of the morphological forms present and the cell (compartment) types that contribute to the overall performance of the culture. 34 The challenge is to balance cellular potential, process design, and economic feasibility.…”

Section: Cell Factory Characteristicsmentioning

confidence: 99%

Integrated Approaches for Assessment of Cellular Performance in Industrially Relevant Filamentous Fungi

WorkmanMhairi

Andersen

ThykaerJette

2013

Industrial Biotechnology

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The performance of filamentous fungi in submerged cultivation determines their suitability for large-scale industrial biotechnology processes and is the result of complex interplay between the physical and chemical parameters of the process and the cellular biology of the fungi. Filamentous fungi have a natural ability to degrade complex substrates through secretion of a large number of diverse enzymes and to produce a number of metabolites that inhibit or prevent the growth of other species in the surroundings. These features have been exploited by industry, resulting in multi-billion dollar processes for producing enzymes and metabolites of value. However, the wealth of diversity of these species is still not fully represented in large-scale bioprocesses, and thus advancement from discovery to application is one of the challenges for modern biotechnology. Acceleration of the process can be achieved through integrated approaches for assessing cellular performance (quantitative physiology), genetic modification of strains (metabolic engineering), and omics analyses and modeling (systems biology). In this review, we evaluate stateof-the-art of filamentous fungal applications in industrial biotechnology , focusing on physiological aspects of the fungi that provide the basis of their cellular performance. We also discuss the advancement of systems biology approaches and how the establishment of these tools for fungal research has begun to reveal the possibilities for further exploitation of these organisms. Increased future focus on multicellular physiology and relevant assays will lead to fungal cells and processes that are customizable to a greater degree, finally allowing the full potential of these complex organisms and their product diversity to unfold.

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Metabolic Engineering of the Morphology of Aspergillus

Cited by 53 publications

References 127 publications

Protein kinase A is involved in the control of morphology and branching during aerobic growth of Mucor circinelloides

Protein kinase A is involved in the control of morphology and branching during aerobic growth of Mucor circinelloides

Metabolic Engineering of the Morphology of Aspergillus oryzae by Altering Chitin Synthesis

Integrated Approaches for Assessment of Cellular Performance in Industrially Relevant Filamentous Fungi

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