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- Knuf, Christoph, 1984, et al.
(författare)
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Aspergilli: Systems biology and industrial applications
- 2012
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Ingår i: Biotechnology journal. - : Wiley. - 1860-6768 .- 1860-7314. ; 7:9, s. 1147-1155
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Forskningsöversikt (refereegranskat)abstract
- Aspergilli are widely used as cell factories for the production of food ingredients, enzymes and antibiotics. Traditionally, improvement of these cell factories has been done using classical methods, that is, random mutagenesis and screening; however, advances in methods for performing directed genetic modifications has enabled the use of metabolic engineering strategies. Genome sequencing of Aspergilli was originally trailing behind developments in the field of bacteria and yeasts, but with the recent availability of genome sequences for several industrially relevant Aspergilli, it has become possible to implement systems biology tools to advance metabolic engineering. These tools include genome-wide transcription analysis and genome-scale metabolic models. Herein, we review achievements in the field and highlight the impact of Aspergillus systems biology on industrial biotechnology.
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- Knuf, Christoph, 1984, et al.
(författare)
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Investigation of Malic Acid Production in Aspergillus oryzae under Nitrogen Starvation Conditions
- 2013
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Ingår i: Applied and Environmental Microbiology. - 1098-5336 .- 0099-2240. ; 79:19, s. 6050-6058
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Tidskriftsartikel (refereegranskat)abstract
- Malic acid has great potential for replacing petrochemical building blocks in the future. For this application, high yields, rates, and titers are essential in order to sustain a viable biotechnological production process. Natural high-capacity malic acid producers like the malic acid producer Aspergillus flavus have so far been disqualified because of special growth requirements or the production of mycotoxins. As A. oryzae is a very close relative or even an ecotype of A. flavus, it is likely that its high malic acid production capabilities with a generally regarded as safe (GRAS) status may be combined with already existing large-scale fermentation experience. In order to verify the malic acid production potential, two wild-type strains, NRRL3485 and NRRL3488, were compared in shake flasks. As NRRL3488 showed a volumetric production rate twice as high as that of NRRL3485, this strain was selected for further investigation of the influence of two different nitrogen sources on malic acid secretion. The cultivation in lab-scale fermentors resulted in a higher final titer, 30.27 +/- 1.05 g liter(-1), using peptone than the one of 22.27 +/- 0.46 g liter(-1) obtained when ammonium was used. Through transcriptome analysis, a binding site similar to the one of the Saccharomyces cerevisiae yeast transcription factor Msn2/4 was identified in the upstream regions of glycolytic genes and the cytosolic malic acid production pathway from pyruvate via oxaloacetate to malate, which suggests that malic acid production is a stress response. Furthermore, the pyruvate carboxylase reaction was identified as a target for metabolic engineering, after it was confirmed to be transcriptionally regulated through the correlation of intracellular fluxes and transcriptional changes.
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3. |
- Knuf, Christoph, 1984
(författare)
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Malic acid production by Aspergillus oryzae
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Malic acid is a C4 dicarboxylic acid which is used as an acidulant in food and beverages. It isalso considered as a bio-building block to replace petrochemically derived compounds in thepost oil era. This organic acid can be biotechnologically derived from fermentation usingrenewable feedstocks as carbon source. Aspergilli are among the best producers of organicacid and A. flavus/oryzae is the best natural producer of malic acid.The mechanism of malic acid production in A. oryzae was first assessed by transcriptomeanalysis. A nitrogen starvation response, probably regulated by a transcription factor relatedto the S. cerevisiae Msn2/4 transcriptional activator of stress related genes, was found toresult in high malic acid production. Furthermore the pyruvate carboxylase reaction wasidentified as a metabolic engineering target. This gene, together with the malatedehydrogenase and a malic acid exporter was overexpressed in the strain 2103a-68, whichwas characterized in a second project. The overexpression led to an 80% increase in yieldduring the starvation phase (1.49 mol (mol gluc)-1) and a triplication of the specificproduction rate. The increase in citric acid production in the engineered strain and itsevaluation through model simulations led to the curation of the A. oryzae GEM. The existingmodel was curated with special emphasis on the mitochondrial transport reactions and let toa more defined network around the production of organic acids. Furthermore, theperformance of the strain 2103a-68 on xylose as carbon source was evaluated as well andthe good results led to the final project of manipulating the carbon source utilization bydeleting the carbon catabolite repressor CreA.This work contributed to the understanding of the regulation of malic acid production. Thisknowledge was used for the development of A. oryzae as an organic acid producer throughmetabolic engineering. Furthermore, the evaluation of xylose as an alternative carbonsource paved the way towards the use of lignucellulosic feedstocks and showed thesuitability of A. oryzae for the biorefinery of the future.
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4. |
- Knuf, Christoph, 1984, et al.
(författare)
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Physiological characterization of the high malic acid-producing Aspergillus oryzae strain 2103a-68
- 2014
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Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 98:8, s. 3517-3527
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Tidskriftsartikel (refereegranskat)abstract
- Malic acid is a C-4 dicarboxylic acid that is currently mainly used in the food and beverages industry as an acidulant. Because of the versatility of the group of C-4 dicarboxylic acids, the chemical industry has a growing interest in this chemical compound. As malic acid will be considered as a bulk chemical, microbial production requires organisms that sustain high rates, yields, and titers. Aspergillus oryzae is mainly known as an industrial enzyme producer, but it was also shown that it has a very competitive natural production capacity for malic acid. Recently, an engineered A. oryzae strain, 2103a-68, was presented which overexpressed pyruvate carboxylase, malate dehydrogenase, and a malic acid transporter. In this work, we report a detailed characterization of this strain including detailed rates and yields under malic acid production conditions. Furthermore, transcript levels of the genes of interest and corresponding enzyme activities were measured. On glucose as carbon source, 2103a-68 was able to secrete malic acid at a maximum specific production rate during stationary phase of 1.87 mmol (g dry weight (DW))(-1) h(-1) and with a yield of 1.49 mol mol(-1). Intracellular fluxes were obtained using C-13 flux analysis during exponential growth, supporting the success of the metabolic engineering strategy of increasing flux through the reductive cytosolic tricarboxylic acid (rTCA) branch. Additional cultivations using xylose and a glucose/xylose mixture demonstrated that A. oryzae is able to efficiently metabolize pentoses and hexoses to produce malic acid at high titers, rates, and yields.
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