Optimization of L ‐malic acid production by Aspergillus flavus in a stirred fermentor

A recent effort to improve malic acid production by Saccharomyces cerevisiae by means of metabolic engineering resulted in a strain that produced up to 59 g liter ؊1 of malate at a yield of 0.42 mol (mol glucose) ؊1 in calcium carbonate-buffered shake flask cultures. With shake flasks, process parameters that are important for scaling up this process cannot be controlled independently. In this study, growth and product formation by the engineered strain were studied in bioreactors in order to separately analyze the effects of pH, calcium, and carbon dioxide and oxygen availability. A near-neutral pH, which in shake flasks was achieved by adding CaCO 3 , was required for efficient C 4 dicarboxylic acid production. Increased calcium concentrations, a side effect of CaCO 3 dissolution, had a small positive effect on malate formation. Carbon dioxide enrichment of the sparging gas (up to 15% [vol/vol]) improved production of both malate and succinate. At higher concentrations, succinate titers further increased, reaching 0.29 mol (mol glucose) ؊1, whereas malate formation strongly decreased. Although fully aerobic conditions could be achieved, it was found that moderate oxygen limitation benefitted malate production. In conclusion, malic acid production with the engineered S. cerevisiae strain could be successfully transferred from shake flasks to 1-liter batch bioreactors by simultaneous optimization of four process parameters (pH and concentrations of CO 2 , calcium, and O 2 ). Under optimized conditions, a malate yield of 0.48 ؎ 0.01 mol (mol glucose) ؊1 was obtained in bioreactors, a 19% increase over yields in shake flask experiments.

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Key Process Conditions for Production of C ₄ Dicarboxylic Acids in Bioreactor Batch Cultures of an Engineered Saccharomyces cerevisiae Strain

Zelle

Hulster

Kloezen

et al. 2010

“…Isoenzymes of malate dehydrogenase were shown to be active in both the cytosol and mitochondrion compartments for Aspergillus flavus (3). After optimization of the fermentation process, yields of 1.26 mol mol Ϫ1 on glucose and a productivity of 0.59 g liter Ϫ1 h Ϫ1 were achieved in fermentors after 190 h of fermentation (5).…”

mentioning

confidence: 99%

Investigation of Malic Acid Production in Aspergillus oryzae under Nitrogen Starvation Conditions

Knuf

Nookaew

Brown

et al. 2013

bMalic 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.

“…Since then, malic acid production has been observed for a wide range of microorganisms. Fermentative production of malic acid has been most successfully demonstrated with Aspergillus flavus, achieving 63% of the maximum theoretical yield of malic acid on glucose at high production rates and titers (4). Since its potential aflatoxin production disqualified A. flavus as a producer of food-grade chemicals (16), malic acid production was studied with other organisms, including the yeast Saccharomyces cerevisiae ( Table 1).…”

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confidence: 99%

Malic Acid Production by Saccharomyces cerevisiae : Engineering of Pyruvate Carboxylation, Oxaloacetate Reduction, and Malate Export

Zelle

Hulster

Winden

et al. 2008

322

247

Malic acid is a potential biomass-derivable "building block" for chemical synthesis. Since wild-type Saccharomyces cerevisiae strains produce only low levels of malate, metabolic engineering is required to achieve efficient malate production with this yeast. A promising pathway for malate production from glucose proceeds via carboxylation of pyruvate, followed by reduction of oxaloacetate to malate. This redox-and ATP-neutral, CO 2 -fixing pathway has a theoretical maximum yield of 2 mol malate (mol glucose) ؊1 . A previously engineered glucose-tolerant, C 2 -independent pyruvate decarboxylase-negative S. cerevisiae strain was used as the platform to evaluate the impact of individual and combined introduction of three genetic modifications: (i) overexpression of the native pyruvate carboxylase encoded by PYC2, (ii) high-level expression of an allele of the MDH3 gene, of which the encoded malate dehydrogenase was retargeted to the cytosol by deletion of the C-terminal peroxisomal targeting sequence, and (iii) functional expression of the Schizosaccharomyces pombe malate transporter gene SpMAE1. While single or double modifications improved malate production, the highest malate yields and titers were obtained with the simultaneous introduction of all three modifications. In glucose-grown batch cultures, the resulting engineered strain produced malate at titers of up to 59 g liter ؊1 at a malate yield of 0.42 mol (mol glucose)؊1 . Metabolic flux analysis showed that metabolite labeling patterns observed upon nuclear magnetic resonance analyses of cultures grown on 13 C-labeled glucose were consistent with the envisaged nonoxidative, fermentative pathway for malate production. The engineered strains still produced substantial amounts of pyruvate, indicating that the pathway efficiency can be further improved.