Abstract
Malic acid is a C4 dicarboxylic acid that is currently mainly used in the food and beverages industry as an acidulant. Because of the versatility of the group of C4 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 13C 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.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Abe S, Furuya A, Saito T, Takayama K (1962) Method of producing l-malic acid by fermentation. US Patent 3,063,910
Battat E, Peleg Y, Bercovitz A, Rokem JS, Goldberg I (1991) Optimization of l-malic acid production by Aspergillus flavus in a stirred fermentor. Biotechnol Bioeng 37(11):1108–1116. doi:10.1002/bit.260371117
Becker J, Reinefeld J, Stellmacher R, Schäfer R, Lange A, Meyer H, Lalk M, Zelder O, von Abendroth G, Schröder H, Haefner S, Wittmann C (2013) Systems-wide analysis and engineering of metabolic pathway fluxes in bio-succinate producing Basfia succiniciproducens. Biotechnol Bioeng 110(11):3013–3023. doi:10.1002/bit.24963
Bercovitz A, Peleg Y, Battat E, Rokem JS, Goldberg I (1990) Localization of pyruvate carboxylase in organic acid-producing Aspergillus strains. Appl Environ Microbiol 56(6):1594–1597
Brown SH, Bashkirova L, Berka R, Chandler T, Doty T, McCall K, McCulloch M, McFarland S, Thompson S, Yaver D, Berry A (2013) Metabolic engineering of Aspergillus oryzae NRRL 3488 for increased production of l-malic acid. Appl Microbiol Biotechnol 97(20):8903–8912. doi:10.1007/s00253-013-5132-2
Buschke N, Schröder H, Wittmann C (2011) Metabolic engineering of Corynebacterium glutamicum for production of 1,5-diaminopentane from hemicellulose. Biotechnol J 6(3):306–317. doi:10.1002/biot.201000304
Camarasa C, Bidard F, Bony M, Barre P, Dequin S (2001) Characterization of Schizosaccharomyces pombe malate permease by expression in Saccharomyces cerevisiae. Appl Environ Microbiol 67(9):4144–4151. doi:10.1128/aem.67.9.4144-4151.2001
Cvijovic M, Olivares-Hernández R, Agren R, Dahr N, Vongsangnak W, Nookaew I, Patil KR, Nielsen J (2010) BioMet Toolbox: genome-wide analysis of metabolism. Nucleic Acids Res 38(Suppl 2):W144–W149. doi:10.1093/nar/gkq404
de Jongh WA, Nielsen J (2008) Enhanced citrate production through gene insertion in Aspergillus niger. Metab Eng 10(2):87–96. doi:10.1016/j.ymben.2007.11.002
Goldberg I, Rokem JS, Pines O (2006) Organic acids: old metabolites, new themes. J Chem Technol Biotechnol 81(10):1601–1611. doi:10.1002/jctb.1590
Gombert AK, Moreira dos Santos M, Christensen B, Nielsen J (2001) Network identification and flux quantification in the central metabolism of Saccharomyces cerevisiae under different conditions of glucose repression. J Bacteriol 183(4):1441–1451. doi:10.1128/jb.183.4.1441-1451.2001
Grotkjaer T, Christakopoulos P, Nielsen J, Olsson L (2005) Comparative metabolic network analysis of two xylose fermenting recombinant Saccharomyces cerevisiae strains. Metab Eng 7(5–6):437–444. doi:10.1016/j.ymben.2005.07.003
Karaffa L, Kubicek C (2003) Aspergillus niger citric acid accumulation: do we understand this well working black box? Appl Microbiol Biotechnol 61(3):189–196. doi:10.1007/s00253-002-1201-7
Kawaguchi H, Vertès AA, Okino S, Inui M, Yukawa H (2006) Engineering of a xylose metabolic pathway in Corynebacterium glutamicum. Appl Environ Microbiol 72(5):3418–3428. doi:10.1128/aem.72.5.3418-3428.2006
Knuf C, Nookaew I, Brown SH, McCulloch M, Berry A, Nielsen J (2013) Investigation of malic acid production in Aspergillus oryzae under nitrogen starvation conditions. Appl Environ Microbiol 79(19):6050–6058. doi:10.1128/aem.01445-13
Liu R, Liang L, Chen K, Ma J, Jiang M, Wei P, Ouyang P (2012) Fermentation of xylose to succinate by enhancement of ATP supply in metabolically engineered Escherichia coli. Appl Microbiol Biotechnol 94(4):959–968. doi:10.1007/s00253-012-3896-4
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25(4):402–408. doi:10.1006/meth.2001.1262
Meijer S, Otero J, Olivares R, Andersen MR, Olsson L, Nielsen J (2009) Overexpression of isocitrate lyase—glyoxylate bypass influence on metabolism in Aspergillus niger. Metab Eng 11(2):107–116. doi:10.1016/j.ymben.2008.12.002
Nolan T, Hands RE, Bustin SA (2006) Quantification of mRNA using real-time RT-PCR. Nat Protoc 1(3):1559–1582
Panagiotou G, Andersen MR, Grotkjaer T, Regueira TB, Hofmann G, Nielsen J, Olsson L (2008) Systems analysis unfolds the relationship between the phosphoketolase pathway and growth in Aspergillus nidulans. PLoS One 3(12):e3847. doi:10.1371/journal.pone.0003847
Papini M, Nookaew I, Siewers V, Nielsen J (2012) Physiological characterization of recombinant Saccharomyces cerevisiae expressing the Aspergillus nidulans phosphoketolase pathway: validation of activity through 13C-based metabolic flux analysis. Appl Microbiol Biotechnol 95(4):1001–1010. doi:10.1007/s00253-012-3936-0
Payne GA, Nierman WC, Wortman JR, Pritchard BL, Brown D, Dean RA, Bhatnagar D, Cleveland TE, Machida M, Yu J (2006) Whole genome comparison of Aspergillus flavus and A. oryzae. Med Mycol 44(s1):9–11. doi:10.1080/13693780600835716
Pedersen H, Carlsen M, Nielsen J (1999) Identification of enzymes and quantification of metabolic fluxes in the wild type and in a recombinant Aspergillus oryzae strain. Appl Environ Microbiol 65(1):11–19
Peleg Y, Barak A, Scrutton M, Goldberg I (1989) Malic acid accumulation by Aspergillus flavus. Appl Microbiol Biotechnol 30(2):176–183. doi:10.1007/bf00264008
Regev-Rudzki N, Battat E, Goldberg I, Pines O (2009) Dual localization of fumarase is dependent on the integrity of the glyoxylate shunt. Mol Microbiol 72(2):297–306. doi:10.1111/j.1365-2958.2009.06659.x
Sakai K, Kinoshita H, Nihira T (2012) Heterologous expression system in Aspergillus oryzae for fungal biosynthetic gene clusters of secondary metabolites. Appl Microbiol Biotechnol 93(5):2011–2022. doi:10.1007/s00253-011-3657-9
Scalcinati G, Otero JM, Van Vleet JRH, Jeffries TW, Olsson L, Nielsen J (2012) Evolutionary engineering of Saccharomyces cerevisiae for efficient aerobic xylose consumption. FEMS Yeast Res 12(5):582–597. doi:10.1111/j.1567-1364.2012.00808.x
Schmidt K, Marx A, de Graaf AA, Wiechert W, Sahm H, Nielsen J, Villadsen J (1998) 13C Tracer experiments and metabolite balancing for metabolic flux analysis: comparing two approaches. Biotechnol Bioeng 58(2–3):254–257. doi:10.1002/(sici)1097-0290(19980420)58:2/3<254::aid-bit19>3.0.co;2-c
Shen Y, Chen X, Peng B, Chen L, Hou J, Bao X (2012) An efficient xylose-fermenting recombinant Saccharomyces cerevisiae strain obtained through adaptive evolution and its global transcription profile. Appl Microbiol Biotechnol 96(4):1079–1091. doi:10.1007/s00253-012-4418-0
Udvardi MK, Czechowski T, Scheible W-R (2008) Eleven golden rules of quantitative RT-PCR. Plant Cell 20(7):1736–1737. doi:10.1105/tpc.108.061143
Untergasser A, Cutcutache I, Koressaar T, Ye J, Faircloth BC, Remm M, Rozen SG (2012) Primer3—new capabilities and interfaces. Nucleic Acids Res 40(15):e115. doi:10.1093/nar/gks596
van Winden W, Wittmann C, Heinzle E, Heijnen J (2002) Correcting mass isotopomer distributions for naturally occurring isotopes. Biotechnol Bioeng 80:477–479
Vongsangnak W, Olsen P, Hansen K, Krogsgaard S, Nielsen J (2008) Improved annotation through genome-scale metabolic modeling of Aspergillus oryzae. BMC Genomics 9(1):245
Wiechert W (2001) 13C metabolic flux analysis. Metab Eng 3:195–206
Wittmann C (2007) Fluxome analysis using GC-MS. Microb Cell Fact 6(1):6
Zelle RM, de Hulster E, van Winden WA, de Waard P, Dijkema C, Winkler AA, Geertman J-MA, van Dijken JP, Pronk JT, van Maris AJA (2008) Malic acid production by Saccharomyces cerevisiae: engineering of pyruvate carboxylation, oxaloacetate reduction, and malate export. Appl Environ Microbiol 74(9):2766–2777. doi:10.1128/aem.02591-07
Acknowledgments
We acknowledge funding for our research activities from the Knut and Alice Wallenberg Foundation, the European Research Council (grant no. 247013), Novozymes, and the Novo Nordisk Foundation. Scientific discussions with Eugenio Meza and Sergio Bordel were much appreciated.
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
Online resource 1
(XLSX 20 kb)
Online resource 2
(PDF 307 kb)
Rights and permissions
About this article
Cite this article
Knuf, C., Nookaew, I., Remmers, I. et al. Physiological characterization of the high malic acid-producing Aspergillus oryzae strain 2103a-68. Appl Microbiol Biotechnol 98, 3517–3527 (2014). https://doi.org/10.1007/s00253-013-5465-x
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-013-5465-x