Skip to main content
SpringerLink
Log in

Enhanced succinic acid production in Aspergillus saccharolyticus by heterologous expression of fumarate reductase from Trypanosoma brucei

  • Applied genetics and molecular biotechnology
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

Aspergillus saccharolyticus exhibits great potential as a cell factory for industrial production of dicarboxylic acids. In the analysis of the organic acid profile, A. saccharolyticus was cultivated in an acid production medium using two different pH conditions. The specific activities of the enzymes, pyruvate carboxylase (PYC), malate dehydrogenase (MDH), and fumarase (FUM), involved in the reductive tricarboxylic acid (rTCA) branch, were examined and compared in cells harvested from the acid production medium and a complete medium. The results showed that ambient pH had a significant impact on the pattern and the amount of organic acids produced by A. saccharolyticus. The wild-type strain produced higher amount of malic acid and succinic acid in the pH buffered condition (pH 6.5) compared with the pH non-buffered condition. The enzyme assays showed that the rTCA branch was active in the acid production medium as well as the complete medium, but the measured enzyme activities were different depending on the media. Furthermore, a soluble NADH-dependent fumarate reductase gene (frd) from Trypanosoma brucei was inserted and expressed in A. saccharolyticus. The expression of the frd gene led to an enhanced production of succinic acid in frd transformants compared with the wild-type in both pH buffered and pH non-buffered conditions with highest amount produced in the pH buffered condition (16.2 ± 0.5 g/L). This study demonstrates the feasibility of increasing succinic acid production through the cytosolic reductive pathway by genetic engineering in A. saccharolyticus.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Arikawa Y, Kuroyanagi T, Shimosaka M, Muratsubaki H, Enomoto K, Kodaira R, Okazaki M (1999) Effect of gene disruptions of the TCA cycle on production of succinic acid in Saccharomyces cerevisiae. J Biosci Bioeng 87:28–36

    Article  CAS  PubMed  Google Scholar 

  • 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 37:1108–1116

    Article  CAS  PubMed  Google Scholar 

  • Beauprez JJ, De Mey M, Soetaert WK (2010) Microbial succinic acid production: natural versus metabolic engineered producers. Process Biochem 45:1103–1114

    Article  CAS  Google Scholar 

  • Bercovitz A, Peleg Y, Battat E, Rokem JS, Goldberg I (1990) Localization of pyruvate carboxylase in organic acid-producing Aspergillus strains. Appl Environ Microbiol 56:1594–1597

    PubMed Central  CAS  PubMed  Google Scholar 

  • Besteiro S, Biran M, Biteau N, Coustou V, Baltz T, Canioni P, Bringaud F (2002) Succinate secreted by Trypanosoma brucei is produced by a novel and unique glycosomal enzyme, NADH-dependent fumarate reductase. J Biol Chem 277:38001–38012

    Article  CAS  PubMed  Google Scholar 

  • 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 97:8903–8912

    Article  CAS  PubMed  Google Scholar 

  • Cao Y, Zhang R, Sun C, Cheng T, Liu Y, Xian M (2013) Fermentative succinate production: an emerging technology to replace the traditional petrochemical processes. BioMed Research International

  • Cecchini G, Schröder I, Gunsalus RP, Maklashina E (2002) Succinate dehydrogenase and fumarate reductase from Escherichia coli. Biochim Biophys Acta Bioenerg 1553:140–157

    Article  CAS  Google Scholar 

  • Cheng K, Zhao X, Zeng J, Zhang J (2012) Biotechnological production of succinic acid: current state and perspectives. Biofuels Bioprod Biorefin 6:302–318

    Article  CAS  Google Scholar 

  • Cleland WW, Johnson MJ (1956) Studies on the formation of oxalic acid by Aspergillus niger. The J Biol Chem 220:595–606

    CAS  PubMed  Google Scholar 

  • Corona-González RI, Bories A, González-Álvarez V, Pelayo-Ortiz C (2008) Kinetic study of succinic acid production by Actinobacillus succinogenes ZT-130. Process Biochem 43:1047–1053

    Article  Google Scholar 

  • Coustou V, Besteiro S, Rivière L, Biran M, Biteau N, Franconi J, Boshart M, Baltz T, Bringaud F (2005) A mitochondrial NADH-dependent fumarate reductase involved in the production of succinate excreted by procyclic Trypanosoma brucei. J Biol Chem 280:16559–16570

    Article  CAS  PubMed  Google Scholar 

  • de Jongh WA, Nielsen J (2008) Enhanced citrate production through gene insertion in Aspergillus niger. Metab Eng 10:87–96

    Article  PubMed  Google Scholar 

  • Ding Y, Li S, Dou C, Yu Y, He H (2011) Production of fumaric acid by Rhizopus oryzae: role of carbon-nitrogen ratio. Appl Biochem Biotechnol 164:1461–1467

    Article  CAS  PubMed  Google Scholar 

  • Dole S, Yocum RR (2014) Organic acid production in microorganisms by combined reductive and oxidative tricaboxylic acid cycle pathways. U.S. Patent and Trademark Office NO.8: 778–656

  • Gallmetzer M, Meraner J, Burgstaller W (2002) Succinate synthesis and excretion by Penicillium simplicissimum under aerobic and anaerobic conditions. FEMS Microbiol Lett 210:221–225

    Article  CAS  PubMed  Google Scholar 

  • Hansen NB, Lübeck M, Lübeck PS (2014) Advancing USER cloning into simpleUSER and nicking cloning. J Microbiol Methods 96:42–49

    Article  CAS  PubMed  Google Scholar 

  • Hatch MD (1978) A simple spectrophotometric assay for fumarate hydratase in crude tissue extracts. Anal Biochem 85:271–275

    Article  CAS  PubMed  Google Scholar 

  • Hatti-Kaul R, Törnvall U, Gustafsson L, Börjesson P (2007) Industrial biotechnology for the production of bio-based chemicals—a cradle-to-grave perspective. Trends Biotechnol 25:119–124

    Article  CAS  PubMed  Google Scholar 

  • Jansen MLA, Segueilha L, Verwaal R, Louchart M (2011) Dicarboxylic acid production process. U.S. Patent Application 13:821–125

    Google Scholar 

  • Jantama K, Zhang X, Moore JC, Shanmugam KT, Svoronos SA, Ingram LO (2008) Eliminating side products and increasing succinate yields in engineered strains of Escherichia coli C. Biotechnol Bioeng 101:881–893

    Article  CAS  PubMed  Google Scholar 

  • Kenealy W, Zaady E, Du Preez JC (1986) Biochemical aspects of fumaric acid accumulation by Rhizopus arrhizus. Appl Environ Microbiol 52:128–133

    PubMed Central  CAS  PubMed  Google Scholar 

  • Kim P, Laivenieks M, Vieille C, Zeikus JG (2004) Effect of overexpression of Actinobacillus succinogenes phosphoenolpyruvate carboxykinase on succinate production in Escherichia coli. Appl Environ Microbiol 70:1238–1241

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • 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 79:6050–6058

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Knuf C, Nookaew I, Remmers I, Khoomrung S, Brown S, Berry A, Nielsen J (2014) Physiological characterization of the high malic acid-producing Aspergillus oryzae strain 2103a-68. Appl Microbiol Biotechnol 98:3517–3527

    Article  CAS  PubMed  Google Scholar 

  • Liaud N, Giniés C, Navarro D, Fabre N, Crapart S, Herpoël-Gimbert I, Levasseur A, Raouche S, Sigoillot J (2014) Exploring fungal biodiversity: organic acid production by 66 strains of filamentous fungi. Fungal Biol Biotechnol 1: 1-1-

  • Ma R, Guo M, Zhang X (2014) Selective conversion of biorefinery lignin into dicarboxylic acids. ChemSusChem 7:412–415

    Article  CAS  PubMed  Google Scholar 

  • Magnuson JK, Lasure LL (2004) Organic acid production by filamentous fungi. Advances in Fungal Biotechnology for Industry, Agriculture, and Medicine : 307-340

  • Mandal SK, Banerjee PC (2005) Submerged production of oxalic acid from glucose by immobilized Aspergillus niger. Process Biochem 40:1605–1610

    Article  CAS  Google Scholar 

  • May G (1992) Fungal technology. Applied Molecular Genetics of Filamentous Fungi: 1-27

  • 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 11:107–116

    Article  CAS  PubMed  Google Scholar 

  • Miura A, Kameya M, Arai H, Ishii M, Igarashi Y (2008) A soluble NADH-dependent fumarate reductase in the reductive tricarboxylic acid cycle of Hydrogenobacter thermophilus TK-6. J Bacteriol 190:7170–7177

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Muzumdar AV, Sawant SB, Pangarkar VG (2004) Reduction of maleic acid to succinic acid on titanium cathode. Org Process Res Dev 8:685–688

    Article  CAS  Google Scholar 

  • Papagianni M (2007) Advances in citric acid fermentation by Aspergillus niger: biochemical aspects, membrane transport and modeling. Biotechnol Adv 25:244–263

    Article  CAS  PubMed  Google Scholar 

  • Peleg Y, Barak A, Scrutton MC, Goldberg I (1989) Malic acid accumulation by Aspergillus flavus—III. 13C NMR and isoenzyme analyses. Appl Microbiol Biotechnol 30:176–183

    Article  CAS  Google Scholar 

  • Peleg Y, Rahamim E, Kessel M, Goldberg I (1988a) Malic acid accumulation by Aspergillus flavus—II. Crystals and hair-like processes formed by A. flavus in a l-malic acid production medium. Appl Microbiol Biotechnol 28:76–79

    Article  CAS  Google Scholar 

  • Peleg Y, Stieglitz B, Goldberg I (1988b) Malic acid accumulation by Aspergillus flavus—I. Biochemical aspects of acid biosynthesis. Appl Microbiol Biotechnol 28:69–75

    Article  CAS  Google Scholar 

  • Raab AM, Lang C (2011) Oxidative versus reductive succinic acid production in the yeast Saccharomyces cerevisiae. Bioengineered Bugs 2

  • Raab AM, Gebhardt G, Bolotina N, Weuster-Botz D, Lang C (2010) Metabolic engineering of Saccharomyces cerevisiae for the biotechnological production of succinic acid. Metab Eng 12:518–525

    Article  CAS  PubMed  Google Scholar 

  • Ramachandran S, Fontanille P, Pandey A, Larroche C (2006) Gluconic acid: properties, applications and microbial production. Food Technol Biotechnol 44:185–195

    CAS  Google Scholar 

  • Rhodes RA, Moyer AJ, Smith ML, Kelley SE (1959) Production of fumaric acid by Rhizopus arrhizus. Appl Microbiol 7:74–80

    PubMed Central  CAS  PubMed  Google Scholar 

  • Sauer M, Porro D, Mattanovich D, Branduardi P (2008) Microbial production of organic acids: expanding the markets. Trends Biotechnol 26:100–108

    Article  CAS  PubMed  Google Scholar 

  • Singh OV, Jain RK, Singh RP (2003) Gluconic acid production under varying fermentation conditions by Aspergillus niger. J Chem Technol Biotechnol 78:208–212

    Article  CAS  Google Scholar 

  • Song H, Jang SH, Park JM, Lee SY (2008) Modeling of batch fermentation kinetics for succinic acid production by Mannheimia succiniciproducens. Biochem Eng J 40:107–115

    Article  CAS  Google Scholar 

  • Sørensen A, Andersen JJ, Ahring BK, Teller PJ, Lübeck M (2014) Screening of carbon sources for beta-glucosidase production by Aspergillus saccharolyticus. Int Biodeterior Biodegrad 93:78–83

    Article  Google Scholar 

  • Sørensen A, Lübeck PS, Lübeck M, Teller PJ, Ahring BK (2011) ß-Glucosidases from a new Aspergillus species can substitute commercial ß-glucosidases for saccharification of lignocellulosic biomass. Can J Microbiol 57:638–650

    Article  PubMed  Google Scholar 

  • van Heerden CD, Nicol W (2013) Continuous and batch cultures of Escherichia coli KJ134 for succinic acid fermentation: metabolic flux distributions and production characteristics. Microb Cell Factories 12

  • Wang X, Gong CS, Tsao GT (1998) Bioconversion of fumaric acid to succinic acid by recombinant E. coli. In: Applied Biochemistry and Biotechnology—Part A Enzyme Engineering and Biotechnology, vol 70-72, pp. 919–928

    Google Scholar 

  • Werpy T, Frye J, Holladay J (2008) Succinic acid—a model building block for chemical production from renewable resources. In: Biorefineries-Industrial Processes and Products: Status Quo and Future Directions, vol 2, pp. 367–379

    Google Scholar 

  • Yang L, Lübeck M, Lübeck PS (2014) Deletion of glucose oxidase changes the pattern of organic acid production in Aspergillus carbonarius. AMB Express 4

  • Zeikus JG, Jain MK, Elankovan P (1999) Biotechnology of succinic acid production and markets for derived industrial products. Appl Microbiol Biotechnol 51:545–552

    Article  CAS  Google Scholar 

  • Zhang B, Skory CD, Yang S (2012) Metabolic engineering of Rhizopus oryzae: effects of overexpressing pyc and pepc genes on fumaric acid biosynthesis from glucose. Metab Eng 14:512–520

    Article  CAS  PubMed  Google Scholar 

  • Zhao F, Yan F, Qian Y, Chu Y, Ma C (2012) Electrochemical synthesis of succinic acid at a TiO2 film electrode prepared by in-situ anodic oxidation. Int J Electrochem Sci 7:12931–12940

    CAS  Google Scholar 

Download references

Acknowledgments

Financial support from SUPRA-BIO EU grant 241640-2 and BIOREF DSF grant 09-065165 is greatly acknowledged. We thank Prof. Israel Goldberg for providing the recipe of acid production medium and cultivation conditions.

Author information

Authors and Affiliations

  1. Section for Sustainable Biotechnology, Department of Chemistry and Bioscience, Aalborg University Copenhagen, A. C. Meyers Vaenge 15, DK-2450, Copenhagen SV, Denmark

    Lei Yang, Mette Lübeck, Birgitte K. Ahring & Peter S. Lübeck

  2. Bioproducts, Sciences & Engineering Laboratory (BSEL), Washington State University, Tri-Cities, 2710 Crimson Way, Richland, WA, 99354, USA

    Birgitte K. Ahring

Authors
  1. Lei Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mette Lübeck
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Birgitte K. Ahring
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Peter S. Lübeck
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Peter S. Lübeck.

Ethics declarations

Conflict of interest

The authors declare that they have no competing interests.

Ethical approval

This study does not contain any experiment with human participants or animals performed by any of the authors.

Electronic supplementary material

ESM 1

(PDF 124 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Yang, L., Lübeck, M., Ahring, B.K. et al. Enhanced succinic acid production in Aspergillus saccharolyticus by heterologous expression of fumarate reductase from Trypanosoma brucei . Appl Microbiol Biotechnol 100, 1799–1809 (2016). https://doi.org/10.1007/s00253-015-7086-z

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-015-7086-z

Keywords

Search

Navigation