Selection of 5-fluorocytosine-resistant mutants from an Aspergillus niger citric acid-producing strain

Seleção de mutantes resistentes à 5-fluorocitosina isolados de uma linhagem de Aspergillus niger produtora de ácido cítrico

Selection of 5-fluorocytosine-resistant mutants from an Aspergillus niger citric acid-producing strain

Seleção de mutantes resistentes à 5-fluorocitosina isolados de uma linhagem de Aspergillus niger produtora de ácido cítrico

Ana Paula de F. Conte^I; José M. MarinII^*

^IFaculdade de Ciências Farmacêuticas de Ribeirão Preto, São Paulo, SP, Brasil

IIDepartamento de Morfologia, Estomatologia e Fisiologia da Faculdade de Odontologia de Ribeirão Preto, Universidade de São Paulo, Campus Ribeirão Preto, SP, Brasil

SHORT COMMUNICATION

ABSTRACT

Mutants of Aspergillus niger N402, induced by UV mutagenesis, were selected and tested for resistance or sensitivity to 5-fluorocytosine. Some mutants showed increased citric acid production, which did not correlate with the intracellular amount of protein or ammonium ion. The resistance to 5-fluorocytosine proved to be a rational approach for isolation of new mutants with improved production of citric acid. The best mutant (FR13) accumulated double the amount of citric acid produced by the parental strain.

Key words:Aspergillus niger, citric acid, 5-fluorocytosine

RESUMO

Através de mutagênese com UV, foram isolados mutantes de Aspergillus niger, verificando-se sua resistência ou sensibilidade à 5-fluorocitosina. Alguns mutantes apresentaram um aumento na produção de ácido cítrico sem um aumento correspondente da quantidade de proteína intracelular ou de ions amônio. A resistência à 5-fluorocitosina mostrou ser uma abordagem racional para o isolamento de mutantes com maior capacidade de produção de ácido cítrico. O melhor mutante selecionado, FR13, produziu o dobro da quantidade de ácido cítrico em relação à linhagem parental.

Palavras-chave:Aspergillus niger, ácido cítrico, 5-fluorocitosina

Because of its high solubility, palatability and low toxicity, citric acid is one of the most commonly used acids in the food and pharmaceutical industry. There are many microorganisms, including fungi, yeasts and bacteria, that can produce citric acid by fermentation. Since Aspergillus niger is one of the best known citric acid producers, it is widely used for industrial-scale production (14).

In spite of the large number of producer strains available, it is still important to generate strains with some advantageous characteristics, such as enhanced citric acid production and increased rate of fermentation. To improve citric acid producing strains, selection after random mutation has been generally carried out (10,13).

Since A. niger shows impaired protein synthesis during citric acid fermentation, the disturbance of protein synthesis has been claimed to be an important phenomenon (5,12). The idea is that mutants having impaired protein synthesis should accumulate large amounts of citric acid and this property has thus been proposed as a new tool for mutant screening (15). Antibiotics and metabolic antagonists are the first choice for sensitive or resistant mutant isolation with impaired protein synthesis. The nucleotide analogue 5-fluorocytosine (5-FC) is a metabolic antagonist which promotes impaired protein, RNA and DNA synthesis (3,4), and has been used to test the hypothesis that other mechanisms besides disturbance in protein synthesis might be involved in citric acid production by A. niger.

The A. niger strain N402, a cspA1 (conferring short conidiophores) derivative of ATCC 9029 (1) is not a hyper-accumulating strain used by the citric acid industry. This strain was used in the present work as a model strain for the selection of mutants. Strain N402 was kept on yeast extract-glucose agar slants and subcultured each month. For citric acid production, and for selection of mutants, the medium and conditions described by Rugsaseel et al. (15), was used, except for the isolation medium, which was 0.1 ug 5-FC/mL. All cultivations were carried out at 30ºC.

After UV mutagenesis (8), a conidial suspension was transferred to the isolation medium. For the isolation of resistant mutants, after 24, 48 and 72 h, the broth was filtered through a glass wool filter to remove the germinated conidia, which were immediately plated onto medium containing 5-FC. To confirm resistance, the colonies were replica plated onto the same medium and cultured for seven days. The sensitive mutants were collected as conidia in the filtrate, which was centrifuged, concentrated and plated. To confirm sensitivity, the mutants were replica plated onto media with and without 5-FC and cultivated for 72 h. Twenty-two mutants were isolated, 20 of them resistant and 2 sensitive, and inoculated in the presence of 5-FC to test growth impairment. Most of the mutants showed almost the same radial growth as the parental strain after 72 hours of incubation (data not shown). Citric acid was determined in the broth of cultures inoculated with 10⁶ conidia/mL after nine days of cultivation. The amount of acid in broth was measured by titration with 0.1 M NaOH against phenolphthalein. After thrichloroacetic acid (TCA)-insoluble protein removal, glucose in the broth was measured enzymatically using the glucose GO-test kit (Sigma, St Louis, MO). The resultant mycelium was treated as described before (15) and intracellular protein was measured in the pellet kept at -20^oC (11). Growth was estimated by measurement of mycelial dry weight.

Mycelium (20 mg) plus 200 mg of washed sand, 200 mg of glass beads and 400 uL of 0.1 M NaOH was placed in a microcentrifuge tube and submitted to maximum speed vortexing for 10 min. The homogenate was then centrifuged at 14,000 g for 5 min and the supernatant (250 uL) was placed in a tube with 250 uL of 10% (W/V) TCA. The mixture was homogenized and centrifuged at 14,000 g for 5 min. The free protein supernatant was used to measure the ammonium ion (NH₄⁺) with the Nessler reagent (19). Nucleic acids were extracted from mycelium (2) and the extracted amount was determined with a Gene Quant spectrophotometer (Pharmacia Biotech).

Citric-acid-accumulation tests were applied to the parental strain and also to the selected mutants. The sensitive mutants were extremely difficult to obtain and always showed a smaller production of citric acid than the parental N402. Five mutants selected for 5-FC resistance showed a small increase in the rate of citric acid accumulation as shown in

Table 1. A. niger mutants resistant to the metabolic antagonist 2 deoxy-D-glucose (8) also showed a small increase in citric acid accumulation, although in this case some mutants showed a reduction in the fermentation time to five days, while another mutant, a cycloheximide-sensitive mutant of the hyper-accumulating citric acid strain WU-2223L, showed a four-fold increase in citric acid production after nine days of fermentation (15).

5-FC is taken up by fungal cells through a cytosine permease, deaminated to 5-fluorouracil, converted into the nucleoside triphosphate and incorporated into RNA, where it causes miscoding. In addition, 5-fluorouracil is converted into deoxynucleoside, which inhibits thymidylate synthase and thereby DNA biosynthesis (4).

It was previously shown that protein content decreases in A. niger under conditions of citric acid accumulation (6,9) while the content of intracellular NH₄⁺ increases due to protein degradation (12). This was also observed by Rugsaseel et al (15). However, as shown in

Table 2, the 5-FC-resistant mutants presented a higher level of intracellular protein than the parental strain N402, while the ammonium ion concentration was always lower than in the parental one. Also, the content of nucleic acids in these mutants was two to three times higher relative to the parental. This may represent an advantage by permiting a more extended period of citric acid fermentation, an interesting economic characteristic.

It is important to consider that the generally observed decrease in the total amount of intracellular protein, as well as the consequent increase in the amount of NH₄⁺ ions could not be sufficient to explain the whole mechanism of citric acid accumulation, which remains controversial for different authors. It is widely accepted that there is an increase in the carbon flux through the glycolytic pathway under the conditions of citric acid fermentation, and many authors have attempted to explain this increase in glycolysis as being caused by phosphofructokinase stimulation by NH₄⁺ ions (18). Such a stimulus would then result in enhanced production of pyruvate, from which both the precursors of citric acid are produced: acetyl-CoA and oxaloacetic acid, whose condensation in the citric acid cycle is the major route of citrate synthesis.

However, several reports have shown that the single stimulation of phosphofructokinase is not the unique aspect leading to citric acid production. In fact, it was shown that overexpression of the glycolitic enzymes phosphofructokinase and/or pyruvate kinase in A. niger did not increase citric acid production by the fungus significantly (16). It ultimately means that the increase in the activity of one or two enzymes would not explain the strong accumulation of citric acid because other components of the pathway could then turn into a blockage to the metabolic flux. The same authors concluded that phosphofructokinase and pyruvate kinase seem not to contribute in a major way to flux control of the metabolism involved in the conversion of glucose to citric acid.

Furthermore, other authors verified that addition of 5-fluorouracil to the fermentation media resulted in citric acid production even when Mn²⁺ ions are present (12). It should be noticed that Mn²⁺ absence or limitation is considered to be responsible for protein degradation and NH₄⁺ accumulation during citric acid fermentation. Thus, it is possible to conclude that 5-FC and ultimately 5-fluorouracil might have other metabolic effects leading to citric acid accumulation which are not dependent on protein decrease. Besides, Mn²⁺ deficiency might have another role, e.g. in decreasing the formation of the by-product oxalic acid, since a recent report showed that oxalate production is mediated by the Mn²⁺ dependent enzyme oxaloacetate acetylhydrolase in an A. niger citric acid producing strain (17).

Several mechanisms can result in resistance to 5-FC, as verified in the model organism Saccharomyces cerevisiae (7). Hyper production of pyrimidines is one of these mechanisms that might be occurring in the mutants isolated in the present work and that helps to explain the increase in the total amount of nucleic acid and protein in the mutants studied. This could mean that such mutants have a more efficient metabolism, and therefore grow better than the parental strain under the limiting conditions of citric acid fermentation, what in fact is shown by their increase in biomass production. Thus, it is possible that the metabolic advantage of the 5-FC-resistant mutants result in a better turnover and reposition of anabolic components (enzymes, cofactors) ensuring the maintenance of an adequate flux through the pathways involved in citric acid accumulation.

Taken together, the results indicate that 5-FC resistance might be a useful criterium for the rational screening of A. niger mutants in order to increase citric acid accumulation.

ACKNOWLEDGEMENTS

The authors thank Dr. V. Ede, Roche Quim. Farm. Brasil, for the gift of 5-fluorocytosine. Thanks are also due to Prof. Suraia Said for her support and encouragement. This work is part of a thesis submittted by Ana Paula F. Conte to the Faculdade de Ciências Farmacêuticas de Ribeirão Preto-USP as part of the requirements for the Masterdegree.

Submitted: September 25, 2001; Returned to authors for corrections: April 18, 2002; Approved: February 21, 2003

• 1. Bos, C.J.; Debets, A.J.; Swart, K.; Huybers, A.; Kobus, G.; Slakhorst, S.M. Genetic analysis and the construction of master strains for assignment of genes to six-linkage groups in Aspergillus niger Curr. Genet, 14: 437-443, 1988.
• 2. Chow, T.Y.K.; Käfer, E. A rapid method for isolation of total nucleic acids from Aspergillus nidulans. Fungal Genet. Newslett., 40: 25-27, 1993.
• 3. Diasio, R.B.; Benett, J.E.; Myers, C.E. Mode of action of 5-fluorocytosine. Biochem. Pharmacol., 27: 703-707, 1978.
• 4. Georgopapadakou, N.H.; Walsh, T.J. Antifungal agents: chemotherapeutic targets and immunologic strategies. Antimicrob. Agents Chemother., 40: 279-291, 1996.
• 5. Habison, A.; Kubicek, C.P.; Rohr, M. Partial purification and regulatory properties of phosphofructokinase from Aspergillus niger Biochem. J, 209: 669-676, 1983.
• 6. Jernejc, K.; Cimerman, A.A. Composition of Aspergillus niger mycelium during growth on productive and unproductive substrates. J. Bacteriol., 25: 341-348, 1992.
• 7. Jundt, R.; Lacroute, F. Genetic and physiological aspects of resistance to 5-fluoropyrimidines in Sacccharomyces cerevisiae J. Bacteriol., 102: 607-615, 1970.
• 8. Kirimura, K.; Sarangbin, S.; Rugsaseel, S.; Usami, S. Citric acid production by 2-deoxyglucose-resistant mutant strains of Aspergillus niger. Appl. Microbiol. Biotechnol., 36: 573-577, 1992.
• 9. Kubicek, C.P.; Hampel, W.; Röhr, M. Manganese deficiency leads to elevated amino acid pools in citric acid accumulating Aspergillus niger. Arch. Microbiol., 123: 73-79, 1979.
• 10. Kubicek, C.P.; Röhr, M. Citric acid fermentation.CRC Crit. Rev. Biotechnol., 3: 331-373, 1986.
• 11. Lowry, O.H.; Rosebrough, N.J.; Farr, A.L.; Randall, R.J. Protein measurement with the Folin phenol reagent. J. Biol Chem, 193: 267-275, 1951.
• 12. Ma, H.; Kubicek, C.P.; Röhr, M. Metabolic effects of manganese deficiency in Aspergillus niger: evidence for increased protein degradation. Arch. Microbiol., 141: 266-268, 1985.
• 13. Röhr, M.; Kubicek, C.P.; Kominek, J. Citric acid. In: Rehm, H.J.; Reed, G. (eds.) Biotechnology, Vol 3, Verlag Chemie, Weinheim, 1983, p. 419-454.
• 14. Röhr, M.; Kubicek, C.P.; Kominek, J. Industrial acids and other small molecules. In: Bennet, J.W.; Klich, M.A. (eds.) Aspergillus: Biology and Industrial Applications, Boston, Butterworth-Heinemann, 1992, p. 91-131.
• 15. Rugsaseel, S.; Kirimura, K.; Usami, S. Citric acid accumulation by cycloheximide-sensitive mutant strains of Aspergillus niger. Appl. Microbiol. Biotechnol., 45: 28-35, 1996.
• 16. Ruijter, G.J.G.; Panneman, J.; Visser, J. Overexpression of phosphofructokinase and pyruvate kinase in citric acid-producing Aspergillus niger. Biochem. Biophys. Acta, 1334: 317-326, 1997.
• 17. Ruijter, G.J.G.; van de Vondervoort, P.J.I.; Visser, J. Oxalic acid production by Aspergillus niger: an oxalate-non-producing mutant produces citric acid at pH 5 and in the presence of manganese. Microbiology, 145: 2569-2576, 1999.
• 18. Schreferl, G.; Kubicek, C.P.; Rohr, M. Inhibition of citric acid accumulation by manganese ions in Aspergillus niger mutants with reduced citrate control of phosphofructokinase. J. Bacteriol., 165: 1019-1022, 1986.
• 19. Seligson, D.; Seligson, H. A microdiffusion method for the determination of nitrogen liberated as ammonia. J. Lab. Clin. Med, 38: 324-330, 1951.

Publication Dates

• Publication in this collection
  30 June 2003
• Date of issue
  Apr 2003

History

• Accepted
  21 Feb 2003
• Reviewed
  18 Mar 2002
• Received
  25 Sept 2001