Appl Microbiol Biotechnol (2014) 98:5145–5152 DOI 10.1007/s00253-014-5615-9
APPLIED MICROBIAL AND CELL PHYSIOLOGY
Alternative respiration and fumaric acid production of Rhizopus oryzae Shuai Gu & Qing Xu & He Huang & Shuang Li
Received: 7 November 2013 / Revised: 11 February 2014 / Accepted: 12 February 2014 / Published online: 19 March 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Under the conditions of fumaric acid fermentation, Rhizopus oryzae ME-F14 possessed at least two respiratory systems. The respiration of mycelia was partially inhibited by the cytochrome respiration inhibitor antimycin A or the alternative respiration inhibitor salicylhydroxamic acid and was completely inhibited in the presence of both antimycin A and salicylhydroxamic acid. During fumaric acid fermentation process, the activity of alternative respiration had a great correlation with fumaric acid productivity; both of them reached peak at the same time. The alternative oxidase gene, which encoded the mitochondrial alternative oxidase responsible for alternative respiration in R. oryzae ME-F14, was cloned and characterized in Escherichia coli. The activity of alternative respiration, the alternative oxidase gene transcription level, as well as the fumaric acid titer were measured under different carbon sources and different carbon-nitrogen ratios. The activity of alternative respiration was found to be comparable to the transcription level of the alternative oxidase gene and the fumaric acid titer. These results indicated that the activity of the alternative oxidase was regulated at the transcription stage under the conditions tested for R. oryzae ME-F14. Keywords Alternative respiration . SHAM-sensitive respiration . Fumaric acid . Rhizopus oryzae . Alternative oxidase
S. Gu : Q. Xu : H. Huang (*) : S. Li (*) State Key Laboratory of Material-oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, No. 5 Xinmofan Road, Nanjing 210009, People’s Republic of China e-mail: [email protected] e-mail: [email protected]
Introduction Fumaric acid is a four-carbon unsaturated dicarboxylic acid widely used in chemical, food, and pharmaceutical industries. The filamentous fungi Rhizopus oryzae is one of the bestknown producers of fumaric acid (Engel et al. 2008). To get an efficient fumaric acid production, many efforts have been done on the strain improvement, the morphology control, and the optimization of fermentation process (Xu et al. 2012). Fumaric acid is one of the primary metabolic products, whose accumulation is significantly affected by both the carbon metabolism and the energy metabolism. Thus, more attention should be focused on how to manipulate and balance the energy metabolism and carbon metabolism to achieve an efficient fumaric acid production. Alternative respiration pathway, characterized as sensitive to salicylhydroxamic acid (SHAM) and insensitive to conventional inhibitors of the cytochrome respiration pathway, has been widely found in plants, fungi, and yeast (Costa–de– Oliveira et al. 2012; Kirimura et al. 1999; Li et al. 2008). Alternative oxidase (AOX), a quinol oxidase localized in the inner mitochondrial membrane, is a terminal oxidase responsible for the activity of the alternative respiration (Moore et al. 2002). Alternative respiration pathway transfers electrons directly from the ubiquinone pool to oxygen, bypassing complex III and cytochrome c oxidase, two sites of energy conservation in the cell, thus producing fewer ATP than that via the cytochrome pathway (Vanlerberghe and McIntosh 1997). The alternative route is essential in every system, where the ATP production due to the regeneration of reduced coenzymes inhibits the overproduction of metabolites (Jeppsson et al. 1995; Kubicek et al. 1980). The alternative respiration pathway has been shown to be important for citric acid and cephalosporin C overproduction (Kirimura et al. 2000;
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Kozma and Karaffa 1996), and it plays an important role in oxidative stress in fungal fermentation processes (Costa–de–Oliveira et al. 2012). In the present study, we described, for the first time, the presence of an alternative respiration pathway in R. oryzae under the conditions of fumaric acid accumulation. The alternative oxidase gene (aox) was cloned and characterized; the relationship among the transcription level of aox and the activity of alternative respiration, as well as the fumaric acid accumulation, were examined in R. oryzae under several conditions. This work will help us understand the contribution of alternative respiration pathway to fumaric acid production in R. oryzae.
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Inhibitors at appropriate concentrations were added into the suspension, and 2 mL of the suspension was used for the measurement of respiration at 25 °C. The respiration rates were defined as oxygen consumption in micromoles O2 per minute per gram (dry weight) in the presence or absence of inhibitors. Cytochrome respiration was sensitive to antimycin A, and the alternative respiration was sensitive to SHAM (Costa–de–Oliveira et al. 2012). To distinguish between the cytochrome respiration and the alternative respiration, 0.25 mM antimycin A and 5 mM SHAM were added to the suspension, respectively. The ratio of the cytochrome and alternative respiration was determinate by the method of Bahr and Bonner (1973). The rate of inhibition by antimycin A alone was calculated as the cytochrome respiration, and that by SHAM alone was calculated as the alternative respiration.
Materials and methods Strain and growth condition R. oryzae ME-F14, a mutant of R. oryzae ME-F12 (Ding et al. 2011; Xu et al. 2010) with higher fumaric acid productivity, derived from R. oryzae ATCC 20344, was used in this study. R. oryzae ME-F14 was grown on potato-dextrose agar (PDA) slants at 35 °C for 7 days. The fungal spores in the slants were suspended in sterilized water and maintained at 4 °C (Fu et al. 2010). The spores (at a final concentration of 107 spores/mL) were grown for 30 h in 50 mL of seed culture medium containing 30 g/L glucose, 2 g/L urea, 0.6 g/L KH2PO4, 0.5 g/L MgSO4 · 7H2O, 0.11 g/L ZnSO4 · 7H2O, and 0.0088 g/L FeSO4 ·7H2O with pH 2.5, in 250-mL Erlenmeyer flasks at 35 °C and 200 rpm. The seed culture was then inoculated by 10 % (v/v) into the fermentation medium containing 80 g/L glucose, 0.1 g/L (for the study of carbon-nitrogen ratio, the urea concentrations used were 0.1, 0.4, and 2 g/L), and 50 g/L CaCO3, supplemented with other ingredients same to those of the seed medium. All the media were autoclaved at 115 °C for 30 min. Quantitative determination of fumaric acid Fumaric acid and biomass were determined as described by Xu et al. (2010).
Cloning and characterization of R. oryzae alternative oxidase gene The plasmids pMD18T-simple and pET22b were purchased from Novagen and TaKaRa, respectively. Escherichia coli JM109 and E. coli BL21 (DE3) were used as cloning host and expression host, respectively. For the extraction of mRNA, mycelia at 24 h (at the fermentation stage) were used. Mycelia were collected on Whatman GF/A and washed with distilled water. According to the sequence of the hypothetical aox of R. oryzae (GenBank accession no. CH476747.1, NCBI), the hypothetical aox of R. oryzae ME-F14 was amplified by reverse transcription PCR (RT-PCR) using specific primers that included restriction sites Nde I: 5′-TGCCATATGCCGTACGACATGTACCGT GA-3′ (NdeI site underlined, primer 1) and Sac I: 5′AGCGAGCTCTTATGCTGTCGATGGGTGAG-3′ (SacI site underlined, primer 2). The RT-PCR product was inserted into a pMD18T-simple vector and sequenced by GENEWIZ Corporation (Suzhou, China). All manipulations with recombinant DNA in E. coli were carried out according to standard procedures and as specified by enzyme manufacturers. Restriction endonucleases, T4 DNA ligase, and buffer components (TaKaRa Bio) were used as recommended by the supplier.
Respiration activity analysis
Quantification of aox mRNAs using quantitative real-time reverse transcriptase PCR
The respiration of cells was measured by a Clark electrode connected to a polarization and measurement device (Hansatech, England), and the data were collected by computer. During the fermentation process, mycelia were harvested on a Whatman GF/A filter with suction and suspended directly in the incubation buffer (50 mL) containing 8 % (w/v) glucose in 10 mM potassium phosphate buffer (pH 7.0). The suspension was constantly stirred for enough dissolve oxygen.
The aox expression in R. oryzae was analyzed by quantitative real-time RT-PCR (qRT-PCR) using a pair of specific primers: primer 3: 5′-CGACTGCTTCCGGACACTTA-3′, primer 4: 5′-TTGTCTTCAGACATGCGACGT-3′, which were designed according to the sequence of the hypothetical aox of R. oryzae. Methodology of RNA isolation, cDNA synthesis, qRT-PCR, relative quantification of gene expression ,and statistical analysis were described by Mhadhbi et al. (2011).
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Results Presence of an alternative respiration pathway in R. oryzae During the fumaric acid fermentation, the oxygen consumption rate was determined using a Clark electrode at 24 h of the fermentation. As Fig. 1 showed, the oxygen consumption of R. oryzae was partially inhibited by antimycin A (33 %), suggesting that there was an antimycin A-insensitive respiration pathway in R. oryzae. Then, we measured the oxygen consumption in the presence of SHAM, and the oxygen consumption rate was also partially inhibited with the addition of SHAM alone (60 %). And only in the presence of both antimycin A and SHAM, the respiration could be almost completely inhibited (Fig. 1). The results indicated that there existed at least two respiratory systems in the mycelia of R. oryzae, one antimycin A-sensitive and the other SHAMsensitive. It has been reported that alternative respiration pathway is insensitive to cytochrome pathway inhibitors, such as antimycin A and cyanide, but it is specifically inhibited by SHAM (Vanlerberghe and McIntosh 1997). We therefore supposed that the SHAM-sensitive respiration is an alternative respiration pathway in R. oryzae.
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alternative respiration increased significantly during the first 24 h and then decreased sharply. As shown in Fig. 2b, during the first 24 h, the alternative respiration ratio increased from 15 to 60 %, and the fumaric acid productivity also increased; both the alternative respiration ratio (60 %) and fumaric acid productivity (1.1 g/L/h) reached the highest at 24 h. After 24 h, the fumaric acid productivity decreased, and the alternative respiration ratio also decreased. At last, the alternative respiration ratio maintained at 40 %, and the fumaric acid productivity maintained at 0.4–0.5 g/L/h. Addition of SHAM to repress the alternative respiration resulted in the decrease of fumaric acid accumulation (Table 1). When adding 1.0 mM SHAM in the fermentation, fumaric acid production and productivity decreased by 27 and 30 %, respectively. The results indicated that the alternative respiration pathway in R. oryzae was important for fumaric acid productivity. Molecular cloning and expression characteristics of the alternative oxidase gene of R. oryzae
To examine the relationship between fumaric acid productivity and the activity of the alternative respiration, the respiratory rates, the alternative respiration ratios, and the fumaric acid productivity during the fermentation process were determined. At the first 36 h, the total respiratory rate of mycelia decreased from 2.73 to 1.15 μmol O2/min/g and thereafter remained almost constant (Fig. 2a); however, the activity of
The RT-PCR amplification of the hypothetical aox of R. oryzae ME-F14 generated single bands of approximately 800 bp (Fig. 3). The sequence of the RT-PCR product showed 100 % homology with that of the hypothetical aox in R. oryzae (GenBank accession no. CH476747.1, NCBI). The molecular mass of the R. oryzae AOX was estimated to be 29.6 kDa with a theoretical isoelectric point of 6.04. To verify if the hypothetical protein was functional as an alternative oxidase, the hypothetical aox cDNA fragment was inserted into the expression vector pET22b. The recombinant plasmid pET22b-aox was transformed into E. coli BL21 (DE3) strain. SDS-PAGE analysis demonstrated an expressed 29.6-kDa AOX protein in E. coli BL21 (DE3)/pET22b-aox
Fig. 1 Effect of antimycin A and salicylhydroxamic acid (SHAM) on the mycelia respiration of R. oryzae ME-F14. Mycelia at 24 h (at the fermentation stage) were used. At the points indicated by arrows, antimycin A or SHAM was added to the reaction mixture at final concentration of 0.25 or
5 mM, respectively. The values shown in the figure were the respiratory rates expressed as oxygen consumption in micromoles O2 per minute per gram (dry weight). The rate of inhibition by antimycin A alone was 33.21 ±3.41 % (a) and that by SHAM alone was 60.08±2.82 % (b)
Alternative respiration pathway and productivity of fumaric acid
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Fig. 3 Agarose gel electrophoresis of RT-PCR-amplified aox DNA fragments. RT-PCR was carried out with sets of primers 1 and 2, as shown in “Materials and methods.” Lanes 1–4 were the products of RTPCR-amplified aox DNA fragments. Lane 5 was the molecular weight marker
Fig. 2 Time courses of the total respiratory rate (open rectangles), the alternative respiratory rate (open circles), the fumaric acid productivity (filled circles), and the alternative respiration ratio (filled rectangles) of R. oryzae. In b, the alternative respiration (percentage of total respiration) was shown as the rate of inhibition by salicylhydroxamic acid (SHAM) and calculated as the ratio of SHAM-sensitive respiration in total respiration. The final concentration of SHAM in the reaction mixture was 5 mM. The productivity of fumaric acid (grams per liter per hour) is expressed as grams of fumaric acid produced per liter per hour. Data were presented as the mean values of triplicates (n=3)
inhibited oxygen consumption in the parent strain E. coli BL21 (DE3) (Fig. 5a1), whereas antimycin A only partially inhibited oxygen consumption in the recombinant strain (Fig. 5b1). Moreover, the addition of SHAM had no effect on oxygen consumption in the parent strain (Fig. 5a2), whereas SHAM could partially inhibit oxygen consumption in the recombinant strain (Fig. 5b2). The results clearly indicated that the introduction of the hypothetical aox resulted in the generation of an antimycin A-resistant and SHAM-sensitive respiration pathway in E. coli. Moreover, it was confirmed that the alternative respiration pathway in R. oryzae was catalyzed by AOX which was encoded by the hypothetical aox.
(Fig. 4), indicating that the hypothetical aox of R. oryzae was properly expressed in aox-deficient E. coli. The oxygen consumption of E. coli BL21 (DE3) and E. coli BL21 (DE3)/pET22b-aox was measured by a Clark electrode. As shown in Fig. 5, antimycin A almost completely
Table 1 Influence of SHAM on fumaric acid production and productivity SHAM (mM)
Fumaric acid (g/L)
Productivity (g/L/h)
Fermentation time (h)
0 0.5 1.0
40.1±1.8 34.1±2.1 29.3±1.7
0.56±0.025 0.47±0.029 0.39±0.022
72 72 75
SHAM was added in the fermentation medium at the final concentration of 0.5 and 1.0 mM,respectively. Fumaric acid was measured at the end of fermentation. The productivity of fumaric acid is expressed as grams of fumaric acid produced per liter per hour. Data were presented as the mean values of triplicates (n=3)
Fig. 4 SDS-PAGE analysis of alternative oxidase. The gene of R. oryzae hypothesis alternative oxidase was expressed in E. coli BL21(DE3). Lane 1: host E. coli BL21(DE3) (control), Lane 2 molecular weight marker, Lanes 3 and 4 recombinant E. coli BL21(DE3) carrying pET22b-aox induced by 1 mM IPTG
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Fig. 5 Effect of antimycin A and salicylhydroxamic acid (SHAM) on oxygen consumption of parent strain E. coli BL21(DE3) and recombinant strains grown on LB medium. At the points indicated by arrows, antimycin A or SHAM was added to the reaction mixture to final concentration of 0.25 or 5 mM, respectively. The values shown in the figure were the respiratory rates expressed in micromoles O2 per minute per gram (dry weight). a1 and a2: host BL21(DE3) (control), b1 and b2: recombinant BL21(DE3) carrying pET22b-aox induced by 1 mM IPTG
Effect of the carbon source on the alternative respiration pathway With xylose as a carbon source for fumaric acid production by R. oryzae ME-F14, the final fumaric acid titer was only about 5 g/L (data not shown), much lower than that in the glucose medium. The oxygen consumption of mycelia was measured. As shown in Fig. 6, the oxygen consumption was remarkably inhibited (almost 90 %) by the addition of antimycin A (Fig. 6b), while the addition of SHAM almost did not inhibit the oxygen consumption (Fig. 6a). This result indicated that the alternative respiration pathway almost did not exist in R. oryzae when using xylose as a carbon source; it also illustrated that the alternative respiration pathway played an important role in the accumulation of fumaric acid. Effect of the carbon-nitrogen ratio on the alternative respiration pathway Glucose (80 g/L) was used as a carbon source, and three different concentrations (0.1, 0.4, and 2 g/L) of urea were used as the nitrogen source for the fermentation. We measured oxygen consumption of mycelia, aox transcription level,
biomass, and fumaric acid titer under different carbonnitrogen ratios. As shown in Table 2, biomass increased with the increase of urea concentration; in reverse, aox transcription level, activity of alternative respiration, and fumaric acid titer sharply decreased. Moreover, both the activity of alternative respiration and the yield of fumaric acid production agreed well with the transcription levels of aox. As a result, a lower urea concentration was preferable for aox transcription and activity of alternative respiration. In addition, the activity of the alternative oxidase was regulated at the transcription stage under conditions tested for R. oryzae.
Discussion The presence of an alternative respiration pathway in R. oryzae Alternative respiration pathway occurs in all higher plants, many fungi, and some yeast (Magnani et al. 2007; Panda et al. 2013; Veiga et al. 2003). Several authors have stressed the relevance of the mitochondrial respiration and its influence upon metabolic behavior, stress environment adaptability, and
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Fig. 6 Effect of antimycin A and salicylhydroxamic acid (SHAM) on oxygen consumption of R. oryzae ME-F14 using xylose as a carbon source. Mycelia at 24 h (at the fermentation stage) were used. At the points indicated by arrows, antimycin A or SHAM was added to the reaction mixture to final concentration of 0.25 or 5 mM, respectively. The
values shown in the figure were the respiratory rates expressed in micromoles O2 per minute per gram (dry weight). The rate of inhibition by SHAM alone was 1.9±1.1 % (a) and that by antimycin A alone was 90.35±5.2 % (b)
morphological control (Costa–de–Oliveira et al. 2012; Karaffa et al. 1996; Kozma and Karaffa 1996). AOX encoded by aox is the key enzyme for the activity of alternative respiration. The whole-genome sequencing projects of R. oryzae were completed, and a hypothetical aox in the filamentous R. oryzae was first found by Ma et al. (2009). In this study, we described, for the first time, the presence of an alternative respiration pathway in R. oryzae and the relationship among the yield of fumaric acid, the activity of alternative respiration, and the transcription level of aox. We concluded that the alternative respiration pathway of R. oryzae was mediated by AOX encoded by the hypothetical aox (GenBank accession no. CH476747.1, NCBI).
fumaric acid, especially in the fumaric acid production using xylose as a carbon source. Many reports have showed that R. oryzae can grow well but produce fumaric acid poorly using xylose as a carbon source (Kautola and Linko 1989; Xu et al. 2010); our research, for the first time, demonstrated that the deficiency of alternative respiration pathway had a relation to the failure in fumaric acid accumulation. It is reported that with a high carbohydrate concentration, the cytochrome respiratory system generates high levels of ATP, which are enough to inhibit the glycolytic pathway and the production of metabolites (Kozma et al. 1991). However, the alternative respiration pathway transfers electrons directly from the ubiquinone pool to oxygen, without the generation of ATP. So the existence of an alternative respiratory system overcomes the above problem; the cells are able to degrade glucose (Lambers 1982) and probably are able to produce metabolites (Kozma et al. 1991). The definite relationship between alternative respiration pathway and productivity of fumaric acid showed the importance of energy metabolic regulation for fumaric acid accumulation in R. oryzae.
The alternative respiration pathway stimulates the productivity of fumaric acid In our study, we showed that the activity of the alternative respiration has a great relation to the productivity of fumaric acid during the fermentation process; moreover, the lack of alternative respiration activity inhibited the production of
Table 2 Effect of carbon-nitrogen ratio on aox transcription level, activity of alternative respiration, and fumaric acid titer Carbon source
Concentration of urea (g/L)
aox transcription levels
Alternative respiration (%total respiration)
Biomass (g/L)
Fumaric acid (g/L)
Fermentation time (h)
Glucose (80 g/L)
2 0.4 0.1
1.0 2.5 3.72
17.71±3.51 34.24±5.13 60.08±2.82
12.91±1.1 5.23±0.8 4.26±0.9
13.8±1.3 22.4±1.8 40.5±1.5
36 48 72
Mycelia at 24 h (at the fermentation stage) were used for the measurement of the aox transcription level and the activity of alternative respiration. The fumaric acid and biomass were measured at the end of fermentation. To calculate the relative transcription level, the signal intensity of aox was normalized using the signal intensity of 18 s, and the relative transcription level of aox on the concentration of urea of 2.0 g/L was represented as 1.0. The calculation of the alternative respiration was the same as in Fig. 2 Data with sign “±” are the average of triplicates with standard deviation (n=3)
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Nitrogen starve stress stimulates the alternative respiration of R. oryzae Studies with different species have demonstrated that aox is induced by a variety of treatments usually labeled as stresses, such as oxidative stress and cold (Vanlerberghe and McIntosh 1997). Additionally, stress appears to be a common requirement for the unusually high accumulation of organic acids by all producing organisms. The most important factor is nitrogen limitation (Moresi et al. 1991); fungi starved of nitrogen produce mainly fumaric acid and/or L-malic acid, but not biomass, from glucose. In fermentations, only after full depletion of the limiting nitrogen source do the acids be accumulated by secretion into the medium (Goldberg et al. 2006). In our previous study, we found that nitrogen source limitation can enhance the activity of cytosolic fumarase, and cytosolic fumarase played an important role in the high accumulation of fumaric acid (Ding et al. 2011). In this study, we found that the aox transcription level, the activity of SHAMsensitive respiration, and the yield of fumaric acid reached the highest under lower urea concentration; as a result, nitrogen starve stress stimulated the activity of alternative respiration, thus leading to the accumulation of fumaric acid. Regulation of alternative oxidase in R. oryzae In Aspergillus niger, alternative respiration pathway is constitutive (Hattori et al. 2009). In contrast, in Neurospora crassa, the alternative respiratory system is induced when the cytochrome chain functions are insufficient due to mutation or inhibition of mitochondrial protein synthesis (Li et al. 1996). In A. niger WU-2223 L, the activity of alternative oxidase under the conditions of citric acid production is regulated at the transcription stage (Hattori et al. 2009). In this study, the aox transcription and the activity of the alternative respiration in R. oryzae were found to be induced by nitrogen starvation stress, and the alternative respiration could not be induced by xylose. In the xylose culture, neither aox transcription (data not shown) nor alternative respiration could be detected. Moreover, we found that the changes in the activity of alternative respiration and the fumaric acid production titer agreed well with that in the aox transcription level. It means that activity of alternative oxidase in R. oryzae was regulated at the transcription stage. Acknowledgments This work was financially supported by the National Natural Science Foundation of China (nos. 21076104 and 21106065), National Science Foundation for Distinguished Young Scholars of China (no. 21225626), the National High Technology Research and Development Program of China (863 Plan) (no. 2011AA02A206), and the National Basic Research Program of China (973 Plan) (no. 2013CB733605).
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APPLIED MICROBIAL AND CELL PHYSIOLOGY
Alternative respiration and fumaric acid production of Rhizopus oryzae Shuai Gu & Qing Xu & He Huang & Shuang Li
Received: 7 November 2013 / Revised: 11 February 2014 / Accepted: 12 February 2014 / Published online: 19 March 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Under the conditions of fumaric acid fermentation, Rhizopus oryzae ME-F14 possessed at least two respiratory systems. The respiration of mycelia was partially inhibited by the cytochrome respiration inhibitor antimycin A or the alternative respiration inhibitor salicylhydroxamic acid and was completely inhibited in the presence of both antimycin A and salicylhydroxamic acid. During fumaric acid fermentation process, the activity of alternative respiration had a great correlation with fumaric acid productivity; both of them reached peak at the same time. The alternative oxidase gene, which encoded the mitochondrial alternative oxidase responsible for alternative respiration in R. oryzae ME-F14, was cloned and characterized in Escherichia coli. The activity of alternative respiration, the alternative oxidase gene transcription level, as well as the fumaric acid titer were measured under different carbon sources and different carbon-nitrogen ratios. The activity of alternative respiration was found to be comparable to the transcription level of the alternative oxidase gene and the fumaric acid titer. These results indicated that the activity of the alternative oxidase was regulated at the transcription stage under the conditions tested for R. oryzae ME-F14. Keywords Alternative respiration . SHAM-sensitive respiration . Fumaric acid . Rhizopus oryzae . Alternative oxidase
S. Gu : Q. Xu : H. Huang (*) : S. Li (*) State Key Laboratory of Material-oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, No. 5 Xinmofan Road, Nanjing 210009, People’s Republic of China e-mail: [email protected] e-mail: [email protected]
Introduction Fumaric acid is a four-carbon unsaturated dicarboxylic acid widely used in chemical, food, and pharmaceutical industries. The filamentous fungi Rhizopus oryzae is one of the bestknown producers of fumaric acid (Engel et al. 2008). To get an efficient fumaric acid production, many efforts have been done on the strain improvement, the morphology control, and the optimization of fermentation process (Xu et al. 2012). Fumaric acid is one of the primary metabolic products, whose accumulation is significantly affected by both the carbon metabolism and the energy metabolism. Thus, more attention should be focused on how to manipulate and balance the energy metabolism and carbon metabolism to achieve an efficient fumaric acid production. Alternative respiration pathway, characterized as sensitive to salicylhydroxamic acid (SHAM) and insensitive to conventional inhibitors of the cytochrome respiration pathway, has been widely found in plants, fungi, and yeast (Costa–de– Oliveira et al. 2012; Kirimura et al. 1999; Li et al. 2008). Alternative oxidase (AOX), a quinol oxidase localized in the inner mitochondrial membrane, is a terminal oxidase responsible for the activity of the alternative respiration (Moore et al. 2002). Alternative respiration pathway transfers electrons directly from the ubiquinone pool to oxygen, bypassing complex III and cytochrome c oxidase, two sites of energy conservation in the cell, thus producing fewer ATP than that via the cytochrome pathway (Vanlerberghe and McIntosh 1997). The alternative route is essential in every system, where the ATP production due to the regeneration of reduced coenzymes inhibits the overproduction of metabolites (Jeppsson et al. 1995; Kubicek et al. 1980). The alternative respiration pathway has been shown to be important for citric acid and cephalosporin C overproduction (Kirimura et al. 2000;
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Kozma and Karaffa 1996), and it plays an important role in oxidative stress in fungal fermentation processes (Costa–de–Oliveira et al. 2012). In the present study, we described, for the first time, the presence of an alternative respiration pathway in R. oryzae under the conditions of fumaric acid accumulation. The alternative oxidase gene (aox) was cloned and characterized; the relationship among the transcription level of aox and the activity of alternative respiration, as well as the fumaric acid accumulation, were examined in R. oryzae under several conditions. This work will help us understand the contribution of alternative respiration pathway to fumaric acid production in R. oryzae.
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Inhibitors at appropriate concentrations were added into the suspension, and 2 mL of the suspension was used for the measurement of respiration at 25 °C. The respiration rates were defined as oxygen consumption in micromoles O2 per minute per gram (dry weight) in the presence or absence of inhibitors. Cytochrome respiration was sensitive to antimycin A, and the alternative respiration was sensitive to SHAM (Costa–de–Oliveira et al. 2012). To distinguish between the cytochrome respiration and the alternative respiration, 0.25 mM antimycin A and 5 mM SHAM were added to the suspension, respectively. The ratio of the cytochrome and alternative respiration was determinate by the method of Bahr and Bonner (1973). The rate of inhibition by antimycin A alone was calculated as the cytochrome respiration, and that by SHAM alone was calculated as the alternative respiration.
Materials and methods Strain and growth condition R. oryzae ME-F14, a mutant of R. oryzae ME-F12 (Ding et al. 2011; Xu et al. 2010) with higher fumaric acid productivity, derived from R. oryzae ATCC 20344, was used in this study. R. oryzae ME-F14 was grown on potato-dextrose agar (PDA) slants at 35 °C for 7 days. The fungal spores in the slants were suspended in sterilized water and maintained at 4 °C (Fu et al. 2010). The spores (at a final concentration of 107 spores/mL) were grown for 30 h in 50 mL of seed culture medium containing 30 g/L glucose, 2 g/L urea, 0.6 g/L KH2PO4, 0.5 g/L MgSO4 · 7H2O, 0.11 g/L ZnSO4 · 7H2O, and 0.0088 g/L FeSO4 ·7H2O with pH 2.5, in 250-mL Erlenmeyer flasks at 35 °C and 200 rpm. The seed culture was then inoculated by 10 % (v/v) into the fermentation medium containing 80 g/L glucose, 0.1 g/L (for the study of carbon-nitrogen ratio, the urea concentrations used were 0.1, 0.4, and 2 g/L), and 50 g/L CaCO3, supplemented with other ingredients same to those of the seed medium. All the media were autoclaved at 115 °C for 30 min. Quantitative determination of fumaric acid Fumaric acid and biomass were determined as described by Xu et al. (2010).
Cloning and characterization of R. oryzae alternative oxidase gene The plasmids pMD18T-simple and pET22b were purchased from Novagen and TaKaRa, respectively. Escherichia coli JM109 and E. coli BL21 (DE3) were used as cloning host and expression host, respectively. For the extraction of mRNA, mycelia at 24 h (at the fermentation stage) were used. Mycelia were collected on Whatman GF/A and washed with distilled water. According to the sequence of the hypothetical aox of R. oryzae (GenBank accession no. CH476747.1, NCBI), the hypothetical aox of R. oryzae ME-F14 was amplified by reverse transcription PCR (RT-PCR) using specific primers that included restriction sites Nde I: 5′-TGCCATATGCCGTACGACATGTACCGT GA-3′ (NdeI site underlined, primer 1) and Sac I: 5′AGCGAGCTCTTATGCTGTCGATGGGTGAG-3′ (SacI site underlined, primer 2). The RT-PCR product was inserted into a pMD18T-simple vector and sequenced by GENEWIZ Corporation (Suzhou, China). All manipulations with recombinant DNA in E. coli were carried out according to standard procedures and as specified by enzyme manufacturers. Restriction endonucleases, T4 DNA ligase, and buffer components (TaKaRa Bio) were used as recommended by the supplier.
Respiration activity analysis
Quantification of aox mRNAs using quantitative real-time reverse transcriptase PCR
The respiration of cells was measured by a Clark electrode connected to a polarization and measurement device (Hansatech, England), and the data were collected by computer. During the fermentation process, mycelia were harvested on a Whatman GF/A filter with suction and suspended directly in the incubation buffer (50 mL) containing 8 % (w/v) glucose in 10 mM potassium phosphate buffer (pH 7.0). The suspension was constantly stirred for enough dissolve oxygen.
The aox expression in R. oryzae was analyzed by quantitative real-time RT-PCR (qRT-PCR) using a pair of specific primers: primer 3: 5′-CGACTGCTTCCGGACACTTA-3′, primer 4: 5′-TTGTCTTCAGACATGCGACGT-3′, which were designed according to the sequence of the hypothetical aox of R. oryzae. Methodology of RNA isolation, cDNA synthesis, qRT-PCR, relative quantification of gene expression ,and statistical analysis were described by Mhadhbi et al. (2011).
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Results Presence of an alternative respiration pathway in R. oryzae During the fumaric acid fermentation, the oxygen consumption rate was determined using a Clark electrode at 24 h of the fermentation. As Fig. 1 showed, the oxygen consumption of R. oryzae was partially inhibited by antimycin A (33 %), suggesting that there was an antimycin A-insensitive respiration pathway in R. oryzae. Then, we measured the oxygen consumption in the presence of SHAM, and the oxygen consumption rate was also partially inhibited with the addition of SHAM alone (60 %). And only in the presence of both antimycin A and SHAM, the respiration could be almost completely inhibited (Fig. 1). The results indicated that there existed at least two respiratory systems in the mycelia of R. oryzae, one antimycin A-sensitive and the other SHAMsensitive. It has been reported that alternative respiration pathway is insensitive to cytochrome pathway inhibitors, such as antimycin A and cyanide, but it is specifically inhibited by SHAM (Vanlerberghe and McIntosh 1997). We therefore supposed that the SHAM-sensitive respiration is an alternative respiration pathway in R. oryzae.
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alternative respiration increased significantly during the first 24 h and then decreased sharply. As shown in Fig. 2b, during the first 24 h, the alternative respiration ratio increased from 15 to 60 %, and the fumaric acid productivity also increased; both the alternative respiration ratio (60 %) and fumaric acid productivity (1.1 g/L/h) reached the highest at 24 h. After 24 h, the fumaric acid productivity decreased, and the alternative respiration ratio also decreased. At last, the alternative respiration ratio maintained at 40 %, and the fumaric acid productivity maintained at 0.4–0.5 g/L/h. Addition of SHAM to repress the alternative respiration resulted in the decrease of fumaric acid accumulation (Table 1). When adding 1.0 mM SHAM in the fermentation, fumaric acid production and productivity decreased by 27 and 30 %, respectively. The results indicated that the alternative respiration pathway in R. oryzae was important for fumaric acid productivity. Molecular cloning and expression characteristics of the alternative oxidase gene of R. oryzae
To examine the relationship between fumaric acid productivity and the activity of the alternative respiration, the respiratory rates, the alternative respiration ratios, and the fumaric acid productivity during the fermentation process were determined. At the first 36 h, the total respiratory rate of mycelia decreased from 2.73 to 1.15 μmol O2/min/g and thereafter remained almost constant (Fig. 2a); however, the activity of
The RT-PCR amplification of the hypothetical aox of R. oryzae ME-F14 generated single bands of approximately 800 bp (Fig. 3). The sequence of the RT-PCR product showed 100 % homology with that of the hypothetical aox in R. oryzae (GenBank accession no. CH476747.1, NCBI). The molecular mass of the R. oryzae AOX was estimated to be 29.6 kDa with a theoretical isoelectric point of 6.04. To verify if the hypothetical protein was functional as an alternative oxidase, the hypothetical aox cDNA fragment was inserted into the expression vector pET22b. The recombinant plasmid pET22b-aox was transformed into E. coli BL21 (DE3) strain. SDS-PAGE analysis demonstrated an expressed 29.6-kDa AOX protein in E. coli BL21 (DE3)/pET22b-aox
Fig. 1 Effect of antimycin A and salicylhydroxamic acid (SHAM) on the mycelia respiration of R. oryzae ME-F14. Mycelia at 24 h (at the fermentation stage) were used. At the points indicated by arrows, antimycin A or SHAM was added to the reaction mixture at final concentration of 0.25 or
5 mM, respectively. The values shown in the figure were the respiratory rates expressed as oxygen consumption in micromoles O2 per minute per gram (dry weight). The rate of inhibition by antimycin A alone was 33.21 ±3.41 % (a) and that by SHAM alone was 60.08±2.82 % (b)
Alternative respiration pathway and productivity of fumaric acid
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Fig. 3 Agarose gel electrophoresis of RT-PCR-amplified aox DNA fragments. RT-PCR was carried out with sets of primers 1 and 2, as shown in “Materials and methods.” Lanes 1–4 were the products of RTPCR-amplified aox DNA fragments. Lane 5 was the molecular weight marker
Fig. 2 Time courses of the total respiratory rate (open rectangles), the alternative respiratory rate (open circles), the fumaric acid productivity (filled circles), and the alternative respiration ratio (filled rectangles) of R. oryzae. In b, the alternative respiration (percentage of total respiration) was shown as the rate of inhibition by salicylhydroxamic acid (SHAM) and calculated as the ratio of SHAM-sensitive respiration in total respiration. The final concentration of SHAM in the reaction mixture was 5 mM. The productivity of fumaric acid (grams per liter per hour) is expressed as grams of fumaric acid produced per liter per hour. Data were presented as the mean values of triplicates (n=3)
inhibited oxygen consumption in the parent strain E. coli BL21 (DE3) (Fig. 5a1), whereas antimycin A only partially inhibited oxygen consumption in the recombinant strain (Fig. 5b1). Moreover, the addition of SHAM had no effect on oxygen consumption in the parent strain (Fig. 5a2), whereas SHAM could partially inhibit oxygen consumption in the recombinant strain (Fig. 5b2). The results clearly indicated that the introduction of the hypothetical aox resulted in the generation of an antimycin A-resistant and SHAM-sensitive respiration pathway in E. coli. Moreover, it was confirmed that the alternative respiration pathway in R. oryzae was catalyzed by AOX which was encoded by the hypothetical aox.
(Fig. 4), indicating that the hypothetical aox of R. oryzae was properly expressed in aox-deficient E. coli. The oxygen consumption of E. coli BL21 (DE3) and E. coli BL21 (DE3)/pET22b-aox was measured by a Clark electrode. As shown in Fig. 5, antimycin A almost completely
Table 1 Influence of SHAM on fumaric acid production and productivity SHAM (mM)
Fumaric acid (g/L)
Productivity (g/L/h)
Fermentation time (h)
0 0.5 1.0
40.1±1.8 34.1±2.1 29.3±1.7
0.56±0.025 0.47±0.029 0.39±0.022
72 72 75
SHAM was added in the fermentation medium at the final concentration of 0.5 and 1.0 mM,respectively. Fumaric acid was measured at the end of fermentation. The productivity of fumaric acid is expressed as grams of fumaric acid produced per liter per hour. Data were presented as the mean values of triplicates (n=3)
Fig. 4 SDS-PAGE analysis of alternative oxidase. The gene of R. oryzae hypothesis alternative oxidase was expressed in E. coli BL21(DE3). Lane 1: host E. coli BL21(DE3) (control), Lane 2 molecular weight marker, Lanes 3 and 4 recombinant E. coli BL21(DE3) carrying pET22b-aox induced by 1 mM IPTG
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Fig. 5 Effect of antimycin A and salicylhydroxamic acid (SHAM) on oxygen consumption of parent strain E. coli BL21(DE3) and recombinant strains grown on LB medium. At the points indicated by arrows, antimycin A or SHAM was added to the reaction mixture to final concentration of 0.25 or 5 mM, respectively. The values shown in the figure were the respiratory rates expressed in micromoles O2 per minute per gram (dry weight). a1 and a2: host BL21(DE3) (control), b1 and b2: recombinant BL21(DE3) carrying pET22b-aox induced by 1 mM IPTG
Effect of the carbon source on the alternative respiration pathway With xylose as a carbon source for fumaric acid production by R. oryzae ME-F14, the final fumaric acid titer was only about 5 g/L (data not shown), much lower than that in the glucose medium. The oxygen consumption of mycelia was measured. As shown in Fig. 6, the oxygen consumption was remarkably inhibited (almost 90 %) by the addition of antimycin A (Fig. 6b), while the addition of SHAM almost did not inhibit the oxygen consumption (Fig. 6a). This result indicated that the alternative respiration pathway almost did not exist in R. oryzae when using xylose as a carbon source; it also illustrated that the alternative respiration pathway played an important role in the accumulation of fumaric acid. Effect of the carbon-nitrogen ratio on the alternative respiration pathway Glucose (80 g/L) was used as a carbon source, and three different concentrations (0.1, 0.4, and 2 g/L) of urea were used as the nitrogen source for the fermentation. We measured oxygen consumption of mycelia, aox transcription level,
biomass, and fumaric acid titer under different carbonnitrogen ratios. As shown in Table 2, biomass increased with the increase of urea concentration; in reverse, aox transcription level, activity of alternative respiration, and fumaric acid titer sharply decreased. Moreover, both the activity of alternative respiration and the yield of fumaric acid production agreed well with the transcription levels of aox. As a result, a lower urea concentration was preferable for aox transcription and activity of alternative respiration. In addition, the activity of the alternative oxidase was regulated at the transcription stage under conditions tested for R. oryzae.
Discussion The presence of an alternative respiration pathway in R. oryzae Alternative respiration pathway occurs in all higher plants, many fungi, and some yeast (Magnani et al. 2007; Panda et al. 2013; Veiga et al. 2003). Several authors have stressed the relevance of the mitochondrial respiration and its influence upon metabolic behavior, stress environment adaptability, and
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Fig. 6 Effect of antimycin A and salicylhydroxamic acid (SHAM) on oxygen consumption of R. oryzae ME-F14 using xylose as a carbon source. Mycelia at 24 h (at the fermentation stage) were used. At the points indicated by arrows, antimycin A or SHAM was added to the reaction mixture to final concentration of 0.25 or 5 mM, respectively. The
values shown in the figure were the respiratory rates expressed in micromoles O2 per minute per gram (dry weight). The rate of inhibition by SHAM alone was 1.9±1.1 % (a) and that by antimycin A alone was 90.35±5.2 % (b)
morphological control (Costa–de–Oliveira et al. 2012; Karaffa et al. 1996; Kozma and Karaffa 1996). AOX encoded by aox is the key enzyme for the activity of alternative respiration. The whole-genome sequencing projects of R. oryzae were completed, and a hypothetical aox in the filamentous R. oryzae was first found by Ma et al. (2009). In this study, we described, for the first time, the presence of an alternative respiration pathway in R. oryzae and the relationship among the yield of fumaric acid, the activity of alternative respiration, and the transcription level of aox. We concluded that the alternative respiration pathway of R. oryzae was mediated by AOX encoded by the hypothetical aox (GenBank accession no. CH476747.1, NCBI).
fumaric acid, especially in the fumaric acid production using xylose as a carbon source. Many reports have showed that R. oryzae can grow well but produce fumaric acid poorly using xylose as a carbon source (Kautola and Linko 1989; Xu et al. 2010); our research, for the first time, demonstrated that the deficiency of alternative respiration pathway had a relation to the failure in fumaric acid accumulation. It is reported that with a high carbohydrate concentration, the cytochrome respiratory system generates high levels of ATP, which are enough to inhibit the glycolytic pathway and the production of metabolites (Kozma et al. 1991). However, the alternative respiration pathway transfers electrons directly from the ubiquinone pool to oxygen, without the generation of ATP. So the existence of an alternative respiratory system overcomes the above problem; the cells are able to degrade glucose (Lambers 1982) and probably are able to produce metabolites (Kozma et al. 1991). The definite relationship between alternative respiration pathway and productivity of fumaric acid showed the importance of energy metabolic regulation for fumaric acid accumulation in R. oryzae.
The alternative respiration pathway stimulates the productivity of fumaric acid In our study, we showed that the activity of the alternative respiration has a great relation to the productivity of fumaric acid during the fermentation process; moreover, the lack of alternative respiration activity inhibited the production of
Table 2 Effect of carbon-nitrogen ratio on aox transcription level, activity of alternative respiration, and fumaric acid titer Carbon source
Concentration of urea (g/L)
aox transcription levels
Alternative respiration (%total respiration)
Biomass (g/L)
Fumaric acid (g/L)
Fermentation time (h)
Glucose (80 g/L)
2 0.4 0.1
1.0 2.5 3.72
17.71±3.51 34.24±5.13 60.08±2.82
12.91±1.1 5.23±0.8 4.26±0.9
13.8±1.3 22.4±1.8 40.5±1.5
36 48 72
Mycelia at 24 h (at the fermentation stage) were used for the measurement of the aox transcription level and the activity of alternative respiration. The fumaric acid and biomass were measured at the end of fermentation. To calculate the relative transcription level, the signal intensity of aox was normalized using the signal intensity of 18 s, and the relative transcription level of aox on the concentration of urea of 2.0 g/L was represented as 1.0. The calculation of the alternative respiration was the same as in Fig. 2 Data with sign “±” are the average of triplicates with standard deviation (n=3)
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Nitrogen starve stress stimulates the alternative respiration of R. oryzae Studies with different species have demonstrated that aox is induced by a variety of treatments usually labeled as stresses, such as oxidative stress and cold (Vanlerberghe and McIntosh 1997). Additionally, stress appears to be a common requirement for the unusually high accumulation of organic acids by all producing organisms. The most important factor is nitrogen limitation (Moresi et al. 1991); fungi starved of nitrogen produce mainly fumaric acid and/or L-malic acid, but not biomass, from glucose. In fermentations, only after full depletion of the limiting nitrogen source do the acids be accumulated by secretion into the medium (Goldberg et al. 2006). In our previous study, we found that nitrogen source limitation can enhance the activity of cytosolic fumarase, and cytosolic fumarase played an important role in the high accumulation of fumaric acid (Ding et al. 2011). In this study, we found that the aox transcription level, the activity of SHAMsensitive respiration, and the yield of fumaric acid reached the highest under lower urea concentration; as a result, nitrogen starve stress stimulated the activity of alternative respiration, thus leading to the accumulation of fumaric acid. Regulation of alternative oxidase in R. oryzae In Aspergillus niger, alternative respiration pathway is constitutive (Hattori et al. 2009). In contrast, in Neurospora crassa, the alternative respiratory system is induced when the cytochrome chain functions are insufficient due to mutation or inhibition of mitochondrial protein synthesis (Li et al. 1996). In A. niger WU-2223 L, the activity of alternative oxidase under the conditions of citric acid production is regulated at the transcription stage (Hattori et al. 2009). In this study, the aox transcription and the activity of the alternative respiration in R. oryzae were found to be induced by nitrogen starvation stress, and the alternative respiration could not be induced by xylose. In the xylose culture, neither aox transcription (data not shown) nor alternative respiration could be detected. Moreover, we found that the changes in the activity of alternative respiration and the fumaric acid production titer agreed well with that in the aox transcription level. It means that activity of alternative oxidase in R. oryzae was regulated at the transcription stage. Acknowledgments This work was financially supported by the National Natural Science Foundation of China (nos. 21076104 and 21106065), National Science Foundation for Distinguished Young Scholars of China (no. 21225626), the National High Technology Research and Development Program of China (863 Plan) (no. 2011AA02A206), and the National Basic Research Program of China (973 Plan) (no. 2013CB733605).
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