Abstract
Poly(β-L-malic acid) (PMA) is a promising polyester formed from L-malate in microbial cells. Malate biosynthesis is crucial for PMA production. Previous studies have shown that the non-oxidative pathway or oxidative pathway (TCA cycle) is the main biosynthetic pathway of malate in most of PMA-producing strains, while the glyoxylate cycle is only a supplementary pathway. In this study, we investigated the effect of exogenous metabolic intermediates and inhibitors of the malate biosynthetic pathway on PMA production by Aureobasidium melanogenum GXZ-6. The results showed that PMA production was stimulated by maleic acid (a fumarase inhibitor) and sodium malonate (a succinate dehydrogenase inhibitor) but inhibited by succinic acid and fumaric acid. This indicated that the TCA cycle might not be the only biosynthetic pathway of malate. In addition, the PMA titer increased by 18.1% upon the addition of glyoxylic acid after 72 h of fermentation, but the PMA titer decreased by 7.5% when itaconic acid (an isocitrate lyase inhibitor) was added, which indicated that malate for PMA production was synthesized significantly via the glyoxylate cycle rather than the TCA cycle. Furthermore, in vitro enzyme activities of the TCA and glyoxylate cycles suggested that the glyoxylate cycle significantly contributed to the PMA production, which is contradictory to what has been reported previously in other PMA-producing A. pullulans.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
References
Bott, M. and Eikmanns, B.J. 2013. TCA cycle and glyoxylate shunt of Corynebacterium glutamicum, pp. 281–313. In Hideaki, Y. and Masayuki, I. (eds.), Corynebacterium glutamicum, Springer, Berlin, Germany.
Cao, W., Luo, J., Qi, B., Zhao, J., Qiao, C., Ding, L., Su, Y., and Wan, Y. 2014. β-poly(L-malic acid) production by fed-batch culture of Aureobasidium pullulans ipe-1 with mixed sugars. Eng. Life Sci. 14, 180–189.
Chi, Z., Liu, G.L., Liu, C.G., and Chi, Z.M. 2016a. Poly(β-L-malic acid) (PMLA) from Aureobasidium spp. and its current proceedings. Appl. Microbiol. Biotechnol. 100, 3841–3851.
Chi, Z., Wang, Z.P., Wang, G.Y., Khan, I., and Chi, Z.M. 2016b. Microbial biosynthesis and secretion of L-malic acid and its applications. Crit. Rev. Biotechnol. 36, 99–107.
Chinnici, F., Spinabelli, U., Riponi, C., and Amati, A. 2005. Optimization of the determination of organic acids and sugars in fruit juices by ion-exclusion liquid chromatography. J. Food Compos. Anal. 18, 121–130.
Ding, H., Helguera, G., Rodriguez, J.A., Markman, J., Luria-Perez, R., Gangalum, P., Portilla-Arias, J., Inoue, S., Daniels-Wells, T.R., Black, K., et al. 2013. Polymalic acid nanobioconjugate for simultaneous immunostimulation and inhibition of tumor growth in HER2/neu-positive breast cancer. J. Control. Release 171, 322–329.
Fischer, H., Erdmann, S., and Holler, E. 1989. An unusual polyanion from Physarum polycephalum that inhibits homologous DNApolymerasea in vitro. Biochemistry 28, 5219–5226.
Garraway, M.O. and Evans, R.C. 1984. Fungal nutrition and physiology, pp. 298–312. Wiley, New York, USA.
Giachetti, E., Pinzauti, G., Bonaccorsi, R., and Vanni, P. 1988. Isocitrate lyase from Pinus pinea. Characterization of its true substrate and the action of magnesium ions. Eur. J. Biochem. 172, 85–91.
Holler, E., Angerer, B., Achhammer, G., Miller, S., and Windisch, C. 1992. Biological and biosynthetic properties of poly-L-malate. FEMS Microbiol. Rev. 103, 109–118.
Jong-Gubbels, P.D., Vanrolleghem, P., Heijnen, S., Dijken, J.P.V., and Pronk, J.T. 1995. Regulation of carbon metabolism in chemostat cultures of Saccharomyces cerevisiae grown on mixtures of glucose and ethanol. Yeast 11, 407–418.
Lee, B.S. and Holler, E. 2000. β-poly(L-malate) production by nongrowing microplasmodia of Physarum polycephalum. Effects of metabolic intermediates and inhibitors. FEMS Microbiol. Lett. 193, 69–74.
Liu, S.J. and Steinbüchel, A. 1997. Production of poly(malic acid) from different carbon sources and its regulation in Aureobasidium pullulans. Biotechnol. Lett. 19, 11–14.
Livak, K.J. and Schmittgen, T.D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods 25, 402–408.
Massey, V. 1953. Studies on fumarase. II. The effects of inorganic anions on fumarase activity. Biochem. J. 53, 67.
Nagata, N., Nakahara, T., and Tabuchi, T. 1993. Fermentative production of poly(β-L-malic acid), a polyelectrolytic biopolyester, by Aureobasidium sp. Biosci. Biotech. Biochem. 57, 638–642.
Rikhvanov, E.G., Varakina, N.N., Rusaleva, T.M., Rachenko, E.I., and Voinikov, V.K. 2003. The effect of sodium malonate on yeast thermotolerance. Microbiology 72, 548–552.
Schmidt, A., Windisch, C., and Holler, E. 1996. Nuclear accumulation and homeostasis of the unusual polymer β-poly(L-malate) in plasmodia of Physarum polycephalum. Eur. J. Cell Biol. 70, 373–380.
Shimada, K., Matsushima, K.I., Fukumoto, J., and Yamamoto, T. 1969. Poly-(L)-malic acid; A new protease inhibitor from Penicillium cyclopium. Biochem. Biophys. Res. Commun. 35, 619–624.
Wallace, J.C., Jitrapakdee, S., and Chapmansmith, A. 1998. Pyruvate carboxylase. Int. J. Biochem. Cell 30, 1–5.
Yang, E.S., Richter, C., Chun, J.S., Huh, T.L., Kang, S.S., and Park, J.W. 2002. Inactivation of NADP+-dependent isocitrate dehydrogenase by nitric oxide. Free Radic. Biol. Med. 33, 927–937.
Yang, X.T., Guo, Y.M., Zeng, X.F., Man, Y., Zheng, P., Wang, C.L., and Sun, J.B. 2011. Characterization of the hyperproduction process of citric acid by Aspergillus niger and the formation of glyoxylic acid. Mod. Food Sci. Technol. 27, 1183–1186.
Zeng, W., Zhang, B., Chen, G.G., Li, M.X., and Liang, Z.Q. 2018. Efficient production of polymalic acid by a novel isolated Aureobasidium pullulans using metabolic intermediates and inhibitors. Appl. Biochem. Biotechnol. Doi:10.1007/s12010-018-2825-0.
Zou, X., Yang, J., Tian, X., Guo, M., Li, Z., and Li, Y. 2016. Production of polymalic acid and malic acid from xylose and corncob hydrolysate by a novel Aureobasidium pullulans YJ 6–11 strain. Process Biochem. 51, 16–23.
Author information
Authors and Affiliations
Corresponding author
Additional information
Supplemental material for this article may be found at https://doi.org/www.springerlink.com/content/120956.
Electronic supplementary material
Rights and permissions
About this article
Cite this article
Zeng, W., Zhang, B., Liu, Q. et al. Analysis of the L-malate biosynthesis pathway involved in poly(β-L-malic acid) production in Aureobasidium melanogenum GXZ-6 by addition of metabolic intermediates and inhibitors. J Microbiol. 57, 281–287 (2019). https://doi.org/10.1007/s12275-019-8424-0
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12275-019-8424-0