Acetoin is widely used as flavor agent and serves as a precursor for chemical synthesis. Here we focused on identifying the best physiological conditions (initial substrate concentrations, pH, temperature, and agitation) for enhanced acetoin accumulation by Bacillus subtilis 168. The optimal physiological conditions support maximum acetoin accumulation by minimizing byproduct (acetate and butanediol) synthesis and a maximum of 75% enhancement in acetoin yield could be achieved. Additionally, the effect of change in ALS (acetolactate synthase) and ALDC (acetolactate decarboxylase) activities was evaluated on acetoin accumulation. Increasing ALS and ALDC enzyme activities led to efficient utilization of pyruvate towards acetoin accumulation and about 80% enhancement in acetoin accumulation was observed. 1. Introduction Acetoin is a four-carbon hydroxy-keto compound that is secreted by various microorganisms when grown on glycolytic substrates. It is widely used in the food and dairy industry as a preservative [1]. Acetoin has two biological analogues, diacetyl and 2,3-butanediol. The ratio of these metabolites present depends on the culture redox potential [2]. Acetoin is a secondary carbon source synthesized during sporulation in various organisms such as Bacillus subtilis [3, 4], Klebsiella terrigena [5], and Pelobacter carbinolicus [6]. In Bacillus subtilis, acetoin synthesis involves alsSD operon that consists of alsS and alsD genes encoding for acetolactate synthase (ALS; E.C. 2.2.1.6) and acetolactate decarboxylase (ALDC; E.C. 4.1.1.5), respectively [3]. However, in Klebsiella and Pelobacter sp., a butanediol dehydrogenase (bdh) is integrated with alsSD operon that leads to production of 2,3-butanediol as major product instead of acetoin. Therefore, Bacillus subtilis is used as a model organism for study of acetoin synthesis. A number of factors can affect the fate of metabolites by diverse alternative metabolic pathways [7]. Among them, initial substrate concentration [8, 9], temperature [10], pH [11], and oxygen levels [12] are known to affect secondary metabolites production in Klebsiella and Pelobacter sp. To our knowledge, no report exists on the assessment of these physiological factors and activities of acetoin synthesis enzymes (ALS and ALDC) for acetoin accumulation by B. subtilis. Our current interest focuses on identification of important variables to enhance acetoin accumulation and minimizing byproduct formation. For this purpose, we focused on two effects. First, we focus on the systematic evaluation of physiological factors (initial
References
[1]
Z. Xiao and P. Xu, “Acetoin metabolism in bacteria,” Critical Reviews in Microbiology, vol. 33, no. 2, pp. 127–140, 2007.
[2]
E. B. Collins, “Biosynthesis of flavor compounds by microorganisms,” Journal of Dairy Science, vol. 55, pp. 1022–1028, 1972.
[3]
M. C. Renna, N. Najimudin, L. R. Winik, and S. A. Zahler, “Regulation of the Bacillus subtilis alsS, alsD, and alsR genes involved in post-exponential-phase production of acetoin,” Journal of Bacteriology, vol. 175, no. 12, pp. 3863–3875, 1993.
[4]
S. Atsumi, Z. Li, and J. C. Liao, “Acetolactate synthase from Bacillus subtilis serves as a 2-ketoisovalerate decarboxylase for isobutanol biosynthesis in Escherichia coli,” Applied and Environmental Microbiology, vol. 75, no. 19, pp. 6306–6311, 2009.
[5]
D. Mayer, V. Schlensog, and A. Bock, “Identification of the transcriptional activator controlling the butanediol fermentation pathway in Klebsiella terrigena,” Journal of Bacteriology, vol. 177, no. 18, pp. 5261–5269, 1995.
[6]
F. Bernd Oppermann, A. Steinbüchel, and H. G. Schlegel, “Utilization of methylacetoin by the strict anaerobe Pelobacter carbinolicus and consequences for the catabolism of acetoin,” FEMS Microbiology Letters, vol. 55, no. 1, pp. 47–52, 1988.
[7]
S. Klumpp, Z. Zhang, and T. Hwa, “Growth rate-dependent global effects on gene expression in bacteria,” Cell, vol. 139, no. 7, pp. 1366–1375, 2009.
[8]
T. K. Kirk, E. Schultz, and W. J. Connors, “Influence of culture parameters on lignin metabolism by Phanerochaete chrysosporium,” Archives of Microbiology, vol. 117, no. 3, pp. 277–285, 1978.
[9]
K. V. Ramana, A. Tomar, and L. Singh, “Effect of various carbon and nitrogen sources on cellulose synthesis by Acetobacter xylinum,” World Journal of Microbiology and Biotechnology, vol. 16, no. 3, pp. 245–248, 2000.
[10]
P. Perego, A. Converti, and M. Del Borghi, “Effects of temperature, inoculum size and starch hydrolyzate concentration on butanediol production by Bacillus licheniformis,” Bioresource Technology, vol. 89, no. 2, pp. 125–131, 2003.
[11]
G. M. Vignolo, M. N. Kairuz, A. A. P. Ruiz Holgado, and G. Oliver, “Influence of growth conditions on the production of lactocin 705, a bacteriocin produced by Lactobacillus casei CRL 705,” Journal of Applied Bacteriology, vol. 78, no. 1, pp. 5–10, 1995.
[12]
M. Wang, J. Fu, X. Zhang, and T. Chen, “Metabolic engineering of Bacillus subtilis for enhanced production of acetoin,” Biotechnology Letters, vol. 34, pp. 1877–1885, 2012.
[13]
P. Sharma and S. Noronha, “Systematic study of acuABC operon in Bacillus subtilis 168,” in International Proceedings of Chemical, Biological & Environmenta (IPCBEE '12), vol. 31, pp. 17–21.
[14]
H. C. Ramos, T. Hoffmann, M. Marino et al., “Fermentative metabolism of Bacillus subtilis: physiology and regulation of gene expression,” Journal of Bacteriology, vol. 182, no. 11, pp. 3072–3080, 2000.
[15]
W. W. Westerfeld, “A colorimetric determination of blood acetoin,” The Journal of Biological Chemistry, vol. 161, pp. 495–502, 1945.
[16]
H.-M. Blencke, G. Homuth, H. Ludwig, U. M？der, M. Hecker, and J. Stülke, “Transcriptional profiling of gene expression in response to glucose in Bacillus subtilis: regulation of the central metabolic pathways,” Metabolic Engineering, vol. 5, no. 2, pp. 133–149, 2003.
[17]
M. Huang, F. B. Oppermann-Sanio, and A. Steinbüchel, “Biochemical and molecular characterization of the Bacillus subtilis acetoin catabolic pathway,” Journal of Bacteriology, vol. 181, no. 12, pp. 3837–3841, 1999.
[18]
P. D. A. James, C. Edwards, and M. Dawson, “The effects of temperature, pH and growth rate on secondary metabolism in Streptomyces thermoviolaceus grown in a chemostat,” Journal of General Microbiology, vol. 137, no. 7, pp. 1715–1720, 1991.
[19]
T. Hornb？k, M. Jakobsen, J. Dynesen, and A. K. Nielsen, “Global transcription profiles and intracellular pH regulation measured in Bacillus licheniformis upon external pH upshifts,” Archives of Microbiology, vol. 182, no. 6, pp. 467–474, 2004.
[20]
J. C. Wilks, R. D. Kitko, S. H. Cleeton et al., “Acid and base stress and transcriptomic responses in Bacillus subtilis,” Applied and Environmental Microbiology, vol. 75, no. 4, pp. 981–990, 2009.
[21]
W.-B. Yu, S.-H. Gao, C.-Y. Yin, Y. Zhou, and B.-C. Ye, “Comparative transcriptome analysis of Bacillus subtilis responding to dissolved oxygen in adenosine fermentation,” PLoS ONE, vol. 6, no. 5, Article ID e20092, 2011.
[22]
H. D. Nguyen, Q. A. Nguyen, R. C. Ferreira, L. C. S. Ferreira, L. T. Tran, and W. Schumann, “Construction of plasmid-based expression vectors for Bacillus subtilis exhibiting full structural stability,” Plasmid, vol. 54, no. 3, pp. 241–248, 2005.
[23]
T. Kaneko, M. Takahashi, and H. Suzuki, “Acetoin fermentation by citrate-positive Lactococcus lactis subsp. lactis 3022 grown aerobically in the presence of hemin or Cu2+,” Applied and Environmental Microbiology, vol. 56, no. 9, pp. 2644–2649, 1990.
