Evolutionary engineering of Phaffia rhodozyma for astaxanthin-overproducing strain

Abstract

Evolutionary engineering is a novel whole-genome wide engineering strategy inspired by natural evolution for strain improvement. Astaxanthin has been widely used in cosmetics, pharmaceutical and health care food due to its capability of quenching active oxygen. Strain improvement of Phaffia rhodozyma, one of the main sources for natural astaxanthin, is of commercial interest for astaxanthin production. In this study a selection procedure was developed for adaptive evolution of P. rhodozyma strains under endogenetic selective pressure induced by additive in environmental niches. Six agents, which can induce active oxygen in cells, were added to the culture medium respectively to produce selective pressure in process of evolution. The initial strain, P. rhodozyma AS2-1557, was mutagenized to acquire the initial strain population, which was then cultivated for 550 h at selective pressure and the culture was transferred every 48h. Finally, six evolved strains were selected after 150 generations of evolution. The evolved strains produced up to 48.2% more astaxanthin than the initial strain. Our procedure may provide a promising alternative for improvement of high-production strain.

Keywords

evolutionary engineering / astaxanthin / strain improvement

References

[1] Selifonova O, Valle F, Schellenberger V. Rapid evolution of novel traits in microorganisms. Applied and Environmental Microbiology, 2001, 67(8): 3645-3649

[2] Sonderegger M, Sauer U. Evolutionary engineering of Saccharomyces cerevisiae for anaerobic growth on xylose. Applied and Environmental Microbiology, 2003, 69(4): 1990-1998

[3] Steiner P, Sauer U. Long-term continuous evolution of acetate resistant Acetobacter aceti. Biotechnology and Bioengineering, 2003, 84(1): 40-44

[4] van Maris A J A, Geertman J M A, Vermeulen A, Groothuizen M K, Winkler A A, Piper M D W, van Dijken J P, Pronk J T. Directed evolution of pyruvate decarboxylase-negative Saccharomyces cerevisiae, yielding a C-2-independent, glucose-tolerant, and pyruvate-hyperproducing yeast. Applied and Environmental Microbiology, 2004, 70(1): 159-166

[5] Cakar Z P, Seker U O S, Tamerler C, Sonderegger M, Sauer U. Evolutionary engineering of multiple-stress resistant Saccharomyces cerevisiae. FEMS Yeast Research, 2005, 5(6-7): 569-578

[6] Kuyper M, Toirkens M, Diderich J, Winkler A, Vandijken J, Pronk J. Evolutionary engineering of mixed-sugar utilization by a xylose-fermenting Saccharomyces cerevisiae strain. FEMS Yeast Research, 2005, 5: 925-934

[7] Wisselink H W, Toirkens M J, del Rosario Franco Berriel M, Winkler A A, van Dijken J P, Pronk J T, van Maris A J A. Engineering of Saccharomyces cerevisiae for efficient anaerobic alcoholic fermentation of L-arabinose. Applied and Environmental Microbiology, 2007, 73(15): 4881-4891

[8] Guimaraes P M R, Francois J, Parrou J L, Teixeira J A, Domingues L. Adaptive evolution of a lactose-consuming Saccharomyces cerevisiae recombinant. Applied and Environmental Microbiology, 2008, 74(6): 1748-1756

[9] Jantama K, Haupt M J, Svoronos S A, Zhang X, Moore J C, Shanmugam K T, Ingram L O. Combining metabolic engineering and metabolic evolution to develop nonrecombinant strains of Escherichia coli C that produce succinate and malate. Biotechnology and Bioengineering, 2008, 99(5): 1140-1153

[10] Meijnen J P, de Winde J H, Ruijssenaars H J. Engineering pseudomonas putida S12 for efficient utilization of D-xylose and L-arabinose. Applied and Environmental Microbiology, 2008, 74(16): 5031-5037

[11] Cakar Z P, Alkım C, Turanlı B, Tokman N, Akman S, Sarıkaya M, Tamerler C, Benbadis L, François J M. Isolation of cobalt hyper-resistant mutants of Saccharomyces cerevisiae by in vivo evolutionary engineering approach. Journal of Biotechnology, 2009, 143(2): 130-138

[12] Gilbert A, Sangurdekar D P, Srienc F. Rapid strain improvement through optimized evolution in the cytostat. Biotechnology and Bioengineering, 2009, 103(3): 500-512

[13] Wisselink H W, Toirkens M J, Wu Q, Pronk J T, Maris A JA. Novel evolutionary engineering approach for accelerated utilization of glucose, xylose, and arabinose mixtures by engineered Saccharomyces cerevisiae strains. Applied and Environmental Microbiology, 2009, 75(4): 907-914

RIGHTS & PERMISSIONS

Higher Education Press and Springer-Verlag Berlin Heidelberg