Alleviation of metabolic bottleneck by combinatorial engineering enhanced astaxanthin synthesis in Saccharomyces cerevisiae.
Highly efficient biosynthesis of the commercially valuable carotenoid astaxanthin by microbial cells is an attractive alternative to chemical synthesis and microalgae extraction. With the goal of enhancing heterologous astaxanthin production in Saccharomyces cerevisiae, metabolic engineering and protein engineering were integrated to improve both the expression and activity of rate-limiting enzymes. Firstly, to increase the supply of β-carotene as a key precursor for astaxanthin, a positive mutant of GGPP synthase (CrtE03M) was overexpressed together with three other rate-limiting enzymes tHMG1, CrtI and CrtYB. Subsequently, to accelerate the conversion of β-carotene to astaxanthin, a color screening system was developed and adopted for directed evolution of β-carotene ketolase (OBKT), generating a triple mutant OBKTM (H165R/V264D/F298Y) with 2.4-fold improved activity. After adjusting copy numbers of the above-mentioned rate-limiting enzymes to further balance the metabolic flux, a diploid strain YastD-01 was generated by mating two astaxanthin-producing haploid strains carrying the same carotenogenic pathway. Finally, further overexpression of OCrtZ and OBKTM in YastD-01 resulted in accumulation of 8.10mg/g DCW (47.18mg/l) of (3S, 3'S)-astaxanthin in shake-flask cultures. This combinatorial strategy might be also applicable for alleviation of metabolic bottleneck in biosynthesis of other value-added products, especially colored metabolites.
['Zhou|Pingping|P|', 'Xie|Wenping|W|', 'Li|Aipeng|A|', 'Wang|Fan|F|', 'Yao|Zhen|Z|', 'Bian|Qi|Q|', 'Zhu|Yongqiang|Y|', 'Yu|Hongwei|H|', 'Ye|Lidan|L|']
D018533:Biomass D053898:Biosynthetic Pathways D004171:Diploidy D019020:Directed Molecular Evolution D051231:Farnesyltranstransferase D005285:Fermentation D007218:Industrial Microbiology D060847:Metabolic Engineering D050505:Mutant Proteins D010105:Oxygenases D015202:Protein Engineering D011994:Recombinant Proteins D012441:Saccharomyces cerevisiae D016823:Volvocida D024341:Xanthophylls D019207:beta Carotene
Astaxanthin Saccharomyces cerevisiae Metabolic engineering Directed evolution β-carotene ketolase