期刊
ENZYME AND MICROBIAL TECHNOLOGY
卷 100, 期 -, 页码 28-36出版社
ELSEVIER SCIENCE INC
DOI: 10.1016/j.enzmictec.2017.02.006
关键词
Astaxanthin; Saccharomyces cerevisiae; Metabolic engineering; Directed evolution; beta-carotene ketolase
资金
- Natural Science Foundation of China [21406196, 21576234]
- Qianjiang Talents Project
- National High Technology Research and Development Program (863 Program) of China [SS2015AA020601]
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 beta-carotene as a key precursor for astaxanthin, a positive mutant of GGPP synthase (CrtE03M) was overexpressed together with three other rate-limiting enzymes tHMG1, Crtl and CrtYB. Subsequently, to accelerate the conversion of beta-carotene to astaxanthin, a color screening system was developed and adopted for directed evolution of beta-carotene ketolase (OBKT), generating a triple mutant OBIKTM (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 rhating two astaxanthin-producing haploid strains carrying the same carotenogenic pathway. Finally, further overexpression of OCrtZ and OBKTM in YastD-01 resulted in accumulation of 8.10 mg/g DCW (47.18 mg/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. (C) 2017 Elsevier Inc. All rights reserved.
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