title: Alleviation of metabolic bottleneck by combinatorial engineering enhanced astaxanthin synthesis in Saccharomyces cerevisiae.
abstract: 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.
authors.affiliation: Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, PR China.
authors.forename: Pingping
authors.initials: P
authors.lastname: Zhou
authors.affiliation: Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, PR China.
authors.forename: Wenping
authors.initials: W
authors.lastname: Xie
authors.affiliation: Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, PR China.
authors.forename: Aipeng
authors.initials: A
authors.lastname: Li
authors.affiliation: Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, PR China.
authors.forename: Fan
authors.initials: F
authors.lastname: Wang
authors.affiliation: Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, PR China.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Yao
authors.affiliation: Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, PR China.
authors.forename: Qi
authors.initials: Q
authors.lastname: Bian
authors.affiliation: Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, PR China.
authors.forename: Yongqiang
authors.initials: Y
authors.lastname: Zhu
authors.affiliation: Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, PR China.
authors.forename: Hongwei
authors.initials: H
authors.lastname: Yu
authors.affiliation: Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, PR China; Key Laboratory of Biomass Chemical Engineering of Ministry of Education, Zhejiang University, Hangzhou 310027, PR China. Electronic address: yelidan@zju.edu.cn.
authors.forename: Lidan
authors.initials: L
authors.lastname: Ye
mesh_terms: D018533:Biomass; D053898:Biosynthetic Pathways; D004171:Diploidy; D019020:Directed Molecular Evolution; D051231:Farnesyltranstransferase / Q000235:genetics / Q000378:metabolism; D005285:Fermentation; D007218:Industrial Microbiology; D060847:Metabolic Engineering / Q000379:methods*; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D010105:Oxygenases / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D016823:Volvocida / Q000201:enzymology / Q000235:genetics; D024341:Xanthophylls / Q000096:biosynthesis; D019207:beta Carotene / Q000378:metabolism
chemical_list: D050505:Mutant Proteins; D011994:Recombinant Proteins; D024341:Xanthophylls; D019207:beta Carotene; C005948:astaxanthine; D010105:Oxygenases; C093259:beta-carotene ketolase; D051231:Farnesyltranstransferase
keywords: Astaxanthin; Directed evolution; Metabolic engineering; Saccharomyces cerevisiae; β-carotene ketolase