Alleviation of metabolic bottleneck by combinatorial engineering enhanced astaxanthin synthesis in Saccharomyces cerevisiae
Alleviation of metabolic bottleneck by combinatorial engineering enhanced astaxanthin synthesis in Saccharomyces cerevisiae
复制标题
通过组合工程增强酿酒酵母中虾青素合成缓解代谢瓶颈
DOI：
10.1016/j.enzmictec.2017.02.006
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发表时间：
2017-05-01
影响因子：
3.4
通讯作者：
Ye, Lidan
中科院分区：
文献类型：
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作者：
Zhou, Pingping;Xie, Wenping;Ye, Lidan
利用微生物细胞高效合成具有商业价值的类胡萝卜素虾青素是化学合成和微藻提取的一种有吸引力的替代方法。为了提高酿酒酵母中异源虾青素的产量，将代谢工程和蛋白质工程相结合，提高限速酶的表达和活性。首先，为了增加作为虾青素的关键前体的β-胡萝卜素的供应，GGPP合酶的阳性突变体（CrtE 03 M）与其他三种限速酶tHMG 1、Crt 1和CrtYB一起过表达。随后，为了加速β-胡萝卜素向虾青素的转化，开发了颜色筛选系统并用于β-胡萝卜素酮醇酶（OBKT）的定向进化，产生了具有2.4倍提高的活性的三重突变体OBIKTM（H165 R/V264 D/F298 Y）。通过调节上述限速酶的拷贝数以进一步平衡代谢流，将两株具有相同胡萝卜素生成途径的产虾青素单倍体菌株进行杂交，获得二倍体菌株YastD-01。最后，在YastD-01中OCrtZ和OBKTM的进一步过表达导致在摇瓶培养物中积累8.10 mg/g DCW（47.18 mg/l）的（3S，3 ′ S）-虾青素。这种组合策略也可用于缓解其他增值产品，特别是有色代谢产物的生物合成中的代谢瓶颈。(C)2017爱思唯尔公司All rights reserved.
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.