Directed evolution of the fusion enzyme for improving astaxanthin biosynthesis in Saccharomyces cerevisiae

虾青素 角黄素 生物合成 生物化学 玉米黄质 酿酒酵母 化学 突变体 类胡萝卜素 叶黄素 基因
作者
Yong-Wen Ding,Chuanzhen Lü,yan zheng,Han-Zhang Ma,Jin Jin,Bin Jia,Ying‐Jin Yuan
出处
期刊:Synthetic and Systems Biotechnology [Elsevier]
卷期号:8 (1): 46-53 被引量:21
标识
DOI:10.1016/j.synbio.2022.10.005
摘要

The accumulation of the intermediate zeaxanthin and canthaxanthin in the astaxanthin biosynthesis pathway catalyzed by β-carotene hydroxylase (crtZ) and β-carotene ketolase (crtW) decreases the content of the astaxanthin. Here, we exploited directed evolution of the fusion of crtZ and crtW for improving astaxanthin biosynthesis in Saccharomyces cerevisiae. The results demonstrated that the fusion enzyme of crtZ-crtW with 2 X GGGGS peptides linker can effectively reduce the accumulation of intermediates and improves the content of astaxanthin. Compared with the control strain, the fusion enzyme of ketase and hydroxylase reduced zeaxanthin and canthaxanthin by 7 and 14 times and increased astaxanthin by 1.6 times, respectively. Moreover, 9 variant fusion mutants with improved astaxanthin production were generated through directed evolution. Combining these dominant mutants generated a variant, L95S + I206L, which increased the astaxanthin content of 3.8 times than the control strain. The AlphaFold2 assisted structural analysis indicated that these two mutations alter the interaction between the substrate and the enzymes pocket. Our research provided an efficient idea to reduce the accumulation of the intermediate products in complex biosynthesis pathway.
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