Institute of Bioengineering, College of Chemical and Biological Engineering , Zhejiang University , Hangzhou 310027 , P.R. China.
Joint International Research Laboratory of Agriculture and Agri-Product Safety/Key Laboratory of Prevention and Control of Biological Hazard Factors (Animal Origin) for Agrifood Safety and Quality, The Ministry of Education of China , Yangzhou University , Yangzhou 225009 , P.R. China.
J Agric Food Chem. 2019 Jan 30;67(4):1072-1080. doi: 10.1021/acs.jafc.8b05003. Epub 2019 Jan 16.
Because it is an outstanding antioxidant with wide applications, biotechnological production of astaxanthin has attracted increasing research interest. However, the astaxanthin titer achieved to date is still rather low, attributed to the poor efficiency of β-carotene ketolation and hydroxylation, as well as the adverse effect of astaxanthin accumulation on cell growth. To address these problems, we constructed an efficient astaxanthin-producing Saccharomyces cerevisiae strain by combining protein engineering and dynamic metabolic regulation. First, superior mutants of β-carotene ketolase and β-carotene hydroxylase were obtained by directed coevolution to accelerate the conversion of β-carotene to astaxanthin. Subsequently, the Gal4M9-based temperature-responsive regulation system was introduced to separate astaxanthin production from cell growth. Finally, 235 mg/L of (3 S,3' S)-astaxanthin was produced by two-stage, high-density fermentation. This study demonstrates the power of combining directed coevolution and temperature-responsive regulation in astaxanthin biosynthesis and may provide methodological reference for biotechnological production of other value-added chemicals.
由于其具有广泛应用的出色抗氧化性能，生物技术生产虾青素引起了越来越多的研究兴趣。然而，迄今为止实现的虾青素产量仍然相当低，这归因于β-胡萝卜素酮化和羟化效率低下，以及虾青素积累对细胞生长的不利影响。为了解决这些问题，我们通过结合蛋白质工程和动态代谢调控，构建了一种高效的产虾青素酿酒酵母菌株。首先，通过定向共进化获得了优良的β-胡萝卜素酮化酶和β-胡萝卜素羟化酶突变体，以加速β-胡萝卜素向虾青素的转化。随后，引入基于 Gal4M9 的温度响应调控系统将虾青素的生产与细胞生长分离。最后，通过两段式高密度发酵生产出了 235mg/L 的（3S,3'S）-虾青素。本研究证明了定向共进化和温度响应调控在虾青素生物合成中的结合的强大功能，可能为其他有价值化学品的生物技术生产提供方法学参考。
