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Systematic Optimization of HPO-CPR to Boost (+)-Nootkatone Synthesis in Engineered Saccharomyces cerevisiae
Journal of Agricultural and Food Chemistry ( IF 5.7 ) Pub Date : 2022-12-05 , DOI: 10.1021/acs.jafc.2c07068 Tong Liu 1 , Wen Li 1 , Hefeng Chen 1 , Tao Wu 1 , Chaoyi Zhu 1 , Min Zhuo 1 , Shuang Li 1
Journal of Agricultural and Food Chemistry ( IF 5.7 ) Pub Date : 2022-12-05 , DOI: 10.1021/acs.jafc.2c07068 Tong Liu 1 , Wen Li 1 , Hefeng Chen 1 , Tao Wu 1 , Chaoyi Zhu 1 , Min Zhuo 1 , Shuang Li 1
Affiliation
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As an important and expensive natural sesquiterpene compound in grapefruit, the interest in (+)-nootkatone is stimulated by its strong grapefruit-like odor and physiological activities, which induce efforts for its microbial production. However, the low catalytic efficiency of the cytochrome P450-P450 reductase (HPO-CPR) system is the main challenge. We developed a high-throughput screening (HTS) method using the principle of the color reaction between carbonyl compounds and 2,4-dinitrophenylhydrazine (DNPH), which could rapidly screen the activity of candidate HPO mutants. After optimizing the pairing of HPO and CPR and through semirational design, the optimal mutant HPO_M18 with catalytic performance 2.54 times that of the initial was obtained. An encouraging (+)-nootkatone titer of 2.39 g/L was achieved through two-stage fed-batch fermentation after metabolic engineering and endoplasmic reticulum engineering, representing the highest titer reported to date. Our findings lay the foundation for the development of an economically viable bioprocess for (+)-nootkatone.
中文翻译：
系统优化 HPO-CPR 以促进工程化酿酒酵母中 (+)-Nootkatone 的合成
作为葡萄柚中重要且昂贵的天然倍半萜化合物，(+)-nootkatone 因其强烈的葡萄柚样气味和生理活性而引起人们的兴趣，这促使其微生物生产。然而，细胞色素 P450-P450 还原酶 (HPO-CPR) 系统的低催化效率是主要挑战。我们利用羰基化合物与 2,4-二硝基苯肼 (DNPH) 显色反应的原理开发了一种高通量筛选 (HTS) 方法，可以快速筛选候选 HPO 突变体的活性。优化HPO与CPR配对后，通过半理性设计，得到催化性能是初始2.54倍的最优突变体HPO_M18。令人鼓舞的 (+)-nootkatone 效价为 2。通过代谢工程和内质网工程后的两阶段补料分批发酵达到39 g/L，是迄今为止报道的最高滴度。我们的研究结果为 (+)-nootkatone 的经济可行生物工艺的开发奠定了基础。
更新日期：2022-12-05
中文翻译：
系统优化 HPO-CPR 以促进工程化酿酒酵母中 (+)-Nootkatone 的合成
作为葡萄柚中重要且昂贵的天然倍半萜化合物，(+)-nootkatone 因其强烈的葡萄柚样气味和生理活性而引起人们的兴趣，这促使其微生物生产。然而，细胞色素 P450-P450 还原酶 (HPO-CPR) 系统的低催化效率是主要挑战。我们利用羰基化合物与 2,4-二硝基苯肼 (DNPH) 显色反应的原理开发了一种高通量筛选 (HTS) 方法，可以快速筛选候选 HPO 突变体的活性。优化HPO与CPR配对后，通过半理性设计，得到催化性能是初始2.54倍的最优突变体HPO_M18。令人鼓舞的 (+)-nootkatone 效价为 2。通过代谢工程和内质网工程后的两阶段补料分批发酵达到39 g/L，是迄今为止报道的最高滴度。我们的研究结果为 (+)-nootkatone 的经济可行生物工艺的开发奠定了基础。
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