Systematic Optimization of HPO-CPR to Boost (+)-Nootkatone Synthesis in Engineered Saccharomyces cerevisiae

Abstract

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 highthroughput screening (HTS) method using the principle of the color reaction between carbonyl compounds and 2,4dinitrophenylhydrazine (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.

Green Production of a High-value Mosquito Insecticide of Nootkatone from Seaweed Hydrolysates

“…Nootkatone is a natural protective compound with higher commercial value than valencene and has been widely used in the food, fragrance, and cosmetics industries. , The prerequisite for the application of nootkatone is obtaining a high-purity product. However, current studies on de novo nootkatone synthesis often produce other byproducts, particularly valencene and nootkatol precursors, , posing challenges for downstream isolation and purification. Therefore, finding a new way to obtain a high-purity nootkatone is crucial.…”

“…The mutants used in this study, HPO/AtCPR1 and ADH, were previously generated by our research team. 16 BJ5464-01 strain was precultured in 5 mL of SD medium for 24 h at 25 °C and 200 rpm. The seed culture was scaled up in a 250 mL baffled culture flask containing 50 mL of SD liquid medium with a starter OD 600 of 0.05.…”

“…cerevisiae BJ5464-01, harboring plasmid Yep 352 containing HPO/AtCPR1 genes and plasmid p414 containing the ADH gene, was used for whole-cell biotransformation. The mutants used in this study, HPO/AtCPR1 and ADH, were previously generated by our research team . BJ5464-01 strain was precultured in 5 mL of SD medium for 24 h at 25 °C and 200 rpm.…”

Complete pathway elucidation and heterologous reconstitution of (+)‐nootkatone biosynthesis from Alpinia oxyphylla

“…Among the five previously reported VOs, HPO showed the highest activity and it is the most widely adopted VO enzyme in metabolic engineering in yeast platforms (Gavira et al ., 2013; Meng et al ., 2020; Liu et al ., 2022). To study the catalytic activity of the novel AoCYP18, we launched a parallel comparison experiment in our previously constructed valencene‐producing yeast strain JGH72 (Ye et al ., 2022).…”

“…The development of microbial cell factories to produce (+)‐nootkatone is attractive because of its short microbial growth cycle and low costs associated with this process. Therefore, considerable efforts have been made in metabolic engineering to this purpose (Gavira et al ., 2013; Wriessnegger et al ., 2014; Meng et al ., 2020; Cha et al ., 2022; Liu et al ., 2022; Ye et al ., 2022). The biosynthesis of (+)‐nootkatone is a three‐step reaction.…”