Enhanced Production of Sulforaphane by Heterologous Expression of Aphid Myrosinase in Escherichia coli

Myrosinase plays a pivotal role in the hydrolysis of glucosinolates in plants, generating a diverse array of biologically active metabolites. This study reports the successful heterologous expression of the gene encoding aphid myrosinase (BbMyr) in Escherichia coli, yielding an engineered strain with potent hydrolytic activity towards glucoraphanin (GRA). Following purification, the specific enzyme activity was determined to be 15.45 U/mg. Under optimized reaction conditions (pH 4.5 and 45 ﾰC), the engineered E. coli whole-cell effectively degraded glucoraphanin, producing sulforaphane (SFN) at a concentration of 1.6 g/L with a remarkable yield of 20.8 mg/g broccoli seeds, the highest yield documented per gram of broccoli seeds to date. This achievement signifies a significant advancement in the sustainable production of SFN, a highly valued bioactive compound with numerous health benefits. Furthermore, the immobilized engineered E. coli demonstrated an impressive 64% activity retention after seven consecutive reaction cycles, in contrast to only 29% for the free cells. This remarkable stability underscores the potential of immobilized E. coli as a robust biocatalyst for industrial-scale SFN production. To gain deeper insights into the catalytic mechanism of BbMyr, molecular dynamics simulations were employed to elucidate the protonation state of Glu167, the enzyme's active center. This information provides a solid foundation for future molecular modification endeavors aimed at enhancing the catalytic efficiency and stability of BbMyr. In conclusion, this study presents a novel and efficient platform for the production of SFN, paving the way for the development of sustainable and scalable biocatalytic processes for the production of this valuable bioactive compound.