Systems metabolic engineering for hydroxytyrosol production in Escherichia coli

ABSTRACT Hydroxytyrosol (HT), the primary functional component of olive oil, presents significant challenges for microbial biosynthesis due to its inherent chemical properties and fermentation requirements. In this study, we describe the modular engineering of HT production in Escherichia coli (E. coli ). We initially employed a highly efficient 4-hydroxyphenylacetic acid 3-monooxygenase (encoded by the HpaBC gene) from E. coli , phenylpyruvate decarboxylase (encoded by the ARO10 gene), and alcohol dehydrogenase (encoded by the ADH6 gene) from Saccharomyces cerevisiae (S. cerevisiae ) to establish the de novo biosynthetic pathway for HT in E. coli . To enhance carbon flux toward HT production, we attenuated the competing metabolic pathway while reinforcing the synthetic route and optimized the gene copy numbers of key enzymes involved. Further strain engineering involved strengthening the membrane-bound pyridine nucleotide transhydrogenase (encoded by the pntAB gene) and incorporating a heterologous riboflavin biosynthesis pathway to improve the supply of the cofactors NADPH and FADH₂, culminating in the generation of the high-producing strain HT32-3. Then, a two-stage potential of hydrogen (pH) and two-stage dissolved oxygen (DO) control strategy was implemented to mitigate oxidative degradation of HT. In addition, a fed-batch supplementation process was developed to address the dependency of the key enzyme ARO10 on the cofactor vitamin B₁ (VB₁). These combined strategies enabled the achievement of a final HT titer of 9.22 g/L in a 5 L bioreactor. The present study provides a practical strategy for the modification of HT strains and a fermentation strategy, which lays the foundation for the development of engineered strains of high-value derivatives of tyrosine. IMPORTANCE In this study, a strain with high hydroxytyrosol (HT) production capacity was constructed by means of metabolic engineering modification. A two-stage pH and two-stage DO fermentation strategy was developed based on its physicochemical properties. Combined with the VB 1 replenishment strategy, the fermentation process of this strain was optimized, and the retention of HT was significantly improved, laying the foundation for the large-scale production of HT. This study explored the metabolic synthesis pathway and efficient fermentation strategy of HT, providing an innovative fermentation strategy and a practical strategy for the fermentation and production of HT and its related products.