The synthetic biology strategy of hydroxytyrosol

The following are the strategies for constructing high-yield strains and optimizing metabolic engineering in the synthetic biology of hydroxytyrosol:

I. Construction of High-yield Strains

Gene Editing: Modify the host strain through gene editing technology. For example, the research group led by Tang Shuangyan from the Institute of Microbiology, Chinese Academy of Sciences, used protein structure-guided modeling and directed evolution technology to engineer the Escherichia coli monooxygenase HpaBC, and obtained an HpaBC mutant that can efficiently catalyze the production of L-dopa from tyrosine. By replacing the rate-limiting murine tyrosine hydroxylase in the original pathway, the synthesis efficiency of hydroxytyrosol is improved.

Gene Integration: Integrate relevant genes into the genome of the host strain to ensure the stable expression of the genes. For instance, in a study, the HpaBC gene expression cassette regulated by the tac promoter was integrated into the genome of the pre-constructed tyrosol high-yield strain YMG5A^*R through CRISPR-Cas9 technology. At the same time, the synthesis pathway of the by-product acetic acid was deleted, and the Escherichia coli metabolic engineering strain YMGRD1H1 that can directly produce hydroxytyrosol using glucose was obtained.

Screening and Breeding: Screen and breed strains using tools such as whole-cell biosensors. The team led by Tang Shuangyan analyzed the complex crystal structure of the regulatory protein VanR from Corynebacterium glutamicum and its natural inducer vanillic acid. Through the modification and screening of the induction specificity of the VanR protein, a VanR protein mutant that can specifically respond to the induction of hydroxytyrosol was obtained and was developed into the first whole-cell biosensor for hydroxytyrosol. Furthermore, the rate-limiting enzyme tyramine oxidase (TYO) in the new biosynthetic pathway of hydroxytyrosol was subjected to in vivo directed evolution modification and screening, and a high-yield strain of hydroxytyrosol was obtained.

II. Optimization of Metabolic Engineering

Balancing Precursor Supply: Overexpress enzymes related to precursor synthesis to increase the supply of precursors required for the synthesis of hydroxytyrosol. For example, in Saccharomyces cerevisiae, the transketolase and ribose-5-phosphate ketol isomerase are overexpressed to balance the precursor supply and promote the metabolic flux towards the synthesis of hydroxytyrosol.

Optimizing the Metabolic Pathway: Analyze and optimize the entire metabolic pathway, relieve the feedback inhibition of key enzymes, and improve the efficiency of the metabolic pathway. For instance, in Saccharomyces cerevisiae, the feedback inhibition of tyrosol production is relieved, and the chorismate synthase and chorismate mutase are overexpressed to maximize the metabolic flux towards tyrosol synthesis, thereby increasing the production of hydroxytyrosol, as tyrosol is a precursor of hydroxytyrosol.

Blocking Competitive Pathways: Knock out the metabolic pathways that compete for carbon sources or intermediates in the synthesis of hydroxytyrosol, so that more carbon flux enters the synthesis pathway of hydroxytyrosol. For example, genes such as abz1, trp2, or pha2 are knocked out in relevant strains to block the competitive pathways and further guide the carbon flux into tyrosol synthesis, thus increasing the production of hydroxytyrosol.

Regulation of Cofactor Balance: The balance of cofactors within the cell has an important impact on the efficiency of the metabolic pathway. By regulating the cofactors, the synthesis of hydroxytyrosol can be optimized. For example, in Saccharomyces cerevisiae, through combined modification strategies such as cofactor balance regulation, the production of hydroxytyrosol has been increased.