Presentation Information
[P01-002]Dynamic regulation of a toxic intermediate pathway for hydroxytyrosol production
○Koko Nakata1, Toshiki Saito1, Shunsuke Takahashi2 (1. Dept. Life Sci. Eng., Grad. Sch. Sci. Eng., Tokyo Denki Univ. (Japan), 2. Div. Life Sci. Eng., Sch. Sci. Eng., Tokyo Denki Univ. (Japan))
Keywords:
Synthetic biology,Metabolic engineering,Hydroxytyrosol,Catecholamine
[Background] Constructing efficient metabolic pathways requires precise, gene-by-gene control over both the timing and level of expression. However, conventional metabolic engineering typically relies on a single shared inducer — such as IPTG — or constitutive promoters that apply a common regulatory signal to all pathway genes simultaneously. Although operationally simple, this "one-signal-controls-all" architecture fundamentally prevents the independent tuning of individual genes, a limitation that becomes critical when a pathway harbors reactive or toxic intermediates.
To overcome this limitation, we developed a multi-input gene expression platform that enables independent, orthogonal, and tunable control of each pathway gene by precisely specifying when and how strongly each component is expressed.
[Model System] To rigorously validate this platform, we targeted microbial production of hydroxytyrosol (HT) via an oxidative cascade through 3,4-dihydroxyphenylacetaldehyde (DOPAL). This four-step pathway proceeds through L-DOPA and dopamine before reaching DOPAL; each step involves oxidative chemistry, and each intermediate is chemically reactive and potentially cytotoxic. Unlike the conventional HT route through tyrosol, which is chemically stable and compatible with static, uniform control, this fully oxidative cascade presents compounding risks at every node. DOPAL is a highly electrophilic catecholaldehyde that rapidly forms covalent adducts with cellular proteins and nucleic acids; any accumulation is acutely lethal. The pathway thus demands not merely tight control of one bottleneck, but precise, coordinated management of flux across an entire reactive landscape — exactly the kind of challenge that single-inducer strategies cannot meet.
[Strategy] Using our platform, we independently controlled upstream catecholamine biosynthesis (L-Tyr → L-DOPA → dopamine) and downstream DOPAL detoxification under orthogonal induction conditions, temporally decoupling the "generate" and "consume" phases of this reactive intermediate. Systematic ribosome binding site (RBS) engineering eliminated leaky basal expression, achieving tight module-level control throughout.
[Results] We achieved up to 3.0 mM HT was produced from 10 mM L-tyrosine, representing the first successful demonstration of HT biosynthesis via the DOPAL oxidative pathway. This work demonstrates that dynamic, modular gene regulation is not merely advantageous but essential for accessing metabolic routes that conventional engineering cannot safely navigate, and provides a broadly applicable platform for next-generation engineering of high-risk, high-value biosynthetic targets.
[Acknowledgement]The authors thank the Analytical Center of Tokyo Denki University for analytical support.
To overcome this limitation, we developed a multi-input gene expression platform that enables independent, orthogonal, and tunable control of each pathway gene by precisely specifying when and how strongly each component is expressed.
[Model System] To rigorously validate this platform, we targeted microbial production of hydroxytyrosol (HT) via an oxidative cascade through 3,4-dihydroxyphenylacetaldehyde (DOPAL). This four-step pathway proceeds through L-DOPA and dopamine before reaching DOPAL; each step involves oxidative chemistry, and each intermediate is chemically reactive and potentially cytotoxic. Unlike the conventional HT route through tyrosol, which is chemically stable and compatible with static, uniform control, this fully oxidative cascade presents compounding risks at every node. DOPAL is a highly electrophilic catecholaldehyde that rapidly forms covalent adducts with cellular proteins and nucleic acids; any accumulation is acutely lethal. The pathway thus demands not merely tight control of one bottleneck, but precise, coordinated management of flux across an entire reactive landscape — exactly the kind of challenge that single-inducer strategies cannot meet.
[Strategy] Using our platform, we independently controlled upstream catecholamine biosynthesis (L-Tyr → L-DOPA → dopamine) and downstream DOPAL detoxification under orthogonal induction conditions, temporally decoupling the "generate" and "consume" phases of this reactive intermediate. Systematic ribosome binding site (RBS) engineering eliminated leaky basal expression, achieving tight module-level control throughout.
[Results] We achieved up to 3.0 mM HT was produced from 10 mM L-tyrosine, representing the first successful demonstration of HT biosynthesis via the DOPAL oxidative pathway. This work demonstrates that dynamic, modular gene regulation is not merely advantageous but essential for accessing metabolic routes that conventional engineering cannot safely navigate, and provides a broadly applicable platform for next-generation engineering of high-risk, high-value biosynthetic targets.
[Acknowledgement]The authors thank the Analytical Center of Tokyo Denki University for analytical support.
Comment
To browse or post comments, you must log in.Log in