Princeton: Yeast Fermentation Enhanced by Light-Controlled Nanobody Inhibition

Transcriptional control is widely used in dynamic metabolic engineering, but it suffers from inherent time lags because existing intracellular enzymes remain active even after transcription is halted. This is particularly problematic for stable enzymes with long half-lives.  The Avalos group at Princeton engineered a novel two-layered control system for yeast fermentations that combines optogenetic transcriptional regulation with post-translational inhibition using a genetically encoded anti-pyruvate-decarboxylase (Pdc1p) nanobody. 

Optogenetics is a technique that uses light-responsive proteins to control protein and cellular functions. Using light as an inducible agent offers several advantages: light is highly tunable, reversible, and orthogonal to biological processes (it has low toxicity, it cannot be metabolized, and causes little to no side effects in most non-photosynthetic organisms), and it is easy to interface with computers for automated control. 

Nanobodies are heavy-chain-only antibody fragments from camelids.  They are small in size, have high affinity and specificity, and are used here for post-translational control of enzymes by being genetically encoded and expressed within the cell to inhibit target proteins directly.

The group used a dark-inducible nanobody, NbJRI, specifically inactivating the major yeast pyruvate decarboxylase (Pdc1p), a key enzyme in ethanol fermentation, so it acts as a genetically encoded inhibitor of biomass and ethanol production.  Blue light activates pyruvate decarboxylase transcription during the growth phase, enabling cell growth and ethanol production, while NbJRI expression is repressed.  The light is switched off during the production phase. Pdc1p transcription is halted, and NbJRI expression is activated, inhibiting existing pyruvate decarboxylase and allowing for a faster switch from ethanol production to producing desired chemicals.  The dual control system significantly enhanced the production of 2,3-butanediol (up to 100% increase) and citramalate (up to 92% increase) compared to using transcriptional control alone in dynamic two-phase fermentations.

Tang AY, Gonzalez CL, Mantri KA, Lalwani MA, Avalos JL. Anti-Pdc1p Nanobody as a Genetically Encoded Inhibitor of Ethanol Production Enables Dual Transcriptional and Post-translational Controls of Yeast Fermentations. ACS Synth Biol. 2025 Apr 18;14(4):1072-1083. doi: 10.1021/acssynbio.4c00617. Epub 2025 Mar 17. PMID: 40098243.

https://pubs.acs.org/doi/10.1021/acssynbio.4c00617