Curated Optogenetic Publication Database

Abstract: Targeted protein degradation is a powerful tool for biological research, cell therapy, and synthetic biology. However, conventional methods often depend on pre-fused degrons or chemical degraders, limiting their wider applications. Here we develop a guided protein labeling and degradation system (GPlad) in Escherichia coli, using de novo designed guide proteins and arginine kinase (McsB) for precise degradation of various proteins, including fluorescent proteins, metabolic enzymes, and human proteins. We expand GPlad into versatile tools such as antiGPlad, OptoGPlad, and GPTAC, enabling reversible inhibition, optogenetic regulation, and biological chimerization. The combination of GPlad and antiGPlad allows for programmable circuit construction, including ON/OFF switches, signal amplifiers, and oscillators. OptoGPlad-mediated degradation of MutH accelerates E. coli evolution under protocatechuic acid stress, reducing the required generations from 220 to 100. GPTAC-mediated degradation of AroE enhanced the titer of 3-dehydroshikimic acid to 92.6 g/L, a 23.8% improvement over the conventional CRISPR interference method. We provide a tunable, plug-and-play strategy for straightforward protein degradation without the need for pre-fusion, with substantial implications for synthetic biology and metabolic engineering.

Abstract: Background Biomass formation and product synthesis decoupling have been proven to be promising to increase the titer of desired value add products. Optogenetics provides a potential strategy to develop light-induced circuits that conditionally control metabolic flux redistribution for enhanced microbial production. However, the limited number of light-sensitive proteins available to date hinders the progress of light-controlled tools. Results To address these issues, two optogenetic systems (TPRS and TPAS) were constructed by reprogramming the widely used repressor TetR and protease TEVp to expand the current optogenetic toolkit. By merging the two systems, a bifunctional optogenetic switch was constructed to enable orthogonally regulated gene transcription and protein accumulation. Application of this bifunctional switch to decouple biomass formation and shikimic acid biosynthesis allowed 35 g/L of shikimic acid production in a minimal medium from glucose, representing the highest titer reported to date by E. coli without the addition of any chemical inducers and expensive aromatic amino acids. This titer was further boosted to 76 g/L when using rich medium fermentation. Conclusion The cost effective and light-controlled switch reported here provides important insights into environmentally friendly tools for metabolic pathway regulation and should be applicable to the production of other value-add chemicals.

Abstract: Cell division can perturb the metabolic performance of industrial microbes. The C period of cell division starts from the initiation to the termination of DNA replication, whereas the D period is the bacterial division process. Here, we first shorten the C and D periods of E. coli by controlling the expression of the ribonucleotide reductase NrdAB and division proteins FtsZA through blue light and near-infrared light activation, respectively. It increases the specific surface area to 3.7 μm-1 and acetoin titer to 67.2 g·L-1. Next, we prolong the C and D periods of E. coli by regulating the expression of the ribonucleotide reductase NrdA and division protein inhibitor SulA through blue light activation-repression and near-infrared (NIR) light activation, respectively. It improves the cell volume to 52.6 μm3 and poly(lactate-co-3-hydroxybutyrate) titer to 14.31 g·L-1. Thus, the optogenetic-based cell division regulation strategy can improve the efficiency of microbial cell factories.