Bifunctional optogenetic switch for improving shikimic acid production in E. coli

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.

Metabolic Engineering of Shikimic Acid Biosynthesis Pathway for the Production of Shikimic Acid and Its Branched Products in Microorganisms: Advances and Prospects

“…Unlike the traditional top-down process that focuses on optimizing endogenous pathways or reconstructing heterologous natural pathways to produce metabolites, it is possible for synthetic biology to design new specific metabolic pathways for the desired compounds. So far, synthetic biology technology has played an important role in regulation strategies, such as quorum sensing, temperature-sensitive dynamic systems, photogenetic dynamic systems, metabolite response circuits, and so on [ 63 ]. It is also worth mentioning that the new genetic tools involved in synthetic biology will greatly shorten the modification cycle and be more economical.…”

“…When this bifunctional switch is applied to the biosynthesis of SA, SA of 35 g/L is produced in the minimum glucose medium without adding any chemical inducers and expensive aromatic amino acids, which is the highest titer reported by E. coli so far. When fermented with a rich culture medium, the titer was further increased to 76 g/L, which provides a promising method for the construction of economically attractive microbial cell factories [ 63 ]. Obviously, the dynamic switching module can solve the problem of carbon flux imbalance in traditional metabolic engineering.…”

Light-regulated gene expression in Bacteria: Fundamentals, advances, and perspectives

“…In the corresponding pLATR setups, TetR repression was enhanced by blue light, and up to 75-fold reduction of gene expression resulted. In another study, the repression by TetR was subjected to light control by splitting the repressor into two parts (Komera et al, 2022). Linkage of the resultant fragments to NcVVD provided the TRU system which mediated the light-induced reconstitution of the repressor and a 13-fold downregulation of a fluorescent reporter.…”

“…It has however not been reported if or to what extent iLID can be reconfigured for inducing protein degradation by light in bacteria. Against this backdrop, the PRU approach pursues a different strategy based on split TEV protease and the NcVVD module (Komera et al, 2022). Blue light prompted reconstitution of the TEV protease and enabled the on-demand cleavage of target proteins.…”

Perspectives in growth production trade-off in microbial bioproduction

“…Further optimized version, OptoAMP based control of LDH was reported for lactic acid production at 6 g/L in S. cerevisiae even at low light intensities 51 . In another study optogenetic tools were developed using the TetR system along with the tobacco etch virus protease (TEVp) for production of shikimic acid in E. coli at 35 g/L using glucose minimal media 52 . The major bottlenecks with optogenetic tools include limited number of photo-switchable proteins, restricted implementation in popular industrial hosts, insufficient and heterogenous lighting at high cell density in large-scale bioreactors and cost associated with specifically designed industrial-scale lightbioreactors (up to 5000-10 000 l) for production of commodity chemicals.…”