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

Irene Komera,Xiulai Chen,Guipeng Hu,Cong Gao,Liang Guo,Liming Liu

doi:10.1186/s13068-022-02111-3

Abstract

BackgroundBiomass 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.ResultsTo 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.ConclusionThe 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.

Highlights

Biomass formation and product synthesis decoupling have been proven to be promising to increase the titer of desired value add products
Design and characterization of the light‐inducible transcription regulation unit To assemble a bifunctional metabolic flux control tool, two units, the transcription activation unit and the transcription repression unit were constructed by mining the TetR repressor (Fig. 1)
To achieve lightinduced recombination and splitting, blue light-sensitive VVD dimers[24] were fused to eleven TetR splits (26, 36, 46, 68, 99, 104, 124, 166, 167,169, and 179), which were designed using computer-based simulations and protein homology modeling according to the TetR crystal structure (PDB: 4V2G) (Fig. 1a, Additional file 1: Fig. S1)

Summary

Introduction

Biomass formation and product synthesis decoupling have been proven to be promising to increase the titer of desired value add products. While some popular static regulation strategies, such as gene knockout [3], promoter engineering [4], RBS shuffling [5], etc., have been successfully used to redirect the carbon flux towards desired metabolites, they cause cell growth defects by either blocking the synthesis of essential metabolites or accumulating toxic metabolites. To this end, dynamic regulation tools are needed to finetune cellular carbon flux without sacrificing cell growth [6].

Methods

Results

Conclusion