Engineering a Controllable Targeted Protein Degradation System and a Derived OR-GATE-Type Inducible Gene Expression System in Synechococcus elongatus PCC 7942

Abstract

Cyanobacteria are important model organisms for exploring the mechanisms of photosynthesis and are considered as promising microbial platforms for photosynthetic biomanufacturing. The development of efficient cyanobacteria cell factories requires efficient and convenient tools to dynamically regulate and manipulate target proteins, modules, and pathways. Targeted protein degradation is important to achieve rapid responses of cellular metabolic networks to artificial or environmental signals, and there are currently limited approaches to induce protein degradation in cyanobacteria. In this work, we developed an Escherichia coli sourced ssrA-tagging system in an important cyanobacteria strain, Synechococcus elongatus PCC 7942, to achieve inducible degradation of target proteins. A modified version of the E. coli ssrA tag (ssrADAS) proved to be immune to the native ClpXP system in Synechococcus elongatus PCC 7942, while induced expression of the E. coli sourced adaptor SspB and ClpXP resulted in effective degradation of the tagged proteins. Compared to the previously developed down-regulation approaches, the inducible ssrADAS–SspB-ClpXPEc system facilitated the smart and rapid degradation of target proteins in PCC7942 cells at different growth stages. Furthermore, when used to regulate the degradation of LacI, the repressor element of LacO-LacI transcription regulation system, an efficient and stringent inducible gene expression system was obtained based on an OR-GATE type genetic circuit design. The tools developed in this work expanded the cyanobacteria synthetic biology toolbox and will facilitate the success of future dynamic metabolic engineering.

State-of-the-art in engineering small molecule biosensors and their applications in metabolic engineering

“…Apart from E. coli, the degron tagging system has been employed in other bacterial species [104,106,[119][120][121][122]. However, the specificity to the ClpXP protease is low in bacteria, resulting in recognition by other proteases, which hampers the ability to tightly control degradation.…”

Bacterial degrons in synthetic circuits

“…Most synthetic circuits to date, especially dynamic circuits, exploit almost exclusively this tag and variants for targeted degradation. Apart from E. coli, synthetic circuits using the tag have been tested in other bacterial species, including bacillus [106,107], pseudomonas [108], salmonella [109], mycobacteria [110][111][112] or cyanobacteria [113]. The most common design includes fusing the SsrA tag to the C-terminal of the proteins of interest [37].…”

Improved salt tolerance of Synechococcus elongatus PCC 7942 by heterologous synthesis of compatible solute ectoine

“…Cyanobacteria can use solar energy and CO 2 in the air to synthesize organic compounds, realizing the negative carbon economy in the process, which have received widespread attention as autotrophic cell factories ( Kato et al, 2022 ; Liu et al, 2022 ; Tan et al, 2022 ). As a model cyanobacterium, Synechococcus elongatus PCC 7942 (hereafter Syn7942), is amenable to genetic manipulation with the development of toolboxes and has been employed as a biological chassis for chemical production ( Kim et al, 2017 ; Sun et al, 2018 ; Sengupta et al, 2019 ; Zhang M. et al, 2022 ). Nowadays, it is expected to carry out industrialized production of biofuels and chemicals through the large-scale cultivation of cyanobacteria ( Farrokh et al, 2019 ; Davies et al, 2021 ; Wang et al, 2021 ), which requires the employment of seawater resources with rich reserves ( Cui et al, 2020 ).…”