Rewiring Metabolic Flux in Corynebacterium glutamicum Using a CRISPR/dCpf1-Based Bifunctional Regulation System

Corynebacterium glutamicum, a microorganism classified as generally recognized as safe for use in the industrial production of food raw materials and additives, has encountered challenges in achieving widespread adoption and popularization as microbial cell factories. These obstacles arise from the intricate nature of manipulating metabolic flux through conventional methods, such as gene knockout and enzyme overexpression. To address this challenge, we developed a CRISPR/dCpf1-based bifunctional regulation system to bidirectionally regulate the expression of multiple genes in C. glutamicum. Specifically, through fusing various transcription factors to the C-terminus of dCpf1, the resulting dCpf1-SoxS exhibited both CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) capabilities in C. glutamicum by altering the binding sites of crRNAs. The bifunctional regulation system was used to fine-tune metabolic flux from shikimic acid (SA) and l-serine biosynthesis, resulting in 27-fold and 10-fold increases in SA and l-serine production, respectively, compared to the original strain. These findings highlight the potential of the CRISPR/dCpf1-based bifunctional regulation system in effectively enhancing the yield of target products in C. glutamicum

Protein content (a), POD activity (b), SOD activity (c), CAT activity (d), Soluble sugar content (e) and MDA content (f).

<p>Protein content (a), POD activity (b), SOD activity (c), CAT activity (d), Soluble sugar content (e) and MDA content (f).</p

The Chl a content (a), Chl b content (b), Chl a+b content (c) and Chl a/b (d).

<p>The values represented mean ± SE, and different letters mark significant differences among shade treatments on the same day (P<0.05).</p

Net photosynthetic rate (P_n) (a), stomatal conductance (G_s) (b) and intercellular CO₂ concentration (C_i) (c).

<p>The values represented mean ± SE, and different letters mark significant differences among shade treatments on the same day (P<0.05).</p

Curves of diurnal variation of photosynthetically active radiation (PAR) under 50%, 30%, 20% and 5% light irradiances during one day in June 2012 in Lin’an, China.

<p>Curves of diurnal variation of photosynthetically active radiation (PAR) under 50%, 30%, 20% and 5% light irradiances during one day in June 2012 in Lin’an, China.</p

The chloroplast ultrastructure observed in the leaves of Anoectochilus roxburghii at 40 DOT.

<p>(a), (b), (c) the plants under 50% irradiance treatment; (d), (e), (f) the plants under 30% irradiance treatment; (g), (h), (i) the plants under 20% irradiance treatment; (j), (k), (l) the plants under 5% irradiance treatment. Notice the differences of the number of SG (indicated by arrow heads) and the number of grana lamella (indicated by arrows) between different irradiances. Abbreviation: Ch, chloroplast; CW, cell wall; OG, osmiophilic globules.</p

The appearance of whole plants (a) and leaves (b) exposed to 40 d of various levels of shading.

<p>The appearance of whole plants (a) and leaves (b) exposed to 40 d of various levels of shading.</p

Electron transport rate (ETR) (a), photochemical quenching (qP) (b) and nonphotochemical quenching (NPQ) (c).

<p>The values represented mean ± SE, and different letters mark significant differences among shade treatments on the same day (P<0.05).</p