Development of a Type I-E CRISPR-Based Programmable Repression System for Fine-Tuning Metabolic Flux toward D-Pantothenic Acid in .

The CRISPR-based regulation tools enable fine-tuning of gene transcription, showing potential in areas of biomanufacturing and live therapeutics. However, the cell toxicity and PAM specificity of existing CRISPR-based regulation systems limit their broad application. The development of new and less-toxic CRISPR-controlled expression systems remains highly desirable for expanding the application scope of CRISPR-based tools. Here, we reconstituted the type I CRISPR-Cas system from to finely tune gene expression in . Through engineering the 5' untranslated region (UTR) of mRNAs of genes, we remarkably improved the efficacy of the type I CRISPRi system. The improved type I CRISPRi system was applied in engineering the D-pantothenic acid (DPA)-producing , which was generated by strengthening the metabolic flux toward β-alanine and ()-pantoate via enhancing expression of key enzymes at both transcriptional and translational levels. Through controlling the expression of with the CRISPRi system for fine-tuning the metabolic flux toward DPA and the TCA cycle, we elevated the DPA titer to 0.88 g/L in shake flasks and 12.81 g/L in fed-batch fermentations without the addition of the precursor β-alanine. The type I CRISPRi system and the strategy for fine-tuning metabolic flux reported here not only enrich the CRISPR toolbox in and facilitate DPA production through microbial fermentation but also provide a paradigm for programming important organisms to produce value-added chemicals with cheap raw materials.