CRISPR-based transcriptional regulation systems have been widely employed for precise metabolic network control in microbial cell factories.However, the development of CRISPR activation (CRISPRa) systems in Gram-positive bacteria, particularly Bacillus subtilis, remains limited due to unclear activation domains and narrow effective windows.In this study, we constructed a bidirectional CRISPR-based regulation system in B.subtilis, capable of simultaneously activating and repressing multiple target genes.First, we established CRISPRa and CRISPR interference (CRISPRi) systems based on dCpf1 fused with activation domains (dCpf1-AD) and identified RemA as a highly efficient activation domain, enabling bidirectional regulation of multiple target genes.Second, we improved the transcriptional activation efficiency by optimizing protein scaffold structures, enhancing expression levels, and introducing random mutagenesis within the activation domain, resulting in a 36.7% and 40.5% increase in sfGFP and mCherry expression, respectively.Finally, we applied this system to a riboflavin-producing strain and simultaneously activated and repressed three key genes involved in riboflavin biosynthesis, leading to an 8.7-fold increase in riboflavin production, reaching 227.5 mg/L.This study provides a novel approach for constructing B.subtilis-based microbial cell factories with enhanced metabolic productivity.
LIU Jifu
,
YU Wenwen
,
XU Xianhao
,
WU Yaokang
,
LIU Yanfeng
,
LI Jianghua
,
DU Guocheng
,
LYU Xueqin
,
LIU Long
. Construction of a CRISPR-based bidirectional transcriptional regulation system in Bacillus subtilis for metabolic network controls[J]. Food and Fermentation Industries, 2025
, 51(24)
: 35
-42
.
DOI: 10.13995/j.cnki.11-1802/ts.042725
[1] ROK C, DAE J, YANG D, et al.Systems metabolic engineering strategies:Integrating systems and synthetic biology with metabolic engineering[J].Trends in Biotechnology, 2019, 37(8):817-837.
[2] DING Q, DIAO W W, GAO C, et al.Microbial cell engineering to improve cellular synthetic capacity[J].Biotechnology Advances, 2020, 45:107649.
[3] ZHOU C Y, YE B, CHENG S, et al.Promoter engineering enables overproduction of foreign proteins from a single copy expression cassette in Bacillus subtilis[J].Microbial Cell Factories, 2019, 18(1):111.
[4] JIANG Z, NIU T F, LYU X Q, et al.Secretory expression fine-tuning and directed evolution of diacetylchitobiose deacetylase by Bacillus subtilis[J].Applied and Environmental Microbiology, 2019, 85(17):e01076-19.
[5] ZHAO L, TIAN R Z, SHEN Q Y, et al.Pathway engineering of Bacillus subtilis for enhanced N-acetylneuraminic acid production via whole-cell biocatalysis[J].Biotechnology Journal, 2019, 14(7):e1800682.
[6] JIAO X, LYU L T, ZHANG Y, et al.Reduction of lipid-accumulation of oleaginous yeast Rhodosporidium toruloides through CRISPR/Cas9-mediated inactivation of lipid droplet structural proteins[J].FEMS Microbiology Letters, 2021, 368(16):fnab111.
[7] WU Y K, LIU Y F, LV X Q, et al.CAMERS-B:CRISPR/Cpf1 assisted multiple-genes editing and regulation system for Bacillus subtilis[J].Biotechnology and Bioengineering, 2020, 117(6):1817-1825.
[8] LIU Y, WAN X Y, WAN B J.Engineered CRISPRa enables programmable eukaryote-like gene activation in bacteria[J].Nature Communications, 2019, 10:3693.
[9] GU Y, XU X H, WU Y K, et al.Advances and prospects of Bacillus subtilis cellular factories:From rational design to industrial applications[J].Metabolic Engineering, 2018, 50:109-121.
[10] MASSAIU I, PASOTTI L, SONNENSCHEIN N, et al.Integration of enzymatic data in Bacillus subtilis genome-scale metabolic model improves phenotype predictions and enables in silico design of poly-γ-glutamic acid production strains[J].Microbial Cell Factories, 2019, 18(1):3.
[11] DONG C, FONTANA J, PATEL A, et al.Synthetic CRISPR-Cas gene activators for transcriptional reprogramming in bacteria[J].Nature Communications, 2018, 9:2489.
[12] FONTANA J, SPARKMAN-YAGER D, ZALATAN J G, et al.Challenges and opportunities with CRISPR activation in bacteria for data-driven metabolic engineering[J].Current Opinion in Biotechnology, 2020, 64:190-198.
[13] PHAN T T P, NGUYEN H D, SCHUMANN W.Development of a strong intracellular expression system for Bacillus subtilis by optimizing promoter elements[J].Journal of Biotechnology, 2012, 157(1):167-172.
[14] WU Y K, LI Y, JIN K, et al.CRISPR-dCas12a-mediated genetic circuit cascades for multiplexed pathway optimization[J].Nature Chemical Biology, 2023, 19(3):367-377.
[15] TANENBAUM M E, GILBERT L A, QI L S, et al.A protein-tagging system for signal amplification in gene expression and fluorescence imaging[J].Cell, 2014, 159(3):635-646.
[16] ZHAI H T, CUI L, XIONG Z, et al.CRISPR-mediated protein-tagging signal amplification systems for efficient transcriptional activation and repression in Saccharomyces cerevisiae[J].Nucleic Acids Research, 2022, 50(10):5988-6000.
[17] MURAYAMA S, ISHIKAWA S, CHUMSAKUL O, et al.The role of α-CTD in the genome-wide transcriptional regulation of the Bacillus subtilis cells[J].PLoS One, 2015, 10(7):e0131588.
[18] SCHWECHHEIMER S K, PARK E Y, REVUELTA J L, et al.Biotechnology of riboflavin[J].Applied Microbiology and Biotechnology, 2016, 100(5):2107-2119.
[19] MACK M, VAN LOON A P,HOHMANN H P, et al.Regulation of riboflavin biosynthesis in Bacillus subtilis is affected by the activity of the flavokinase/flavin adenine dinucleotide synthetase encoded by ribC[J].Journal of Bacteriology, 1998, 180(4).
[20] WANG Z W, CHEN T, MA X H, et al.Enhancement of riboflavin production with Bacillus subtilis by expression and site-directed mutagenesis of zwf and gnd gene from Corynebacterium glutamicum[J].Bioresource Technology, 2011, 102(4):3934-3940.
