Development of a CRISPRi system in Fusarium fujikuroi and its application in gibberellic acid production

Abstract

Gene knockdown is a pivotal genetic manipulation technique, particularly when targeting lethal genes or genes involved in product synthesis pathways, where complete gene knockout is not a viable option. This approach is particularly valuable in multinucleate species, such as Fusarium fujikuroi, where generating homogeneous gene knockouts is notoriously difficult. To address these limitations, we first screened a set of repression domains, and then leveraged the optimal candidates to construct a CRISPR/dCas9-mediated knockdown platform for F. fujikuroi. By targeting erg9, which encodes squalene synthase, the first committed enzyme in the mevalonate pathway for ergosterol biosynthesis, we successfully diverted a portion of the metabolic flux from sterol production to gibberellic acid (GA) biosynthesis. This strategy minimizes carbon loss to competing pathways while retaining phenotypically normal growth. Additionally, CRISPR/dCas9-mediated knockdown of the dehydrogenase gene des enhanced GA4 production by 2.62-fold and eliminated the intermediate GA7, generating a GA3 + 4-producing strain and fine-tuning its metabolic profile. Using our CRISPRi system, we achieved a 70–89 % reduction in erg9 mRNA levels and a 67– 84 % reduction in des mRNA levels. Our findings establish a tailored CRISPRi platform for effective gene repression in F. fujikuroi.

Keywords

CRISPRi / Gibberellic acid / F. fujikuroi / Essential genes / Metabolic redirection

Data availability statement

The data supporting the findings of this study are included in the published article as well as in the Supplementary Materials available online.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

CRediT authorship contribution statement

Yuke Cen: Writing – review & editing, Writing – original draft, Methodology. Hang Xiao: Writing – review & editing, Writing – original draft, Investigation. Jingwen Jia: Data curation, Conceptualization. Jiajia Mou: Formal analysis. Haoyang Li: Validation. Jialiang Wang: Visualization. Yaling Yi: Software. Minghan Li: Data curation. Zhiqiang Liu: Writing – review & editing, Supervision, Resources, Project administration. Yuguo Zheng: Resources, Project administration.

References

[1] S. Janevska, B. Tudzynski, Secondary metabolism in Fusarium fujikuroi: strategies to unravel the function of biosynthetic pathways , Appl. Microbiol. Biotechnol. 102 (2) (2018) 615-630, doi: 10.1007/s00253—017—8679—5.

[2] R. Mäkilä, B. Wybouw, O. Smetana, et al., Gibberellins promote polar auxin transport to regulate stem cell fate decisions in cambium, Nat. Plants. 9 (4) (2023) 631-644, doi: 10.1038/s41477—023—01360—w.

[3] J. An, R. Xu, X. Wang, X. Zhang, C. You, Y. Han, MdbHLH162 connects the gibberellin and jasmonic acid signals to regulate anthocyanin biosynthesis in apple, JIPB 66 (2) (2024) 265-284, doi: 10.1111/jipb.13608.

[4] A. Hernández Rodríguez, A. Díaz Pacheco, S.E. Martínez Tolibia, Y. Melendez Xicohtencatl, S.Y. Granados Balbuena, Y. López, V.E López, Bioprocess of gibberellic acid by fusarium fujikuroi: the challenge of regulation, raw materials, and product yields , JoF 10 (6) (2024) 418, doi: 10.3390/jof10060418.

[5] R. Gupta, S.K. Chakrabarty, Gibberellic acid in plant: still a mystery unresolved, Plant Signal. Behav. 8 (9) (2013) e25504, doi: 10.4161/psb.25504.

[6] B. Tudzynski, Gibberellin biosynthesis in fungi: genes, enzymes, evolution, and impact on biotechnology, Appl. Microbiol. Biotechnol. 66 (6) (2005) 597-611, doi: 10.1007/s00253—004—1805—1.

[7] P. Hedden, S.G. Thomas, Gibberellin biosynthesis and its regulation, Biochem. J. 444 (1) (2012) 11-25, doi: 10.1042/BJ20120245.

[8] Y.K. Cen, Li M han , Q. Wang, et al. , Evolutionary engineering of Fusarium fujikuroi for enhanced production of gibberellic acid , Process. Biochem. 125 (2023) 7-14, doi: 10.1016/j.procbio.2022.12.009.

[9] B. Tudzynski, Gibberellin biosynthesis in fungi: genes, enzymes, evolution, and impact on biotechnology, Appl. Microbiol. Biotechnol. 66 (6) (2005) 597-611, doi: 10.1007/s00253—004—1805—1.

[10] L. Song, S. Wang, H. Zou, et al., Regulation of ergosterol biosynthesis in pathogenic fungi: opportunities for therapeutic development, Microorganisms 13 (4) (2025) 862, doi: 10.3390/microorganisms13040862.

[11] Y.K. Cen, J.G. Lin, Y.L. Wang, J.Y. Wang, Z.Q. Liu, Y.G. Zheng, The gibberellin producer fusarium fujikuroi: methods and technologies in the current toolkit , Front. Bioeng. Biotechnol. 8 (2020) 232, doi: 10.1016/j.procbio.2022.12.009.

[12] T.Q. Shi, C.L. Yang, D.X. Li, Y.T. Wang, Z.K. Nie, Establishment of a selectable marker recycling system for iterative gene editing in Fusarium fujikuroi , Synth. Syst. Biotechnol. 9 (1) (2024) 159-164, doi: 10.1016/j.synbio.2024.01.010.

[13] L. Huang, N. Li, Y. Song, J. Gao, L. Nian, J. Zhou, B. Zhang, Z. Liu, Y. Zheng, Development of a marker recyclable CRISPR/Cas9 system for scarless and multigene editing in Fusarium fujikuroi , Biotechnol. J. 19 (2024) e2400164, doi: 10.1002/biot.202400164.

[14] T.Q. Shi, J. Gao, W.J. Wang, et al. , CRISPR/Cas9—based genome editing in the filamentous fungus fusarium fujikuroi and its application in strain engineering for gibberellic acid production , ACS. Synth. Biol. 8 (2) (2019) 445-454, doi: 10.1021/acssynbio.8b00478.

[15] L. Huang, Y. Song, N. Li, et al., Deletion of bikaverin and fusarubin biosynthesis gene clusters via CRISPR/Cas9 system in Fusarium fujikuroi and its effect on GA3 biosynthesis , J. Biotechnol 405 (2025) 229-237, doi: 10.1016/j.jbiotec.2025.06.004.

[16] M. Larson, L. Gilbert, X. Wang, et al., CRISPR interference (CRISPRi) for sequence—specific control of gene expression, Nat. Protoc. 8 (11) (2013) 2180-2196, doi: 10.1038/nprot.2013.132.

[17] S. Vercauteren, S. Fiesack, L. Maroc, et al., The rise and future of CRISPR—based approaches for high—throughput genomics, FEMS Microbiol. Rev. 48 (5) (2024) fuae020, doi: 10.1093/femsre/fuae020.

[18] A. Vojta, P. Dobrini ć, V. Tadi ć, et al., Repurposing the CRISPR—Cas9 system for targeted DNA methylation, Nucleic. Acids. Res. 44 (12) (2016) 5615-5628, doi: 10.1093/nar/gkw159.

[19] A. Cebrian—Serrano, B. Davies, CRISPR—Cas orthologues and variants: optimizing the repertoire, specificity and delivery of genome engineering tools, Mamm. Genome 28 (7—8) (2017) 247-261, doi: 10.1007/s00335—017—9697—4.

[20] Y. Gao, X. Xiong, S. Wong, E.J. Charles, W.A. Lim, L.S. Qi, Complex transcriptional modulation with orthogonal and inducible dCas9 regulators, Nat. Methods 13 (12) (2016) 1043-1049, doi: 10.1038/nmeth.4042.

[21] I. Zukher, G. Dujardin, R. Sousa—Luís, N.J. Proudfoot, Elongation roadblocks mediated by dCas9 across human genes modulate transcription and nascent RNA processing, Nat. Struct. Mol. Biol. 30 (10) (2023) 1536-1548, doi: 10.1038/s41594—023—01090—9.

[22] Y. Li, L. Zhou, dCas9 techniques for transcriptional repression in mammalian cells: progress, applications and challenges, Bioessays 43 (9) (2021) 2100086, doi: 10.1002/bies.202100086.

[23] L.A. Gilbert, M.H. Larson, L. Morsut, et al. , CRISPR—mediated modular RNA—guided regulation of transcription in eukaryotes, Cell 154 (2) (2013) 442-451, doi: 10.1016/j.cell.2013.06.044.

[24] M.W. Gander, J.D. Vrana, W.E. Voje, J.M. Carothers, E. Klavins, Digital logic circuits in yeast with CRISPR/dCas9 NOR gates , Nat. Commun. 8 (1) (2017) 15459, doi: 10.1038/ncomms15459.

[25] J. Lian, M. HamediRad, S. Hu, H. Zhao, Combinatorial metabolic engineering using an orthogonal tri—functional CRISPR system, Nat. Commun. 8 (1) (2017) 1688, doi: 10.1038/s41467—017—01695—x.

[26] Y.K. Cen, M.H. Li, J.T. Li, et al. , Characterization of the G protein—coupled receptor FfGpr1—g 𝛼—AC signal transduction in Fusarium fujikuroi and its role in gibberellin biosynthesis , J. Appl. Microbiol. 136 (8) (2025) lxaf191, doi: 10.1093/jambio/lxaf191.

[27] L.S. Qi, M.H. Larson, L.A. Gilbert, et al. , Repurposing CRISPR as an RNA—guided platform for sequence—specific control of gene expression, Cell 152 (5) (2013) 1173-1183, doi: 10.1016/j.cell.2013.06.044.

[28] T.D. Schmittgen, K.J. Livak, Analyzing real—time PCR data by the comparative CT method, Nat. Protoc. 3 (6) (2008) 1101-1108, doi: 10.1038/nprot.2008.73.

[29] H. Wang, X. Ke, R. Jia, et al. , Gibberellic acid overproduction in Fusarium fujikuroi using regulatory modification and transcription analysis , Appl. Microbiol. Biotechnol. 107 (2023) 3071-3084, doi: 10.1007/s00253—023—12498—0.

[30] D. Gao, S. Smith, M. Spagnuolo, G. Rodriguez, M. Blenner, Dual CRISPR—Cas9 cleavage mediated gene excision and targeted integration in Yarrowia lipolytica , Biotechnol. J. 13 (9) (2018) 1700590, doi: 10.1002/biot.201700590.

[31] Y. Zhao, L. Li, G. Zheng, et al., CRISPR/dCas9—mediated multiplex gene repression in streptomyces , Biotechnol. J. 13 (9) (2018) 1800121, doi: 10.1002/biot.201800121.

[32] Y. Wang, Z. Zhang, S. Seo, et al., Gene transcription repression in Clostridium beijerinckii using CRISPR—dCas9 , Biotechnol. Bioeng. 113 (12) (2016) 2739-2743, doi: 10.1002/bit.26020.

[33] Y. Li, L. Zhou, dCas9 techniques for transcriptional repression in mammalian cells: progress, applications and challenges, Bioessays 43 (9) (2021) 2100086, doi: 10.1002/bies.202100086.

[34] C. Pan, S. Sretenovic, Y. Qi, CRISPR/dCas—mediated transcriptional and epigenetic regulation in plants, Curr. Opin. Plant Biol. 60 (2021) 101980, doi: 10.1016/j.pbi.2020.101980.

[35] Z. Wang, H. Pan, S. Ni, Z. Li, J. Lian, Establishing CRISPRi for programmable gene repression and genome evolution in Cupriavidus necator , ACS. Synth. Biol. 13 (3) (2024) 851-861, doi: 10.1021/acssynbio.3c00664.

[36] Q. Du, Y. Wei, L. Zhang, et al., An improved CRISPR and CRISPR interference (CRISPRi) toolkit for engineering the model methanogenic archaeon methanococcus maripaludis , Microb. Cell Fact. 23 (1) (2024) 239, doi: 10.1186/s12934—024—02492—0.

[37] X. Bu, J. Lin, C. Duan, M.A.G. Koffas, G. Yan, Dual regulation of lipid droplet—triacylglycerol metabolism and ERG9 expression for improved 𝛽—carotene production in Saccharomyces cerevisiae , Microb. Cell Fact. 21 (1) (2022) 3, doi: 10.1186/s12934—021—01723—y.

[38] D. Tzamarias, K. Struhl, Functional dissection of the yeast Cyc8—Tupl transcriptional co—repressor complex , Nature 369 (6483) (1994) 758-761, doi: 10.1038/369758a0.