Engineering Aspergillus oryzae for enhanced amylase production via multi-copy integration and microfluidic screening

Abstract

Aspergillus oryzae is an industrial filamentous fungus possessing high-efficiency protein expression and secretion capacity, widely used in enzyme preparation, food fermentation, and other fields. The industrial production strain A. oryzae F1 was selected for this study. Although already used for amylase production, its yield still fails to meet industrial demand. To improve the production of α-amylase, targeted engineering of the strain was performed using CRISPR/Cas9-mediated gene editing combined with high-throughput screening strategies. To enhance the site-specific integration efficiency of exogenous genes in A. oryzae F1, the gene lig4 encoding DNA ligase was knocked out, which increased the efficiency of homology-directed repair (HDR) from 13.4% to 34.7%. On this basis, α-amylase activity was improved by 15.2% through multi-copy gene integration. Furthermore, an excellent mutant strain was obtained by combining ARTP mutagenesis and droplet microfluidic high-throughput screening. In a 50 L fermentation system, the α-amylase activity of this mutant reached 4,052 U g⁻¹, representing a 37.4% increase compared with the parental strain F1. These results not only provide a feasible technical strategy for constructing high-efficiency amylase-producing strains, but also offer technical references for the engineering of filamentous fungal chassis cells.

Keywords

Aspergillus oryzae / CRISPR/Cas9 / Homology-directed repair / Microfluidic screening

References

[1] Beneyton T, Wijaya IP, Postros P, Najah M, Leblond P, Couvent A, Mayot E, Griffiths AD, Drevelle A. High-throughput screening of filamentous fungi using nanoliter-range droplet-based microfluidics. Sci Rep, 2016, 6: 27223

[2] Chang HHY, Pannunzio NR, Adachi N, Lieber MR. Non-homologous DNA end joining and alternative pathways to double-strand break repair. Nat Rev Mol Cell Biol, 2017, 18(8): 495-506

[3] Frisvad JC, Møller LLH, Larsen TO, Kumar R, Arnau J. Safety of the fungal workhorses of industrial biotechnology: update on the mycotoxin and secondary metabolite potential of Aspergillus niger, Aspergillus oryzae, and Trichoderma reesei. Appl Microbiol Biotechnol, 2018, 102(22): 9481-515

[4] Frit P, Amin H, Zahid S, Barboule N, Hall C, Matharu G, Hardwick SW, Chauvat J, Britton S, Chirgadze DY, Ropars V, Charbonnier JB, Calsou P, Chaplin AK. Structural and functional insights into the interaction between Ku70/80 and Pol X family polymerases in NHEJ. Nat Commun, 2025, 16(1): 4208

[5] Furuse M, Nagase Y, Tsubouchi H, Murakami-Murofushi K, Shibata T, Ohta K. Distinct roles of two separable in vitro activities of yeast Mre11 in mitotic and meiotic recombination. EMBO J, 1998, 17(21): 6412-25

[6] Gong YX, Li SF, Liu QR, Chen FS, Shao YC. CRISPR/Cas9 system is a suitable gene targeting editing tool to filamentous fungus Monascus pilosus. Appl Microbiol Biotechnol, 2024, 108(1): 154

[7] Grewal SIS. The molecular basis of heterochromatin assembly and epigenetic inheritance. Mol Cell, 2023, 83(11): 1767-85

[8] Gutschner T, Haemmerle M, Genovese G, Draetta, Giulio F, Chin L. Post-translational regulation of Cas9 during G1 enhances homology-directed repair. Cell Rep, 2016, 14(6): 1555-66

[9] Hertzberg RP, Pope AJ. High-throughput screening: new technology for the 21st century. Curr Opin Chem Biol, 2000, 4(4): 445-51

[10] Jasin M, Rothstein R. Repair of strand breaks by homologous recombination. Cold Spring Harb Perspect Biol, 2013, 5(11): a012740-012740

[11] Jia X, Song JY, Wu YJ, Feng S, Sun ZA, Hu Y, Yu MX, Han R, Zeng B. Strategies for the enhancement of secondary metabolite production via biosynthesis gene cluster regulation in. J Fungi, 2024, 10(5): 312

[12] Jin FJ, Wang BT, Wang ZD, Jin L, Han P. CRISPR/Cas9-based genome editing and its application in. J Fungi, 2022, 8(5): 467

[13] Jin YY, Zhang P, Liu LL, Zhao X, Hu XQ, Liu SZ, Li ZK, Liu Q, Wang JQ, Hao DL, Zhang ZQ, Chen HZ, Liu DP. Enhancing homology-directed repair efficiency with HDR-boosting modular ssDNA donor. Nat Commun, 2024, 15(1): 6843

[14] Lehmbeck J, Andersen B, Sáez-Sáez J, Frisvad JC, Arnau J. Mycotoxin-free Aspergillus oryzae strain lineage for alternative and novel protein production at industrial scale. Appl Microbiol Biotechnol, 2025, 109(1): 94

[15] Lei T, Du S, Peng Z, Chen L. Multifaceted regulation and functions of 53BP1 in NHEJ-mediated DSB repair (Review). Int J Mol Med, 2022, 50(1): 90

[16] Li QH, Lu JC, Liu JY, Li J, Zhang G, Du G, Chen J. High-throughput droplet microfluidics screening and genome sequencing analysis for improved amylase-producing Aspergillus oryzae. Biotechnol Biofuels Bioprod, 2023, 16(1): 185

[17] Li QH, Lu JC, Liu JY, Li JH, Zhang GQ, Du GC, Chen J. High-throughput droplet microfluidics screening and genome sequencing analysis for improved amylase-producing. Biotechnol Biofuels Bioprod, 2023, 16(1): 185

[18] Li QH, Lu JC, Zhang GQ, Zhou JW, Li JH, Du GC, Chen J. CRISPR/Cas9-mediated multiplexed genome editing in. J Fungi, 2023, 9(1): 109

[19] Liu JJ, Li JH, Liu YF, Shin HD, Ledesma Amaro R, Du GC, Chen J, Liu L. Synergistic rewiring of carbon metabolism and redox metabolism in cytoplasm and mitochondria of for increased l-malate production. ACS Synth Biol, 2018, 7(9): 2139-47

[20] Liu GX, Huang KY, Liu SY, Sheng ZY, Zhang PM. UHRF1 controls the timing of RAD51 removal during DNA damage repair through suppressing RFWD3. Adv Sci, 2025, 8: e09901

[21] Miné-Hattab J, Rothstein R. DNA in motion during double-strand break repair. Trends Cell Biol, 2013, 23(11): 529-36

[22] Moncalian G, Lengsfeld B, Bhaskara V, Hopfner KP, Karcher A, Alden E, Tainer JA, Paull TT. The rad50 signature motif: essential to ATP binding and biological function. J Mol Biol, 2004, 335(4): 937-51

[23] Nam HJ, Han JH, Yu J, Cho CS, Kim D, Kim YE, Kim MJ, Kim JH, Jo DH, Bae S. Autophagy induction enhances homologous recombination-associated CRISPR-Cas9 gene editing. Nucleic Acids Res, 2025, 53(7): gkaf258

[24] Nødvig CS, Hoof JB, Kogle ME, Jarczynska ZD, Lehmbeck J, Klitgaard DK, Mortensen UH. Efficient oligo nucleotide mediated CRISPR-Cas9 gene editing in Aspergilli. Fungal Genet Biol, 2018, 115: 78-89

[25] Ntana F, Mortensen UH, Sarazin C, Figge R. Aspergillus: a powerful protein production platform. Catalysts, 2020, 10(9): 1064

[26] Radzisheuskaya A, Shlyueva D, Müller I, Helin K. Optimizing sgRNA position markedly improves the efficiency of CRISPR/dCas9-mediated transcriptional repression. Nucleic Acids Res, 2016, 44(18): e141

[27] Robert F, Barbeau M, Éthier S, Dostie J, Pelletier J. Pharmacological inhibition of DNA-PK stimulates Cas9-mediated genome editing. Genome Med, 2015, 7(1): 93

[28] Sakai K, Kinoshita H, Nihira T. Heterologous expression system in Aspergillus oryzae for fungal biosynthetic gene clusters of secondary metabolites. Appl Microbiol Biotechnol, 2011, 93(5): 2011-22

[29] Selvaraj S, Feist WN, Viel S, Vaidyanathan S, Dudek AM, Gastou M, Rockwood SJ, Ekman FK, Oseghale AR, Xu L, Pavel-Dinu M, Luna SE, Cromer MK, Sayana R, Gomez-Ospina N, Porteus MH. High-efficiency transgene integration by homology-directed repair in human primary cells using DNA-PKcs inhibition. Nat Biotechnol, 2023, 42(5): 731-44

[30] Shioi T, Hatazawa S, Oya E, Hosoya N, Kobayashi W, Ogasawara M, Kobayashi T, Takizawa Y, Kurumizaka H. Cryo-EM structures of RAD51 assembled on nucleosomes containing a DSB site. Nature, 2024, 628(8006): 212-20

[31] Song J, Yang DS, Xu J, Zhu TQ, Chen YE, Zhang JF. RS-1 enhances CRISPR/Cas9- and TALEN-mediated knock-in efficiency. Nat Commun, 2016, 7(1): 10548

[32] Svobodová Kovaříková A, Legartová S, Krejčí J, Bártová E. H3K9me3 and H4K20me3 represent the epigenetic landscape for 53BP1 binding to DNA lesions. Aging, 2018, 10(10): 2585-605

[33] Syed A, Tainer JA. The MRE11-RAD50-NBS1 complex conducts the orchestration of damage signaling and outcomes to stress in DNA replication and repair. Annu Rev Biochem, 2018, 87(1): 263-94

[34] Teo SH, Jackson SP. Identification of Saccharomyces cerevisiae DNA ligase IV: involvement in DNA double-strand break repair. EMBO J, 1997, 16(15): 4788-95

[35] Todokoro T, Hata Y, Ishida H. CRISPR/Cas9 improves targeted knock-in efficiency in Aspergillus oryzae. Biotechnol Notes, 2024, 5: 58-63

[36] Tseng HM, Tomkinson AE. A physical and functional interaction between yeast Pol4 and Dnl4-Lif1 links DNA synthesis and ligation in nonhomologous end joining. J Biol Chem, 2002, 277(47): 45630-7

[37] Wakai S, Nakashima N, Ogino C, Tsutsumi H, Hata Y, Kondo A. Modified expression of multi-cellulases in a filamentous fungus Aspergillus oryzae. Bioresour Technol, 2019, 276: 146-53

[38] Wallis GL, Swift RJ, Hemming FW, Trinci AP, Peberdy JF. Glucoamylase overexpression and secretion in Aspergillus niger: analysis of glycosylation. Biochim Biophys Acta, 1999, 1472(3): 576-86

[39] Wang SN, Xue YB, Zhang P, Yan QJ, Li YX, Jiang ZQ. CRISPR/Cas9 system-mediated multi-copy expression of an alkaline serine protease in for the production of XOD-inhibitory peptides. J Agric Food Chem, 2023, 71(41): 15194-203

[40] Yang H, Song CN, Liu CW, Wang PC. Synthetic biology tools for engineering. J Fungi, 2024, 10(1): 34

[41] Yang QY, Lu SQ, Wu HY, Zhao DS, Wei W, Yin HR, Li X, Ye C, Shi TQ, Wang Z, Wang YT. Advanced droplet microfluidic platform for high-throughput screening of industrial fungi. Biosens Bioelectron, 2025, 285: 117594

[42] Yu C, Liu YX, Ma TH, Liu K, Xu SH, Zhang Y, Liu HL, La Russa M, Xie M, Ding S, Qi, Lei S. Small molecules enhance CRISPR genome editing in pluripotent stem cells. Cell Stem Cell, 2015, 16(2): 142-7

[43] Zeng WZ, Guo LK, Xu S, Chen J, Zhou JW. High-throughput screening technology in industrial biotechnology. Trends Biotechnol, 2020, 38(8): 888-906

[44] Zhang HX, Liu HL, Chen YL, Yang X, Wang PF, Liu TZ, Deng M, Qin B, Correia C, Lee S, Kim J, Sparks M, Nair AA, Evans DL, Kalari KR, Zhang PM, Wang LW, You ZS, Kaufmann SH, Lou ZK, Pei HD. A cell cycle-dependent BRCA1-UHRF1 cascade regulates DNA double-strand break repair pathway choice. Nat Commun, 2016, 7(1): 10201

[45] Zhang ZK, Ge MY, Guo Q, Jiang Y, Jia WD, Gao L, Hu JH. Ultrahigh-throughput screening of high-β-xylosidase-producing and investigation of the novel β-xylosidase characteristics. J Fungi, 2022, 8(4): 325

[46] Zhang CT, Wu XH, Song FQ, Liu S, Yu SQ, Zhou JW. Core-shell droplet-based microfluidic screening system for filamentous fungi. ACS Sens, 2023, 8(9): 3468-77

Funding

Basic Research Program of Jiangsu Province(BK20251600)

Jiangsu Funding Program for Excellent Postdoctoral Talent(2025ZB888)

National Key Research and Development Program of China(2024YFA0918300)

Jiangsu Basic Research Center for Synthetic Biology(BK20233003)

RIGHTS & PERMISSIONS

Jiangnan University