Strategy of employing plug-and-play vectors and LC–MS screening to facilitate the discovery of natural products using Aspergillus oryzae

Abstract

Aspergillus oryzae is a widely used host for heterologous expression of fungal natural products. However, the vectors previously developed are not convenient for use and screening positive transformants by PCR and fermentation is time- and effort-consuming. Hence, three plug-and-play vectors were developed here for multi-gene expression and liquid chromatography mass spectrometry detection was introduced to screen positive transformants. Using rug BGC for verification, we demonstrated that the vectors we developed perform well and liquid chromatography mass spectrometry detection is feasible to screen positive transformants. For deleterious gene expression, PxyrA rather than PamyB was employed. Utilizing the toolkit described here to express natural products, dozen days can be saved.

Graphical Abstract

References

[1] Alberti F, Foster GD, Bailey AM. Natural products from filamentous fungi and production by heterologous expression Appl Microbiol Biotechnol, 2017, 101: 493-500.

[2] Aleksenko A, Clutterbuck A. Autonomous plasmid replication in Aspergillus nidulans: AMA1 and MATE elements Fungal Genet Biol, 1997, 21: 373-387.

[3] Awakawa T, Abe I. Reconstitution of polyketide-derived meroterpenoid biosynthetic pathway in Aspergillus oryzae J Fungi, 2021, 7: 486.

[4] Baldin C, Kühbacher A, Merschak P, Wagener J, Gsaller F. Modular inducible multigene expression system for filamentous fungi Microbiol Spectr, 2022, 10: e03670-e13622.

[5] Blin K, Shaw S, Medema MH, Weber T. The antiSMASH database version 4: additional genomes and BGCs, new sequence-based searches and more Nucleic Acids Res, 2024, 52: D586-D589.

[6] Chiang Y-M, Lee K-H, Sanchez JF, Keller NP, Wang CC. Unlocking fungal cryptic natural products Nat Prod Commun, 2009.

[7] Endo A, Kuroda M, Tsujita Y. ML-236A, ML-236B, and ML-236C, new inhibitors of cholesterogenesis produced by Penicillium citrinum J Antibiot (Tokyo), 1976, 29: 1346-1348.

[8] Fleming A. On the antibacterial action of cultures of a penicillium, with special reference to their use in the isolation of B. influenzae Br J Exp Pathol, 1929, 10: 226

[9] Fujii T, Yamaoka H, Gomi K, Kitamoto A, Kumagai C. Cloning and nucleotide sequence of the ribonuclease T1 gene (rntA) from Aspergillus oryzae and its expression in Saccharomyces cerevisiae and Aspergillus oryzae Biosci Biotechnol Biochem, 1995, 59: 1869-1874.

[10] Gomi K, Iimura Y, Hara S. Integrative transformation of Aspergillus oryzae with a plasmid containing the Aspergillus nidulans argB gene Agric Biol Chem, 1987, 51: 2549-2555.

[11] Han YB, Bai W, Ding CX, Liang J, Wu S-H, Tan RX. Intertwined biosynthesis of skyrin and rugulosin A underlies the formation of cage-structured bisanthraquinones J Am Chem Soc, 2021, 143: 14218-14226.

[12] Inoue T, Toji H, Tanaka M, Takama M, Hasegawa-Shiro S, Yamaki Y, Shintani T, Gomi K. Alternative transcription start sites of the enolase-encoding gene enoA are stringently used in glycolytic/gluconeogenic conditions in Aspergillus oryzae Curr Genet, 2020, 66: 729-747.

[13] Ishida H, Hata Y, Ichikawa E, Kawato A, Suginami K, Imayasu S. Regulation of the glucoamylase-encoding gene (glaB), expressed in solid-state culture (koji) of Aspergillus oryzae J Ferment Bioeng, 1998, 86: 301-307.

[14] Ishida H, Hata Y, Kawato A, Abe Y, Suginami K, Imayasu S. Identification of functional elements that regulate the glucoamylase-encoding gene (glaB) expressed in solid-state culture of Aspergillus oryzae Curr Genet, 2000, 37: 373-379.

[15] Ishida H, Hata Y, Kawato A, Abe Y, Kashiwagi Y. Isolation of a novel promoter for efficient protein production in Aspergillus oryzae Biosci Biotechnol Biochem, 2004, 68: 1849-1857.

[16] Jeennor S, Anantayanon J, Chutrakul C, Panchanawaporn S, Laoteng K. Novel pentose-regulated promoter of Aspergillus oryzae with application in controlling heterologous gene expression Biotechnol Rep (Amst), 2022, 33. e00695

[17] Jiang L, Lv K, Zhu G, Lin Z, Zhang X, Xing C, Yang H, Zhang W, Wang Z, Liu C. Norditerpenoids biosynthesized by variediene synthase-associated P450 machinery along with modifications by the host cell Aspergillus oryzae Synth Syst Biotechnol, 2022, 7: 1142-1147.

[18] Jin FJ, Maruyama J-i, Juvvadi PR, Arioka M, Kitamoto K. Development of a novel quadruple auxotrophic host transformation system by argB gene disruption using adeA gene and exploiting adenine auxotrophy in Aspergillus oryzae FEMS Microbiol Lett, 2004, 239: 79-85.

[19] Jin FJ, Maruyama J-i, Juvvadi PR, Arioka M, Kitamoto K. Adenine auxotrophic mutants of Aspergillus oryzae: development of a novel transformation system with triple auxotrophic hosts Biosci Biotechnol Biochem, 2004, 68: 656-662.

[20] Katayama T, Tanaka Y, Okabe T, Nakamura H, Fujii W, Kitamoto K, Maruyama J-i. Development of a genome editing technique using the CRISPR/Cas9 system in the industrial filamentous fungus Aspergillus oryzae Biotechnol Lett, 2016, 38: 637-642.

[21] Khaldi N, Seifuddin FT, Turner G, Haft D, Nierman WC, Wolfe KH, Fedorova ND. SMURF: genomic mapping of fungal secondary metabolite clusters Fungal Genet Biol, 2010, 47: 736-741.

[22] Kubodera T, Yamashita N, Nishimura A. Pyrithiamine resistance gene (ptrA) of Aspergillus oryzae: cloning, characterization and application as a dominant selectable marker for transformation Biosci Biotechnol Biochem, 2000, 64: 1416-1421.

[23] Lazarus CM, Williams K, Bailey AM. Reconstructing fungal natural product biosynthetic pathways Nat Prod Rep, 2014, 31: 1339-1347.

[24] Lee KKM, Da Silva NA, Kealey JT. Determination of the extent of phosphopantetheinylation of polyketide synthases expressed in Escherichia coli and Saccharomyces cerevisiae Anal Biochem, 2009, 394: 75-80.

[25] Liu C, Minami A, Ozaki T, Wu J, Kawagishi H, Maruyama J-i, Oikawa H. Efficient reconstitution of basidiomycota diterpene erinacine gene cluster in ascomycota host Aspergillus oryzae based on genomic DNA sequences J Am Chem Soc, 2019, 141: 15519-15523.

[26] Lv J-M, Hu D, Gao H, Kushiro T, Awakawa T, Chen G-D, Wang C-X, Abe I, Yao X-S. Biosynthesis of helvolic acid and identification of an unusual C-4-demethylation process distinct from sterol biosynthesis Nat Commun, 2017, 8: 1644.

[27] Maruyama J-I, Kitamoto K. Multiple gene disruptions by marker recycling with highly efficient gene-targeting background (ΔligD) in Aspergillus oryzae Biotechnol Lett, 2008, 30: 1811-1817.

[28] Matsuda Y, Wakimoto T, Mori T, Awakawa T, Abe I. Complete biosynthetic pathway of anditomin: nature’s sophisticated synthetic route to a complex fungal meroterpenoid J Am Chem Soc, 2014, 136: 15326-15336.

[29] Meng X, Fang Y, Ding M, Zhang Y, Jia K, Li Z, Collemare J, Liu W. Developing fungal heterologous expression platforms to explore and improve the production of natural products from fungal biodiversity Biotechnol Adv, 2022, 54. 107866

[30] Mori N, Katayama T, Saito R, Iwashita K, Maruyama J-I. Inter-strain expression of sequence-diverse HET domain genes severely inhibits growth of Aspergillus oryzae Biosci Biotechnol Biochem, 2019, 83: 1557-1569.

[31] Mózsik L, Iacovelli R, Bovenberg RA, Driessen AJ. Transcriptional activation of biosynthetic gene clusters in filamentous fungi Front Bioeng Biotechnol, 2022, 10. 901037

[32] Nagamine S, Liu C, Nishishita J, Kozaki T, Sogahata K, Sato Y, Minami A, Ozaki T, Schmidt-Dannert C, Maruyama J-i. Ascomycete Aspergillus oryzae is an efficient expression host for production of basidiomycete terpenes by using genomic DNA sequences Appl Environ Microbiol, 2019, 85: e00409-00419.

[33] Oda K, Terado S, Toyoura R, Fukuda H, Kawauchi M, Iwashita K. Development of a promoter shutoff system in Aspergillus oryzae using a sorbitol-sensitive promoter Biosci Biotechnol Biochem, 2016, 80: 1792-1801.

[34] Pahirulzaman KA, Williams K, Lazarus CM (2012) A toolkit for heterologous expression of metabolic pathways in Aspergillus oryzae. In: Hopwood DA (ed) Methods in enzymology. Academic Press. https://doi.org/10.1016/B978-0-12-404634-4.00012-7

[35] Shoji J-y, Maruyama J-i, Arioka M, Kitamoto K. Development of Aspergillus oryzae thiA promoter as a tool for molecular biological studies FEMS Microbiol Lett, 2005, 244: 41-46.

[36] Tagami K, Minami A, Fujii R, Liu C, Tanaka M, Gomi K, Dairi T, Oikawa H. Rapid reconstitution of biosynthetic machinery for fungal metabolites in Aspergillus oryzae: total biosynthesis of aflatrem ChemBioChem, 2014, 15: 2076-2080.

[37] Tsuchiya K, Tada S, Gomi K, Kitamoto K, Kumagai C, Tamura G. Deletion analysis of the Taka-amylase A gene promoter using a homologous transformation system in Aspergillus oryzae Biosci Biotechnol Biochem, 1992, 56: 1849-1853.

[38] Yamada O, Lee BR, Gomi K, Iimura Y. Cloning and functional analysis of the Aspergillus oryzae conidiation regulator gene brlA by its disruption and misscheduled expression J Biosci Bioeng, 1999, 87: 424-429.

[39] Yamada O, Nan SN, Akao T, Tominaga M, Watanabe H, Satoh T, Enei H, Akita O. dffA Gene from Aspergillus oryzae encodes L-ornithineN⁵-oxygenase and is indispensable for deferriferrichrysin biosynthesis J Biosci Bioeng, 2003, 95: 82-88.

[40] Yamane M, Minami A, Liu C, Ozaki T, Takeuchi I, Tsukagoshi T, Tokiwano T, Gomi K, Oikawa H. Biosynthetic machinery of diterpene pleuromutilin isolated from basidiomycete fungi ChemBioChem, 2017, 18: 2317-2322.

[41] Yuan Y, Cheng S, Bian G, Yan P, Ma Z, Dai W, Chen R, Fu S, Huang H, Chi H. Efficient exploration of terpenoid biosynthetic gene clusters in filamentous fungi Nat Catal, 2022, 5: 277-287.

Funding

Open Research Fund Program of State Key Laboratory of Bioreactor Engineering

Shanghai Collaborative Innovation Center for Biomanufacturing Technology

National Key R&D Program of China(2023YFA0914102)

RIGHTS & PERMISSIONS

The Author(s)