Screening and genetic engineering of marine-derived Aspergillus terreus for high-efficient production of lovastatin.

Lovastatin / biosynthesis Aspergillus / metabolism Genetic Engineering Fermentation Aquatic Organisms / metabolism
Genetic engineering LovE Lovastatin Marine-derived Aspergillus terreus Strong promoter

Journal

Microbial cell factories
ISSN: 1475-2859
Titre abrégé: Microb Cell Fact
Pays: England
ID NLM: 101139812

Informations de publication

Date de publication:
09 May 2024
Historique:
received: 18 01 2024
accepted: 17 04 2024
medline: 10 5 2024
pubmed: 10 5 2024
entrez: 9 5 2024
Statut: epublish

Résumé

Lovastatin has widespread applications thanks to its multiple pharmacological effects. Fermentation by filamentous fungi represents the major way of lovastatin production. However, the current lovastatin productivity by fungal fermentation is limited and needs to be improved. In this study, the lovastatin-producing strains of Aspergillus terreus from marine environment were screened, and their lovastatin productions were further improved by genetic engineering. Five strains of A. terreus were isolated from various marine environments. Their secondary metabolites were profiled by metabolomics analysis using Ultra Performance Liquid Chromatography-Mass spectrometry (UPLC-MS) with Global Natural Products Social Molecular Networking (GNPS), revealing that the production of secondary metabolites was variable among different strains. Remarkably, the strain of A. terreus MJ106 could principally biosynthesize the target drug lovastatin, which was confirmed by High Performance Liquid Chromatography (HPLC) and gene expression analysis. By one-factor experiment, lactose was found to be the best carbon source for A. terreus MJ106 to produce lovastatin. To improve the lovastatin titer in A. terreus MJ106, genetic engineering was applied to this strain. Firstly, a series of strong promoters was identified by transcriptomic and green fluorescent protein reporter analysis. Then, three selected strong promoters were used to overexpress the transcription factor gene lovE encoding the major transactivator for lov gene cluster expression. The results revealed that compared to A. terreus MJ106, all lovE over-expression mutants exhibited significantly more production of lovastatin and higher gene expression. One of them, LovE-b19, showed the highest lovastatin productivity at a titer of 1512 mg/L, which represents the highest production level reported in A. terreus. Our data suggested that combination of strain screen and genetic engineering represents a powerful tool for improving the productivity of fungal secondary metabolites, which could be adopted for large-scale production of lovastatin in marine-derived A. terreus.

Sections du résumé

BACKGROUND BACKGROUND
Lovastatin has widespread applications thanks to its multiple pharmacological effects. Fermentation by filamentous fungi represents the major way of lovastatin production. However, the current lovastatin productivity by fungal fermentation is limited and needs to be improved.
RESULTS RESULTS
In this study, the lovastatin-producing strains of Aspergillus terreus from marine environment were screened, and their lovastatin productions were further improved by genetic engineering. Five strains of A. terreus were isolated from various marine environments. Their secondary metabolites were profiled by metabolomics analysis using Ultra Performance Liquid Chromatography-Mass spectrometry (UPLC-MS) with Global Natural Products Social Molecular Networking (GNPS), revealing that the production of secondary metabolites was variable among different strains. Remarkably, the strain of A. terreus MJ106 could principally biosynthesize the target drug lovastatin, which was confirmed by High Performance Liquid Chromatography (HPLC) and gene expression analysis. By one-factor experiment, lactose was found to be the best carbon source for A. terreus MJ106 to produce lovastatin. To improve the lovastatin titer in A. terreus MJ106, genetic engineering was applied to this strain. Firstly, a series of strong promoters was identified by transcriptomic and green fluorescent protein reporter analysis. Then, three selected strong promoters were used to overexpress the transcription factor gene lovE encoding the major transactivator for lov gene cluster expression. The results revealed that compared to A. terreus MJ106, all lovE over-expression mutants exhibited significantly more production of lovastatin and higher gene expression. One of them, LovE-b19, showed the highest lovastatin productivity at a titer of 1512 mg/L, which represents the highest production level reported in A. terreus.
CONCLUSION CONCLUSIONS
Our data suggested that combination of strain screen and genetic engineering represents a powerful tool for improving the productivity of fungal secondary metabolites, which could be adopted for large-scale production of lovastatin in marine-derived A. terreus.

Identifiants

DOI: 10.1186/s12934-024-02396-z PMID: 38724934
pubmed: 38724934
doi: 10.1186/s12934-024-02396-z
pii: 10.1186/s12934-024-02396-z
doi:

Substances chimiques

Lovastatin 9LHU78OQFD

Types de publication

Journal Article

Langues

eng

Sous-ensembles de citation

IM

Pagination

134

Subventions

Organisme : the Major Project of Qingdao Marine Science and Technology Center
ID : 2022QNLM030003-1
Organisme : Shandong Provincial Natural Science Foundation (Major Basic Research Projects)
ID : ZR2019ZD18
Organisme : the National Natural Science Foundations of China
ID : 41830535
Organisme : the National Natural Science Foundations of China
ID : 32100059
Organisme : Natural Science Foundation of Fujian Province
ID : 2021J011043
Organisme : the grants from Minjiang University
ID : MYK19011
Organisme : the grants from Minjiang University
ID : JAT190622

Informations de copyright

© 2024. The Author(s).

Références

Barrios-González J, Miranda RU. Biotechnological production and applications of statins. Appl Microbiol Biotechnol. 2010;85:869–83.
pubmed: 19820926 doi: 10.1007/s00253-009-2239-6
Xie L, Zhu G, Shang J, Chen X, Zhang C, Ji X, Zhang Q, Wei Y. An overview on the biological activity and anti-cancer mechanism of lovastatin. Cell Signal. 2021;87:110–22.
doi: 10.1016/j.cellsig.2021.110122
Robson A. Lovastatin improves endothelial cell function in LMNA-related DCM. Nat Rev Cardiol. 2020;17:613.
pubmed: 32782396 doi: 10.1038/s41569-020-0432-6
Osterweil EK, Chuang SC, Chubykin AA, Sidorov M, Bianchi R, Wong RK, Bear MF. Lovastatin corrects excess protein synthesis and prevents epileptogenesis in a mouse model of fragile X syndrome. Neuron. 2013;77:243–50.
pubmed: 23352161 pmcid: 3597444 doi: 10.1016/j.neuron.2012.01.034
Huelsenbeck J, Henninger C, Schad A, Lackner KJ, Kaina B, Fritz G. Inhibition of Rac1 signaling by lovastatin protects against anthracycline-induced cardiac toxicity. Cell Death Dis. 2011;2: e190.
pubmed: 21833028 pmcid: 3181415 doi: 10.1038/cddis.2011.65
Xiong Z, Cao X, Wen Q, Chen Z, Cheng Z, Huang X, Zhang Y, Long C, Zhang Y, Huang Z. An overview of the bioactivity of monacolin K/lovastatin. Food Chem Toxicol. 2019;131: 110585.
pubmed: 31207306 doi: 10.1016/j.fct.2019.110585
Sadowska A, Osiński P, Roztocka A, Kaczmarz-Chojnacka K, Zapora E, Sawicka D, Car H. Statins—from fungi to pharmacy. Int J Mol Sci. 2024;25:466.
doi: 10.3390/ijms25010466
Hong XK, Guo TL, Xu XQ, Lin J. Multiplex metabolic pathway engineering of Monascus pilosus enhances lovastatin production. Appl Microbiol Biotechnol. 2023;107:6541–52.
pubmed: 37672068 doi: 10.1007/s00253-023-12747-2
Ryngajllo M, Boruta T, Bizukojc M. Complete genome sequence of lovastatin producer Aspergillus terreus ATCC 20542 and evaluation of genomic diversity among A. terreus strains. Appl Microbiol Biotechnol. 2021;105:1615–27.
pubmed: 33515286 pmcid: 7880949 doi: 10.1007/s00253-021-11133-0
Mulder KC, Mulinari F, Franco OL, Soares MS, Magalhaes BS, Parachin NS. Lovastatin production: from molecular basis to industrial process optimization. Biotechnol Adv. 2015;33:648–65.
pubmed: 25868803 doi: 10.1016/j.biotechadv.2015.04.001
Wang J, Liang J, Chen L, Zhang W, Kong L, Peng C, Su C, Tang Y, Deng Z, Wang Z. Structural basis for the biosynthesis of lovastatin. Nat Commun. 2021;12:867.
pubmed: 33558520 pmcid: 7870829 doi: 10.1038/s41467-021-21174-8
Xu W, Chooi YH, Choi JW, Li S, Vederas JC, Da Silva NA, Tang Y. LovG: the thioesterase required for dihydromonacolin L release and lovastatin nonaketide synthase turnover in lovastatin biosynthesis. Angew Chem Int Ed Engl. 2013;52:6472–5.
pubmed: 23653178 doi: 10.1002/anie.201302406
Huang X, Tang S, Zheng L, Teng Y, Yang Y, Zhu J, Lu X. Construction of an efficient and robust Aspergillus terreus cell factory for monacolin J production. ACS Synth Biol. 2019;8:818–25.
pubmed: 30856313 doi: 10.1021/acssynbio.8b00489
Damare S, Singh P, Raghukumar S. Biotechnology of marine fungi. Prog Mol Subcell Biol. 2012;53:277–97.
pubmed: 22222837 doi: 10.1007/978-3-642-23342-5_14
Yao G, Chen X, Han Y, Zheng H, Wang Z, Chen J. Development of versatile and efficient genetic tools for the marine-derived fungus Aspergillus terreus RA2905. Curr Genet. 2022;68:153–64.
pubmed: 35043238 doi: 10.1007/s00294-021-01218-8
Wu JS, Shi XH, Zhang YH, Shao CL, Fu XM, Li X, Yao GS, Wang CY. Benzyl furanones and pyrones from the marine-derived fungus Aspergillus terreus induced by chemical epigenetic modification. Molecules. 2020;25:3927.
pubmed: 32867374 pmcid: 7503933 doi: 10.3390/molecules25173927
Zheng YY, Ma ZL, Wu JS, Shao CL, Yao GS, Wang CY. Induction of secondary metabolite biosynthesis by deleting the histone deacetylase hdaA in the marine-derived fungus Aspergillus terreus RA2905. J Fungi (Basel). 2022;8:1024.
pubmed: 36294591 doi: 10.3390/jof8101024
Wu JS, Shi XH, Yao GS, Shao CL, Fu XM, Zhang XL, Guan HS, Wang CY. New thiodiketopiperazine and 3,4-dihydroisocoumarin derivatives from the marine-derived fungus Aspergillus terreus. Mar Drugs. 2020;18:132.
pubmed: 32110865 pmcid: 7143538 doi: 10.3390/md18030132
Shannon P, Markiel A, Ozier O, Baliga NS, Wang JT, Ramage D, Amin N, Schwikowski B, Ideker T. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 2003;13:2498–504.
pubmed: 14597658 pmcid: 403769 doi: 10.1101/gr.1239303
Ying Y, Tu S, Ni J, Lu X, Hu X, Lei P, Li X, Wang Y, Jin G, Wang H. Secondary metabolites from Aspergillus terreus F6-3, a marine fungus associated with Johnius belengerii. Fitoterapia. 2023;170: 105662.
pubmed: 37648028 doi: 10.1016/j.fitote.2023.105662
Ahn J, Hong J, Lee H, Park M, Lee E, Kim C, Choi E, Jung J, Lee H. Translation elongation factor 1-alpha gene from Pichia pastoris: molecular cloning, sequence, and use of its promoter. Appl Microbiol Biotechnol. 2007;74:601–8.
pubmed: 17124582 doi: 10.1007/s00253-006-0698-6
Tong S, An K, Zhou W, Chen W, Sun Y, Wang Q, Li D. Establishment of high-efficiency screening system for gene deletion in Fusarium venenatum TB01. J Fungi. 2022;8:169.
doi: 10.3390/jof8020169
Mukhtar H, Ijaz SS. Ikram-Ul-Haq: upstream and downstream processing of lovastatin by Aspergillus terreus. Cell Biochem Biophys. 2014;70:309–20.
pubmed: 24671671 doi: 10.1007/s12013-014-9914-7
Porcel ER. Effects of the sporulation conditions on the lovastatin production by Aspergillus terreus. Bioprocess Biosyst Eng. 2006;29:1–5.
pubmed: 16491374 doi: 10.1007/s00449-006-0048-1
Lai LS, Hung CS, Lo CC. Effects of lactose and glucose on production of itaconic acid and lovastatin by Aspergillus terreus ATCC 20542. J Biosci Bioeng. 2007;104:9–13.
pubmed: 17697977 doi: 10.1263/jbb.104.9
Jia Z. Enhancement of lovastatin production by supplementing polyketide antibiotics to the submerged culture of Aspergillus terreus. Appl Biochem Biotechnol. 2010;160:2014–25.
pubmed: 19728167 doi: 10.1007/s12010-009-8762-1
Kaur H. Response surface methodology for lovastatin production by Aspergillus terreus GD13 strain. Acta Microbiol Immunol Hung. 2010;57:377–91.
pubmed: 21183423 doi: 10.1556/AMicr.57.2010.4.4
Patil RH, Krishnan P, Maheshwari VL. Production of lovastatin by wild strains of Aspergillus terreus. Nat Prod Commun. 2011;6:183–6.
pubmed: 21425670
Hanan H, Hafiz M. Improved lovastatin production by inhibiting (+)-geodin biosynthesis in Aspergillus terreus. N Biotechnol. 2019;52:19–24.
doi: 10.1016/j.nbt.2019.04.003
Hasan H. Increasing lovastatin production by re-routing the precursors flow of Aspergillus terreus via metabolic engineering. Mol Biotechnol. 2022;64:90–9.
pubmed: 34546548 doi: 10.1007/s12033-021-00393-w
Hasan H, Abd Rahim MH, Campbell L. Overexpression of acetyl-CoA carboxylase in Aspergillus terreus to increase lovastatin production. N Biotechnol. 2018;44:64–71.
pubmed: 29727712 doi: 10.1016/j.nbt.2018.04.008
Steenwyk JL, Mead ME, Knowles SL, Raja HA, Roberts CD, Bader O, Houbraken J, Goldman GH, Oberlies NH, Rokas A. Variation among biosynthetic gene clusters, secondary metabolite profiles, and cards of virulence across Aspergillus species. Genetics. 2020;216:481–97.
pubmed: 32817009 pmcid: 7536862 doi: 10.1534/genetics.120.303549
Lind AL, Wisecaver JH, Lameiras C, Wiemann P, Palmer JM, Keller NP, Rodrigues F, Goldman GH, Rokas A. Drivers of genetic diversity in secondary metabolic gene clusters within a fungal species. PLoS Biol. 2017;15: e2003583.
pubmed: 29149178 pmcid: 5711037 doi: 10.1371/journal.pbio.2003583
Chang PK, Horn BW, Dorner JW. Sequence breakpoints in the aflatoxin biosynthesis gene cluster and flanking regions in nonaflatoxigenic Aspergillus flavus isolates. Fungal Genet Biol. 2005;42:914–23.
pubmed: 16154781 doi: 10.1016/j.fgb.2005.07.004
Chiara M, Fanelli F, Mule G, Logrieco AF, Pesole G, Leslie JF, Horner DS, Toomajian C. Genome sequencing of multiple isolates highlights subtelomeric genomic diversity within Fusarium fujikuroi. Genome Biol Evol. 2015;7:3062–9.
pubmed: 26475319 pmcid: 5635591 doi: 10.1093/gbe/evv198
Yao G, Yue Y, Fu Y, Fang Z, Xu Z, Ma G, Wang S. Exploration of the regulatory mechanism of secondary metabolism by comparative transcriptomics in Aspergillus flavus. Front Microbiol. 2018;9:1568.
pubmed: 30131770 pmcid: 6090018 doi: 10.3389/fmicb.2018.01568
Drott MT, Bastos RW, Rokas A, Ries LNA, Gabaldon T, Goldman GH, Keller NP, Greco C. Diversity of secondary metabolism in Aspergillus nidulans clinical isolates. mSphere. 2020;5:2.
doi: 10.1128/mSphere.00156-20
Bergmann S, Funk AN, Scherlach K, Schroeckh V, Shelest E, Horn U, Hertweck C, Brakhage AA. Activation of a silent fungal polyketide biosynthesis pathway through regulatory cross talk with a cryptic nonribosomal peptide synthetase gene cluster. Appl Environ Microbiol. 2010;76:8143–9.
pubmed: 20952652 pmcid: 3008269 doi: 10.1128/AEM.00683-10
Zhgun AA. Fungal BGCs for production of secondary metabolites: main types, central roles in strain improvement, and regulation according to the piano principle. Int J Mol Sci. 2023;24:13.
doi: 10.3390/ijms241311184
Andrie RM, Martinez JP, Ciuffetti LM. Development of ToxA and ToxB promoter-driven fluorescent protein expression vectors for use in filamentous ascomycetes. Mycologia. 2005;97:1152–61.
pubmed: 16596965 doi: 10.1080/15572536.2006.11832763
Yu JH, Hamari Z, Han KH, Seo JA, Reyes-Dominguez Y, Scazzocchio C. Double-joint PCR: a PCR-based molecular tool for gene manipulations in filamentous fungi. Fungal Genet Biol. 2004;41:973–81.
pubmed: 15465386 doi: 10.1016/j.fgb.2004.08.001
Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method. Methods. 2001;25:402–8.
pubmed: 11846609 doi: 10.1006/meth.2001.1262
Aron AT, Gentry EC, McPhail KL, Nothias LF, Nothias-Esposito M, Bouslimani A, Petras D, Gauglitz JM, Sikora N, Vargas F. Reproducible molecular networking of untargeted mass spectrometry data using GNPS. Nat Protoc. 2020;15:1954–91.
pubmed: 32405051 doi: 10.1038/s41596-020-0317-5

Auteurs

Han Na (H)

Key Laboratory of Marine Drugs and Key Laboratory of Evolution and Marine Biodiversity (the Ministry of Education of China), Institute of Evolution & Marine Biodiversity, School of Medicine and Pharmacy, Ocean University of China, Qingdao, 266003, China.
Fujian Key Laboratory on Conservation and Sustainable Utilization of Marine Biodiversity, Institute of Oceanography, Minjiang University, Fuzhou, 350108, China.

Yao-Yao Zheng (YY)

Key Laboratory of Marine Drugs and Key Laboratory of Evolution and Marine Biodiversity (the Ministry of Education of China), Institute of Evolution & Marine Biodiversity, School of Medicine and Pharmacy, Ocean University of China, Qingdao, 266003, China.
Laboratory for Marine Drugs and Bioproducts, Qingdao National Laboratory for Marine Science and Technology, Qingdao, 266237, China.

Yaoning Jia (Y)

Key Laboratory of Marine Drugs and Key Laboratory of Evolution and Marine Biodiversity (the Ministry of Education of China), Institute of Evolution & Marine Biodiversity, School of Medicine and Pharmacy, Ocean University of China, Qingdao, 266003, China.
Fujian Key Laboratory on Conservation and Sustainable Utilization of Marine Biodiversity, Institute of Oceanography, Minjiang University, Fuzhou, 350108, China.

Jingzhao Feng (J)

Fujian Key Laboratory on Conservation and Sustainable Utilization of Marine Biodiversity, Institute of Oceanography, Minjiang University, Fuzhou, 350108, China.
College of Life Science, Fujian Agriculture and Forestry University, Fuzhou, 350002, China.

Jizi Huang (J)

Fujian Key Laboratory on Conservation and Sustainable Utilization of Marine Biodiversity, Institute of Oceanography, Minjiang University, Fuzhou, 350108, China.
School of Future Technology, Fujian Agriculture and Forestry University, Fuzhou, China.

Jihao Huang (J)

Fujian Key Laboratory on Conservation and Sustainable Utilization of Marine Biodiversity, Institute of Oceanography, Minjiang University, Fuzhou, 350108, China.

Chang-Yun Wang (CY)

Key Laboratory of Marine Drugs and Key Laboratory of Evolution and Marine Biodiversity (the Ministry of Education of China), Institute of Evolution & Marine Biodiversity, School of Medicine and Pharmacy, Ocean University of China, Qingdao, 266003, China. changyun@ouc.edu.cn.
Laboratory for Marine Drugs and Bioproducts, Qingdao National Laboratory for Marine Science and Technology, Qingdao, 266237, China. changyun@ouc.edu.cn.

Guangshan Yao (G)

Fujian Key Laboratory on Conservation and Sustainable Utilization of Marine Biodiversity, Institute of Oceanography, Minjiang University, Fuzhou, 350108, China. 2616@mju.edu.cn.
School of Future Technology, Fujian Agriculture and Forestry University, Fuzhou, China. 2616@mju.edu.cn.

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Classifications MeSH

questionsmedicales.fr › Composés polycycliques › Hydrocarbures aromatiques polycycliques › Naphtalènes › Lovastatine › Screening and genetic engineering of...
questionsmedicales.fr › Eucaryotes › Champignons › Deuteromycota › Aspergillus › Screening and genetic engineering of...
questionsmedicales.fr › Techniques d'investigation › Techniques génétiques › Génie génétique › Screening and genetic engineering of...
questionsmedicales.fr › Métabolisme › Métabolisme glucidique › Fermentation › Screening and genetic engineering of...
questionsmedicales.fr › Phénomènes biologiques › Phénomènes écologiques et environnementaux › Eaux salées › Eau de mer › Organismes aquatiques › Screening and genetic engineering of...