Abstract
Recent technical advances regarding filamentous fungi have accelerated the engineering of fungal-based production and benefited basic science. However, challenges still remain and limit the speed of fungal applications. For example, high-throughput technologies tailored to filamentous fungi are not yet commonly available for genetic modification. The currently used fungal genetic manipulations are time-consuming and laborious. Here, we developed a flow cytometry-based plating-free system to directly screen and isolate the transformed protoplasts in industrial fungi Myceliophthora thermophila and Aspergillus niger. This system combines genetic engineering via the 2A peptide and the CRISPR–Cas9 system, strain screening by flow cytometry, and direct sorting of colonies for deep-well-plate incubation and phenotypic analysis while avoiding culturing transformed protoplasts in plates, colony picking, conidiation, and cultivation. As a proof of concept, we successfully applied this system to generate the glucoamylase-hyperproducing strains MtYM6 and AnLM3 in M. thermophila and A. niger, respectively. Notably, the protein secretion level and enzyme activities in MtYM6 were 17.3- and 25.1-fold higher than in the host strain. Overall, these findings suggest that the flow cytometry-based plating-free system can be a convenient and efficient tool for strain engineering in fungal biotechnology. We expect this system to facilitate improvements of filamentous fungal strains for industrial applications.
Key points
• Development of a flow cytometry-based plating-free (FCPF) system is presented.
• Application of FCPF system in M. thermophila and A. niger for glucoamylase platform.
• Hyper-produced strains MtYM6 and AnLM3 for glucoamylase production are generated.
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References
Allman R (1992) Characterization of fungal spores using flow cytometry. Mycol Res 96:1016–1018
Andersen MR, Salazar MP, Schaap PJ, van de Vondervoort PJ, Culley D, Thykaer J, Frisvad JC, Nielsen KF, Albang R, Albermann K, Berka RM, Braus GH, Braus-Stromeyer SA, Corrochano LM, Dai Z, van Dijck PW, Hofmann G, Lasure LL, Magnuson JK, Menke H, Meijer M, Meijer SL, Nielsen JB, Nielsen ML, van Ooyen AJ, Pel HJ, Poulsen L, Samson RA, Stam H, Tsang A, van den Brink JM, Atkins A, Aerts A, Shapiro H, Pangilinan J, Salamov A, Lou Y, Lindquist E, Lucas S, Grimwood J, Grigoriev IV, Kubicek CP, Martinez D, van Peij NN, Roubos JA, Nielsen J, Baker SE (2011) Comparative genomics of citric-acid-producing Aspergillus niger ATCC 1015 versus enzyme-producing CBS 513.88. Genome Res 21(6):885–97. https://doi.org/10.1101/gr.112169.110
Bergquist PL, Hardiman EM, Ferrari BC, Winsley T (2009) Applications of flow cytometry in environmental microbiology and biotechnology. Extremophiles 13(3):389–401. https://doi.org/10.1007/s00792-009-0236-4
Berka RM, Grigoriev IV, Otillar R, Salamov A, Grimwood J, Reid I, Ishmael N, John T, Darmond C, Moisan MC, Henrissat B, Coutinho PM, Lombard V, Natvig DO, Lindquist E, Schmutz J, Lucas S, Harris P, Powlowski J, Bellemare A, Taylor D, Butler G, de Vries RP, Allijn IE, van den Brink J, Ushinsky S, Storms R, Powell AJ, Paulsen IT, Elbourne LD, Baker SE, Magnuson J, Laboissiere S, Clutterbuck AJ, Martinez D, Wogulis M, de Leon AL, Rey MW, Tsang A (2011) Comparative genomic analysis of the thermophilic biomass-degrading fungi Myceliophthora thermophila and Thielavia terrestris. Nat Biotechnol 29(10):922–927. https://doi.org/10.1038/nbt.1976
Bleichrodt RJ, Read ND (2019) Flow cytometry and FACS applied to filamentous fungi. Fungal Biol Rev 33(1):1–15. https://doi.org/10.1016/j.fbr.2018.06.001
Blum A, Benfield AH, Stiller J, Kazan K, Batley J, Gardiner DM (2016) High-throughput FACS-based mutant screen identifies a gain-of-function allele of the Fusarium graminearum adenylyl cyclase causing deoxynivalenol over-production. Fungal Genet Biol 90:1–11. https://doi.org/10.1016/j.fgb.2016.02.005
Cairns TC, Nai C, Meyer V (2018) How a fungus shapes biotechnology: 100 years of Aspergillus niger research. Fungal Biol Biotechnol 5:13. https://doi.org/10.1186/s40694-018-0054-5
Chroumpi T, Makela MR, de Vries RP (2020) Engineering of primary carbon metabolism in filamentous fungi. Biotechnol Adv 43:107551. https://doi.org/10.1016/j.biotechadv.2020.107551
Druzhinina IS, Kubicek CP (2017) Genetic engineering of Trichoderma reesei cellulases and their production. Microb Biotechnol 10(6):1485–1499. https://doi.org/10.1111/1751-7915.12726
Ehgartner D, Herwig C, Fricke J (2017) Morphological analysis of the filamentous fungus Penicillium chrysogenum using flow cytometry-the fast alternative to microscopic image analysis. Appl Microbiol Biot 101(20):7675–7688. https://doi.org/10.1007/s00253-017-8475-2
Fan FY, Ma GL, Li JG, Liu Q, Benz JP, Tian CG, Ma YH (2015) Genome-wide analysis of the endoplasmic reticulum stress response during lignocellulase production in Neurospora crassa. Biotechnol Biofuels 8:66. https://doi.org/10.1186/S13068-015-0248-5
Gabriel R, Thieme N, Liu Q, Li F, Kohler LT, Harth S, Jecmenica M, Ramamurthy M, Gorman J, Simmons BA, McCluskey K, Baker SE, Tian C, Schuerg T, Singer SW, Fleissner A, Benz JP (2021) The F-box protein gene exo-1 is a target for reverse engineering enzyme hypersecretion in filamentous fungi. Proc Natl Acad Sci U S A 118(26):e2025689118. https://doi.org/10.1073/pnas.2025689118
Gao F, Hao Z, Sun X, Qin L, Zhao T, Liu W, Luo H, Yao B, Su X (2018) A versatile system for fast screening and isolation of Trichoderma reesei cellulase hyperproducers based on DsRed and fluorescence-assisted cell sorting. Biotechnol Biofuels 11:261. https://doi.org/10.1186/s13068-018-1264-z
He R, Ding R, Heyman JA, Zhang D, Tu R (2019) Ultra-high-throughput picoliter-droplet microfluidics screening of the industrial cellulase-producing filamentous fungus Trichoderma reesei. J Ind Microbiol Biotechnol 46(11):1603–1610. https://doi.org/10.1007/s10295-019-02221-2
Ishii S, Tago K, Senoo K (2010) Single-cell analysis and isolation for microbiology and biotechnology: methods and applications. Appl Microbiol Biotechnol 86(5):1281–1292. https://doi.org/10.1007/s00253-010-2524-4
Lencastre Fernandes R, Carlquist M, Lundin L, Heins AL, Dutta A, Sorensen SJ, Jensen AD, Nopens I, Lantz AE, Gernaey KV (2013) Cell mass and cell cycle dynamics of an asynchronous budding yeast population: experimental observations, flow cytometry data analysis, and multi-scale modeling. Biotechnol Bioeng 110(3):812–826. https://doi.org/10.1002/bit.24749
Karnaouri A, Topakas E, Antonopoulou I, Christakopoulos P (2014) Genomic insights into the fungal lignocellulolytic system of Myceliophthora thermophila. Front Microbiol 5:281. https://doi.org/10.3389/fmicb.2014.00281
Kim JH, Lee SR, Li LH, Park HJ, Park JH, Lee KY, Kim MK, Shin BA, Choi SY (2011) High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice. PLoS ONE 6(4):e18556. https://doi.org/10.1371/journal.pone.0018556
Kolbusz MA, Di Falco M, Ishmael N, Marqueteau S, Moisan MC, Baptista CD, Powlowski J, Tsang A (2014) Transcriptome and exoproteome analysis of utilization of plant-derived biomass by Myceliophthora thermophila. Fungal Genet Biol 72:10–20. https://doi.org/10.1016/j.fgb.2014.05.006
Le Crom S, Schackwitz W, Pennacchio L, Magnuson JK, Culley DE, Collett JR, Martin J, Druzhinina IS, Mathis H, Monot F, Seiboth B, Cherry B, Rey M, Berka R, Kubicek CP, Baker SE, Margeot A (2009) Tracking the roots of cellulase hyperproduction by the fungus Trichoderma reesei using massively parallel DNA sequencing. Proc Natl Acad Sci USA 106(38):16151–16156. https://doi.org/10.1073/pnas.0905848106
Li F, Liu Q, Li X, Zhang C, Li J, Sun W, Liu D, Xiao D, Tian C (2020a) Construction of a new thermophilic fungus Myceliophthora thermophila platform for enzyme production using a versatile 2A peptide strategy combined with efficient CRISPR-Cas9 system. Biotechnol Lett 42(7):1181–1191. https://doi.org/10.1007/s10529-020-02882-5
Li JG, Lin LC, Sun T, Xu J, Ji JX, Liu Q, Tian CG (2020b) Direct production of commodity chemicals from lignocellulose using Myceliophthora thermophila. Metab Eng 61:416–426. https://doi.org/10.1016/j.ymben.2019.05.007
Li J, Zhang Y, Li J, Sun T, Tian C (2020c) Metabolic engineering of the cellulolytic thermophilic fungus Myceliophthora thermophila to produce ethanol from cellobiose. Biotechnol Biofuels 13:23. https://doi.org/10.1186/s13068-020-1661-y
Liu G, Qu Y (2019) Engineering of filamentous fungi for efficient conversion of lignocellulose: tools, recent advances and prospects. Biotechnol Adv 37(4):519–529. https://doi.org/10.1016/j.biotechadv.2018.12.004
Liu Q, Gao R, Li J, Lin L, Zhao J, Sun W, Tian C (2017) Development of a genome-editing CRISPR/Cas9 system in thermophilic fungal Myceliophthora species and its application to hyper-cellulase production strain engineering. Biotechnol Biofuels 10:1. https://doi.org/10.1186/s13068-016-0693-9
Liu Q, Zhang Y, Li F, Li J, Sun W, Tian C (2019) Upgrading of efficient and scalable CRISPR-Cas-mediated technology for genetic engineering in thermophilic fungus Myceliophthora thermophila. Biotechnol Biofuels 12:293. https://doi.org/10.1186/s13068-019-1637-y
Longin C, Petitgonnet C, Guilloux-Benatier M, Rousseaux S, Alexandre H (2017) Application of flow cytometry to wine microorganisms. Food Microbiol 62:221–231. https://doi.org/10.1016/j.fm.2016.10.023
Marín-Navarro J, Polaina J (2011) Glucoamylases: structural and biotechnological aspects. Appl Microbiol Biotechnol 89(5):1267–1273. https://doi.org/10.1007/s00253-010-3034-0
Mathis H, Margeot A, Bouix M (2020) Optimization of flow cytometry parameters for high-throughput screening of spores of the filamentous fungus Trichoderma reesei. J Biotechnol 321:78–86. https://doi.org/10.1016/j.jbiotec.2020.05.015
Meng J, Makela MR, de Vries RP (2021) Molecular engineering to improve lignocellulosic biomass based applications using filamentous fungi. Adv Appl Microbiol 114:73–109. https://doi.org/10.1016/bs.aambs.2020.09.001
Meyer V, Andersen MR, Brakhage AA, Braus GH, Caddick MX, Cairns TC, de Vries RP, Haarmann T, Hansen K, Hertz-Fowler C, Krappmann S, Mortensen UH, Penalva MA, Ram AFJ, Head RM (2016) Current challenges of research on filamentous fungi in relation to human welfare and a sustainable bio-economy: a white paper. Fungal Biol Biotechnol 3:6. https://doi.org/10.1186/s40694-016-0024-8
Meyer V, Basenko EY, Benz JP, Braus GH, Caddick MX, Csukai M, de Vries RP, Endy D, Frisvad JC, Gunde-Cimerman N, Haarmann T, Hadar Y, Hansen K, Johnson RI, Keller NP, Krasevec N, Mortensen UH, Perez R, Ram AFJ, Record E, Ross P, Shapaval V, Steiniger C, van den Brink H, van Munster J, Yarden O, Wösten HAB (2020) Growing a circular economy with fungal biotechnology: a white paper. Fungal Biol Biotechnol 7:5. https://doi.org/10.1186/s40694-020-00095-z
Nevalainen H, Peterson R, Curach N (2018) Overview of gene expression using filamentous fungi. Curr Protoc Protein Sci 92(1):e55. https://doi.org/10.1002/cpps.55
Nielsen BR, Lehmbeck J, Frandsen TP (2002) Cloning, heterologous expression, and enzymatic characterization of a thermostable glucoamylase from Talaromyces emersonii. Protein Expr Purif 26(1):1–8. https://doi.org/10.1016/s1046-5928(02)00505-3
Opitz C, Schade G, Kaufmann S, Di Berardino M, Ottiger M, Grzesiek S (2019) Rapid determination of general cell status, cell viability, and optimal harvest time in eukaryotic cell cultures by impedance flow cytometry. Appl Microbiol Biotechnol 103(20):8619–8629. https://doi.org/10.1007/s00253-019-10046-3
Pei X, Fan F, Lin L, Chen Y, Sun W, Zhang S, Tian C (2015) Involvement of the adaptor protein 3 complex in lignocellulase secretion in Neurospora crassa revealed by comparative genomic screening. Biotechnol Biofuels 8:124. https://doi.org/10.1186/s13068-015-0302-3
Pel HJ, de Winde JH, Archer DB, Dyer PS, Hofmann G, Schaap PJ, Turner G, de Vries RP, Albang R, Albermann K, Andersen MR, Bendtsen JD, Benen JA, van den Berg M, Breestraat S, Caddick MX, Contreras R, Cornell M, Coutinho PM, Danchin EG, Debets AJ, Dekker P, van Dijck PW, van Dijk A, Dijkhuizen L, Driessen AJ, d’Enfert C, Geysens S, Goosen C, Groot GS, de Groot PW, Guillemette T, Henrissat B, Herweijer M, van den Hombergh JP, van den Hondel CA, van der Heijden RT, van der Kaaij RM, Klis FM, Kools HJ, Kubicekvan Kuyk CPPA, Lauber J, Lu X, van der Maarel MJ, Meulenberg R, Menke H, Mortime MA, Nielsen J, Oliver SG, Olsthoorn M, Pal K, van Peij NN, Ram AF, Rinas U, Roubos JA, Sagt CM, Schmoll M, Sun J, Ussery D, Varga J, Vervecken W, van de Vondervoort PJ, Wedler H, Wösten HA, Zeng AP, van Ooyen AJ, Visser J, Stam H (2007) Genome sequencing and analysis of the versatile cell factory Aspergillus niger CBS 513.88. Nat Biotechnol 25(2):221–31. https://doi.org/10.1038/nbt1282
Posch AE, Herwig C, Spadiut O (2013) Science-based bioprocess design for filamentous fungi. Trends Biotechnol 31(1):37–44. https://doi.org/10.1016/j.tibtech.2012.10.008
Qin L, Jiang X, Dong Z, Huang J, Chen X (2018) Identification of two integration sites in favor of transgene expression in Trichoderma reesei. Biotechnol Biofuels 11:142. https://doi.org/10.1186/s13068-018-1139-3
Rieseberg M, Kasper C, Reardon KF, Scheper T (2001) Flow cytometry in biotechnology. Appl Microbiol Biotechnol 56(3–4):350–360. https://doi.org/10.1007/s002530100673
Schmoll M, Zeilinger S (2021) Resistance marker- and gene gun-mediated transformation of Trichoderma reesei. Methods Mol Biol 2234:55–62. https://doi.org/10.1007/978-1-0716-1048-0_4
Schuetze T, Meyer V (2017) Polycistronic gene expression in Aspergillus niger. Microb Cell Fact 16(1):162. https://doi.org/10.1186/s12934-017-0780-z
Singh B (2016) Myceliophthorathermophila syn. Sporotrichum thermophile: a thermophilic mould of biotechnological potential. Crit Rev Biotechnol 36:159–69. https://doi.org/10.3109/07388551.2014.923985
Song R, Zhai Q, Sun L, Huang E, Zhang Y, Zhu Y, Guo Q, Tian Y, Zhao B, Lu H (2019) CRISPR/Cas9 genome editing technology in filamentous fungi: progress and perspective. Appl Microbiol Biotechnol 103(17):6919–6932. https://doi.org/10.1007/s00253-019-10007-w
Subramanian V, Schuster LA, Moore KT, Taylor LE 2nd, Baker JO, Vander Wall TA, Linger JG, Himmel ME, Decker SR (2017) A versatile 2A peptide-based bicistronic protein expressing platform for the industrial cellulase producing fungus. Trichoderma Reesei Biotechnol Biofuels 10:34. https://doi.org/10.1186/s13068-017-0710-7
Suntar I, Cetinkaya S, Haydaroglu US, Habtemariam S (2021) Bioproduction process of natural products and biopharmaceuticals: biotechnological aspects. Biotechnol Adv 50:107768. https://doi.org/10.1016/j.biotechadv.2021.107768
Tracy BP, Gaida SM, Papoutsakis ET (2010) Flow cytometry for bacteria: enabling metabolic engineering, synthetic biology and the elucidation of complex phenotypes. Curr Opin Biotech 21(1):85–99. https://doi.org/10.1016/j.copbio.2010.02.006
Tisch D, Pomraning KR, Collett JR, Freitag M, Baker SE, Chen CL, Hsu PW, Chuang YC, Schuster A, Dattenbock C, Stappler E, Sulyok M, Bohmdorfer S, Oberlerchner J, Wang TF, Schmoll M (2017) Omics analyses of Trichoderma reesei CBS999.97 and QM6a indicate the relevance of female fertility to carbohydrate-active enzyme and transporter levels. Appl Environ Microbiol 83(22):e01578-17. https://doi.org/10.1128/AEM.01578-17
Turgeon BG, Condon B, Liu J, Zhang N (2010) Protoplast transformation of filamentous fungi. Methods Mol Biol 638:3–19. https://doi.org/10.1007/978-1-60761-611-5_1
Veiter L, Herwig C (2019) The filamentous fungus Penicillium chrysogenum analysed via flow cytometry-a fast and statistically sound insight into morphology and viability. Appl Microbiol Biotechnol 103(16):6725–6735. https://doi.org/10.1007/s00253-019-09943-4
Vlaardingerbroek I, Beerens B, Shahi S, Rep M (2015) Fluorescence assisted selection of transformants (FAST): using flow cytometry to select fungal transformants. Fungal Genet Biol 76:104–109. https://doi.org/10.1016/j.fgb.2015.02.003
Vogel HJ (1956) A convenient growth medium for Neurospora (Medium N). Microbiol Genet Bull 13:42–43
Wang G, Jia W, Chen N, Zhang K, Wang L, Lv P, He R, Wang M, Zhang D (2018) A GFP-fusion coupling FACS platform for advancing the metabolic engineering of filamentous fungi. Biotechnol Biofuels 11:232. https://doi.org/10.1186/s13068-018-1223-8
Wang S, Chen H, Tang X, Zhang H, Chen W, Chen YQ (2017) Molecular tools for gene manipulation in filamentous fungi. Appl Microbiol Biotechnol 101(22):8063–8075. https://doi.org/10.1007/s00253-017-8486-z
Ward OP (2012) Production of recombinant proteins by filamentous fungi. Biotechnol Adv 30(5):1119–1139. https://doi.org/10.1016/j.biotechadv.2011.09.012
Wösten HAB (2019) Filamentous fungi for the production of enzymes, chemicals and materials. Curr Opin Biotechnol 59:65–70. https://doi.org/10.1016/j.copbio.2019.02.010
Xu G, Li J, Liu Q, Sun W, Jiang M, Tian C (2018) Transcriptional analysis of Myceliophthora thermophila on soluble starch and role of regulator AmyR on polysaccharide degradation. Bioresour Technol 265:558–562. https://doi.org/10.1016/j.biortech.2018.05.086
Zhang C, Meng X, Wei X, Lu L (2016) Highly efficient CRISPR mutagenesis by microhomology-mediated end joining in Aspergillus fumigatus. Fungal Genet Biol 86:47–57. https://doi.org/10.1016/j.fgb.2015.12.007
Zheng F, Cao Y, Lv X, Wang L, Li C, Zhang W, Chen G, Liu W (2017) A copper-responsive promoter replacement system to investigate gene functions in Trichoderma reesei: a case study in characterizing SAGA genes. Appl Microbiol Biotechnol 101(5):2067–2078. https://doi.org/10.1007/s00253-016-8036-0
Zou G, Shi S, Jiang Y, van den Brink J, de Vries RP, Chen L, Zhang J, Ma L, Wang C, Zhou Z (2012) Construction of a cellulase hyper-expression system in Trichoderma reesei by promoter and enzyme engineering. Microb Cell Fact 11:21. https://doi.org/10.1186/1475-2859-11-21
Zou G, Xiao M, Chai S, Zhu Z, Wang Y, Zhou Z (2020) Efficient genome editing in filamentous fungi via an improved CRISPR-Cas9 ribonucleoprotein method facilitated by chemical reagents. Microb Biotechnol. https://doi.org/10.1111/1751-7915.13652
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This study was supported financially by the National Key Research & Developmental Program of China (2018YFA0900500), the National Natural Science Foundation of China (31771386, 31972878, and 31972879), Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project (Grant No. TSBICIP-KJGG-006), and Youth Innovation Promotion Association of the Chinese Academy of Sciences (Grant No. 2019180). National Major Science and Technology Projects of China,2018YFA0900500,Chao-Guang Tian,National Natural Science Foundation of China,31771386,Qian Liu,31972878,Qian Liu,31972879,Chao-Guang Tian,Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project,TSBICIP-KJGG-006,Chao-Guang Tian,Youth Innovation Promotion Association of the Chinese Academy of Sciences,2019180,Qian Liu
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QL and CGT designed the project. YJY, YL, QL, DDL, WZG, and LXW carried out the experiments. QL, XJW, HXL, and YY analyzed the data. QL and CGT wrote the manuscript. All authors read and approved the final manuscript. All authors contributed to read and approved the manuscript.
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Yang, YJ., Liu, Y., Liu, DD. et al. Development of a flow cytometry-based plating-free system for strain engineering in industrial fungi. Appl Microbiol Biotechnol 106, 713–727 (2022). https://doi.org/10.1007/s00253-021-11733-w
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DOI: https://doi.org/10.1007/s00253-021-11733-w