Abstract
Neurospora crassa is a filamentous fungus potent in secreting cellulase and degrading lignocellulosic materials. Here, heterologous cis-aconitic acid decarboxylase was expressed in N. crassa to synthesize itaconic acid which is a high potential platform chemical with applications as an alternative for petroleum-based products. The present study demonstrated that itaconic acid can be produced directly from cellulose and other lignocellulosic materials by the engineered strain, with the highest production of 20.414 ± 0.674 mg/L. The multivariate data analysis methods were used in the parameter analysis of the conversion process. It was found by the hot map analysis that itaconic acid production can promote the secretion of cellulase in N. crassa. Principal component analysis suggested that itaconic acid production was closely related to the concentration of the glucose degraded from lignocelluloses, indicating that the secretion of cellulase is key to the direct conversion of cellulose to itaconic acid in the engineered N. crassa. This work demonstrates that N. crassa could be considered as a new platform in the application of cellulose conversion to itaconic acid.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Abdi H, Williams LJ (2010) Principal component analysis. Comput Stat 2(4):433–459. https://doi.org/10.1002/wics.101
Benz JP, Chau BH, Zheng D, Bauer S, Glass NL, Somerville CR (2014) A comparative systems analysis of polysaccharide-elicited responses in Neurospora crassa reveals carbon source-specific cellular adaptations. Mol Microbiol 91(2):275–299. https://doi.org/10.1111/mmi.12459
Blazeck J, Miller J, Pan A, Gengler J, Holden C, Jamoussi M, Alper HS (2014) Metabolic engineering of Saccharomyces cerevisiae for itaconic acid production. Appl Microbiol Biotechnol 98(19):8155–8164. https://doi.org/10.1007/s00253-014-5895-0
Blazeck J, Hill A, Jamoussi M, Pan A, Miller J, Alper HS (2015) Metabolic engineering of Yarrowia lipolytica for itaconic acid production. Metab Eng 32:66–73. https://doi.org/10.1016/j.ymben.2015.09.005
Coradetti ST, Craig JP, Xiong Y, Shock T, Tian C, Glass NL (2012) Conserved and essential transcription factors for cellulase gene expression in ascomycete fungi. Proc Natl Acad Sci U S A 109(19):7397–7402. https://doi.org/10.1073/pnas.1200785109
Fang H, Xia L (2013) High activity cellulase production by recombinant Trichoderma reesei ZU-02 with the enhanced cellobiohydrolase production. Bioresour Technol 144:693–697. https://doi.org/10.1016/j.biortech.2013.06.120
Fang H, Zhao C, Song XY (2010) Optimization of enzymatic hydrolysis of steam-exploded corn stover by two approaches: response surface methodology or using cellulase from mixed cultures of Trichoderma reesei RUT-C30 and Aspergillus niger NL02. Bioresour Technol 101(11):4111–4119. https://doi.org/10.1016/j.biortech.2010.01.078
Fang H, Zhao C, Chen S (2016a) Single cell oil production by Mortierella isabellina from steam exploded corn stover degraded by three-stage enzymatic hydrolysis in the context of on-site enzyme production. Bioresour Technol 216:988–995. https://doi.org/10.1016/j.biortech.2016.06.051
Fang H, Zhao C, Kong Q, Zou Z, Chen N (2016b) Comprehensive utilization and conversion of lignocellulosic biomass for the production of long chain α,ω-dicarboxylic acids. Energy 116:177–189. https://doi.org/10.1016/j.energy.2016.09.110
Galbe M, Zacchi G (2012) Pretreatment: the key to efficient utilization of lignocellulosic materials. Biomass Bioenergy 46:70–78. https://doi.org/10.1016/j.biombioe.2012.03.026
Ghose TK (1987) Measurement of cellulase activities. Pure Appl Chem 59(2):257–268. https://doi.org/10.1351/pac198759020257
Glass NL, Schmoll M, Cate JH, Coradetti S (2013) Plant cell wall deconstruction by ascomycete fungi. Annu Rev Microbiol 67:477–498. https://doi.org/10.1146/annurev-micro-092611-150044
Harder BJ, Bettenbrock K, Klamt S (2016) Model-based metabolic engineering enables high yield itaconic acid production by Escherichia coli. Metab Eng 38:29–37. https://doi.org/10.1016/j.ymben.2016.05.008
Hevekerl A, Kuenz A, Vorlop KD (2014) Influence of the pH on the itaconic acid production with Aspergillus terreus. Appl Microbiol Biotechnol 98(24):10005–10012. https://doi.org/10.1007/s00253-014-6047-2
Hildebrand A, Kasuga T, Fan Z (2015) Production of cellobionate from cellulose using an engineered Neurospora crassa strain with laccase and redox mediator addition. PLoS One 10(4):e0123006. https://doi.org/10.1371/journal.pone.0123006
Horton P, Park K, Obayashi T, Nakai K (2006) Protein subcellular localization prediction with WoLF PSORT. In: Proceedings of the 4th Annual Asia Pacific Bioinformatics Conferece APBC06 Taipei:39–48
Huberman LB, Coradetti ST, Glass NL (2017) Network of nutrient-sensing pathways and a conserved kinase cascade integrate osmolarity and carbon sensing in Neurospora crassa. Proc Natl Acad Sci U S A 114(41):E8665–E8674. https://doi.org/10.1073/pnas.1707713114
Jimenez-Quero A, Pollet E, Zhao M, Marchioni E, Averous L, Phalip V (2016) Fungal fermentation of lignocellulosic biomass for itaconic and fumaric acid production. J Microbiol Biotechnol 27:1–8. https://doi.org/10.4014/jmb.1607.07057
Klement T, Büchs J (2013) Itaconic acid – a biotechnological process in change. Bioresour Technol 135:422–431. https://doi.org/10.1016/j.biortech.2012.11.141
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 U S A 106(38):16151–16156. https://doi.org/10.1073/pnas.0905848106
Margolin BS, Freitag M, Selker EU (1997) Improved plasmids for gene targeting at the his-3 locus of Neurospora crassa by electroporation. Fungal Genet Newslett 44:34–36
McNally MT, Free SJ (1988) Isolation and characterization of a Neurospora glucose-repressible gene. Curr Genet 14(6):545–551. https://doi.org/10.1007/bf00434079
Nevalainen H, Peterson R (2014) Making recombinant proteins in filamentous fungi- are we expecting too much? Front Microbiol 5:75. https://doi.org/10.3389/fmicb.2014.00075
Okabe M, Lies D, Kanamasa S, Park EY (2009) Biotechnological production of itaconic acid and its biosynthesis in Aspergillus terreus. Appl Microbiol Biotechnol 84(4):597–606. https://doi.org/10.1007/s00253-009-2132-3
Phillips CM, Beeson WT, Cate JH, Marletta MA (2011) Cellobiose dehydrogenase and a copper-dependent polysaccharide monooxygenase potentiate cellulose degradation by Neurospora crassa. ACS Chem Biol 6(12):1399–1406. https://doi.org/10.1021/cb200351y
Reilly MC, Qin L, Craig JP, Starr TL, Glass NL (2015) Deletion of homologs of the SREBP pathway results in hyper-production of cellulases in Neurospora crassa and Trichoderma reesei. Biotechnology for Biofuels 8:121. https://doi.org/10.1186/s13068-015-0297-9
Roche CM, Glass NL, Blanch HW, Clark DS (2014a) Engineering the filamentous fungus Neurospora crassa for lipid production from lignocellulosic biomass. Biotechnol Bioeng 111(6):1097–1107. https://doi.org/10.1002/bit.25211
Roche CM, Loros JJ, McCluskey K, Glass NL (2014b) Neurospora crassa: looking back and looking forward at a model microbe. Am J Bot 101(12):2022–2035. https://doi.org/10.3732/ajb.1400377
Schmoll M, Dattenböck C (2016) Gene expression systems in fungi: advancements and applications. Fungal Biology Springer International Publishing. https://doi.org/10.1007/978-3-319-27951-0
Smart KF, Aggio RB, Van Houtte JR, Villas-Boas SG (2010) Analytical platform for metabolome analysis of microbial cells using methyl chloroformate derivatization followed by gas chromatography-mass spectrometry. Nat Protoc 5(10):1709–1729. https://doi.org/10.1038/nprot.2010.108
Vogel HJ (1956) A convenient growth medium for Neurospora (medium N). Microb Genet Bull 13:42–43
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
Werpy TA, Holladay JE, White JF (2004) Results of Screening for Potential Candidates from Sugars and Synthesis Gas. In: Top Value Added Chemicals from Biomass I Pacific Northwest National Lab (PNNL), Richland, WA (United States). https://doi.org/10.2172/926125
Xiong Y, Coradetti ST, Li X, Gritsenko MA, Clauss T, Petyuk V, Camp D, Smith R, Cate JHD, Yang F, Glass NL (2014) The proteome and phosphoproteome of Neurospora crassa in response to cellulose, sucrose and carbon starvation. Fungal Genet Biol 72:21–33. https://doi.org/10.1016/j.fgb.2014.05.005
Zhao C, Deng L, Fang H, Chen S (2017) Microbial oil production by Mortierella isabellina from corn stover under different pretreatments. RSC Adv 7(89):56239–56246. https://doi.org/10.1039/c7ra11900c
Znameroski EA, Glass NL (2013) Using a model filamentous fungus to unravel mechanisms of lignocellulose deconstruction. Biotechnol Biofuels 6(1):6. https://doi.org/10.1186/1754-6834-6-6
Znameroski EA, Coradetti ST, Roche CM, Tsai JC, Iavarone AT, Cate JH, Glass NL (2012) Induction of lignocellulose-degrading enzymes in Neurospora crassa by cellodextrins. Proc Natl Acad Sci U S A 109(16):6012–6017. https://doi.org/10.1073/pnas.1118440109
Funding
This work was financially supported by the Youth Fund from Jiangsu Province (grant number BK20150130), the Youth Fund from Shaanxi Province (grant number 2018JQ2022), the second-class General Financial Grant from the China Postdoctoral Science Foundation (grant number 2017M613213), the Fund for Doctoral Scientific Research (grant number Z109021632), and the Start-up Fund for Talent Introduction (grant number Z111021602) from Northwest A&F University.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Ethical approval
This article does not contain any studies with human participants or animals performed by any of the authors.
Electronic supplementary material
ESM 1
(PDF 94 kb)
Rights and permissions
About this article
Cite this article
Zhao, C., Chen, S. & Fang, H. Consolidated bioprocessing of lignocellulosic biomass to itaconic acid by metabolically engineering Neurospora crassa. Appl Microbiol Biotechnol 102, 9577–9584 (2018). https://doi.org/10.1007/s00253-018-9362-1
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-018-9362-1