Transcriptomic analysis of Aspergillus niger strains reveals the mechanism underlying high citric acid productivity

Abstract

Background

Aspergillus niger is a highly important industrial microorganism because of its amazing capacity to produce citric acid (CA). To explore the metabolic mechanism and physiological phenotype associated with high CA productivity, the transcriptomes of high CA-producing A. niger YX-1217 and degenerative strain YX-1217G were investigated using A. niger ATCC1015 as a control.

Results

These strains showed distinct transcriptional differences in CA production. By contrast, the genes encoding glycoside hydrolases, aspartyl endoproteases, and carboxypeptidases were unusually upregulated in CA-producing strain YX-1217, which involved the carbohydrate hydrolysis and polypeptide degradation pathways, and should be related to its powerful capacity to utilize cornmeal fluidified liquid as raw material for the production of CA. In central metabolism of YX-1217, gene 9.735.1, which encodes glyceraldehyde 3-phosphate dehydrogenase, and two transcriptionally outstanding genes, 6000119 (An15g01920) and 3.2152.1 (An08g10920) that encode citrate synthase, were upregulated, thereby ensuring CA accumulation. In addition, a relatively strong electron transport chain, a regeneration system for NAD⁺/NADP⁺, and an efficient resistance mechanism may have contributed to the high CA production rate of YX-1217.

Conclusions

These comparisons have shed light on the mechanism underlying high CA yield in A. niger YX-1217 as well as provide insights into the development of novel strains that produce other organic acids.

Keywords

Transcriptome / Aspergillus niger / Citric acid / Metabolism

References

[1] Adav SS, Li AA, Manavalan A, Punt P, Sze SK. Quantitative iTRAQ secretome analysis of Aspergillus niger reveals novel hydrolytic enzymes. J Proteome Res, 2010, 9: 3932-3940.

[2] Alsheikh H, Watson AJ, Lacey GA, Punt PJ, MacKenzie DJ, Jeenes DJ, Pakula T, Penttilä M, Alcocer MJC, Archer DB. Endoplasmic reticulum stress leads to the selective transcriptional downregulation of the glucoamylase gene in Aspergillus niger. Mol Microbiol, 2004, 53: 1731-1742.

[3] Andersen MR, Nielsen ML, J Nielsen. Metabolic model integration of the bibliome, genome, metabolome and reactome of Aspergillus niger. Mol Syst Biol, 2008, 4: 178.

[4] Andersen MR, Lehmann L, Nielsen J. Systemic analysis of the response of Aspergillus niger to ambient pH. Genome Biol, 2009, 10: R47.

[5] Andersen MR, Salazar MP, Schaap PJ, . Comparative genomics of citric-acid-producing Aspergillus niger ATCC 1015 versus enzyme-producing CBS 513.88. Genome Res, 2011, 21: 885-897.

[6] Augustine GJ, Charlton MP, Smith SJ. Calcium action in synaptic transmitter release. Annu Rev Neurosci, 1987, 10: 633-693.

[7] de Oliveira JM, van Passel MW, Schaap PJ, de Graaff LH. Proteomic analysis of the secretory response of Aspergillus niger to d-maltose and d-xylose. PLoS ONE, 2011, 6: e20865.

[8] Devasagayam T, Tilak JC, Boloor KK, Sane KS, Ghaskadbi SS, Lele RD. Free radicals and antioxidants in human health: current status and future prospects. JAPI, 2004, 52: 794-804.

[9] Fan FY, Ma GL, Li JG, Liu Q, Benz JP, Tian CG, Ma YH. Genome-wide analysis of the endoplasmic reticulum stress response during lignocellulase production in Neurospora crassa. Biotechnol Biofuels, 2015, 8: 1-17.

[10] Fountain JC, Bajaj P, Pandey M, Nayak SN, Yang L, Kumar V, Jayale AS, Citikineni A, Zhang W, Scully BT, Lee RD, Kemerait RC, Varshney RK, Guo B. Oxidative stress and carbon metabolism influence aspergillus flavus transcriptome composition and secondary metabolite production. Sci Rep, 2016, 6: 38747.

[11] Ghulam M, Aisha T, Muhammad A, Mehboob-Ur R, Amer J. Comparative sequence analysis of citrate synthase and 18s ribosomal DNA from a wild and mutant strains of Aspergillus niger with various fungi. Bioinformation, 2014, 10: 1-7.

[12] Haq I-UL, Ali S, Qadeer MA. Influence of dissolved oxygen concentration on intracellular pH for regulation of Aspergillus niger growth rate during citric acid fermentation in a stirred tank bioreactor. Int J Biol Sci, 2005, 1(1): 34-41.

[13] Janz R, Hofmann K, Südhof TC. Svop, an evolutionarily conserved synaptic vesicle protein, suggests novel transport functions of synaptic vesicles. J Neurosci, 1998, 18: 9269-9281.

[14] Jørgensen TR, Goosen T, van den Hondel C, Lversen JJL. Transcriptomic comparison of Aspergillus niger growing on two different sugars reveals coordinated regulation of the secretory pathway. BMC Genomics, 2009, 10: 44.

[15] Jørgensen TR, Nitsche BM, Lamers GE, Arentshorst M, Ca VDH, Ram AF. Transcriptomic insights into the physiology of Aspergillus niger approaching a specific growth rate of zero. Appl Environ Microbiol, 2010, 76: 5344-5355.

[16] Krijgsheld P, Bleichrodt R, vanVeluw GJ, Wang F, Müller WH, Dijksterhuis J, Wösten HAB. Development in Aspergillus. Stud Mycol, 2013, 74: 1-29.

[17] Kroneck PMH, Torres MES (2015) Sustaining life on planet earth: metalloenzymes mastering dioxygen and other chewy gases. Metal ions in life sciences, vol 15, pp 1–12

[18] Nitsche BM, Jørgensen TR, Akeroyd M, Meyer V, Ram AFJ. The carbon starvation response of Aspergillus niger during submerged cultivation. BMC Genomics, 2012, 13: 318.

[19] Novodvorska M, Hayer K, Pullan ST, Wilson R, Blythe MJ, Stam H, Stratford M, Archer DB. Trancriptional landscape of Aspergillus niger at breaking of conidial dormancy revealed by RNA-sequencing. BMC Genomics, 2013, 14: 1-18.

[20] Pel HJ, de Winde JH, Archer DB, . Genome sequencing and analysis of the versatile cell factory Aspergillus niger CBS 513.88. Nat Biotechnol, 2007, 25: 221-231.

[21] Peñalva MA, Arst HN Jr. Regulation of gene expression by ambient pH in filamentous fungi and yeasts. Microbiol Mol Biol Rev, 2002, 66: 426-446.

[22] Ruijter GJ, Panneman H, Visser J. Overexpression of phosphofructokinase and pyruvate kinase in citric acid-producing Aspergillus niger. Biochem Biophys Acta, 1997, 1334: 317-326.

[23] Ruijter GJG, Panneman H, Xu DB, Visser J. Properties of Aspergillus niger citrate synthase and effects of cita overexpression on citric acid production. FEMS Microbiol Lett, 2000, 184: 35-40.

[24] Steiger MG, Mach RL, Mach-Aigner AR. An accurate normalization strategy for RT-qPCR in Hypocrea jecorina (Trichoderma reesei). J Biotechnol, 2009, 145: 30-37.

[25] Sun JB, Lu X, Rinas U, Zeng AP. Metabolic peculiarities of Aspergillus niger disclosed by comparative metabolic genomics. Genome Biol, 2007, 8: R182.

[26] Tarze A, Deniaud A, Bras ML, Maillier E, Molle D, Larochette N, Zamzami N, Jan G, Kroemer G, Brenner C. GAPDH, a novel regulator of the pro-apoptotic mitochondrial membrane permeabilization. Oncogene, 2007, 26: 2606-2620.

[27] van Leeuwen MRV, Krijgsheld P, Bleichrodt R, Menke H, Stam H, Stark J, Wösten HAB, Dijksterhuis J. Germination of conidia of Aspergillus niger, is accompanied by major changes in RNA profiles. Stud Mycol, 2013, 74: 59-70.

[28] Waditee R, Hossain GS, Tanaka Y, Nakamura T, Shikata M, Takano J, Takabe T, Takabe T. Isolation and functional characterization of Ca2+/H+ antiporters from cyanobacteria. J Biol Chem, 2004, 279: 4330-4338.

[29] Wang B, Guo GW, Wang C, Lin Y, Wang XN, Zhao MM, GuoY He MH, Zhang Y, Pan L. Survey of the transcriptome of Aspergillus oryzae via massively parallel mRNA sequencing. Nuleic Acids Res, 2010, 38: 5075-5087.

[30] Wang ZL, Zhang LB, Ying SH, Feng MG. Catalases play differentiated roles in the adaptation of a fungal entomopathogen to environmental stresses. Environ Microbiol, 2013, 15: 409-418.

[31] Xie XQ, Wang J, Huang BF, Ying SH, Feng MG. A new manganese superoxide dismutase identified from Beauveria bassiana enhances virulence and stress tolerance when overexpressed in the fungal pathogen. Appl Microbiol Biotechnol, 2010, 86: 1543-1553.

[32] Xie XQ, Ying SH, Feng MG. Characterization of a new Cu/Zn-superoxide dismutase from Beauveria bassiana and two site-directed mutations crucial to its antioxidation activity without chaperon. Enzyme Microbiol Technol, 2010, 46: 217-222.

[33] Yin WB, Reinke AW, Szilágyi M, Emri T, Chiang YM, Keating AE, Pócsi I, Wang CCC, Keller NP. bZIP transcription factors affecting secondary metabolism, sexual development and stress responses in Aspergillus nidulans. Microbiol, 2013, 159: 77-88.

[34] Yuan XL, Kaaij RMVD, Cees AMJJ, van den Hondel Punt PJ, van der Marc JEC, Maarel Dijkhuizen L, Ram AFJ. Aspergillus niger, genome-wide analysis reveals a large number of novel alpha-glucan acting enzymes with unexpected expression profiles. Mol Genet Genomics, 2008, 279: 545-561.

[35] Zhang R, Zhao M, Ji HJ, Yuan YH, Chen NH. Study on the dynamic changes in synaptic vesicle-associated protein and axonal transport protein combined with LPS neuroinflammation model. ISRN Neurol, 2013, 2: 496079.

Funding

the National Special Fund for State Key Laboratory of Bioreactor Engineering