Display of Both N- and C-terminal Target Fusion Proteins on the Aspergillus Oryzae Cell Surface Using a Chitin-binding Module

Overview

Journal Appl Microbiol Biotechnol

Specialties Biomedical Engineering
Biotechnology
Microbiology

Date 2010 May 26

PMID 20499230

Citations 7

Authors

Soichiro Tabuchi

Junji Ito

Takashi Adachi

Hiroki Ishida

Yoji Hata

Fumiyoshi Okazaki

Tsutomu Tanaka

Chiaki Ogino

Akihiko Kondo

Affiliations

Soon will be listed here.

Abstract

A novel cell surface display system in Aspergillus oryzae was established by using a chitin-binding module (CBM) from Saccharomyces cerevisiae as an anchor protein. CBM was fused to the N or C terminus of green fluorescent protein (GFP) and the fusion proteins (GFP-CBM and CBM-GFP) were expressed using A. oryzae as a host. Western blotting and fluorescence microscopy analysis showed that both GFP-CBM and CBM-GFP were successfully expressed on the cell surface. In addition, cell surface display of triacylglycerol lipase from A. oryzae (tglA), while retaining its activity, was also successfully demonstrated using CBM as an anchor protein. The activity of tglA was significantly higher when tglA was fused to the C terminus than N terminus of CBM. Together, these results show that CBM used as a first anchor protein enables the fusion of both the N and/or C terminus of a target protein.

Citing Articles

Identification and Immobilization of an Invertase With High Specific Activity and Sucrose Tolerance Ability of sp. w5 for High Fructose Syrup Preparation.

Zhou G, Peng C, Liu X, Chang F, Xiao Y, Liu J Front Microbiol. 2020; 11:633.

PMID: 32328053 PMC: 7160231. DOI: 10.3389/fmicb.2020.00633.

Constitutive expression and cell-surface display of a bacterial β-mannanase in Lactobacillus plantarum.

Nguyen H, Pham M, Stelzer E, Plattner E, Grabherr R, Mathiesen G Microb Cell Fact. 2019; 18(1):76.

PMID: 31023309 PMC: 6482533. DOI: 10.1186/s12934-019-1124-y.

Engineering E. coli cell surface in order to develop a one-step purification method for recombinant proteins.

Fasehee H, Rostami A, Ramezani F, Ahmadian G AMB Express. 2018; 8(1):107.

PMID: 29961206 PMC: 6026487. DOI: 10.1186/s13568-018-0638-8.

Display of a β-mannanase and a chitosanase on the cell surface of Lactobacillus plantarum towards the development of whole-cell biocatalysts.

Nguyen H, Mathiesen G, Stelzer E, Pham M, Kuczkowska K, Mackenzie A Microb Cell Fact. 2016; 15(1):169.

PMID: 27716231 PMC: 5050953. DOI: 10.1186/s12934-016-0570-z.

Arming Technology in Yeast-Novel Strategy for Whole-cell Biocatalyst and Protein Engineering.

Kuroda K, Ueda M Biomolecules. 2014; 3(3):632-50.

PMID: 24970185 PMC: 4030959. DOI: 10.3390/biom3030632.

References

Hansson M, Samuelson P, Gunneriusson E, Stahl S . Surface display on gram positive bacteria. Comb Chem High Throughput Screen. 2001; 4(2):171-84. DOI: 10.2174/1386207013331183. View

Kotaka A, Bando H, Kaya M, Kato-Murai M, Kuroda K, Sahara H . Direct ethanol production from barley beta-glucan by sake yeast displaying Aspergillus oryzae beta-glucosidase and endoglucanase. J Biosci Bioeng. 2008; 105(6):622-7. DOI: 10.1263/jbb.105.622. View

Murai T, Ueda M, Yamamura M, Atomi H, Shibasaki Y, Kamasawa N . Construction of a starch-utilizing yeast by cell surface engineering. Appl Environ Microbiol. 1997; 63(4):1362-6. PMC: 168429. DOI: 10.1128/aem.63.4.1362-1366.1997. View

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(6):373-9. DOI: 10.1007/s002940000118. View

Okano K, Zhang Q, Kimura S, Narita J, Tanaka T, Fukuda H . System using tandem repeats of the cA peptidoglycan-binding domain from Lactococcus lactis for display of both N- and C-terminal fusions on cell surfaces of lactic acid bacteria. Appl Environ Microbiol. 2007; 74(4):1117-23. PMC: 2258587. DOI: 10.1128/AEM.02012-07. View

Hama S, Tamalampudi S, Shindo N, Numata T, Yamaji H, Fukuda H . Role of N-terminal 28-amino-acid region of Rhizopus oryzae lipase in directing proteins to secretory pathway of Aspergillus oryzae. Appl Microbiol Biotechnol. 2008; 79(6):1009-18. DOI: 10.1007/s00253-008-1502-6. View

Kitamoto K . Molecular biology of the Koji molds. Adv Appl Microbiol. 2002; 51:129-53. DOI: 10.1016/s0065-2164(02)51004-2. View

Raha A, Varma N, Yusoff K, Ross E, Foo H . Cell surface display system for Lactococcus lactis: a novel development for oral vaccine. Appl Microbiol Biotechnol. 2005; 68(1):75-81. DOI: 10.1007/s00253-004-1851-8. View

Boder E, Wittrup K . Yeast surface display for screening combinatorial polypeptide libraries. Nat Biotechnol. 1997; 15(6):553-7. DOI: 10.1038/nbt0697-553. View

10.

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(9):1849-57. DOI: 10.1271/bbb.68.1849. View

11.

Washida M, Takahashi S, Ueda M, Tanaka A . Spacer-mediated display of active lipase on the yeast cell surface. Appl Microbiol Biotechnol. 2001; 56(5-6):681-6. DOI: 10.1007/s002530100718. View

12.

Tateno T, Fukuda H, Kondo A . Production of L-Lysine from starch by Corynebacterium glutamicum displaying alpha-amylase on its cell surface. Appl Microbiol Biotechnol. 2007; 74(6):1213-20. DOI: 10.1007/s00253-006-0766-y. View

13.

Machida M, Asai K, Sano M, Tanaka T, Kumagai T, Terai G . Genome sequencing and analysis of Aspergillus oryzae. Nature. 2005; 438(7071):1157-61. DOI: 10.1038/nature04300. View

14.

Toida J, Fukuzawa M, Kobayashi G, Ito K, Sekiguchi J . Cloning and sequencing of the triacylglycerol lipase gene of Aspergillus oryzae and its expression in Escherichia coli. FEMS Microbiol Lett. 2000; 189(2):159-64. DOI: 10.1111/j.1574-6968.2000.tb09223.x. View

15.

Higuchi Y, Shoji J, Arioka M, Kitamoto K . Endocytosis is crucial for cell polarity and apical membrane recycling in the filamentous fungus Aspergillus oryzae. Eukaryot Cell. 2008; 8(1):37-46. PMC: 2620746. DOI: 10.1128/EC.00207-08. View

16.

Shigechi H, Koh J, Fujita Y, Matsumoto T, Bito Y, Ueda M . Direct production of ethanol from raw corn starch via fermentation by use of a novel surface-engineered yeast strain codisplaying glucoamylase and alpha-amylase. Appl Environ Microbiol. 2004; 70(8):5037-40. PMC: 492352. DOI: 10.1128/AEM.70.8.5037-5040.2004. View

17.

Akao T, Sano M, Yamada O, Akeno T, Fujii K, Goto K . Analysis of expressed sequence tags from the fungus Aspergillus oryzae cultured under different conditions. DNA Res. 2007; 14(2):47-57. PMC: 2779895. DOI: 10.1093/dnares/dsm008. View

18.

Satoh E, Niimura Y, Uchimura T, Kozaki M, Komagata K . Molecular cloning and expression of two alpha-amylase genes from Streptococcus bovis 148 in Escherichia coli. Appl Environ Microbiol. 1993; 59(11):3669-73. PMC: 182515. DOI: 10.1128/aem.59.11.3669-3673.1993. View

19.

Hama S, Tamalampudi S, Fukumizu T, Miura K, Yamaji H, Kondo A . Lipase localization in Rhizopus oryzae cells immobilized within biomass support particles for use as whole-cell biocatalysts in biodiesel-fuel production. J Biosci Bioeng. 2006; 101(4):328-33. DOI: 10.1263/jbb.101.328. View

20.

Lee S, Choi J, Xu Z . Microbial cell-surface display. Trends Biotechnol. 2002; 21(1):45-52. DOI: 10.1016/s0167-7799(02)00006-9. View