Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Development of Miniaturized Culture Systems for Large Screening of Mycelial Fungal Cells of Aspergillus terreus Producing Itaconic Acid

  • Shin, Woo-Shik (Green Materials and Process R&BD Group, Korea Institute of Industrial Technology) ;
  • Lee, Dohoon (Green Materials and Process R&BD Group, Korea Institute of Industrial Technology) ;
  • Kim, Sangyong (Green Materials and Process R&BD Group, Korea Institute of Industrial Technology) ;
  • Jeong, Yong-Seob (Department of Food Science and Technology, Chonbuk National University) ;
  • Chun, Gie-Taek (College of Biomedical Science, Kangwon National University)
  • Received : 2016.10.13
  • Accepted : 2016.11.04
  • Published : 2017.01.28
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Abstract

The task of improving a fungal strain is highly time-consuming due to the requirement of a large number of flasks in order to obtain a library with enough diversity. In addition, fermentations (particularly those for fungal cells) are typically performed in high-volume (100-250 ml) shake-flasks. In this study, for large and rapid screening of itaconic acid (IA) high-yielding mutants of Aspergillus terreus, a miniaturized culture method was developed using 12-well and 24-well microtiter plates (MTPs, working volume = 1-2 ml). These miniaturized MTP fermentations were successful, only when highly filamentous forms were induced in the growth cultures. Under these conditions, loose-pelleted morphologies of optimum sizes (less than 0.5 mm in diameter) were casually induced in the MTP production cultures, which turned out to be the prerequisite for the active IA biosynthesis by the mutated strains in the miniaturized fermentations. Another crucial factor for successful MTP fermentation was to supply an optimal amount of dissolved oxygen into the fermentation broth through increasing the agitation speed (240 rpm) and reducing the working volume (1 ml) of each 24-well microtiter plate. Notably, almost identical fermentation physiologies resulted in the 250 ml shake-flasks, as well as in the 12-well and 24-well MTP cultures conducted under the respective optimum conditions, as expressed in terms of the distribution of IA productivity of each mutant. These results reveal that MTP cultures could be considered as viable alternatives for the labor-intensive shake-flask fermentations even for filamentous fungal cells, leading to the rapid development of IA high-yield mutant strains.

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References

  1. Tevz G, Bencina M, Legisa M. 2010. Enhancing itaconic acid production by Aspergillus terreus. Appl. Microbiol. Biotechnol. 87: 1657-1664. https://doi.org/10.1007/s00253-010-2642-z
  2. Willke T, Vorlop KD. 2001. Biotechnological production of itaconic acid. Appl. Microbiol. Biotechnol. 56: 289-295. https://doi.org/10.1007/s002530100685
  3. Tong X, Ma Y, Li Y. 2010. Biomass into chemicals: conversion of sugars to furan derivatives by catalytic processes. Appl. Catal. A Gen. 385: 1-13. https://doi.org/10.1016/j.apcata.2010.06.049
  4. Steiger MG, Blumhoff ML, Mattanovich D, Sauer M. 2013. Biochemistry of microbial itaconic acid production. Front. Microbiol. 4: 23.
  5. Gyamerah M. 1995. Factors affecting the growth form of Aspergillus terreus NRRL 1960 in relation to itaconic acid fermentation. Appl. Microbiol. Biotechnol. 44: 356-361. https://doi.org/10.1007/BF00169929
  6. Alberto F, Navarro D, de Vries RP, Asther M, Record E. 2009. Technical advance in fungal biotechnology: development of a miniaturized culture method and an automated highthroughput screening. Lett. Appl. Microbiol. 49: 278-282. https://doi.org/10.1111/j.1472-765X.2009.02655.x
  7. Fernandes P, Cabral JMS. 2006. Microlitre/millilitre shaken bioreactors in fermentative and biotransformation processes - a review. Biocatal. Biotransformation 24: 237-252. https://doi.org/10.1080/10242420600667684
  8. Studer MH, DeMartini JD, Brethauer S, McKenzie HL, Wyman CE. 2010. Engineering of a high-throughput screening system to identify cellulosic biomass, pretreatments, and enzyme formulations that enhance sugar release. Biotechnol. Bioeng. 105: 231-238. https://doi.org/10.1002/bit.22527
  9. Markert S, Joeris K. 2017. Establishment of a fully automated microtiter plate-based system for suspension cell culture and its application for enhanced process optimization. Biotechnol. Bioeng. 114: 113-121. https://doi.org/10.1002/bit.26044
  10. Betts JI, Baganz F. 2006. Miniature bioreactors: current practices and future opportunities. Microb. Cell Fact. 5: 21. https://doi.org/10.1186/1475-2859-5-21
  11. Archer DB, Connerton IF, MacKenzie DA. 2008. Filamentous fungi for production of food additives and processing aids. Adv. Biochem. Eng. Biotechnol. 111: 99-147.
  12. Doran PM. 1995. Mass transfer, pp. 190-217. In Doran PM(ed.). Bioprocess Engineering Principles. Academic Press, London.
  13. Doran PM. 1995. Heterogeneous reactions, pp. 297-332. In Doran PM (ed.). Bioprocess Engineering Principles. Academic Press, London.
  14. Margaritis A, Zajic JE. 1978. Mixing, mass transfer, and scale-up of polysaccharide fermentations. Biotechnol. Bioeng. 20: 939-1001. https://doi.org/10.1002/bit.260200702
  15. Hong HP. 2010. Enhanced production of polysaccharides through strain improvement and medium optimization in suspended mycelial fermentations of Inonotus obliquus. M.Sc. Thesis. Kangwon National University, Korea.
  16. Song SK. 2006. Development of lovastatin high yielding transformants through introduction of a regulatory gene of lovastatin biosynthesis, and establishment of computercontrolled bioprocess system for enhanced production of lovastatin by immobilized Aspergillus terreus cells. Kangwon National Univerisity, Korea.
  17. Song SK, Jeong YS, Kim PH, Chun GT. 2006. Effects of dissolved oxygen level on avermectin B1a production by Streptomyces avermitilis in computer-controlled bioreactor cultures. J. Microbiol. Biotechnol. 16: 1690-1698.
  18. Shin WS. 2007. Control of morphology in multi-stage bioreactor suspended cultures for the enhanced production of ${\beta}$-D-glucan from fungal mycelia of Phellinus linteus. Kangwon National University, Korea.
  19. Shin WS, Kwon YJ, Jeong YS, Chun GT. 2009. Importance of strain improvement and control of fungal cells morphology for enhanced production of protein-bound polysaccharides (${\beta}$-D-glucan) in suspended cultures of Phellinus linteus mycelia. Kor. Chem. Eng. Res. 47: 220-229.
  20. Gbewonyo K, Wang DI. 1983. Enhancing gas-liquid mass transfer rates in non-newtonian fermentations by confining mycelial growth to microbeads in a bubble column. Biotechnol. Bioeng. 25: 2873-2887. https://doi.org/10.1002/bit.260251206
  21. Cianchetta S, Galletti S, Burzi PL, Cerato C. 2010. A novel microplate-based screening strategy to assess the cellulolytic potential of Trichoderma strains. Biotechnol. Bioeng. 107: 461-468. https://doi.org/10.1002/bit.22816
  22. McConnell SJ, Dinh T, Le MH, Spinella DG. 1999. Biopanning phage display libraries using magnetic beads vs. polystyrene plates. Biotechniques 26: 208-210, 214.
  23. Rowlands RT. 1984. Industrial strain improvement: mutagenesis and random screening procedures. Enzyme Microb. Technol. 6: 3-10. https://doi.org/10.1016/0141-0229(84)90070-X
  24. Zhang X, Yang ST. 2011. High-throughput 3-D cell-based proliferation and cytotoxicity assays for drug screening and bioprocess development. J. Biotechnol. 151: 186-193. https://doi.org/10.1016/j.jbiotec.2010.11.012
  25. Baltz RH, Demain AL, Davies JE. 2010. Manual of Industrial Microbiology and Biotechnology, pp. 103-113. ASM Press, Washington, DC.
  26. Demain AL. 1971. Overproduction of microbial metabolites and enzymes due to alteration of regulation, pp. 113-142. In Ghose TK, Fiechter A (eds.). Advances in Biochemical Engineering, Vol. 1. Springer Berlin-Heidelberg.
  27. Gyamerah MH. 1995. Oxygen requirement and energy relations of itaconic acid fermentation by Aspergillus terreus NRRL 1960. Appl. Microbiol. Biotechnol. 44: 20-26. https://doi.org/10.1007/BF00164475
  28. Crueger W, Crueger A. 1990. Biotechnology: A Textbook of Industrial Microbiology. Sinauer, Sunderland, MA.
  29. Omstead MN, Kaplan L, Buckland BC. 1989. Fermentation development and process improvement, pp. 33-54. In Campbell WC (ed.). Ivermectin and Abamectin. Springer, New York, NY.
  30. Kwon HK. 1999. The effect of morphology on production of lovastatin, cholesterol lowering agent, by Aspergillus terreus. Kangwon National University, Korea.
  31. Levinson WE, Kurtzman CP, Kuo TM. 2006. Production of itaconic acid by Pseudozyma antarctica NRRL Y-7808 under nitrogen-limited growth conditions. Enzyme Microb. Technol. 39: 824-827. https://doi.org/10.1016/j.enzmictec.2006.01.005
  32. John GT, Klimant I, Wittmann C, Heinzle E. 2003. Integrated optical sensing of dissolved oxygen in microtiter plates: a novel tool for microbial cultivation. Biotechnol. Bioeng. 81: 829-836. https://doi.org/10.1002/bit.10534
  33. Shin WS, Lee D, Kim S, Jeong YS, Chun GT. 2013. Application of scale-up criterion of constant oxygen mass transfer coefficient (kLa) for production of itaconic acid in a 50 L pilot-scale fermentor by fungal cells of Aspergillus terreus. J. Microbiol. Biotechnol. 23: 1445-1453. https://doi.org/10.4014/jmb.1307.07084
  34. Li A, Pfelzer N, Zuijderwijk R, Brickwedde A, van Zeijl C, Punt P. 2013. Reduced by-product formation and modified oxygen availability improve itaconic acid production in Aspergillus niger. Appl. Microbiol. Biotechnol. 97: 3901-3911. https://doi.org/10.1007/s00253-012-4684-x

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