Inulinase hyperproduction by Kluyveromyces marxianus through codon optimization, selection of the promoter, and high-cell-density fermentation for efficient inulin hydrolysis View Full Text


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Article Info

DATE

2019-03-22

AUTHORS

Yu Zhang, Yan-Feng Li, Zhe Chi, Guang-Lei Liu, Hong Jiang, Zhong Hu, Zhen-Ming Chi

ABSTRACT

This study aimed to overexpress an inulinase gene in Kluyveromyces marxianus to achieve the inulinase overproduction and preparation of ultra-high-fructose syrup. First, the inulinase gene (INU1 gene) was overexpressed through codon optimization and selection of a suitable promoter. Then, the inulinase was overproduced by high-cell-density fermentation. Finally, ultra-high-fructose syrup was prepared. It was found that optimization of the codons of the native INU1 gene encoding inulinase from Kluyveromyces marxianus KM-0 made a recombinant strain KM-N70 carrying the optimized INU1N gene produce 251.4 U/mL of the inulinase activity. Furthermore, inulinase activity produced by a recombinant KM-KN16 strain carrying the optimized INU1N gene directed by the native TPS1 promoter from K. marxianus KM-0 reached 338.5 U/mL and expression level of the optimized INU1N gene in the recombinant KM-KN16 strain was also greatly enhanced. During a 10-L fermentation, the recombinant KM-KN16 strain could produce 374.3 U/mL of inulinase activity within 24 h, while during a high-cell-density fed-batch fermentation, the recombinant KM-KN16 strain could produce 896.1 U/mL of inulinase activity and OD600nm value of its culture reached 108. The crude inulinase preparation obtained in this study had an inulinase activity of 18,699.8 ± 736.4 U/g of the crude preparation. It was found that 90.3% of 332.4 g/L of inulin was hydrolyzed to produce 41.0 g/L of glucose and 256.0 g/L of fructose and 91.1% of 328.2 g/L of inulin in the extract of the tubers of Jerusalem artichoke was hydrolyzed to produce 48.3 g/L of glucose and 250.5 g/L of fructose by the crude inulinase preparation (75 U/g of the substrate) within 8 h. The hydrolysates contained major monosaccharides and a trace amount of trisaccharides and the monosaccharides were composed of around 85% fructose and 15% glucose. So far, any other yeasts available have produced only up to 120 U/mL of inulinase activity. Together, this made the recombinant KM-KN16 strain be the best inulinase producer at this moment. The inulinase activity of 18,699.8 ± 736.4 U/g of the crude preparation and the ultra-high-fructose syrup with 41.0 g/L of glucose and 256.0 g/L of fructose were obtained. The inulinase activity obtained in this study was the highest among all the inulinase activities produced by yeast, fungal, and bacterial strains obtained so far. More... »

PAGES

1-11

References to SciGraph publications

  • 2007-03. Inulinase production by a marine yeast Pichia guilliermondii and inulin hydrolysis by the crude inulinase in JOURNAL OF INDUSTRIAL MICROBIOLOGY & BIOTECHNOLOGY
  • 1994-12. Production of extracellular inulinase in high-cell-density fed-batch cultures of Kluyveromyces marxianus in APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
  • 2015-08. Genetic Modification of the Marine-Isolated Yeast Aureobasidium melanogenum P16 for Efficient Pullulan Production from Inulin in JOURNAL OF MARINE BIOTECHNOLOGY
  • 2013-10. Expression of exoinulinase genes in Saccharomyces cerevisiae to improve ethanol production from inulin sources in BIOTECHNOLOGY LETTERS
  • 2015-04. Cloning and Characterization of an Inulinase Gene From the Marine Yeast Candida membranifaciens subsp. flavinogenie W14-3 and Its Expression in Saccharomyces sp. W0 for Ethanol Production in MOLECULAR BIOTECHNOLOGY
  • 2013-11. Citric acid production from extract of Jerusalem artichoke tubers by the genetically engineered yeast Yarrowia lipolytica strain 30 and purification of citric acid in BIOPROCESS AND BIOSYSTEMS ENGINEERING
  • 2014-11. Enhanced expression of the codon-optimized exo-inulinase gene from the yeast Meyerozyma guilliermondii in Saccharomyces sp. W0 and bioethanol production from inulin in APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
  • 2013-03. Optimization of the production of Aspergillus niger α-glucosidase expressed in Pichia pastoris in WORLD JOURNAL OF MICROBIOLOGY AND BIOTECHNOLOGY
  • 2013-08. Improved ethanol production in Jerusalem artichoke tubers by overexpression of inulinase gene in Kluyveromyces marxianus in BIOTECHNOLOGY AND BIOPROCESS ENGINEERING
  • 2016-04. Functional analysis of the binding model of microbial inulinases using docking and molecular dynamics simulation in JOURNAL OF MOLECULAR MODELING
  • 2009-04. Bioproducts from Aureobasidium pullulans, a biotechnologically important yeast in APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
  • 2014-01. Enhanced expression of endoinulinase from Aspergillus niger by codon optimization in Pichia pastoris and its application in inulooligosaccharide production in JOURNAL OF INDUSTRIAL MICROBIOLOGY & BIOTECHNOLOGY
  • 2013-03. The changes in Tps1 activity, trehalose content and expression of TPS1 gene in the psychrotolerant yeast Guehomyces pullulans 17-1 grown at different temperatures in EXTREMOPHILES
  • 2012-09. Molecular Modeling and Docking of Microbial Inulinases Towards Perceptive Enzyme–Substrate Interactions in INDIAN JOURNAL OF MICROBIOLOGY
  • Journal

    TITLE

    Annals of Microbiology

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    N/A

    VOLUME

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    Identifiers

    URI

    http://scigraph.springernature.com/pub.10.1007/s13213-019-01457-8

    DOI

    http://dx.doi.org/10.1007/s13213-019-01457-8

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    https://app.dimensions.ai/details/publication/pub.1112943252


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