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

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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.

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Funding

This research was supported by the National Natural Science Foundation of China and the Grant No. is 31500029.

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Authors and Affiliations

  1. Key Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, Yushan Road, No. 5, Qingdao, China

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

  2. Department of Life Science, Shantou University, Shantou, China

    Zhong Hu

Authors
  1. Yu Zhang
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  2. Yan-Feng Li
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  3. Zhe Chi
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  4. Guang-Lei Liu
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  5. Hong Jiang
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  6. Zhong Hu
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  7. Zhen-Ming Chi
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Correspondence to Zhen-Ming Chi.

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Supplementary File 1

The primers used in this study (DOC 46 kb)

Supplementary File 2

The contruction of the expression plasmid carrying the optimized INU1 gene (DOC 731 kb)

Supplementary File 3

Comparison of the native INU1 gene from K. marxianus KM-0 and the optimized INU1N gene obtained in this study. (DOC 1622 kb)

Supplementary File 4.

Thin-layer chromatogram of the hydrolysis products of inulin and the extract of the tubers of of Jerusalem artichoke by the crude inulinase preparation. Lane 1: glucose; Lane 2, Fructose; Lane 3, Sucrose; Lane 4, Raffinose; Lane 5, Inulin solution; Lane 6, The hydrolysate of inulin; Lane 7, The hysrolysate of the extract of the tubers of Jerusalem artichoke; Lane 8, The decoloured hydrolysate of the extract of the tubers of Jerusalem artichoke. (DOC 256 kb)

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Zhang, Y., Li, YF., Chi, Z. et al. Inulinase hyperproduction by Kluyveromyces marxianus through codon optimization, selection of the promoter, and high-cell-density fermentation for efficient inulin hydrolysis. Ann Microbiol 69, 647–657 (2019). https://doi.org/10.1007/s13213-019-01457-8

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  • DOI: https://doi.org/10.1007/s13213-019-01457-8

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