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Cloning, deletion, and overexpression of a glucose oxidase gene in Aureobasidium sp. P6 for Ca2+-gluconic acid overproduction

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Abstract

This study aimed to overexpress a glucose oxidase gene (GOD1) in Aureobasidium sp. P6 to achieve Ca2+-gluconic acid (GA) overproduction. The GOD1 gene was cloned, deleted, and overexpressed. A protein deduced from the GOD1 gene of Aureobasidium sp. P6 strain had 1824 bp that encoded a protein with 606 amino acids, with a conserved NADB-ROSSMAN domain and a GMC-oxred domain. Deleting the GOD1 gene made the disruptant GOK1 completely lose the ability to produce GA and GOD1 activity, whereas overexpressing the GOD1 gene rendered the transformant GOEX8 to produce considerably more Ca2+-GA (160.5 ± 5.6 g/L) and higher GOD1 activity (1438.6 ± 73.2 U/mg of protein) than its parent P6 strain (118.7 ± 4.3 g/L of Ca2+-GA and 1100.0 ± 23.6 U/mg of GOD1 protein). During a 10-L fermentation, the transformant GOEX8 grown in the medium containing 160.0 g/L of glucose produced 186.8 ± 6.0 g/L of Ca2+-GA, the yield was 1.2 g/g of glucose, and the volumetric productivity was 1.7 g/L/h. Most of the produced GOD1 were located in the yeast cell wall. The purified product was identified to be a GA. The transformant GOEX8 overexpressing the GOD1 gene could produce considerably more Ca2+-GA (186.8 ± 6.0 g/L) than its wild-type strain P6.

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Funding

This research was supported by 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, Ocean University of China, Ministry of Education, Yushan Road, No. 5, Qingdao, China

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

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

    Zhong Hu

Authors
  1. Yan Ma
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  2. Zhe Chi
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  3. Yan-Feng Li
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  4. Hong Jiang
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  5. Guang-Lei Liu
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  6. Zhong Hu
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  7. Zhen-Ming Chi
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Corresponding author

Correspondence to Zhe Chi.

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The authors declare that they have no competing interests.

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This article does not contain any studies with human participants or animals performed by any of the authors.

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Informed consent was obtained from all individual participants included in the study.

Electronic supplementary material

Supplementary File 1

Primers used in this study (DOC 32 kb)

Supplementary File 2

Construction of the vector for disruption of the GOD1 gene (DOC 509 kb)

Supplementary File 3

Construction of a vector for over-expression of the GOD1 gene (DOC 295 kb)

Supplementary File 4

Nucleotide sequence of the Aureobasidium sp. P6 strain GOD1 gene, upstream regions, and deduced amino acid sequence. The promoter is underlined, two spn-1 elements, one CAAT box, one TATA box are boxed, the transcriptional start site is indicated by a big arrow. The amino acids in shade are the GMC-oxred domain of the oxidoreductase family, the amino acids double-underlined are the FAD-binding site, the amino acids in both italics and bold are the signal peptide and the cleavage site is indicated by an arrow, N-glycosylation sites are shown by the oval frames (DOC 950 kb)

Supplementary File 5

Ca2+-GA production by different transformants and their parent strain P6. Data are given as mean ± SD, n = 3. ** (p < 0.01) extremely significant difference compared with that of Aureobasidium sp. P6 strain (DOC 371 kb)

Supplementary File 6

The ribbon representation of the secondary structure elements of the GOD1 from Aureobasidium sp. P6 strain, showing a homodimer (orange and blue) (color figure online) (DOC 184 kb)

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Ma, Y., Chi, Z., Li, YF. et al. Cloning, deletion, and overexpression of a glucose oxidase gene in Aureobasidium sp. P6 for Ca2+-gluconic acid overproduction. Ann Microbiol 68, 871–879 (2018). https://doi.org/10.1007/s13213-018-1393-4

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  • DOI: https://doi.org/10.1007/s13213-018-1393-4

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