Enhanced hypocrellin production of Shiraia sp. SUPER-H168 by overexpression of alpha-amylase gene

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Abstract

Relative expression levels of twenty-four amylase genes in Shiraia sp. SUPER-H168 were investigated by real-time quantitative PCR when various carbohydrates, including glucose, sucrose, maltose, amylose, amylopectin and corn flour, were used as carbon source. Genes, including an α-glucosidase gene Amy33 (2997 bp), an α-amylase gene Amy365-1 (1749 bp) and a glycogen debranching enzyme gene Amy130 (2487 bp), were overexpressed, and four overexpression transformants were constructed, respectively. When Amy365-1 and Amy130 were co-overexpressed, relative expression levels of seven hypocrellin biosynthesis genes and four related genes in central carbon catabolism were all increased. Expression of Amy33 was also increased along with increase of Amy365-1 and Amy130. Under liquid state fermentation, biomasses and hypocrellin productions were both gradually increased in four overexpression strains than those of wild type strain. Under SSF, hypocrellin production of Amy365-1 and Amy130 co-expression strain reached 71.85 mg/ gds, which was 2.83 fold than that of wild type strain, and residual sugar was decreased from 35.47% to 16.68%. These results can provide a practical approach for other secondary metabolites by filamentous fungi under SSF when raw starch material is used as carbon source.

Figures

  • Fig 1. Phenomena involved in the growth of biofilm, penetrative, and aerial hyphae. “Long triangles” represent diffusion down concentration gradients. “Dotted arrows” represent flow of water or cytoplasm. “Dashed arrows” represent transport of vesicles.
  • Fig 2. Schematic representation of hypocrellin biosynthesis pathway from glucose to hypocrellin.
  • Table 1. Primers and relevant information of reference and target genes.
  • Table 1. (Continued)
  • Fig 3. Constructions of expression vectors and cDNA of amylase gene in this study. (A) PgfpPuro, (B) PhygAmy33, (C) PhygAmy365-1, (D) PhygAmy130 and (E) PhygPgpdAAmy365-1-Amy130. U6, U6 promoter; PgpdA, gpdA promoter; TtrpC, the trpC terminator; hyg, gene of protein resistant to hygromycin B; 2A, 2A peptide as a coexpression linker; Amy33, α-glucosidase gene; Amy365-1, α-amylase gene; Amy130, glycogen debranching enzyme gene. (F) cDNA of amylase gene. Lane M: DL5000 DNA Marker; lanes 1–2: cDNA of Amy33; lanes 3–4: cDNA of Amy365-1; lanes 5–6: cDNA of Amy130.
  • Table 2. Primers were used for construction of overexpression plasmids.
  • Table 3. Primers of hypocrellin biosynthesis genes and related genes in central carbon metabolism.
  • Fig 4. Relative mRNA levels of twenty-four genes when Shiraia sp. SUPER-H168 was cultured on different carbohydrates. Glucose was selected as a control and gpdwas used as the reference gene for data normalization of relative expression level analysis. S, sucrose; M, maltose; L, amylose; B, amylopectin; C, corn flour.

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Gao, R., Xu, Z., Deng, H., Guan, Z., Liao, X., Zhao, Y., … Cai, Y. (2018). Enhanced hypocrellin production of Shiraia sp. SUPER-H168 by overexpression of alpha-amylase gene. PLoS ONE, 13(5). https://doi.org/10.1371/journal.pone.0196519

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