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. 2022 Jan 26;8(2):119.
doi: 10.3390/jof8020119.

Engineered Fungus Thermothelomyces thermophilus Producing Plant Storage Proteins

Larissa Balabanova  1 , Aleksandra Seitkalieva  1 , Yulia Yugay  2 , Tatiana Rusapetova  2 , Lubov Slepchenko  1 , Anna Podvolotskaya  3   4 , Margarita Yatsunskaya  2 , Elena Vasyutkina  2 , Oksana Son  3   4 , Liudmila Tekutyeva  3   4 , Yury Shkryl  2
Affiliations

Engineered Fungus Thermothelomyces thermophilus Producing Plant Storage Proteins

Larissa Balabanova et al. J Fungi (Basel). .

Abstract

An efficient Agrobacterium-mediated genetic transformation based on the plant binary vector pPZP-RCS2 was carried out for the multiple heterologous protein production in filamentous fungus Thermothelomyces thermophilus F-859 (formerly Myceliophthora thermophila F-859). The engineered fungus Th. thermophilus was able to produce plant storage proteins of Zea mays (α-zein Z19) and Amaranthus hypochondriacus (albumin A1) to enrich fungal biomass by valuable nutritional proteins and improved amino acid content. The mRNA levels of z19 and a1 genes were significantly dependent on their driving promoters: the promoter of tryptophan synthase (PtrpC) was more efficient to express a1, while the promoter of translation elongation factor (Ptef) provided much higher levels of z19 transcript abundance. In general, the total recombinant proteins and amino acid contents were higher in the Ptef-containing clones. This work describes a new strategy to improve mycoprotein nutritive value by overexpression of plant storage proteins.

Keywords: Myceliophthora thermophila; amaranth albumin A1; filamentous fungi; maize alpha-zein; mycoprotein; recombinant protein.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Binary vectors pPZP-RCS2-TrpC:Hyg-TrpC:Z19/A1 (A) and pPZP-RCS2-TrpC:Hyg-TEF:Z19/A1 (B) carrying genes for synthesis of Z19 and A1 under the control of PtrpC and Ptef promoters.
Figure 2
Figure 2
Characterization of transgenic Th. thermophilus rF-859 strains. (A)—Agarose gel electrophoresis of the hph gene amplified using DNA from the Th. thermophilus transformants (Nc—negative control (water); WT—wild type strain Th. thermophilus F-859; TrpC1 and TrpC2—Th. thermophilus rF-859/TrpC:Z19/A1 clones; TEF1 and TEF2—Th. thermophilus rF-859/TEF:Z19/A1) clones. (B)—Total protein production in Th. thermophilus rF-859/TEF:Z19/A1 and Th. thermophilus rF-859/TrpC:Z19/A1 mycelia (production was calculated separately for fresh and dried mycelial biomass in 1 L of media). (C)—Phenotypic view of the wild type and transgenic strains. (D)—Microphotographs of the wild type and Th. thermophilus rF-859 hyphae. * p < 0.05, ** p < 0.01 as compared to wild type strain, Student’s t-test.
Figure 3
Figure 3
qPCR analysis of z19, a1, and hph expression in Th. thermophilus rF-859 strains. Two recombinant clones of Th. thermophilus rF-859 harboring the z19 and a1 genes driven by PtrpC (named TrpC1 and TrpC2), as well as two clones expressing the same genes regulated by Ptef (named TEF1 and TEF2) were analyzed. In all studied clones hph gene was driven by PtrpC. Different letters above the bars indicate statistically significant differences of means (p < 0.05) for each of the gene, Fisher’s LSD.
Figure 4
Figure 4
12.5% SDS-PAGE of ethanol-soluble (A) and total water-soluble (C) protein fractions isolated from the wild type and Th. thermophilus rF-859 strains. Detection of Z19 (B) and A1 proteins (D) using Western blotting with anti-6x-His Tag antibodies. Original Western blots are shown in Supplementary Figure S2.
Figure 5
Figure 5
The differences in AA content in Th. thermophilus rF-859 clones TrpC (blue) and TEF (orange) relatively to the wild type strain after 14 (A) and 28 (B) days of cultivation (liquid potato-dextran medium, 45, without shaking).
See this image and copyright information in PMC

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