Inhibitory effects on silk fibroin production by the expression of phenylalanyl-tRNA synthetase mutants in posterior silk glands of Bombyx mori

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  • Teramoto Hidetoshi
    Silk Materials Research Unit, Institute of Agrobiological Sciences, National Agriculture and Food Research Organization (NARO)
  • Kojima Katsura
    Silk Materials Research Unit, Institute of Agrobiological Sciences, National Agriculture and Food Research Organization (NARO)

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  • Inhibitory effects on silk fibroin production by the expression of phenylalanyl-tRNA synthetase mutants in posterior silk glands of <i>Bombyx mori</i>

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Here we report the inhibitory effects on silk fibroin production by the expression of mutants of B. mori phenylalanyl-tRNA synthetase α-subunit (BmPheRS-α) with relaxed amino acid recognition capacities in posterior silk glands (PSG) of transgenic B. mori larvae. We succeeded previously in incorporating unnatural amino acids (UAAs), 4-substituted analogues of phenylalanine (Phe), into silk fibroin by PSG-specific expression of BmPheRS-α mutants bearing a T407A or A450G mutation on its amino acid binding pocket. We had expected that the repertoire of UAAs incorporated into silk fibroin could be further expanded by expressing BmPheRS-α mutants with more highly relaxed amino acid recognition capacities. Hence, in this study, we generated two novel transgenic B. mori lines expressing BmPheRS-α mutants bearing more drastic side-chain mutations, T407G and T407A/A450G. The newly generated transgenic larvae spun thinner cocoons with substantially lower production of silk fibroin. We found that the PSG dissected from 5th instar transgenic larvae were degenerated into smaller sizes compared to the wild-type larvae. Mass analysis of the silk fibroin indicated misincorporation of tryptophan (Trp) in place of some Phe residues. Recognition of Trp by the T407G or T407A/A450G mutant of BmPheRS-α was verified by in vitro aminoacylation assay. From these observations, we speculated that misincorporation of Trp into protein biosynthesis due to excess relaxation of the amino acid recognition capacity of BmPheRS-α caused the degeneration of PSG in the transgenic larvae.<br>

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