A Basic Study on a Raffinose Biosynthetic Pathway Constructed in Saccharomyces cerevisiae

doi:10.13982/j.mfst.1673-9078.2017.5.020

Abstract：A raffinose biosynthetic pathway constructed in Saccharomyces cerevisiae was investigated,and the results form the basis for creation of cell factories for highly efficient biosynthesis of raffinose in the future.First,genes suc2 and mell encoding invertase and α-galactosidase were deleted in S.cerevisiae (BY4741) to construct El(△suc2∷△mell) as a base strain.Single-gene-expressing strains expressing Arabidopsis thaliana galactinol synthetase genes gols1 and gols3 and raffinose synthase genes sip1 and sip5 [E2(△suc2∷△mel1∷gols1),E3(△suc2∷△mel1∷gols3),E4(△suc2∷△mel1∷sip1),and E5(△suc2∷△mel1∷sip5)],and double-gene-coexpressing strains E6(△suc 2∷ △mel 1 ∷ gols 1 ∷ sip 1),E 7 (△suc 2∷ △mel 1 ∷ gols 1 ∷ sip 5),E8(△suc 2∷ △mel 1 ∷ gols 3 ∷ sip 1),and E 9(△suc 2∷ △mel 1 ∷ gols 3 ∷ sip 5) were constructed.Finally,production of raffinose and the intermediate metabolites-uridine diphosphate (UDP)-galactose,galactinol,and sucrose-in the fermentation broth of these engineered strains was analyzed after induced expression and fermentation to verify the feasibility of construction of the raffinose biosynthetic pathway in S.cerevisiae.The results indicated that construction of the raffinose biosynthetic pathway in S.cerevisiae was achieved via coexpression of heterologous genes encoding exogenous galactinol synthase and raffinose synthase and deletion of genes encoding invertase and α-galactosidase in S.cerevisiae.Different coexpression combinations of the galactinol synthase gene and raffinose synthase gene caused a difference in the raffinose yield.Metabolic flux in the original strain orS.cerevisiae changed after the construction of the raffinose biosynthetic pathway.