在酿酒酵母中重构棉子糖生物合成途径的基础研究

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

摘要:

研究了在酿酒酵母中重构棉子糖生物合成途径，为后续高效生物合成棉子糖细胞工厂的构建奠定基础。敲除酿酒酵母中能降解棉子糖的蔗糖酶与α-半乳糖苷酶的基因构建出E1(Δsuc2::Δmel1)；在此基础上，构建单基因表达拟南芥肌醇半乳糖合成酶基因gols1与gols3及棉子糖合成酶基因sip1与sip5的工程菌株，及构建双基因组合表达（gols1::sip1、gols1::sip5、gols3::sip1与gols3::sip5）工程菌株；比较工程菌株代谢产物如肌醇、UDP-半乳糖、肌醇半乳糖、蔗糖及棉子糖等生成情况，验证在酿酒酵母中重构棉子糖生物合成途径的可行性。研究表明，通过共表达外源肌醇半乳糖合成酶及棉子糖合成酶基因，并敲除能降解棉子糖的蔗糖酶与α-半乳糖苷酶的基因，在酿酒酵母中实现了棉子糖生物合成途径的重构；肌醇半乳糖合成酶与棉子糖合成酶基因的不同共表达组合，棉子糖生成量有差异；重构的棉子糖生物合成途经改变了酿酒酵母原始菌株的代谢流量。

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 mel1 encoding invertase and α-galactosidase were deleted in S. cerevisiae (BY4741) to construct E1(Δsuc2::Δmel1) 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(Δsuc2::Δmel1::gols1), E3(Δsuc2::Δmel1::gols3), E4(Δsuc2::Δmel1::sip1), and E5(Δsuc2::Δmel1::sip5)], and double-gene-coexpressing strains E6(Δsuc2::Δmel1::gols1::sip1), E7(Δsuc2::Δmel1::gols1::sip5), E8(Δsuc2::Δmel1::gols3::sip1), and E9(Δsuc2::Δmel1::gols3::sip5) 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 of S. cerevisiae changed after the construction of the raffinose biosynthetic pathway.