高通量筛选高产酪氨酸的酿酒酵母菌株

High-throughput screening of Saccharomyces cerevisiae efficiently producing tyrosine

酪氨酸是重要的芳香族氨基酸,自身不仅具有重要的营养价值,也是合成香豆素类化合物和黄酮类化合物的重要前体。文中以实验室前期构建的一株解除了酪氨酸反馈抑制的酿酒酵母Saccharomyces cerevisiae LTH0(ARO4K229L,ARO7G141S,Δaro10,Δzwf1,Δura3)为出发菌株,异源表达甜菜黄素合成基因DOD和CYP76AD1,使酿酒酵母产生黄色荧光。然后利用紫外诱变和常压室温等离子体(Atmospheric and room temperature plasma,ARTP)诱变相结合的方法对上述菌株进行随机诱变,并通过流式细胞仪筛选荧光强度显著提高的突变株。其中突变株LTH2-5-DOD-CYP76AD1在激发波长485 nm、发射波长505 nm处荧光强度为(5 941±435) AU/OD,比诱变前提高了8.37倍。对诱变后荧光强度提高较多的14株突变株进行发酵生产酪氨酸,胞外酪氨酸产量最高为26.8 mg/L,比出发菌株提高了3.96倍。进一步异源表达约翰逊黄杆菌Flavobacterium johnsoniae来源的酪氨酸解氨酶Fj TAL,对香豆酸产量达到119.8 mg/L,比出发菌株LTH0-FjTAL提高了1.02倍。

Tyrosine is an important aromatic amino acid.Besides its nutritional value,tyrosine is also an important precursor for the synthesis of coumarins and flavonoids.Previously,our laboratory constructed a Saccharomyces cerevisiae strain LTH0(ARO4K229L,ARO7G141S,Δaro10,Δzwf1,Δura3) where tyrosine feedback inhibition was released.In the present study,heterologous expression of betaxanthins synthesis genes DOD (from Mirabilis jalapa) and CYP76AD1 (from sugar beet B.vulgaris) in strain LTH0 enabled production of yellow fluorescence.The engineered strain LTH0-DOD-CYP76AD1 was subjected to UV combined with ARTP mutagenesis,followed by flow cytometry screening.Among the mutants screened,the fluorescence intensity of the mutant strain LTH2-5-DOD-CYP76AD1 at the excitation wavelength of 485 nm and emission wavelength of 505 nm was (5 941±435) AU/OD,which was 8.37 times higher than that of strain LTH0-DOD-CYP76AD1.Fourteen mutant strains were subjected to fermentation to evaluate their tyrosine producing ability.The highest extracellular tyrosine titer reached 26.8 mg/L,which was 3.96 times higher than that of strain LTH0-DOD-CYP76AD1.Heterologous expression of the tyrosine ammonia lyase Fj TAL derived from Flavobacterium johnsoniae further increased the titer of coumaric acid to 119.8 mg/L,which was 1.02 times higher than that of the original strain LTH0-FjTAL.