引入新型异戊二烯醇利用途径促进解脂耶氏酵母中β-胡萝卜素的合成  

Improving the Biosynthesis ofβ-Carotene in Yarrowia lipolytica by Introducing an Artificial Isopentenol Utilization Pathway

作　　者：朱航志 蒋珊 陈丹 刘鹏阳 万霞[1,3,4] ZHU Hang-zhi;JIANG Shan;CHEN Dan;LIU Peng-yang;WAN Xia(Oil Crops Research Institute of Chinese Academy of Agricultural Sciences,Wuhan 430062,China;School of Chemical Engineering and Pharmacy,Wuhan Institute of Technology,Wuhan 430205,China;Key Laboratory of Biology and Genetie Improvement of Oil Crops,Ministry of Agriculture,Wuhan 430062,China;National and Local Joint Engineeing Laboratory of 0il Lipid Processing Technology,Wuhan 430062,China)

机构地区：[1]中国农业科学院油料作物研究所,武汉430062 [2]武汉工程大学化工与制药学院,武汉430205 [3]农业农村部油料作物生物学与遗传育种重点实验室,武汉430062 [4]农业农村部油料加工重点实验室,武汉430062

出　　处：《中国生物工程杂志》2021年第4期37-46,共10页China Biotechnology

基　　金：中国农业科学院基础前沿培育项目(Y2020XK25);国家重点研发计划(2016YFD0501209);中国农业科学院科技创新工程(CAASASTIP-2016-OCRI)资助项目。

摘　　要：目的:微生物体内异戊二烯类化合物的前体物异戊烯焦磷酸酯的天然合成路径受到严格的代谢调控,因此限制了异戊二烯类化合物的高效生物合成,而新型异戊二烯醇利用途径独立于生物体内源性代谢路径,通过在微生物中引入IUP能够进行异戊烯焦磷酸酯的大量合成,从而促进异戊二烯类化合物的大量合成。方法:在油脂酵母解脂耶氏酵母中引入IUP,强化异戊烯焦磷酸酯生物合成,促进β-胡萝卜素的高效积累。结果:通过生物信息学的方法预测IUP中两个关键蛋白酿酒酵母来源的胆碱激酶Sc CK和拟南芥来源的异戊烯磷酸激酶At IPK,均为酸性亲水性蛋白,无跨膜区和信号肽,二者都具有疏松不稳定的结构特征,显著富集于磷酸类物质的合成通路中。在解脂耶氏酵母中利用同源重组技术引入外源β-胡萝卜素合成关键基因carRP和car B,强化甲羟戊酸途径的关键基因thmgR和ggs1,使工程菌株中积累2.68 mg/Lβ-胡萝卜素。通过Cre-loxP系统回收基因组上的ura标签,再将IUP进一步整合到工程菌株染色体上。当培养基中含有20 m M异戊二烯醇作为底物、碳氮比为4/3且发酵96 h后,重组解脂耶氏酵母中β-胡萝卜素的产量提高到410.2 mg/L,较原始工程菌的产量提高了近200倍。结论:IUP能够促进解脂耶氏酵母中β-胡萝卜素的高效积累,为利用IUP开展β-胡萝卜素和其他异戊二烯类化合物的高效生物合成提供新思路。Objective:The natural synthesis pathway of isopentenyl pyrophosphate,the precursor of isoprene compounds in microorganisms,is subject to strict metabolic regulation,which limits the efficient biosynthesis of isoprene compounds,and the use of the artificial isopentenol utilization pathway(IUP)is independent of the biological endogenous metabolic pathways.By introducing IUP into microorganisms,a large amount of isopentenol pyrophosphate can be synthesized,thereby promoting the large amount of synthesis of isoprene compounds.Methods:Introducing the artificial IUP into the oleaginous yeast Yarrowia lipolytica to strengthen the isopentenyl pyrophosphate biosynthesis and promote the efficient accumulation ofβ-carotene.Results:The tertiary structures of two key proteins involved in IUP,ScCK(Choline kinase from Saccharomyces cerevisiae)and At IPK(isopentenyl phosphate kinase from Arabidopsis thaliana),are predicted to be acidic hydrophilic proteins without transmembrane domain and signal peptide.Both proteins have loose and unstable structural features and contain a variety of post-translational features.Modification sites and multiple interacting proteins are involved in phosphorylation.Theβ-carotene synthesis pathway was constructed by introducing carRP and carB,and strengthening the expression of thmgR and ggs1 in Yarrowia lipolytica by using homologous recombination technique,and 2.68 mg/Lβ-carotene was accumulated in the engineered strain.The ura on the genome were removed by the Cre-loxP system,and then IUP was integrated into the genome of this engineered strain.The yield ofβ-carotene was achieved at the levels of 410.2 mg/L in the engineered strain,which was nearly 200 folds greater than that from the original engineered strain.The fermentation conditions were as follows:cells were incubated for 96 h in the medium containing 20 mmol/L isoprenol and the ratio of carbon to nitrogen was set as4/3 in the medium.Conclusion:IUP can promote the efficient accumulation ofβ-carotene in Y.lipolytica.This study provides

关 键 词：异戊二烯醇利用途径(IUP) 异戊烯焦磷酸酯 Β-胡萝卜素 解脂耶氏酵母 

分 类 号：Q939[生物学—微生物学]