URA3基因影响青蒿酸酿酒酵母工程菌中试发酵产量  

URA3 affects artemisinic acid production by an engineered Saccharomyces cerevisiae in pilot-scale fermentation

作　　者：郭未蔚 艾丽梅 胡栋 陈亚军 耿梦馨 郑玲辉 白利平[1] GUO Weiwei;AI Limei;HU Dong;CHEN Yajun;GENG Mengxin;ZHENG Linghui;BAI Liping(NHC Key Laboratory of Biotechnology of Antibiotics,CAMS Key Laboratory of Synthetic Biology for Drug Innovation,Institute of Medicinal Biotechnology,Chinese Academy of Medical Sciences&Peking Union Medical College,Beijing 100050,China;Zhejiang Key Laboratory of Antifungal Drugs,Zhejiang Hisun Pharmaceutical Co.Ltd.,Taizhou 318000,Zhejiang,China)

机构地区：[1]中国医学科学院北京协和医学院医药生物技术研究所卫健委抗生素生物工程重点实验室/中国医学科学院药物合成生物学重点实验室,北京100050 [2]浙江海正药业股份有限公司浙江抗菌药物重点实验室,浙江台州318000

出　　处：《生物工程学报》2022年第2期737-748,共12页Chinese Journal of Biotechnology

基　　金：国家科技重大专项(2017ZX09101002-003-003);国家自然科学基金(31870059)。

摘　　要：CRISPR/Cas9基因编辑技术已经被广泛应用于工程酿酒酵母的基因插入、基因替换和基因敲除,通过使用选择标记进行基因编辑具有简单高效的特点。前期利用CRISPR/Cas9系统敲除青蒿酸生产菌株酿酒酵母(Saccharomyces cerevisiae)1211半乳糖代谢负调控基因GAL80,获得菌株S.cerevisiae 1211-2,在不添加半乳糖诱导的情况下,青蒿酸摇瓶发酵产量达到了740 mg/L。但在50 L中试发酵实验中,S.cerevisiae 1211-2很难利用对青蒿酸积累起到决定性作用的碳源-乙醇,青蒿酸的产量仅为亲本菌株S.cerevisiae 1211的20%–25%。我们推测因遗传操作所需的筛选标记URA3突变,影响了其生长及青蒿酸产量。随后我们使用重组质粒pML104-KanMx4-u连同90 bp供体DNA成功恢复了URA3基因,获得了工程菌株S.cerevisiae 1211-3。S.cerevisiae 1211-3能够在葡萄糖和乙醇分批补料的发酵罐中正常生长,其青蒿酸产量超过20 g/L,与亲本菌株产量相当。研究不但获得了不加半乳糖诱导的青蒿酸生产菌株,同时首次发现了营养缺陷型标记URA3基因显著影响乙醇补料的中试发酵中青蒿酸的产生,也为酵母作为底盘来进行其他天然产物的生产提供了借鉴。CRISPR/Cas9 has been widely used in engineering Saccharomyces cerevisiae for gene insertion,replacement and deletion due to its simplicity and high efficiency.The selectable markers of CRISPR/Cas9 systems are particularly useful for genome editing and Cas9-plasmids removing in yeast.In our previous research,GAL80 gene has been deleted by the plasmid pML104-mediated CRISPR/Cas9 system in an engineered yeast,in order to eliminate the requirement of galactose supplementation for induction.The maximum artemisinic acid production by engineered S.cerevisiae 1211-2(740 mg/L)was comparable to that of the parental strain 1211 without galactose induction.Unfortunately,S.cerevisiae 1211-2 was inefficient in the utilization of the carbon source ethanol in the subsequent 50 L pilot fermentation experiment.The artemisinic acid yield in the engineered S.cerevisiae 1211-2 was only 20%-25%compared with that of S.cerevisiae 1211.The mutation of the selection marker URA3 was supposed to affect the growth and artemisinic acid production.A ura3 mutant was successfully restored by a recombinant plasmid pML104-KanMx4-u along with a 90 bp donor DNA,resulting in S.cerevisiae 1211-3.This mutant could grow normally in a fed-batch fermentor with mixed glucose and ethanol feeding,and the final artemisinic acid yield(>20 g/L)was comparable to that of the parental strain S.cerevisiae 1211.In this study,an engineered yeast strain producing artemisinic acid without galactose induction was obtained.More importantly,it was the first report showing that the auxotrophic marker URA3 significantly affected artemisinic acid production in a pilot-scale fermentation with ethanol feeding,which provides a reference for the production of other natural products in yeast chassis.

关 键 词：工程酿酒酵母 青蒿酸 CRISPR/Cas9 URA3 

分 类 号：TQ920.1[轻工技术与工程—发酵工程] Q78[生物学—分子生物学]