构建酿酒酵母细胞工厂生产番茄红素  

Construction of Saccharomyces cerevisiae cell factories for lycopene production

作　　者：施明雨 刘怡[2,3] 王冬[1,2,3] 路福平[1] 黄璐琦[4] 戴住波[2,3] 张学礼[2,3] 

机构地区：[1]天津科技大学生物工程学院,天津300457 [2]中国科学院天津工业生物技术研究所,天津300308 [3]中国科学院系统微生物工程重点实验室,天津300308 [4]中国中医科学院中药资源中心,北京100700

出　　处：《中国中药杂志》2014年第20期3978-3985,共8页China Journal of Chinese Materia Medica

基　　金：国家高技术研究发展计划(863)项目(2012AA02A704);国家自然科学基金项目(81202864)

摘　　要：为构建高效生产番茄红素的酿酒酵母细胞工厂,本研究首先在酿酒酵母中引入番茄红素生物合成途径基因Crt B和Crt I,获得能生产0.17 mg·L-1番茄红素的初始工程菌ZD-L-000。在此基础上,在工程菌ZD-L-000中分别过表达MVA途径中的3-羟基-3-甲基戊二酰辅酶A还原酶编码基因t HMG1、萜类合成调控的转录因子编码基因upc2.1、二萜生物合成的牻牛儿基牻牛儿基焦磷酸合酶与法呢基焦磷酸合酶编码基因BTS1-ERG20,以及来自嗜酸热硫化叶菌的牻牛儿基牻牛儿基焦磷酸合酶编码基因Sa GGPS,考察这些基因过表达对番茄红素合成的影响,结果发现过表达upc2.1基因未能提高番茄红素的产量,但过表达t HMG1、BTS1-EGR20和Sa GGPS基因均能显著提高番茄红素的产量,分别为初始菌株的2.0,16.9和20.5倍。在进一步研究中,t HMG1、BTS1-EGR20和Sa GGPS等有效基因被一同整合入工程菌ZD-L-000,获得的高产菌株ZD-L-201中番茄红素的产量提高77.0倍,达到13.23 mg·L-1;在高密度-两相发酵体系中工程菌ZD-L-201的番茄红素产量能进一步提高到135.21 mg·L-1(提高10.2倍)。本研究获得的工程菌为微生物发酵法生产番茄红素提供了基础。For microbial production of lycopene, the lycopene synthetic genes from Pantoea agglomerans were integrated into Saccharomyces cerevisiae strain BY4742, to obtain strain ZD-L-000 for production of 0.17 mg·L-1 lycopene. Improving supplies of isoprenoid precursors was then investigated for increasing lycopene production. Four key genes were chosen to be overexpressed, including truncated 3-hydroxy-3-methylglutaryl-CoA reductase gene （tHMG1）, which is the major rate-limiting enzyme in the mevalonate （MVA） pathway, a mutated global regulatory factor gene （upc2.1）, a fusion gene of FPP synthase （ERG20） and endogenous GGPP synthase （BTS1）, which is a key enzyme in the diterpenoid synthetic pathway, and GGPP synthase gene （SaGGPS） from Sulfolobus acidocaldarius. Over-expression of upc2.1 could not improve lycopene production, while over-expression of tHMG1, BTS1-ERG20 and SaGGPS genes led to 2-, 16.9- and 20.5-fold increase of lycopene production, respectively. In addition, three effective genes, tHMG1, BTS1-ERG20 and SaGGPS, were integrated into rDNA sites of ZD-L-000, resulting in strain ZD-L-201 for production of 13.23 mg·L^-1 lycopene, which was 77-fold higher than that of the parent strain. Finally, two-phase extractive fermentation was performed. The titer of lycopene increased 10-fold to 135.21 mg·L^-1. The engineered yeast strains obtained in this work provided the basis for fermentative production of lycopene.

关 键 词：番茄红素 酿酒酵母 合成生物学 甲羟戊酸途径 

分 类 号：TQ920.6[轻工技术与工程—发酵工程] Q936[生物学—微生物学]