创建酿酒酵母细胞工厂发酵生产视黄醇
Construction of Saccharomyces cerevisiae cell factories for fermentation production of retinol
李文豪 1杨婷婷 2李蕊 1马晓晨 1贾士儒 3张学礼 2王冬 2戴住波4
作者信息
- 1. 天津科技大学 生物工程学院,天津 300457;中国科学院 天津工业生物技术研究所,天津 300308;低碳合成工程生物学重点实验室,天津 300308;国家合成生物技术创新中心,天津 300308
- 2. 中国科学院 天津工业生物技术研究所,天津 300308;低碳合成工程生物学重点实验室,天津 300308;国家合成生物技术创新中心,天津 300308
- 3. 天津科技大学 生物工程学院,天津 300457
- 4. 中国科学院 天津工业生物技术研究所,天津 300308;低碳合成工程生物学重点实验室,天津 300308;国家合成生物技术创新中心,天津 300308;合成生物学海河实验室,天津 300308
- 折叠
摘要
视黄醇是维生素A的主要活性形式之一,对于机体的生长发育、眼部和皮肤功能维持至关重要,被广泛应用于化妆品、医药和饲料添加领域.动物体内不具备完整的维生素A合成途径,但可通过膳食直接摄入或将膳食中获得的β-胡萝卜素转化获得.为了推进视黄醇的生物法合成研究,首先以β-胡萝卜素合成平台CAR∗1 为基础,筛选了 3 种不同来源的醇脱氢酶,确定了大肠杆菌来源的ybbO具有最高的催化活性,转化率可达 95.6%.为了进一步提高反应速率和产量,采用蛋白质融合技术将视黄醇合成模块中 2 个相邻酶blh和ybbO进行融合后,在高产β-胡萝卜素工程菌株CAR∗3 中进行评估,获得了最优组合blh-GGGS-ybbO,其产量较融合前提高了 44.9%,达到(111.1±3.5)mg·L-1.进一步工作中,通过引入人源的视黄醇结合蛋白(RBP4)以及转甲状腺素蛋白(TTR),在酿酒酵母中模拟了人体肝脏细胞分泌视黄醇的过程,视黄醇产量增加至(158.0±13.1)mg·L-1.最后,采用过表达INO2 扩张β-胡萝卜素合成途径反应区室,提高血红蛋白VHb表达量以增强氧气的供给,强化PDR3m表达促进视黄醇的转运和开发 2 个阶段发酵工艺等方法,在 5 L发酵罐中成功将视黄醇的产量提升至(2 320.0±26.0)mg·L-1,为视黄醇产业化发展提供了重要基础.
Abstract
Retinol is one of the main active forms of vitamin A,crucial for the organism's growth,development,and maintenance of eye and skin functions.It is widely used in cosmetics,pharmaceuticals,and feed additives.Although animals lack a complete pathway for synthesizing vitamin A internally,they can obtain vitamin A directly through diet or convert β-carotene acquired from the diet.To boost the research on the biosynthesis of retinol,three different sources of alcohol dehydrogenase were firstly screened based on the β-carotene synthesis platform CAR∗1.It was determined that ybbO from Escherichia coli exhibited the highest catalytic activity,with a conversion rate of 95.6%.To further enhance the reaction rate and yield of retinol,protein fusion technology was employed to merge two adjacent enzymes,blh and ybbO,within the retinol synthesis module.The evaluation was conducted using the high-yield engineered strain CAR∗3 of β-carotene.The optimal combination,blh-GGGS-ybbO,was obtained,with a 44.9%increase in yield after fusion,reaching(111.1±3.5)mg·L-1.Furthermore,through the introduction of human-derived retinol-binding protein(RBP4)and transthyretin(TTR),the process of hepatic cell secreting retinol was simulated in Saccharomyces cerevisiae,leading to an increased retinol yield of(158.0±13.1)mg·L-1.Finally,optimization strategies including overexpressing INO2 to enhance the reaction area for β-carotene synthesis,enhancing hemoglobin VHb expression to improve oxygen supply,and strengthening PDR3m expression to facilitate retinol transport were implemented.A two-stage fermentation process resulted in the successful elevation of retinol production to(2 320.0±26.0)mg·L-1 in the fermentation tank of 5 L,which provided a significant foundation for the industrial development of retinol.
关键词
视黄醇/酿酒酵母/融合蛋白/视黄醇转运Key words
retinol/Saccharomyces cerevisiae/fusion protein/retinol transport引用本文复制引用
基金项目
国家重点研发计划项目(2022YFC2106200)
天津市合成生物技术创新能力提升行动项目(TSBICIP-CXRC-080)
合成生物学海河实验室颠覆性创新(人才)类项目(22HHSWSS00026)
出版年
2024
国家科技期刊平台
NSTL
万方数据