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

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(2320.0±26.0)mg·L^(-1),为视黄醇产业化发展提供了重要基础。

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 convertβ-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 theβ-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 ofβ-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 forβ-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(2320.0±26.0)mg·L^(-1) in the fermentation tank of 5 L,which provided a significant foundation for the industrial development of retinol.