Fermentation and purification strategies for the production of betulinic acid and its lupane-type precursors in Saccharomyces cerevisiae
Fermentation and purification strategies for the production of betulinic acid and its lupane-type precursors in Saccharomyces cerevisiae
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DOI：
10.1002/bit.26377
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发表时间：
2017-11-01
影响因子：
3.8
通讯作者：
Blank, Lars M.
中科院分区：
文献类型：
--
作者：
Czarnotta, Eik;Dianat, Mariam;Blank, Lars M.
微生物生产植物衍生的生物活性化合物有可能为现有的低产植物性工艺提供经济和生态上的替代。目前，具有药理活性的环状三萜白桦酸的生产是通过从梧桐树或桦树皮中提取实现的。在这里，我们对已报道的白桦酸转化为酿酒酵母的途径进行了改造，并使用这种新菌株开发了高效的发酵和产物纯化方法。采用乙醇脉冲补料或控制发酵介质中乙醇浓度恒定的乙醇分批补料培养，可显著提高白桦酸及其三萜类前体的产量。过量乙醇的有益作用在氮限静息细胞发酵中得到了进一步开发，白桦酸浓度达到182 mg/L，总三萜浓度达到854 mg/L，是迄今为止报道的最高浓度。采用丙酮或乙酸乙酯等极性非质子性溶剂，采用固-液萃取法分离纯化羽扇豆烷型三萜类化合物，并用强酸沉淀，实现了高选择性和高产率。本研究通过代谢工程和过程工程相结合，突出了微生物在酿酒酵母中生产植物来源三萜类化合物的潜力。
Microbial production of plant derived, biologically active compounds has the potential to provide economic and ecologic alternatives to existing low productive, plant-based processes. Current production of the pharmacologically active cyclic triterpenoid betulinic acid is realized by extraction from the bark of plane tree or birch. Here, we reengineered the reported betulinic acid pathway into Saccharomyces cerevisiae and used this novel strain to develop efficient fermentation and product purification methods. Fed-batch cultivations with ethanol excess, using either an ethanol-pulse feed or controlling a constant ethanol concentration in the fermentation medium, significantly enhanced production of betulinic acid and its triterpenoid precursors. The beneficial effect of excess ethanol was further exploited in nitrogen-limited resting cell fermentations, yielding betulinic acid concentrations of 182mg/L, and total triterpenoid concentrations of 854mg/L, the highest concentrations reported so far. Purification of lupane-type triterpenoids with high selectivity and yield was achieved by solid-liquid extraction without prior cell disruption using polar aprotic solvents such as acetone or ethyl acetate and subsequent precipitation with strong acids. This study highlights the potential of microbial production of plant derived triterpenoids in S. cerevisiae by combining metabolic and process engineering.